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Sample records for rapamycin-insensitive mtorc2 pathway

  1. Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin.

    Science.gov (United States)

    Bhagwat, Shripad V; Gokhale, Prafulla C; Crew, Andrew P; Cooke, Andy; Yao, Yan; Mantis, Christine; Kahler, Jennifer; Workman, Jennifer; Bittner, Mark; Dudkin, Lorina; Epstein, David M; Gibson, Neil W; Wild, Robert; Arnold, Lee D; Houghton, Peter J; Pachter, Jonathan A

    2011-08-01

    The phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway is frequently activated in human cancers, and mTOR is a clinically validated target. mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, metabolism, proliferation, and survival. Rapamycin and its analogues partially inhibit mTOR through allosteric binding to mTORC1, but not mTORC2, and have shown clinical utility in certain cancers. Here, we report the preclinical characterization of OSI-027, a selective and potent dual inhibitor of mTORC1 and mTORC2 with biochemical IC(50) values of 22 nmol/L and 65 nmol/L, respectively. OSI-027 shows more than 100-fold selectivity for mTOR relative to PI3Kα, PI3Kβ, PI3Kγ, and DNA-PK. OSI-027 inhibits phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1 as well as the mTORC2 substrate AKT in diverse cancer models in vitro and in vivo. OSI-027 and OXA-01 (close analogue of OSI-027) potently inhibit proliferation of several rapamycin-sensitive and -insensitive nonengineered and engineered cancer cell lines and also, induce cell death in tumor cell lines with activated PI3K-AKT signaling. OSI-027 shows concentration-dependent pharmacodynamic effects on phosphorylation of 4E-BP1 and AKT in tumor tissue with resulting tumor growth inhibition. OSI-027 shows robust antitumor activity in several different human xenograft models representing various histologies. Furthermore, in COLO 205 and GEO colon cancer xenograft models, OSI-027 shows superior efficacy compared with rapamycin. Our results further support the important role of mTOR as a driver of tumor growth and establish OSI-027 as a potent anticancer agent. OSI-027 is currently in phase I clinical trials in cancer patients. ©2011 AACR

  2. Architecture of the human mTORC2 core complex.

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    Stuttfeld, Edward; Aylett, Christopher Hs; Imseng, Stefan; Boehringer, Daniel; Scaiola, Alain; Sauer, Evelyn; Hall, Michael N; Maier, Timm; Ban, Nenad

    2018-02-09

    The mammalian target of rapamycin (mTOR) is a key protein kinase controlling cellular metabolism and growth. It is part of the two structurally and functionally distinct multiprotein complexes mTORC1 and mTORC2. Dysregulation of mTOR occurs in diabetes, cancer and neurological disease. We report the architecture of human mTORC2 at intermediate resolution, revealing a conserved binding site for accessory proteins on mTOR and explaining the structural basis for the rapamycin insensitivity of the complex. © 2018, Stuttfeld et al.

  3. CDX2 Stimulates the Proliferation of Porcine Intestinal Epithelial Cells by Activating the mTORC1 and Wnt/β-Catenin Signaling Pathways.

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    Fan, Hong-Bo; Zhai, Zhen-Ya; Li, Xiang-Guang; Gao, Chun-Qi; Yan, Hui-Chao; Chen, Zhe-Sheng; Wang, Xiu-Qi

    2017-11-18

    Caudal type homeobox 2 (CDX2) is expressed in intestinal epithelial cells and plays a role in gut development and homeostasis by regulating cell proliferation. However, whether CDX2 cooperates with the mammalian target of rapamycin complex 1 (mTORC1) and Wnt/β-catenin signaling pathways to stimulate cell proliferation remains unknown. The objective of this study was to investigate the effect of CDX2 on the proliferation of porcine jejunum epithelial cells (IPEC-J2) and the correlation between CDX2, the mTORC1 and Wnt/β-catenin signaling pathways. CDX2 overexpression and knockdown cell culture models were established to explore the regulation of CDX2 on both pathways. Pathway-specific antagonists were used to verify the effects. The results showed that CDX2 overexpression increased IPEC-J2 cell proliferation and activated both the mTORC1 and Wnt/β-catenin pathways, and that CDX2 knockdown decreased cell proliferation and inhibited both pathways. Furthermore, the mTORC1 and Wnt/β-catenin pathway-specific antagonist rapamycin and XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)]-4H -thiopyrano[4,3-d]pyrimidin-4-one) both suppressed the proliferation of IPEC-J2 cells overexpressing CDX2, and that the combination of rapamycin and XAV939 had an additive effect. Regardless of whether the cells were treated with rapamycin or XAV939 alone or in combination, both mTORC1 and Wnt/β-catenin pathways were down-regulated, accompanied by a decrease in CDX2 expression. Taken together, our data indicate that CDX2 stimulates porcine intestinal epithelial cell proliferation by activating the mTORC1 and Wnt/β-catenin signaling pathways.

  4. mTORC2 controls actin polymerization required for consolidation of long-term memory

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    Huang, Wei; Zhu, Ping Jun; Zhang, Shixing; Zhou, Hongyi; Stoica, Loredana; Galiano, Mauricio; Krnjević, Krešimir; Roman, Gregg; Costa-Mattioli, Mauro

    2013-01-01

    A major goal of biomedical research has been the identification of molecular mechanisms that can enhance memory. Here we report a novel signaling pathway that regulates the conversion from short- to long-term memory. The mTOR complex 2 (mTORC2), which contains the key regulatory protein Rictor (Rapamycin-Insensitive Companion of mTOR), was discovered only recently, and little is known about its physiological role. We show that conditional deletion of rictor in the postnatal murine forebrain greatly reduces mTORC2 activity and selectively impairs both long-term memory (LTM) and the late (but not the early) phase of hippocampal long-term potentiation (LTP). Actin polymerization is reduced in the hippocampus of mTORC2-deficient mice and its restoration rescues both L-LTP and LTM. More importantly, a compound that selectively promotes mTORC2 activity converts early-LTP into late-LTP and enhances LTM. These findings indicate that mTORC2 could be a novel therapeutic target for the treatment of cognitive dysfunction. PMID:23455608

  5. Brassinosteriod Insensitive 2 (BIN2) acts as a downstream effector of the Target of Rapamycin (TOR) signaling pathway to regulate photoautotrophic growth in Arabidopsis.

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    Xiong, Fangjie; Zhang, Rui; Meng, Zhigang; Deng, Kexuan; Que, Yumei; Zhuo, Fengping; Feng, Li; Guo, Sundui; Datla, Raju; Ren, Maozhi

    2017-01-01

    The components of the target of rapamycin (TOR) signaling pathway have been well characterized in heterotrophic organisms from yeast to humans. However, because of rapamycin insensitivity, embryonic lethality in tor null mutants and a lack of reliable ways of detecting TOR protein kinase in higher plants, the key players upstream and downstream of TOR remain largely unknown in plants. Using engineered rapamycin-sensitive Binding Protein 12-2 (BP12-2) plants, the present study showed that combined treatment with rapamycin and active-site TOR inhibitors (asTORis) results in synergistic inhibition of TOR activity and plant growth in Arabidopsis. Based on this system, we revealed that TOR signaling plays a crucial role in modulating the transition from heterotrophic to photoautotrophic growth in Arabidopsis. Ribosomal protein S6 kinase 2 (S6K2) was identified as a direct downstream target of TOR, and the growth of TOR-suppressed plants could be rescued by up-regulating S6K2. Systems, genetic, and biochemical analyses revealed that Brassinosteriod Insensitive 2 (BIN2) acts as a novel downstream effector of S6K2, and the phosphorylation of BIN2 depends on TOR-S6K2 signaling in Arabidopsis. By combining pharmacological with genetic and biochemical approaches, we determined that the TOR-S6K2-BIN2 signaling pathway plays important roles in regulating the photoautotrophic growth of Arabidopsis. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  6. Phosphoproteomic profiling of in vivo signaling in liver by the mammalian target of rapamycin complex 1 (mTORC1.

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    Gokhan Demirkan

    Full Text Available Our understanding of signal transduction networks in the physiological context of an organism remains limited, partly due to the technical challenge of identifying serine/threonine phosphorylated peptides from complex tissue samples. In the present study, we focused on signaling through the mammalian target of rapamycin (mTOR complex 1 (mTORC1, which is at the center of a nutrient- and growth factor-responsive cell signaling network. Though studied extensively, the mechanisms involved in many mTORC1 biological functions remain poorly understood.We developed a phosphoproteomic strategy to purify, enrich and identify phosphopeptides from rat liver homogenates. Using the anticancer drug rapamycin, the only known target of which is mTORC1, we characterized signaling in liver from rats in which the complex was maximally activated by refeeding following 48 hr of starvation. Using protein and peptide fractionation methods, TiO(2 affinity purification of phosphopeptides and mass spectrometry, we reproducibly identified and quantified over four thousand phosphopeptides. Along with 5 known rapamycin-sensitive phosphorylation events, we identified 62 new rapamycin-responsive candidate phosphorylation sites. Among these were PRAS40, gephyrin, and AMP kinase 2. We observed similar proportions of increased and reduced phosphorylation in response to rapamycin. Gene ontology analysis revealed over-representation of mTOR pathway components among rapamycin-sensitive phosphopeptide candidates.In addition to identifying potential new mTORC1-mediated phosphorylation events, and providing information relevant to the biology of this signaling network, our experimental and analytical approaches indicate the feasibility of large-scale phosphoproteomic profiling of tissue samples to study physiological signaling events in vivo.

  7. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

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    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Okada, Masato, E-mail: okadam@biken.osaka-u.ac.jp [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. Black-Right-Pointing-Pointer We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. Black-Right-Pointing-Pointer The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. Black-Right-Pointing-Pointer Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. Black-Right-Pointing-Pointer The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.

  8. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

    International Nuclear Information System (INIS)

    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose; Okada, Masato

    2012-01-01

    Highlights: ► p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. ► We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. ► The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. ► Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. ► The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome–lysosome fusion, which is required for processing of various macromolecules.

  9. MNK Controls mTORC1:Substrate Association through Regulation of TELO2 Binding with mTORC1

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    Michael C. Brown

    2017-02-01

    Full Text Available The mechanistic target of rapamycin (mTOR integrates numerous stimuli and coordinates the adaptive response of many cellular processes. To accomplish this, mTOR associates with distinct co-factors that determine its signaling output. While many of these co-factors are known, in many cases their function and regulation remain opaque. The MAPK-interacting kinase (MNK contributes to rapamycin resistance in cancer cells. Here, we demonstrate that MNK sustains mTORC1 activity following rapamycin treatment and contributes to mTORC1 signaling following T cell activation and growth stimuli in cancer cells. We determine that MNK engages with mTORC1, promotes mTORC1 association with the phosphatidyl inositol 3′ kinase-related kinase (PIKK stabilizer, TELO2, and facilitates mTORC1:substrate binding. Moreover, our data suggest that DEPTOR, the endogenous inhibitor of mTOR, opposes mTORC1:substrate association by preventing TELO2:mTORC1 binding. Thus, MNK orchestrates counterbalancing forces that regulate mTORC1 enzymatic activity.

  10. Noncanonical Pathway for Regulation of CCL2 Expression by an mTORC1-FOXK1 Axis Promotes Recruitment of Tumor-Associated Macrophages

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    Hirokazu Nakatsumi

    2017-11-01

    Full Text Available C-C chemokine ligand 2 (CCL2 plays pivotal roles in tumor formation, progression, and metastasis. Although CCL2 expression has been found to be dependent on the nuclear factor (NF-κB signaling pathway, the regulation of CCL2 production in tumor cells has remained unclear. We have identified a noncanonical pathway for regulation of CCL2 production that is mediated by mammalian target of rapamycin complex 1 (mTORC1 but independent of NF-κB. Multiple phosphoproteomics approaches identified the transcription factor forkhead box K1 (FOXK1 as a downstream target of mTORC1. Activation of mTORC1 induces dephosphorylation of FOXK1, resulting in transactivation of the CCL2 gene. Inhibition of the mTORC1-FOXK1 axis attenuated insulin-induced CCL2 production as well as the accumulation of tumor-associated monocytes-macrophages and tumor progression in mice. Our results suggest that FOXK1 directly links mTORC1 signaling and CCL2 expression in a manner independent of NF-κB and that CCL2 produced by this pathway contributes to tumor progression.

  11. Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system.

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    Arriola Apelo, Sebastian I; Neuman, Joshua C; Baar, Emma L; Syed, Faizan A; Cummings, Nicole E; Brar, Harpreet K; Pumper, Cassidy P; Kimple, Michelle E; Lamming, Dudley W

    2016-02-01

    Inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway by the FDA-approved drug rapamycin has been shown to promote lifespan and delay age-related diseases in model organisms including mice. Unfortunately, rapamycin has potentially serious side effects in humans, including glucose intolerance and immunosuppression, which may preclude the long-term prophylactic use of rapamycin as a therapy for age-related diseases. While the beneficial effects of rapamycin are largely mediated by the inhibition of mTOR complex 1 (mTORC1), which is acutely sensitive to rapamycin, many of the negative side effects are mediated by the inhibition of a second mTOR-containing complex, mTORC2, which is much less sensitive to rapamycin. We hypothesized that different rapamycin dosing schedules or the use of FDA-approved rapamycin analogs with different pharmacokinetics might expand the therapeutic window of rapamycin by more specifically targeting mTORC1. Here, we identified an intermittent rapamycin dosing schedule with minimal effects on glucose tolerance, and we find that this schedule has a reduced impact on pyruvate tolerance, fasting glucose and insulin levels, beta cell function, and the immune system compared to daily rapamycin treatment. Further, we find that the FDA-approved rapamycin analogs everolimus and temsirolimus efficiently inhibit mTORC1 while having a reduced impact on glucose and pyruvate tolerance. Our results suggest that many of the negative side effects of rapamycin treatment can be mitigated through intermittent dosing or the use of rapamycin analogs. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  12. The cannabinoid CB1 receptor and mTORC1 signalling pathways interact to modulate glucose homeostasis in mice

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    Francisco J. Bermudez-Silva

    2016-01-01

    Full Text Available The endocannabinoid system (ECS is an intercellular signalling mechanism that is present in the islets of Langerhans and plays a role in the modulation of insulin secretion and expansion of the β-cell mass. The downstream signalling pathways mediating these effects are poorly understood. Mammalian target of rapamycin complex 1 (mTORC1 signalling is a key intracellular pathway involved in energy homeostasis and is known to importantly affect the physiology of pancreatic islets. We investigated the possible relationship between cannabinoid type 1 (CB1 receptor signalling and the mTORC1 pathway in the endocrine pancreas of mice by using pharmacological analysis as well as mice genetically lacking the CB1 receptor or the downstream target of mTORC1, the kinase p70S6K1. In vitro static secretion experiments on islets, western blotting, and in vivo glucose and insulin tolerance tests were performed. The CB1 receptor antagonist rimonabant decreased glucose-stimulated insulin secretion (GSIS at 0.1 µM while increasing phosphorylation of p70S6K1 and ribosomal protein S6 (rpS6 within the islets. Specific pharmacological blockade of mTORC1 by 3 nM rapamycin, as well as genetic deletion of p70S6K1, impaired the CB1-antagonist-mediated decrease in GSIS. In vivo experiments showed that 3 mg/kg body weight rimonabant decreased insulin levels and induced glucose intolerance in lean mice without altering peripheral insulin sensitivity; this effect was prevented by peripheral administration of low doses of rapamycin (0.1 mg/kg body weight, which increased insulin sensitivity. These findings suggest a functional interaction between the ECS and the mTORC1 pathway within the endocrine pancreas and at the whole-organism level, which could have implications for the development of new therapeutic approaches for pancreatic β-cell diseases.

  13. Rac1 Regulates the Activity of mTORC1 and mTORC2 and Controls Cellular Size

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    Saci, Abdelhafid; Cantley, Lewis C.; Carpenter, Christopher L.

    2013-01-01

    SUMMARY Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two separate complexes, mTORC1 and mTORC2, that function to control cell size and growth in response to growth factors, nutrients, and cellular energy levels. Low molecular weight GTP-binding proteins of the Rheb and Rag families are key regulators of the mTORC1 complex, but regulation of mTORC2 is poorly understood. Here, we report that Rac1, a member of the Rho family of GTPases, is a critical regulator of both mTORC1 and mTORC2 in response to growth-factor stimulation. Deletion of Rac1 in primary cells using an inducible-Cre/Lox approach inhibits basal and growth-factor activation of both mTORC1 and mTORC2. Rac1 appears to bind directly to mTOR and to mediate mTORC1 and mTORC2 localization at specific membranes. Binding of Rac1 to mTOR does not depend on the GTP-bound state of Rac1, but on the integrity of its C-terminal domain. This function of Rac1 provides a means to regulate mTORC1 and mTORC2 simultaneously. PMID:21474067

  14. Dynamin-dependent amino acid endocytosis activates mechanistic target of rapamycin complex 1 (mTORC1).

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    Shibutani, Shusaku; Okazaki, Hana; Iwata, Hiroyuki

    2017-11-03

    The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis and potential target for modifying cellular metabolism in various conditions, including cancer and aging. mTORC1 activity is tightly regulated by the availability of extracellular amino acids, and previous studies have revealed that amino acids in the extracellular fluid are transported to the lysosomal lumen. There, amino acids induce recruitment of cytoplasmic mTORC1 to the lysosome by the Rag GTPases, followed by mTORC1 activation by the small GTPase Ras homolog enriched in brain (Rheb). However, how the extracellular amino acids reach the lysosomal lumen and activate mTORC1 remains unclear. Here, we show that amino acid uptake by dynamin-dependent endocytosis plays a critical role in mTORC1 activation. We found that mTORC1 is inactivated when endocytosis is inhibited by overexpression of a dominant-negative form of dynamin 2 or by pharmacological inhibition of dynamin or clathrin. Consistently, the recruitment of mTORC1 to the lysosome was suppressed by the dynamin inhibition. The activity and lysosomal recruitment of mTORC1 were rescued by increasing intracellular amino acids via cycloheximide exposure or by Rag overexpression, indicating that amino acid deprivation is the main cause of mTORC1 inactivation via the dynamin inhibition. We further show that endocytosis inhibition does not induce autophagy even though mTORC1 inactivation is known to strongly induce autophagy. These findings open new perspectives for the use of endocytosis inhibitors as potential agents that can effectively inhibit nutrient utilization and shut down the upstream signals that activate mTORC1. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Prostaglandin E2 activates the mTORC1 pathway through an EP4/cAMP/PKA- and EP1/Ca2+-mediated mechanism in the human pancreatic carcinoma cell line PANC-1.

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    Chang, Hui-Hua; Young, Steven H; Sinnett-Smith, James; Chou, Caroline Ei Ne; Moro, Aune; Hertzer, Kathleen M; Hines, Oscar Joe; Rozengurt, Enrique; Eibl, Guido

    2015-11-15

    Obesity, a known risk factor for pancreatic cancer, is associated with inflammation and insulin resistance. Proinflammatory prostaglandin E2 (PGE2) and elevated insulin-like growth factor type 1 (IGF-1), related to insulin resistance, are shown to play critical roles in pancreatic cancer progression. We aimed to explore a potential cross talk between PGE2 signaling and the IGF-1/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway in pancreatic cancer, which may be a key to unraveling the obesity-cancer link. In PANC-1 human pancreatic cancer cells, we showed that PGE2 stimulated mTORC1 activity independently of Akt, as evaluated by downstream signaling events. Subsequently, using pharmacological and genetic approaches, we demonstrated that PGE2-induced mTORC1 activation is mediated by the EP4/cAMP/PKA pathway, as well as an EP1/Ca(2+)-dependent pathway. The cooperative roles of the two pathways were supported by the maximal inhibition achieved with the combined pharmacological blockade, and the coexistence of highly expressed EP1 (mediating the Ca(2+) response) and EP2 or EP4 (mediating the cAMP/PKA pathway) in PANC-1 cells and in the prostate cancer line PC-3, which also robustly exhibited PGE2-induced mTORC1 activation, as identified from a screen in various cancer cell lines. Importantly, we showed a reinforcing interaction between PGE2 and IGF-1 on mTORC1 signaling, with an increase in IL-23 production as a cellular outcome. Our data reveal a previously unrecognized mechanism of PGE2-stimulated mTORC1 activation mediated by EP4/cAMP/PKA and EP1/Ca(2+) signaling, which may be of great importance in elucidating the promoting effects of obesity in pancreatic cancer. Ultimately, a precise understanding of these molecular links may provide novel targets for efficacious interventions devoid of adverse effects. Copyright © 2015 the American Physiological Society.

  16. PGE2-induced colon cancer growth is mediated by mTORC1

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    Dufour, Marc; Faes, Seraina; Dormond-Meuwly, Anne; Demartines, Nicolas; Dormond, Olivier

    2014-01-01

    Highlights: • PGE 2 activates mTORC1 in colon cancer cells. • Inhibition of mTORC1 blocks PGE 2 induced colon cancer cell growth. • mTORC1 is a signaling intermediary in PGE 2 induced colon cancer cell responses. - Abstract: The inflammatory prostaglandin E 2 (PGE 2 ) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE 2 directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE 2 -induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE 2 increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE 2 EP 4 receptor was responsible for transducing the signal to mTORC1. Moreover, PGE 2 increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE 2 -induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE 2 increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE 2 mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth

  17. mTORC2 Promotes Tumorigenesis via Lipid Synthesis.

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    Guri, Yakir; Colombi, Marco; Dazert, Eva; Hindupur, Sravanth K; Roszik, Jason; Moes, Suzette; Jenoe, Paul; Heim, Markus H; Riezman, Isabelle; Riezman, Howard; Hall, Michael N

    2017-12-11

    Dysregulated mammalian target of rapamycin (mTOR) promotes cancer, but underlying mechanisms are poorly understood. We describe an mTOR-driven mouse model that displays hepatosteatosis progressing to hepatocellular carcinoma (HCC). Longitudinal proteomic, lipidomics, and metabolomic analyses revealed that hepatic mTORC2 promotes de novo fatty acid and lipid synthesis, leading to steatosis and tumor development. In particular, mTORC2 stimulated sphingolipid (glucosylceramide) and glycerophospholipid (cardiolipin) synthesis. Inhibition of fatty acid or sphingolipid synthesis prevented tumor development, indicating a causal effect in tumorigenesis. Increased levels of cardiolipin were associated with tubular mitochondria and enhanced oxidative phosphorylation. Furthermore, increased lipogenesis correlated with elevated mTORC2 activity and HCC in human patients. Thus, mTORC2 promotes cancer via formation of lipids essential for growth and energy production. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. A dynamic network model of mTOR signaling reveals TSC-independent mTORC2 regulation

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    Dalle Pezze, Piero; Sonntag, Annika G; Thien, Antje; Prentzell, Mirja T; Gödel, Markus; Fischer, Sven; Neumann-Haefelin, Elke; Huber, Tobias B; Baumeister, Ralf; Shanley, Daryl P; Thedieck, Kathrin

    2012-01-01

    The kinase mammalian target of rapamycin (mTOR) exists in two multiprotein complexes (mTORC1 and mTORC2) and is a central regulator of growth and metabolism. Insulin activation of mTORC1, mediated by phosphoinositide 3-kinase (PI3K), Akt, and the inhibitory tuberous sclerosis complex 1/2

  19. The dual mTORC1 and mTORC2 inhibitor AZD8055 inhibits head and neck squamous cell carcinoma cell growth in vivo and in vitro

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    Li, Qiang; Song, Xin-mao; Ji, Yang-yang; Jiang, Hui; Xu, Lin-gen, E-mail: drlingenxu@126.com

    2013-11-01

    Highlights: •AZD8055 induces significant cytotoxic effects in cultured HNSCC cells. •AZD8055 blocks mTORC1 and mTORC2 activation in cultured HNSCC cells. •JNK activation is required for AZD8055-induced HNSCC cell death. •AZD8055 inhibits Hep-2 cell growth in vivo, and was more efficient than rapamycin. -- Abstract: The serine/threonine kinase mammalian target of rapamycin (mTOR) promotes cell survival and proliferation, and is constitutively activated in head and neck squamous cell carcinoma (HNSCC). Thus mTOR is an important target for drug development in this disease. Here we tested the anti-tumor ability of AZD8055, the novel mTOR inhibitor, in HNSCC cells. AZD8055 induced dramatic cell death of HNSCC lines (Hep-2 and SCC-9) through autophagy. AZD8055 blocked both mTOR complex (mTORC) 1 and mTORC2 activation without affecting Erk in cultured HNSCC cells. Meanwhile, AZD8055 induced significant c-Jun N-terminal kinase (JNK) activation, which was also required for cancer cell death. JNK inhibition by its inhibitors (SP 600125 and JNK-IN-8), or by RNA interference (RNAi) alleviated AZD8055-induced cell death. Finally, AZD8055 markedly increased the survival of Hep-2 transplanted mice through a significant reduction of tumor growth, without apparent toxicity, and its anti-tumor ability was more potent than rapamycin. Meanwhile, AZD8055 administration activated JNK while blocking mTORC1/2 in Hep-2 tumor engrafts. Our current results strongly suggest that AZD8055 may be further investigated for HNSCC treatment in clinical trials.

  20. Inhibition of Mammalian Target of Rapamycin Complex 1 Attenuates Salt-Induced Hypertension and Kidney Injury in Dahl Salt-Sensitive Rats.

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    Kumar, Vikash; Wollner, Clayton; Kurth, Theresa; Bukowy, John D; Cowley, Allen W

    2017-10-01

    The goal of the present study was to explore the protective effects of mTORC1 (mammalian target of rapamycin complex 1) inhibition by rapamycin on salt-induced hypertension and kidney injury in Dahl salt-sensitive (SS) rats. We have previously demonstrated that H 2 O 2 is elevated in the kidneys of SS rats. The present study showed a significant upregulation of renal mTORC1 activity in the SS rats fed a 4.0% NaCl for 3 days. In addition, renal interstitial infusion of H 2 O 2 into salt-resistant Sprague Dawley rats for 3 days was also found to stimulate mTORC1 activity independent of a rise of arterial blood pressure. Together, these data indicate that the salt-induced increases of renal H 2 O 2 in SS rats activated the mTORC1 pathway. Daily administration of rapamycin (IP, 1.5 mg/kg per day) for 21 days reduced salt-induced hypertension from 176.0±9.0 to 153.0±12.0 mm Hg in SS rats but had no effect on blood pressure salt sensitivity in Sprague Dawley treated rats. Compared with vehicle, rapamycin reduced albumin excretion rate in SS rats from 190.0±35.0 to 37.0±5.0 mg/d and reduced the renal infiltration of T lymphocytes (CD3 + ) and macrophages (ED1 + ) in the cortex and medulla. Renal hypertrophy and cell proliferation were also reduced in rapamycin-treated SS rats. We conclude that enhancement of intrarenal H 2 O 2 with a 4.0% NaCl diet stimulates the mTORC1 pathway that is necessary for the full development of the salt-induced hypertension and kidney injury in the SS rat. © 2017 American Heart Association, Inc.

  1. PGE{sub 2}-induced colon cancer growth is mediated by mTORC1

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    Dufour, Marc, E-mail: Marc.dufour@chuv.ch; Faes, Seraina, E-mail: Seraina.faes@chuv.ch; Dormond-Meuwly, Anne, E-mail: Anne.meuwly-Dormond@chuv.ch; Demartines, Nicolas, E-mail: Demartines@chuv.ch; Dormond, Olivier, E-mail: Olivier.dormond@chuv.ch

    2014-09-05

    Highlights: • PGE{sub 2} activates mTORC1 in colon cancer cells. • Inhibition of mTORC1 blocks PGE{sub 2} induced colon cancer cell growth. • mTORC1 is a signaling intermediary in PGE{sub 2} induced colon cancer cell responses. - Abstract: The inflammatory prostaglandin E{sub 2} (PGE{sub 2}) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE{sub 2} directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE{sub 2}-induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE{sub 2} increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE{sub 2} EP{sub 4} receptor was responsible for transducing the signal to mTORC1. Moreover, PGE{sub 2} increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE{sub 2}-induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE{sub 2} increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE{sub 2} mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth.

  2. mTORC2 activation is regulated by the urokinase receptor (uPAR) in bladder cancer.

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    Hau, Andrew M; Leivo, Mariah Z; Gilder, Andrew S; Hu, Jing-Jing; Gonias, Steven L; Hansel, Donna E

    2017-01-01

    Mammalian target of rapamycin complex 2 (mTORC2) has been identified as a major regulator of bladder cancer cell migration and invasion. Upstream pathways that mediate mTORC2 activation remain poorly defined. Urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored membrane protein and known activator of cell-signaling. We identified increased uPAR expression in 94% of invasive human bladder cancers and in 54-71% of non-invasive bladder cancers, depending on grade. Normal urothelium was uPAR-immunonegative. Analysis of publicly available datasets identified uPAR gene amplification or mRNA upregulation in a subset of bladder cancer patients with reduced overall survival. Using biochemical approaches, we showed that uPAR activates mTORC2 in bladder cancer cells. Highly invasive bladder cancer cell lines, including T24, J82 and UM-UC-3 cells, showed increased uPAR mRNA expression and protein levels compared with the less aggressive cell lines, UROtsa and RT4. uPAR gene-silencing significantly reduced phosphorylation of Serine-473 in Akt, an mTORC2 target. uPAR gene-silencing also reduced bladder cancer cell migration and Matrigel invasion. S473 phosphorylation was observed by immunohistochemistry in human bladder cancers only when the tumors expressed high levels of uPAR. S473 phosphorylation was not controlled by uPAR in bladder cancer cell lines that are PTEN-negative; however, this result probably did not reflect altered mTORC2 regulation. Instead, PTEN deficiency de-repressed alternative kinases that phosphorylate S473. Our results suggest that uPAR and mTORC2 are components of a single cell-signaling pathway. Targeting uPAR or mTORC2 may be beneficial in patients with bladder cancer. Copyright © 2016. Published by Elsevier Inc.

  3. IL-2- and IL-15-induced activation of the rapamycin-sensitive mTORC1 pathway in malignant CD4+ T lymphocytes

    DEFF Research Database (Denmark)

    Marzec, Michal; Liu, Xiaobin; Kasprzycka, Monika

    2008-01-01

    We examined functional status, activation mechanisms, and biologic role of the mTORC1 signaling pathway in malignant CD4(+) T cells derived from the cutaneous T-cell lymphoma (CTCL). Whereas the spontaneously growing CTCL-derived cell lines displayed persistent activation of the TORC1 as well as ...

  4. Rapamycin: An InhibiTOR of Aging Emerges From the Soil of Easter Island.

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    Arriola Apelo, Sebastian I; Lamming, Dudley W

    2016-07-01

    Rapamycin (sirolimus) is a macrolide immunosuppressant that inhibits the mechanistic target of rapamycin (mTOR) protein kinase and extends lifespan in model organisms including mice. Although rapamycin is an FDA-approved drug for select indications, a diverse set of negative side effects may preclude its wide-scale deployment as an antiaging therapy. mTOR forms two different protein complexes, mTORC1 and mTORC2; the former is acutely sensitive to rapamycin whereas the latter is only chronically sensitive to rapamycin in vivo. Over the past decade, it has become clear that although genetic and pharmacological inhibition of mTORC1 extends lifespan and delays aging, inhibition of mTORC2 has negative effects on mammalian health and longevity and is responsible for many of the negative side effects of rapamycin. In this review, we discuss recent advances in understanding the molecular and physiological effects of rapamycin treatment, and we discuss how the use of alternative rapamycin treatment regimens or rapamycin analogs has the potential to mitigate the deleterious side effects of rapamycin treatment by more specifically targeting mTORC1. Although the side effects of rapamycin are still of significant concern, rapid progress is being made in realizing the revolutionary potential of rapamycin-based therapies for the treatment of diseases of aging. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Amino Acids Attenuate Insulin Action on Gluconeogenesis and Promote Fatty Acid Biosynthesis via mTORC1 Signaling Pathway in trout Hepatocytes

    Directory of Open Access Journals (Sweden)

    Weiwei Dai

    2015-06-01

    Full Text Available Background/Aims: Carnivores exhibit poor utilization of dietary carbohydrates and glucose intolerant phenotypes, yet it remains unclear what are the causal factors and underlying mechanisms. We aimed to evaluate excessive amino acids (AAs-induced effects on insulin signaling, fatty acid biosynthesis and glucose metabolism in rainbow trout and determine the potential involvement of mTORC1 and p38 MAPK pathway. Methods: We stimulated trout primary hepatocytes with different AA levels and employed acute administration of rapamycin to inhibit mTORC1 activation. Results: Increased AA levels enhanced the phosphorylation of ribosomal protein S6 kinase (S6K1, S6, and insulin receptor substrate 1 (IRS-1 on Ser302 but suppressed Akt and p38 phosphorylation; up-regulated the expression of genes related to gluconeogenesis and fatty acid biosynthesis. mTORC1 inhibition not only inhibited the phosphorylation of mTORC1 downstream targets, but also blunted IRS-1 Ser302 phosphorylation and restored excessive AAs-suppressed Akt phosphorylation. Rapamycin also inhibited fatty acid biosynthetic and gluconeogenic gene expression. Conclusion: High levels of AAs up-regulate hepatic fatty acid biosynthetic gene expression through an mTORC1-dependent manner, while attenuate insulin-mediated repression of gluconeogenesis through elevating IRS-1 Ser302 phosphorylation, which in turn impairs Akt activation and thereby weakening insulin action. We propose that p38 MAPK probably also involves in these AAs-induced metabolic changes.

  6. Convergence of the mammalian target of rapamycin complex 1- and glycogen synthase kinase 3-β-signaling pathways regulates the innate inflammatory response.

    Science.gov (United States)

    Wang, Huizhi; Brown, Jonathan; Gu, Zhen; Garcia, Carlos A; Liang, Ruqiang; Alard, Pascale; Beurel, Eléonore; Jope, Richard S; Greenway, Terrance; Martin, Michael

    2011-05-01

    The PI3K pathway and its regulation of mammalian target of rapamycin complex 1 (mTORC1) and glycogen synthase kinase 3 (GSK3) play pivotal roles in controlling inflammation. In this article, we show that mTORC1 and GSK3-β converge and that the capacity of mTORC1 to affect the inflammatory response is due to the inactivation of GSK3-β. Inhibition of mTORC1 attenuated GSK3 phosphorylation and increased its kinase activity. Immunoprecipitation and in vitro kinase assays demonstrated that GSK3-β associated with a downstream target of mTORC1, p85S6K, and phosphorylated GSK3-β. Inhibition of S6K1 abrogated the phosphorylation of GSK3-β while increasing and decreasing the levels of IL-12 and IL-10, respectively, in LPS-stimulated monocytes. In contrast, the direct inhibition of GSK3 attenuated the capacity of S6K1 inhibition to influence the levels of IL-10 and IL-12 produced by LPS-stimulated cells. At the transcriptional level, mTORC1 inhibition reduced the DNA binding of CREB and this effect was reversed by GSK3 inhibition. As a result, mTORC1 inhibition increased the levels of NF-κB p65 associated with CREB-binding protein. Inhibition of NF-κB p65 attenuated rapamycin's ability to influence the levels of pro- or anti-inflammatory cytokine production in monocytes stimulated with LPS. These studies identify the molecular mechanism by which mTORC1 affects GSK3 and show that mTORC1 inhibition regulates pro- and anti-inflammatory cytokine production via its capacity to inactivate GSK3.

  7. A new functional role for mechanistic/mammalian target of rapamycin complex 1 (mTORC1 in the circadian regulation of L-type voltage-gated calcium channels in avian cone photoreceptors.

    Directory of Open Access Journals (Sweden)

    Cathy Chia-Yu Huang

    Full Text Available In the retina, the L-type voltage-gated calcium channels (L-VGCCs are responsible for neurotransmitter release from photoreceptors and are under circadian regulation. Both the current densities and protein expression of L-VGCCs are significantly higher at night than during the day. However, the underlying mechanisms of circadian regulation of L-VGCCs in the retina are not completely understood. In this study, we demonstrated that the mechanistic/mammalian target of rapamycin complex (mTORC signaling pathway participated in the circadian phase-dependent modulation of L-VGCCs. The activities of the mTOR cascade, from mTORC1 to its downstream targets, displayed circadian oscillations throughout the course of a day. Disruption of mTORC1 signaling dampened the L-VGCC current densities, as well as the protein expression of L-VGCCs at night. The decrease of L-VGCCs at night by mTORC1 inhibition was in part due to a reduction of L-VGCCα1 subunit translocation from the cytosol to the plasma membrane. Finally, we showed that mTORC1 was downstream of the phosphatidylionositol 3 kinase-protein kinase B (PI3K-AKT signaling pathway. Taken together, mTORC1 signaling played a role in the circadian regulation of L-VGCCs, in part through regulation of ion channel trafficking and translocation, which brings to light a new functional role for mTORC1: the modulation of ion channel activities.

  8. Differential Role of Rapamycin in Epidermis-Induced IL-15-IGF-1 Secretion via Activation of Akt/mTORC2.

    Science.gov (United States)

    Bai, Yang; Xu, Rui; Zhang, Xueyuan; Zhang, Xiaorong; Hu, Xiaohong; Li, Yashu; Li, Haisheng; Liu, Meixi; Huang, Zhenggen; Yan, Rongshuai; He, Weifeng; Luo, Gaoxing; Wu, Jun

    2017-01-01

    Backgroud/Aims: The effects of rapamycin (RPM) on wound healing have been previously studied. However, reciprocal contradictory data have been reported, and the underlying mechanism remains unclear. This study aims to uncover differential role of RPM in regulation of wound healing and explore the possible mechanism. C57BL/6J mice and epidermal cells were treated with different doses of RPM. The wound re-epithelialization was observed by hematoxylin and eosin (HE) staining. The expression of IL-15 and IGF-1 were detected by immunohistochemistry and quantitative real-time PCR. Epidermal cell survival was determined by CCK-8 assays. Moreover, the mTORC1 and mTORC2 pathway were examined by western blot analysis. This study showed that differential doses of RPM could lead to separate consequences in epidermis. Histological analyses showed that low-dose RPM promoted wound healing, and enhanced the expression of IL-15 and IGF-1. Furthermore, western blot analysis showed that the effect of low-dose RPM in epidermis were not through mTORC1 pathway. Instead, activation of the Akt/mTORC2 pathway was involved in low-dose RPM-induced IL-15 and IGF-1 production in epidermis, while high-dose RPM inhibited the expression of IL-15 and IGF-1 and the activity of mTORC1 and mTORC2 pathway. This study for the first time demonstrated that RPM-mediated wound healing was dose-dependent. © 2017 The Author(s). Published by S. Karger AG, Basel.

  9. Acidic tumor microenvironment abrogates the efficacy of mTORC1 inhibitors.

    Science.gov (United States)

    Faes, Seraina; Duval, Adrian P; Planche, Anne; Uldry, Emilie; Santoro, Tania; Pythoud, Catherine; Stehle, Jean-Christophe; Horlbeck, Janine; Letovanec, Igor; Riggi, Nicolo; Demartines, Nicolas; Dormond, Olivier

    2016-12-05

    Blocking the mechanistic target of rapamycin complex-1 (mTORC1) with chemical inhibitors such as rapamycin has shown limited clinical efficacy in cancer. The tumor microenvironment is characterized by an acidic pH which interferes with cancer therapies. The consequences of acidity on the anti-cancer efficacy of mTORC1 inhibitors have not been characterized and are thus the focus of our study. Cancer cell lines were treated with rapamycin in acidic or physiological conditions and cell proliferation was investigated. The effect of acidity on mTORC1 activity was determined by Western blot. The anticancer efficacy of rapamycin in combination with sodium bicarbonate to increase the intratumoral pH was tested in two different mouse models and compared to rapamycin treatment alone. Histological analysis was performed on tumor samples to evaluate proliferation, apoptosis and necrosis. Exposing cancer cells to acidic pH in vitro significantly reduced the anti-proliferative effect of rapamycin. At the molecular level, acidity significantly decreased mTORC1 activity, suggesting that cancer cell proliferation is independent of mTORC1 in acidic conditions. In contrast, the activation of mitogen-activated protein kinase (MAPK) or AKT were not affected by acidity, and blocking MAPK or AKT with a chemical inhibitor maintained an anti-proliferative effect at low pH. In tumor mouse models, the use of sodium bicarbonate increased mTORC1 activity in cancer cells and potentiated the anti-cancer efficacy of rapamycin. Combining sodium bicarbonate with rapamycin resulted in increased tumor necrosis, increased cancer cell apoptosis and decreased cancer cell proliferation as compared to single treatment. Taken together, these results emphasize the inefficacy of mTORC1 inhibitors in acidic conditions. They further highlight the potential of combining sodium bicarbonate with mTORC1 inhibitors to improve their anti-tumoral efficacy.

  10. Quantitative analysis of male germline stem cell differentiation reveals a role for the p53-mTORC1 pathway in spermatogonial maintenance.

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    Xiong, Mulin; Ferder, Ianina C; Ohguchi, Yasuyo; Wang, Ning

    2015-01-01

    p53 protects cells from DNA damage by inducing cell-cycle arrest upon encountering genomic stress. Among other pathways, p53 elicits such an effect by inhibiting mammalian target of rapamycin complex 1 (mTORC1), the master regulator of cell proliferation and growth. Although recent studies have indicated roles for both p53 and mTORC1 in stem cell maintenance, it remains unclear whether the p53-mTORC1 pathway is conserved to mediate this process under normal physiological conditions. Spermatogenesis is a classic stem cell-dependent process in which undifferentiated spermatogonia undergo self-renewal and differentiation to maintain the lifelong production of spermatozoa. To better understand this process, we have developed a novel flow cytometry (FACS)-based approach that isolates spermatogonia at consecutive differentiation stages. By using this as a tool, we show that genetic loss of p53 augments mTORC1 activity during early spermatogonial differentiation. Functionally, loss of p53 drives spermatogonia out of the undifferentiated state and causes a consistent expansion of early differentiating spermatogonia until the stage of preleptotene (premeiotic) spermatocyte. The frequency of early meiotic spermatocytes is, however, dramatically decreased. Thus, these data suggest that p53-mTORC1 pathway plays a critical role in maintaining the homeostasis of early spermatogonial differentiation. Moreover, our FACS approach could be a valuable tool in understanding spermatogonial differentiation.

  11. Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation.

    Science.gov (United States)

    Suryawan, Agus; Jeyapalan, Asumthia S; Orellana, Renan A; Wilson, Fiona A; Nguyen, Hanh V; Davis, Teresa A

    2008-10-01

    Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.

  12. Selective targeting of the mTORC1/2 protein kinase complexes leads to antileukemic effects in vitro and in vivo

    International Nuclear Information System (INIS)

    Schuster, K; Zheng, J; Arbini, A A; Zhang, C C; Scaglioni, P P

    2011-01-01

    The BCR/ABL tyrosine kinase promotes leukemogenesis through activation of several targets that include the phosphoinositide 3-kinase (PI3K). Tyrosine kinase inhibitors (TKIs), which target BCR/ABL, induce striking clinical responses. However, therapy with TKIs is associated with limitations such as drug intolerance, inability to universally eradicate the disease and emergence of BCR/ABL drug-resistant mutants. To overcome these limitations, we tested whether inhibition of the PI3K/target of rapamycin (mTOR) signaling pathway has antileukemic effect in primary hematopoietic stem cells and BA/F3 cells expressing the BCR/ABL oncoprotein. We determined that dual inhibition of PI3K/mTOR causes growth arrest and apoptosis leading to profound antileukemic effects both in vitro and in vivo. We also established that pharmacologic inhibition of the mTORC1/mTORC2 complexes is sufficient to cause these antileukemic effects. Our results support the development of inhibitors of the mTORC1/2 complexes for the therapy of leukemias that either express BCR/ABL or display deregulation of the PI3K/mTOR signaling pathway

  13. The E3 ubiquitin ligase protein associated with Myc (Pam) regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling in vivo through N- and C-terminal domains.

    Science.gov (United States)

    Han, Sangyeul; Kim, Sun; Bahl, Samira; Li, Lin; Burande, Clara F; Smith, Nicole; James, Marianne; Beauchamp, Roberta L; Bhide, Pradeep; DiAntonio, Aaron; Ramesh, Vijaya

    2012-08-31

    Pam and its homologs (the PHR protein family) are large E3 ubiquitin ligases that function to regulate synapse formation and growth in mammals, zebrafish, Drosophila, and Caenorhabditis elegans. Phr1-deficient mouse models (Phr1(Δ8,9) and Phr1(Magellan), with deletions in the N-terminal putative guanine exchange factor region and the C-terminal ubiquitin ligase region, respectively) exhibit axon guidance/outgrowth defects and striking defects of major axon tracts in the CNS. Our earlier studies identified Pam to be associated with tuberous sclerosis complex (TSC) proteins, ubiquitinating TSC2 and regulating mammalian/mechanistic target of rapamycin (mTOR) signaling. Here, we examine the potential involvement of the TSC/mTOR complex 1(mTORC1) signaling pathway in Phr1-deficient mouse models. We observed attenuation of mTORC1 signaling in the brains of both Phr1(Δ8,9) and Phr1(Magellan) mouse models. Our results establish that Pam regulates TSC/mTOR signaling in vitro and in vivo through two distinct domains. To further address whether Pam regulates mTORC1 through two functionally independent domains, we undertook heterozygous mutant crossing between Phr1(Δ8,9) and Phr1(Magellan) mice to generate a compound heterozygous model to determine whether these two domains can complement each other. mTORC1 signaling was not attenuated in the brains of double mutants (Phr1(Δ8,9/Mag)), confirming that Pam displays dual regulation of the mTORC1 pathway through two functional domains. Our results also suggest that although dysregulation of mTORC1 signaling may be responsible for the corpus callosum defects, other neurodevelopmental defects observed with Phr1 deficiency are independent of mTORC1 signaling. The ubiquitin ligase complex containing Pam-Fbxo45 likely targets additional synaptic and axonal proteins, which may explain the overlapping neurodevelopmental defects observed in Phr1 and Fbxo45 deficiency.

  14. Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner.

    Science.gov (United States)

    Newton, Phillip T; Vuppalapati, Karuna K; Bouderlique, Thibault; Chagin, Andrei S

    2015-01-01

    Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H(+)-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A1 or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.

  15. Arctigenin exerts anti-colitis efficacy through inhibiting the differentiation of Th1 and Th17 cells via an mTORC1-dependent pathway.

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    Wu, Xin; Dou, Yannong; Yang, Yan; Bian, Difei; Luo, Jinque; Tong, Bei; Xia, Yufeng; Dai, Yue

    2015-08-15

    Arctigenin, the main effective constituent of Arctium lappa L. fruit, has previously been proven to dramatically attenuate dextran sulfate sodium (DSS)-induced colitis in mice, a frequently used animal model of inflammatory bowel disease (IBD). As Th1 and Th17 cells play a crucial role in the pathogenesis of IBD, the present study addressed whether and how arctigenin exerted anti-colitis efficacy by interfering with the differentiation and activation of Th1/Th17 cells. In vitro, arctigenin was shown to markedly inhibit the differentiation of Th17 cells from naïve T cells, and moderately inhibit the differentiation of Th1 cells, which was accompanied by lowered phosphorylation of STAT3 and STAT4, respectively. In contrast, arctigenin was lack of marked effect on the differentiation of either Th2 or regulatory T cells. Furthermore, arctigenin was shown to suppress the mammalian target of rapamycin complex 1 (mTORC1) pathway in T cells as demonstrated by down-regulated phosphorylation of the downstream target genes p70S6K and RPS6, and it functioned independent of two well-known upstream kinases PI3K/AKT and ERK. Arctigenin was also able to inhibit the activity of mTORC1 by dissociating raptor from mTOR. Interestingly, the inhibitory effect of arctigenin on T cell differentiation disappeared under a status of mTORC1 overactivation via knockdown of tuberous sclerosis complex 2 (TSC2, a negative regulator of mTORC1) or pretreatment of leucine (an agonist of mTOR). In DSS-induced mice, the inhibition of Th1/Th17 responses and anti-colitis effect of arctigenin were abrogated by leucine treatment. In conclusion, arctigenin ameliorates colitis through down-regulating the differentiation of Th1 and Th17 cells via mTORC1 pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Two distinct mTORC2-dependent pathways converge on Rac1 to drive breast cancer metastasis.

    Science.gov (United States)

    Morrison Joly, Meghan; Williams, Michelle M; Hicks, Donna J; Jones, Bayley; Sanchez, Violeta; Young, Christian D; Sarbassov, Dos D; Muller, William J; Brantley-Sieders, Dana; Cook, Rebecca S

    2017-06-30

    The importance of the mTOR complex 2 (mTORC2) signaling complex in tumor progression is becoming increasingly recognized. HER2-amplified breast cancers use Rictor/mTORC2 signaling to drive tumor formation, tumor cell survival and resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapy. Cell motility, a key step in the metastatic process, can be activated by mTORC2 in luminal and triple negative breast cancer cell lines, but its role in promoting metastases from HER2-amplified breast cancers is not yet clear. Because Rictor is an obligate cofactor of mTORC2, we genetically engineered Rictor ablation or overexpression in mouse and human HER2-amplified breast cancer models for modulation of mTORC2 activity. Signaling through mTORC2-dependent pathways was also manipulated using pharmacological inhibitors of mTOR, Akt, and Rac. Signaling was assessed by western analysis and biochemical pull-down assays specific for Rac-GTP and for active Rac guanine nucleotide exchange factors (GEFs). Metastases were assessed from spontaneous tumors and from intravenously delivered tumor cells. Motility and invasion of cells was assessed using Matrigel-coated transwell assays. We found that Rictor ablation potently impaired, while Rictor overexpression increased, metastasis in spontaneous and intravenously seeded models of HER2-overexpressing breast cancers. Additionally, migration and invasion of HER2-amplified human breast cancer cells was diminished in the absence of Rictor, or upon pharmacological mTOR kinase inhibition. Active Rac1 was required for Rictor-dependent invasion and motility, which rescued invasion/motility in Rictor depleted cells. Rictor/mTORC2-dependent dampening of the endogenous Rac1 inhibitor RhoGDI2, a factor that correlated directly with increased overall survival in HER2-amplified breast cancer patients, promoted Rac1 activity and tumor cell invasion/migration. The mTORC2 substrate Akt did not affect RhoGDI2 dampening, but partially

  17. Prenatal Mechanistic Target of Rapamycin Complex 1 (m TORC1) Inhibition by Rapamycin Treatment of Pregnant Mice Causes Intrauterine Growth Restriction and Alters Postnatal Cardiac Growth, Morphology, and Function.

    Science.gov (United States)

    Hennig, Maria; Fiedler, Saskia; Jux, Christian; Thierfelder, Ludwig; Drenckhahn, Jörg-Detlef

    2017-08-04

    Fetal growth impacts cardiovascular health throughout postnatal life in humans. Various animal models of intrauterine growth restriction exhibit reduced heart size at birth, which negatively influences cardiac function in adulthood. The mechanistic target of rapamycin complex 1 (mTORC1) integrates nutrient and growth factor availability with cell growth, thereby regulating organ size. This study aimed at elucidating a possible involvement of mTORC1 in intrauterine growth restriction and prenatal heart growth. We inhibited mTORC1 in fetal mice by rapamycin treatment of pregnant dams in late gestation. Prenatal rapamycin treatment reduces mTORC1 activity in various organs at birth, which is fully restored by postnatal day 3. Rapamycin-treated neonates exhibit a 16% reduction in body weight compared with vehicle-treated controls. Heart weight decreases by 35%, resulting in a significantly reduced heart weight/body weight ratio, smaller left ventricular dimensions, and reduced cardiac output in rapamycin- versus vehicle-treated mice at birth. Although proliferation rates in neonatal rapamycin-treated hearts are unaffected, cardiomyocyte size is reduced, and apoptosis increased compared with vehicle-treated neonates. Rapamycin-treated mice exhibit postnatal catch-up growth, but body weight and left ventricular mass remain reduced in adulthood. Prenatal mTORC1 inhibition causes a reduction in cardiomyocyte number in adult hearts compared with controls, which is partially compensated for by an increased cardiomyocyte volume, resulting in normal cardiac function without maladaptive left ventricular remodeling. Prenatal rapamycin treatment of pregnant dams represents a new mouse model of intrauterine growth restriction and identifies an important role of mTORC1 in perinatal cardiac growth. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  18. Key mediators of intracellular amino acids signaling to mTORC1 activation.

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    Duan, Yehui; Li, Fengna; Tan, Kunrong; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Tang, Yulong; Wu, Guoyao; Yin, Yulong

    2015-05-01

    Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

  19. Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy.

    LENUS (Irish Health Repository)

    Wander, Seth A

    2011-04-01

    Mammalian target of rapamycin (mTOR) is a PI3K-related kinase that regulates cell growth, proliferation, and survival via mTOR complex 1 (mTORC1) and mTORC2. The mTOR pathway is often aberrantly activated in cancers. While hypoxia, nutrient deprivation, and DNA damage restrain mTORC1 activity, multiple genetic events constitutively activate mTOR in cancers. Here we provide a brief overview of the signaling pathways up- and downstream of mTORC1 and -2, and discuss the insights into therapeutic anticancer targets - both those that have been tried in the clinic with limited success and those currently under clinical development - that knowledge of these pathways gives us.

  20. mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR.

    Science.gov (United States)

    Yin, Yancun; Hua, Hui; Li, Minjing; Liu, Shu; Kong, Qingbin; Shao, Ting; Wang, Jiao; Luo, Yuanming; Wang, Qian; Luo, Ting; Jiang, Yangfu

    2016-01-01

    Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor(+/+) MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor(-/-) MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.

  1. Inhibition of mTORC2 Induces Cell-Cycle Arrest and Enhances the Cytotoxicity of Doxorubicin by Suppressing MDR1 Expression in HCC Cells.

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    Chen, Bryan Wei; Chen, Wei; Liang, Hui; Liu, Hao; Liang, Chao; Zhi, Xiao; Hu, Li-Qiang; Yu, Xia-Zhen; Wei, Tao; Ma, Tao; Xue, Fei; Zheng, Lei; Zhao, Bin; Feng, Xin-Hua; Bai, Xue-Li; Liang, Ting-Bo

    2015-08-01

    mTOR is aberrantly activated in hepatocellular carcinoma (HCC) and plays pivotal roles in tumorigenesis and chemoresistance. Rapamycin has been reported to exert antitumor activity in HCC and sensitizes HCC cells to cytotoxic agents. However, due to feedback activation of AKT after mTOR complex 1 (mTORC1) inhibition, simultaneous targeting of mTORC1/2 may be more effective. In this study, we examined the interaction between the dual mTORC1/2 inhibitor OSI-027 and doxorubicin in vitro and in vivo. OSI-027 was found to reduce phosphorylation of both mTORC1 and mTORC2 substrates, including 4E-BP1, p70S6K, and AKT (Ser473), and inhibit HCC cell proliferation. Similar to OSI-027 treatment, knockdown of mTORC2 induced G0-G1 phase cell-cycle arrest. In contrast, rapamycin or knockdown of mTORC1 increased phosphorylation of AKT (Ser473), yet had little antiproliferative effect. Notably, OSI-027 synergized with doxorubicin for the antiproliferative efficacy in a manner dependent of MDR1 expression in HCC cells. The synergistic antitumor effect of OSI-027 and doxorubicin was also observed in a HCC xenograft mouse model. Moreover, AKT was required for OSI-027-induced cell-cycle arrest and downregulation of MDR1. Our findings provide a rationale for dual mTORC1/mTORC2 inhibitors, such as OSI-027, as monotherapy or in combination with cytotoxic agents to treat HCC. Mol Cancer Ther; 14(8); 1805-15. ©2015 AACR. ©2015 American Association for Cancer Research.

  2. Immunohistochemical analysis of the mechanistic target of rapamycin and hypoxia signalling pathways in basal cell carcinoma and trichoepithelioma.

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    Tjinta Brinkhuizen

    Full Text Available BACKGROUND: Basal cell carcinoma (BCC is the most common cancer in Caucasians. Trichoepithelioma (TE is a benign neoplasm that strongly resembles BCC. Both are hair follicle (HF tumours. HFs are hypoxic microenvironments, therefore we hypothesized that hypoxia-induced signalling pathways could be involved in BCC and TE as they are in other human malignancies. Hypoxia-inducible factor 1 (HIF1 and mechanistic/mammalian target of rapamycin (mTOR are key players in these pathways. OBJECTIVES: To determine whether HIF1/mTOR signalling is involved in BCC and TE. METHODS: We used immunohistochemical staining of formalin-fixed paraffin-embedded BCC (n = 45 and TE (n = 35 samples to assess activity of HIF1, mTORC1 and their most important target genes. The percentage positive tumour cells was assessed manually in a semi-quantitative manner and categorized (0%, 80%. RESULTS: Among 45 BCC and 35 TE examined, expression levels were respectively 81% and 57% (BNIP3, 73% and 75% (CAIX, 79% and 86% (GLUT1, 50% and 19% (HIF1α, 89% and 88% (pAKT, 55% and 61% (pS6, 15% and 25% (pMTOR, 44% and 63% (PHD2 and 44% and 49% (VEGF-A. CAIX, Glut1 and PHD2 expression levels were significantly higher in TE when only samples with at least 80% expression were included. CONCLUSIONS: HIF and mTORC1 signalling seems active in both BCC and TE. There are no appreciable differences between the two with respect to pathway activity. At this moment immunohistochemical analyses of HIF, mTORC1 and their target genes does not provide a reliable diagnostic tool for the discrimination of BCC and TE.

  3. The interaction between early life epilepsy and autistic-like behavioral consequences: a role for the mammalian target of rapamycin (mTOR pathway.

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    Delia M Talos

    Full Text Available Early life seizures can result in chronic epilepsy, cognitive deficits and behavioral changes such as autism, and conversely epilepsy is common in autistic children. We hypothesized that during early brain development, seizures could alter regulators of synaptic development and underlie the interaction between epilepsy and autism. The mammalian Target of Rapamycin (mTOR modulates protein translation and is dysregulated in Tuberous Sclerosis Complex, a disorder characterized by epilepsy and autism. We used a rodent model of acute hypoxia-induced neonatal seizures that results in long term increases in neuronal excitability, seizure susceptibility, and spontaneous seizures, to determine how seizures alter mTOR Complex 1 (mTORC1 signaling. We hypothesized that seizures occurring at a developmental stage coinciding with a critical period of synaptogenesis will activate mTORC1, contributing to epileptic networks and autistic-like behavior in later life. Here we show that in the rat, baseline mTORC1 activation peaks during the first three postnatal weeks, and induction of seizures at postnatal day 10 results in further transient activation of its downstream targets phospho-4E-BP1 (Thr37/46, phospho-p70S6K (Thr389 and phospho-S6 (Ser235/236, as well as rapid induction of activity-dependent upstream signaling molecules, including BDNF, phospho-Akt (Thr308 and phospho-ERK (Thr202/Tyr204. Furthermore, treatment with the mTORC1 inhibitor rapamycin immediately before and after seizures reversed early increases in glutamatergic neurotransmission and seizure susceptibility and attenuated later life epilepsy and autistic-like behavior. Together, these findings suggest that in the developing brain the mTORC1 signaling pathway is involved in epileptogenesis and altered social behavior, and that it may be a target for development of novel therapies that eliminate the progressive effects of neonatal seizures.

  4. An emerging role for the mammalian Target of Rapamycin (mTOR in 'pathological' protein translation: relevance to cocaine addiction

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    Christopher V Dayas

    2012-02-01

    Full Text Available Complex neuroadaptations within key nodes of the brain’s ‘reward circuitry’ are thought to underpin long-term vulnerability to relapse. A more comprehensive understanding of the molecular and cellular signalling events that subserve relapse vulnerability may lead to pharmacological treatments that could improve treatment outcomes for psychostimulant-addicted individuals. Recent advances in this regard include findings that drug-induced perturbations to neurotrophin, metabotropic glutamate receptor and dopamine receptor signalling pathways perpetuate plasticity impairments at excitatory glutamatergic synapses on ventral tegmental area (VTA and nucleus accumbens (NAC neurons. In the context of addiction, much previous work, in terms of downstream effectors to these receptor systems, has centered on the extracellular-regulated MAP kinase (ERK signalling pathway. The purpose of the present review is to highlight the evidence of an emerging role for another downstream effector of these addiction-relevant receptor systems - the mammalian target of rapamycin complex 1 (mTORC1. mTORC1 functions to regulate synaptic protein translation and is a potential critical link in our understanding of the neurobiological processes that drive addiction and relapse behavior. The precise cellular and molecular changes that are regulated by mTORC1 and contribute to relapse vulnerability are only just coming to light. Therefore, we aim to highlight evidence that mTORC1 signalling may be dysregulated by drug-exposure and that these changes may contribute to aberrant translation of synaptic proteins that appear critical to increased relapse vulnerability, including AMPARs. The importance of understanding the role of this signalling pathway in the development of addiction vulnerability is underscored by the fact that the mTORC1 inhibitor rapamycin reduces drug-seeking in preclinical models and preliminary evidence indicating that rapamycin suppresses drug craving in

  5. Negative Effects of Chronic Rapamycin Treatment on Behavior in a Mouse Model of Fragile X Syndrome

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    Rachel M. Saré

    2018-01-01

    Full Text Available Fragile X syndrome (FXS, the most common form of inherited intellectual disability, is also highly associated with autism spectrum disorders (ASD. It is caused by expansion of a CGG repeat sequence on the X chromosome resulting in silencing of the FMR1 gene. This is modeled in the mouse by deletion of Fmr1 (Fmr1 KO. Fmr1 KO mice recapitulate many of the behavioral features of the disorder including seizure susceptibility, hyperactivity, impaired social behavior, sleep problems, and learning and memory deficits. The mammalian target of rapamycin pathway (mTORC1 is upregulated in Fmr1 KO mice and is thought to be important for the pathogenesis of this disorder. We treated Fmr1 KO mice chronically with an mTORC1 inhibitor, rapamycin, to determine if rapamycin treatment could reverse behavioral phenotypes. We performed open field, zero maze, social behavior, sleep, passive avoidance, and audiogenic seizure testing. We found that pS6 was upregulated in Fmr1 KO mice and normalized by rapamycin treatment, but, except for an anxiogenic effect, it did not reverse any of the behavioral phenotypes examined. In fact, rapamycin treatment had an adverse effect on sleep and social behavior in both control and Fmr1 KO mice. These results suggest that targeting the mTOR pathway in FXS is not a good treatment strategy and that other pathways should be considered.

  6. Acquired resistance of EGFR-mutant lung adenocarcinomas to afatinib plus cetuximab is associated with activation of mTORC1

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    Pirazzoli, Valentina; Nebhan, Caroline; Song, Xiaoling; Wurtz, Anna; Walther, Zenta; Cai, Guoping; Zhao, Zhongming; Jia, Peilin; de Stanchina, Elisa; Shapiro, Erik M.; Gale, Molly; Yin, Ruonan; Horn, Leora; Carbone, David P.; Stephens, Philip J; Miller, Vincent; Gettinger, Scott; Pao, William; Politi, Katerina

    2014-01-01

    SUMMARY Patients with EGFR-mutant lung adenocarcinomas (LUADs) who initially respond to first-generation TKIs develop resistance to these drugs. A combination of the irreversible TKI afatinib and the EGFR antibody cetuximab can be used to overcome resistance to first-generation TKIs; however, resistance to this drug combination eventually emerges. We identified activation of the mTORC1 signaling pathway as a mechanism of resistance to dual inhibition of EGFR in mouse models. Addition of rapamycin reversed resistance in vivo. Analysis of afatinib+cetuximab-resistant biopsy specimens revealed the presence of genomic alterations in genes that modulate mTORC1 signaling including NF2 and TSC1. These findings pinpoint enhanced mTORC1 activation as a mechanism of resistance to afatinib+cetuximab and identify genomic mechanisms that lead to activation of this pathway, revealing a potential therapeutic strategy for treating patients with resistance to these drugs. PMID:24813888

  7. mTORC1 is a critical mediator of oncogenic Semaphorin3A signaling

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    Yamada, Daisuke; Kawahara, Kohichi; Maeda, Takehiko, E-mail: maeda@nupals.ac.jp

    2016-08-05

    Aberration of signaling pathways by genetic mutations or alterations in the surrounding tissue environments can result in tumor development or metastasis. However, signaling molecules responsible for these processes have not been completely elucidated. Here, we used mouse Lewis lung carcinoma cells (LLC) to explore the mechanism by which the oncogenic activity of Semaphorin3A (Sema3A) signaling is regulated. Sema3A knockdown by shRNA did not affect apoptosis, but decreased cell proliferation in LLCs; both the mammalian target of rapamycin complex 1 (mTORC1) level and glycolytic activity were also decreased. In addition, Sema3A knockdown sensitized cells to inhibition of oxidative phosphorylation by oligomycin, but conferred resistance to decreased cell viability induced by glucose starvation. Furthermore, recombinant SEMA3A rescued the attenuation of cell proliferation and glycolytic activity in LLCs after Sema3A knockdown, whereas mTORC1 inhibition by rapamycin completely counteracted this effect. These results demonstrate that Sema3A signaling exerts its oncogenic effect by promoting an mTORC1-mediated metabolic shift from oxidative phosphorylation to aerobic glycolysis. -- Highlights: •Sema3A knockdown decreased proliferation of Lewis lung carcinoma cells (LLCs). •Sema3A knockdown decreased mTORC1 levels and glycolytic activity in LLCs. •Sema3A knockdown sensitized cells to inhibition of oxidative phosphorylation. •Sema3A promotes shift from oxidative phosphorylation to aerobic glycolysis via mTORC1.

  8. Therapeutic potential of a dual mTORC1/2 inhibitor for the prevention of posterior capsule opacification: An in vitro study.

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    Feng, Hao; Yang, Zhibo; Bai, Xue; Yang, Meirong; Fang, Yuan; Zhang, Xiaonan; Guo, Qiqiang; Ning, Hong

    2018-04-01

    Mammalian target of rapamycin (mTOR) serves a central role in regulating cell growth and survival, and has been demonstrated to be involved in the pathological progression of posterior capsule opacification (PCO). In the present study, the potency of PP242, a novel dual inhibitor of mTOR complex 1/2 (mTORC1/2), in the suppression of the growth of human lens epithelial cells (HLECs) was investigated. Using a Cell Counting Kit‑8 and a wound healing assay, it was demonstrated that PP242 inhibited the proliferation and migration of HLECs. In addition, western blot analysis indicated that PP242 completely inhibited mTORC1 and mTORC2 downstream signaling activities, whereas rapamycin only partially inhibited mTORC1 activity within LECs. Furthermore, PP242 treatment led to an upregulation of the expression levels of p53 and B cell lymphoma‑2 (Bcl‑2)‑associated X and downregulation of Bcl‑2. In addition, flow cytometric analysis demonstrated that PP242 induced the cell cycle arrest at the G0/G1 phase, which may have caused apoptosis and induced autophagy within the LECs. The results of the present study suggested that administration of PP242 may potentially offer a novel therapeutic approach for the prevention of PCO.

  9. Acquired Resistance of EGFR-Mutant Lung Adenocarcinomas to Afatinib plus Cetuximab Is Associated with Activation of mTORC1

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    Valentina Pirazzoli

    2014-05-01

    Full Text Available Patients with EGFR-mutant lung adenocarcinomas (LUADs who initially respond to first-generation tyrosine kinase inhibitors (TKIs develop resistance to these drugs. A combination of the irreversible TKI afatinib and the EGFR antibody cetuximab can be used to overcome resistance to first-generation TKIs; however, resistance to this drug combination eventually emerges. We identified activation of the mTORC1 signaling pathway as a mechanism of resistance to dual inhibition of EGFR in mouse models. The addition of rapamycin reversed resistance in vivo. Analysis of afatinib-plus-cetuximab-resistant biopsy specimens revealed the presence of genomic alterations in genes that modulate mTORC1 signaling, including NF2 and TSC1. These findings pinpoint enhanced mTORC1 activation as a mechanism of resistance to afatinib plus cetuximab and identify genomic mechanisms that lead to activation of this pathway, revealing a potential therapeutic strategy for treating patients with resistance to these drugs.

  10. Transforming Growth Factor-β Is an Upstream Regulator of Mammalian Target of Rapamycin Complex 2-Dependent Bladder Cancer Cell Migration and Invasion.

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    Gupta, Sounak; Hau, Andrew M; Al-Ahmadie, Hikmat A; Harwalkar, Jyoti; Shoskes, Aaron C; Elson, Paul; Beach, Jordan R; Hussey, George S; Schiemann, William P; Egelhoff, Thomas T; Howe, Philip H; Hansel, Donna E

    2016-05-01

    Our prior work identified the mammalian target of rapamycin complex 2 (mTORC2) as a key regulator of bladder cancer cell migration and invasion, although upstream growth factor mediators of this pathway in bladder cancer have not been well delineated. We tested whether transforming growth factor (TGF)-β, which can function as a promotility factor in bladder cancer cells, could regulate mTORC2-dependent bladder cancer cell motility and invasion. In human bladder cancers, the highest levels of phosphorylated SMAD2, a TGF-β signaling intermediate, were present in high-grade invasive bladder cancers and associated with more frequent recurrence and decreased disease-specific survival. Increased expression of TGF-β isoforms, receptors, and signaling components was detected in invasive high-grade bladder cancer cells that expressed Vimentin and lacked E-cadherin. Application of TGF-β induced phosphorylation of the Ser473 residue of AKT, a selective target of mTORC2, in a SMAD2- and SMAD4-independent manner and increased bladder cancer cell migration in a modified scratch wound assay and invasion through Matrigel. Inhibition of TGF-β receptor I using SB431542 ablated TGF-β-induced migration and invasion. A similar effect was seen when Rictor, a key mTORC2 component, was selectively silenced. Our results suggest that TGF-β can induce bladder cancer cell invasion via mTORC2 signaling, which may be applicable in most bladder cancers. Copyright © 2016. Published by Elsevier Inc.

  11. Concurrent inhibition of mTORC1 and mTORC2 by WYE-687 inhibits renal cell carcinoma cell growth in vitro and in vivo.

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    Xiao-Dong Pan

    Full Text Available Mammalian target of rapamycin (mTORin renal cell carcinoma (RCC represents a valuable oncotarget for treatment. We here tested the potential anti-RCC activity by a novel mTOR kinase inhibitor WYE-687in vitro and in vivo.WYE-687 was cytotoxic and anti-proliferative to established RCC cell lines (786-O and A498 and primary human RCC cells. Yet, it was non-cytotoxic toHK-2 tubular epithelial cells.WYE-687 provoked caspase-dependent apoptosis in the RCC cells. At the molecular level, WYE-687 almost completely blocked mTORC1 (p-S6K1 and p-S6 and mTORC2 (p-Akt Ser 473 activation in both 786-Ocells and primary human RCC cells, where it downregulated both hypoxia-inducible factor (HIF-1α and HIF-2α expression. Significantly, oral administration of WYE-687 potently suppressed786-O tumor xenograft growth in nude mice. mTORC1/2 activation and HIF-1α/2α expression were also remarkably downregulated in WYE-687-treated tumor tissues. Thus, our preclinical results imply that WYE-687 may have important translational value for the treatment of RCC.

  12. The mTORC1-Signaling Pathway and Hepatic Polyribosome Profile Are Enhanced after the Recovery of a Protein Restricted Diet by a Combination of Soy or Black Bean with Corn Protein.

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    Márquez-Mota, Claudia C; Rodriguez-Gaytan, Cinthya; Adjibade, Pauline; Mazroui, Rachid; Gálvez, Amanda; Granados, Omar; Tovar, Armando R; Torres, Nimbe

    2016-09-20

    Between 6% and 11% of the world's population suffers from malnutrition or undernutrition associated with poverty, aging or long-term hospitalization. The present work examined the effect of different types of proteins on the mechanistic target of rapamycin (mTORC1)-signaling pathway in: (1) healthy; and (2) protein restricted rats. (1) In total, 200 rats were divided into eight groups and fed one of the following diets: 20% casein (C), soy (S), black bean (B), B + Corn (BCr), Pea (P), spirulina (Sp), sesame (Se) or Corn (Cr). Rats fed C or BCr had the highest body weight gain; rats fed BCr had the highest pS6K1/S6K1 ratio; rats fed B, BCr or P had the highest eIF4G expression; (2) In total, 84 rats were fed 0.5% C for 21 day and protein rehabilitated with different proteins. The S, soy + Corn (SCr) and BCr groups had the highest body weight gain. Rats fed SCr and BCr had the highest eIF4G expression and liver polysome formation. These findings suggest that the quality of the dietary proteins modulate the mTORC1-signaling pathway. In conclusion, the combination of BCr or SCr are the best proteins for dietary protein rehabilitation due to the significant increase in body weight, activation of the mTORC1-signaling pathway in liver and muscle, and liver polysome formation.

  13. Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC1 signaling.

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    Aruna D Balgi

    Full Text Available BACKGROUND: Mammalian target of rapamycin complex 1 (mTORC1 is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy, a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties. METHODOLOGY/PRINCIPAL FINDINGS: Using an automated cell-based assay, we screened a collection of >3,500 chemicals and identified three approved drugs (perhexiline, niclosamide, amiodarone and one pharmacological reagent (rottlerin capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2, which also contains mTOR as a catalytic subunit, suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline, niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2, a negative regulator of mTORC1, was required for inhibition of mTORC1 signaling by rottlerin but not for mTORC1 inhibition by perhexiline, niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions, by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2, an upstream regulator of mTORC1. By contrast, transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition, sustained inhibition of cell growth and no selective cell killing in starvation. CONCLUSION/SIGNIFICANCE: The observation that drugs already

  14. Convergence of the Mammalian Target of Rapamycin Complex 1- and Glycogen Synthase Kinase 3-β–Signaling Pathways Regulates the Innate Inflammatory Response

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    Wang, Huizhi; Brown, Jonathan; Gu, Zhen; Garcia, Carlos A.; Liang, Ruqiang; Alard, Pascale; Beurel, Eléonore; Jope, Richard S.; Greenway, Terrance; Martin, Michael

    2011-01-01

    The PI3K pathway and its regulation of mammalian target of rapamycin complex 1 (mTORC1) and glycogen synthase kinase 3 (GSK3) play pivotal roles in controlling inflammation. In this article, we show that mTORC1 and GSK3-β converge and that the capacity of mTORC1 to affect the inflammatory response is due to the inactivation of GSK3-β. Inhibition of mTORC1 attenuated GSK3 phosphorylation and increased its kinase activity. Immunoprecipitation and in vitro kinase assays demonstrated that GSK3-β associated with a downstream target of mTORC1, p85S6K, and phosphorylated GSK3-β. Inhibition of S6K1 abrogated the phosphorylation of GSK3-β while increasing and decreasing the levels of IL-12 and IL-10, respectively, in LPS-stimulated monocytes. In contrast, the direct inhibition of GSK3 attenuated the capacity of S6K1 inhibition to influence the levels of IL-10 and IL-12 produced by LPS-stimulated cells. At the transcriptional level, mTORC1 inhibition reduced the DNA binding of CREB and this effect was reversed by GSK3 inhibition. As a result, mTORC1 inhibition increased the levels of NF-κB p65 associated with CREB-binding protein. Inhibition of NF-κB p65 attenuated rapamycin’s ability to influence the levels of pro- or anti-inflammatory cytokine production in monocytes stimulated with LPS. These studies identify the molecular mechanism by which mTORC1 affects GSK3 and show that mTORC1 inhibition regulates pro- and anti-inflammatory cytokine production via its capacity to inactivate GSK3. PMID:21422248

  15. Mechanistic Target of Rapamycin Is a Novel Molecular Mechanism Linking Folate Availability and Cell Function.

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    Silva, Elena; Rosario, Fredrick J; Powell, Theresa L; Jansson, Thomas

    2017-07-01

    Folate deficiency has been linked to a wide range of disorders, including cancer, neural tube defects, and fetal growth restriction. Folate regulates cellular function mediated by its involvement in the synthesis of nucleotides, which are needed for DNA synthesis, and its function as a methyl donor, which is critical for DNA methylation. Here we review current data showing that folate sensing by mechanistic target of rapamycin (mTOR) constitutes a novel and distinct pathway by which folate modulates cell functions such as nutrient transport, protein synthesis, and mitochondrial respiration. The mTOR signaling pathway responds to growth factors and changes in nutrient availability to control cell growth, proliferation, and metabolism. mTOR exists in 2 complexes, mTOR complex (mTORC) 1 and mTORC2, which have distinct upstream regulators and downstream targets. Folate deficiency in pregnant mice caused a marked inhibition of mTORC1 and mTORC2 signaling in multiple maternal and fetal tissues, downregulation of placental amino acid transporters, and fetal growth restriction. In addition, folate deficiency in primary human trophoblast (PHT) cells resulted in inhibition of mTORC1 and mTORC2 signaling and decreased the activity of key amino acid transporters. Folate sensing by mTOR in PHT cells is independent of the accumulation of homocysteine and requires the proton-coupled folate transporter (PCFT; solute carrier 46A1). Furthermore, mTORC1 and mTORC2 regulate trophoblast folate uptake by modulating the cell surface expression of folate receptor α and the reduced folate carrier. These findings, which provide a novel link between folate availability and cell function, growth, and proliferation, may have broad biological significance given the critical role of folate in normal cell function and the multiple diseases that have been associated with decreased or excessive folate availability. Low maternal folate concentrations are linked to restricted fetal growth, and we

  16. Rapamycin inhibits mTOR/p70S6K activation in CA3 region of the hippocampus of the rat and impairs long term memory.

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    Lana, D; Di Russo, J; Mello, T; Wenk, G L; Giovannini, M G

    2017-01-01

    The present study was aimed at establishing whether the mTOR pathway and its downstream effector p70S6K in CA3 pyramidal neurons are under the modulation of the cholinergic input to trigger the formation of long term memories, similar to what we demonstrated in CA1 hippocampus. We performed in vivo behavioral experiments using the step down inhibitory avoidance test in adult Wistar rats to evaluate memory formation under different conditions. We examined the effects of rapamycin, an inhibitor of mTORC1 formation, scopolamine, a muscarinic receptor antagonist or mecamylamine, a nicotinic receptor antagonist, on short and long term memory formation and on the functionality of the mTOR pathway. Acquisition was conducted 30min after i.c.v. injection of rapamycin. Recall testing was performed 1h, 4h or 24h after acquisition. We found that (1) mTOR and p70S6K activation in CA3 pyramidal neurons were involved in long term memory formation; (2) rapamycin significantly inhibited mTOR and of p70S6K activation at 4h, and long term memory impairment 24h after acquisition; (3) scopolamine impaired short but not long term memory, with an early increase of mTOR/p70S6K activation at 1h followed by stabilization at longer times; (4) mecamylamine and scopolamine co-administration impaired short term memory at 1h and 4h and reduced the scopolamine-induced increase of mTOR/p70S6K activation at 1h and 4h; (5) mecamylamine and scopolamine treatment did not impair long term memory formation; (6) unexpectedly, rapamycin increased mTORC2 activation in microglial cells. Our results demonstrate that in CA3 pyramidal neurons the mTOR/p70S6K pathway is under the modulation of the cholinergic system and is involved in long-term memory encoding, and are consistent with the hypothesis that the CA3 region of the hippocampus is involved in memory mechanisms based on rapid, one-trial object-place learning and recall. Furthermore, our results are in accordance with previous reports that selective

  17. Selective Activation of mTORC1 Signaling Recapitulates Microcephaly, Tuberous Sclerosis, and Neurodegenerative Diseases

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    Hidetoshi Kassai

    2014-06-01

    Full Text Available Mammalian target of rapamycin (mTOR has been implicated in human neurological diseases such as tuberous sclerosis complex (TSC, neurodegeneration, and autism. However, little is known about when and how mTOR is involved in the pathogenesis of these diseases, due to a lack of animal models that directly increase mTOR activity. Here, we generated transgenic mice expressing a gain-of-function mutant of mTOR in the forebrain in a temporally controlled manner. Selective activation of mTORC1 in embryonic stages induced cortical atrophy caused by prominent apoptosis of neuronal progenitors, associated with upregulation of HIF-1α. In striking contrast, activation of the mTORC1 pathway in adulthood resulted in cortical hypertrophy with fatal epileptic seizures, recapitulating human TSC. Activated mTORC1 in the adult cortex also promoted rapid accumulation of cytoplasmic inclusions and activation of microglial cells, indicative of progressive neurodegeneration. Our findings demonstrate that mTORC1 plays different roles in developmental and adult stages and contributes to human neurological diseases.

  18. LAPTM4b recruits the LAT1-4F2hc Leu transporter to lysosomes and promotes mTORC1 activation.

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    Milkereit, Ruth; Persaud, Avinash; Vanoaica, Liviu; Guetg, Adriano; Verrey, Francois; Rotin, Daniela

    2015-05-22

    Mammalian target of rapamycin 1 (mTORC1), a master regulator of cellular growth, is activated downstream of growth factors, energy signalling and intracellular essential amino acids (EAAs) such as Leu. mTORC1 activation occurs at the lysosomal membrane, and involves V-ATPase stimulation by intra-lysosomal EAA (inside-out activation), leading to activation of the Ragulator, RagA/B-GTP and mTORC1 via Rheb-GTP. How Leu enters the lysosomes is unknown. Here we identified the lysosomal protein LAPTM4b as a binding partner for the Leu transporter, LAT1-4F2hc (SLC7A5-SLAC3A2). We show that LAPTM4b recruits LAT1-4F2hc to lysosomes, leading to uptake of Leu into lysosomes, and is required for mTORC1 activation via V-ATPase following EAA or Leu stimulation. These results demonstrate a functional Leu transporter at the lysosome, and help explain the inside-out lysosomal activation of mTORC1 by Leu/EAA.

  19. The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids

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    Hoxhaj, Gerta; Caddye, Edward; Najafov, Ayaz; Houde, Vanessa P; Johnson, Catherine; Dissanayake, Kumara; Toth, Rachel; Campbell, David G; Prescott, Alan R; MacKintosh, Carol

    2016-01-01

    The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1. DOI: http://dx.doi.org/10.7554/eLife.12278.001 PMID:27244671

  20. E2F1 regulates cellular growth by mTORC1 signaling.

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    Sebastian Real

    2011-01-01

    Full Text Available During cell proliferation, growth must occur to maintain homeostatic cell size. Here we show that E2F1 is capable of inducing growth by regulating mTORC1 activity. The activation of cell growth and mTORC1 by E2F1 is dependent on both E2F1's ability to bind DNA and to regulate gene transcription, demonstrating that a gene induction expression program is required in this process. Unlike E2F1, E2F3 is unable to activate mTORC1, suggesting that growth activity could be restricted to individual E2F members. The effect of E2F1 on the activation of mTORC1 does not depend on Akt. Furthermore, over-expression of TSC2 does not interfere with the effect of E2F1, indicating that the E2F1-induced signal pathway can compensate for the inhibitory effect of TSC2 on Rheb. Immunolocalization studies demonstrate that E2F1 induces the translocation of mTORC1 to the late endosome vesicles, in a mechanism dependent of leucine. E2F1 and leucine, or insulin, together affect the activation of S6K stronger than alone suggesting that they are complementary in activating the signal pathway. From these studies, E2F1 emerges as a key protein that integrates cell division and growth, both of which are essential for cell proliferation.

  1. The human papillomavirus type 16 E6 oncoprotein activates mTORC1 signaling and increases protein synthesis.

    Science.gov (United States)

    Spangle, Jennifer M; Münger, Karl

    2010-09-01

    The mammalian target of rapamycin (mTOR) kinase acts as a cellular rheostat that integrates signals from a variety of cellular signal transduction pathways that sense growth factor and nutrient availability as well as intracellular energy status. It was previously reported that the human papillomavirus type 16 (HPV16) E6 oncoprotein may activate the S6 protein kinase (S6K) through binding and E6AP-mediated degradation of the mTOR inhibitor tuberous sclerosis complex 2 (TSC2) (Z. Lu, X. Hu, Y. Li, L. Zheng, Y. Zhou, H. Jiang, T. Ning, Z. Basang, C. Zhang, and Y. Ke, J. Biol. Chem. 279:35664-35670, 2004; L. Zheng, H. Ding, Z. Lu, Y. Li, Y. Pan, T. Ning, and Y. Ke, Genes Cells 13:285-294, 2008). Our results confirmed that HPV16 E6 expression causes an increase in mTORC1 activity through enhanced phosphorylation of mTOR and activation of downstream signaling pathways S6K and eukaryotic initiation factor binding protein 1 (4E-BP1). However, we did not detect a decrease in TSC2 levels in HPV16 E6-expressing cells. We discovered, however, that HPV16 E6 expression causes AKT activation through the upstream kinases PDK1 and mTORC2 under conditions of nutrient deprivation. We show that HPV16 E6 expression causes an increase in protein synthesis by enhancing translation initiation complex assembly at the 5' mRNA cap and an increase in cap-dependent translation. The increase in cap-dependent translation likely results from HPV16 E6-induced AKT/mTORC1 activation, as the assembly of the translation initiation complex and cap-dependent translation are rapamycin sensitive. Lastly, coexpression of the HPV16 E6 and E7 oncoproteins does not affect HPV16 E6-induced activation of mTORC1 and cap-dependent translation. HPV16 E6-mediated activation of mTORC1 signaling and cap-dependent translation may be a mechanism to promote viral replication under conditions of limited nutrient supply in differentiated, HPV oncoprotein-expressing proliferating cells.

  2. mTORC1 Targets the Translational Repressor 4E-BP2, but Not S6 Kinase 1/2, to Regulate Neural Stem Cell Self-Renewal In Vivo

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    Nathaniel W. Hartman

    2013-10-01

    Full Text Available The mammalian target of rapamycin complex 1 (mTORC1 integrates signals important for cell growth, and its dysregulation in neural stem cells (NSCs is implicated in several neurological disorders associated with abnormal neurogenesis and brain size. However, the function of mTORC1 on NSC self-renewal and the downstream regulatory mechanisms are ill defined. Here, we found that genetically decreasing mTORC1 activity in neonatal NSCs prevented their differentiation, resulting in reduced lineage expansion and aborted neuron production. Constitutive activation of the translational repressor 4E-BP1, which blocked cap-dependent translation, had similar effects and prevented hyperactive mTORC1 induction of NSC differentiation and promoted self-renewal. Although 4E-BP2 knockdown promoted NSC differentiation, p70 S6 kinase 1 and 2 (S6K1/S6K2 knockdown did not affect NSC differentiation but reduced NSC soma size and prevented hyperactive mTORC1-induced increase in soma size. These data demonstrate a crucial role of mTORC1 and 4E-BP for switching on and off cap-dependent translation in NSC differentiation.

  3. Lycopene Protects Keratinocytes Against UVB Radiation-Induced Carcinogenesis via Negative Regulation of FOXO3a Through the mTORC2/AKT Signaling Pathway.

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    Chen, Ping; Xu, Shina; Qu, Jinlong

    2018-01-01

    Lycopene, one of the most potent anti-oxidants, has been reported to exhibit potent anti-proliferative properties in a wide range of cancer cells through modulation of the cell cycle and apoptosis. Forkhead box O3 (FOXO3a) plays a pivotal role in modulating the expression of genes involved in cell death. Herein, we investigated the role of FOXO3a signaling in the anti-cancer effects of lycopene. Results showed that lycopene pretreatment attenuated UVB-induced cell hyper-proliferation and promoted apoptosis, accompanied by decreased cyclin-dependent kinase 2 (CDK2) and CDK4 complex in both human keratinocytes and SKH-1 hairless mice. FOXO3a is phosphorylated in response to UVB irradiation and sequestered in the cytoplasm, while lycopene pretreatment rescued this sensitization. Gene ablation of FOXO3a attenuated lycopene-induced decrease in cell hyper-proliferation, CDK2, and CDK4 complex, indicating a critical role of FOXO3a in the lycopene-induced anti-proliferative effect of keratinocytes during UVB irradiation. Transfection with FOXO3a siRNA inhibited the lycopene-induced increase in cell apoptosis, BAX and cleaved PARP expression. Moreover, loss of AKT induced further accelerated lycopene-induced FOXO3a dephosphorylation, while loss of mechanistic target of rapamycin complex 2 (mTORC2) by transfection with RICTOR siRNA induced levels of AKT phosphorylation comparable to those obtained with lycopene. In contrast, overexpression of AKT or mTORC2 decreased the effects of lycopene on the expression of FOXO3a as well as AKT phosphorylation, suggesting that lycopene depends on the negative modulation of mTORC2/AKT signaling. Taken together, our findings demonstrate that the mTORC2/AKT/FOXO3a axis plays a critical role in the anti-proliferative and pro-apoptotic effects of lycopene in UVB-induced photocarcinogenesis. J. Cell. Biochem. 119: 366-377, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  4. Transforming Growth Factor β1-induced Apoptosis in Podocytes via the Extracellular Signal-regulated Kinase-Mammalian Target of Rapamycin Complex 1-NADPH Oxidase 4 Axis.

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    Das, Ranjan; Xu, Shanhua; Nguyen, Tuyet Thi; Quan, Xianglan; Choi, Seong-Kyung; Kim, Soo-Jin; Lee, Eun Young; Cha, Seung-Kuy; Park, Kyu-Sang

    2015-12-25

    TGF-β is a pleiotropic cytokine that accumulates during kidney injuries, resulting in various renal diseases. We have reported previously that TGF-β1 induces the selective up-regulation of mitochondrial Nox4, playing critical roles in podocyte apoptosis. Here we investigated the regulatory mechanism of Nox4 up-regulation by mTORC1 activation on TGF-β1-induced apoptosis in immortalized podocytes. TGF-β1 treatment markedly increased the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream targets p70S6K and 4EBP1. Blocking TGF-β receptor I with SB431542 completely blunted the phosphorylation of mTOR, p70S6K, and 4EBP1. Transient adenoviral overexpression of mTOR-WT and constitutively active mTORΔ augmented TGF-β1-treated Nox4 expression, reactive oxygen species (ROS) generation, and apoptosis, whereas mTOR kinase-dead suppressed the above changes. In addition, knockdown of mTOR mimicked the effect of mTOR-KD. Inhibition of mTORC1 by low-dose rapamycin or knockdown of p70S6K protected podocytes through attenuation of Nox4 expression and subsequent oxidative stress-induced apoptosis by TGF-β1. Pharmacological inhibition of the MEK-ERK cascade, but not the PI3K-Akt-TSC2 pathway, abolished TGF-β1-induced mTOR activation. Inhibition of either ERK1/2 or mTORC1 did not reduce the TGF-β1-stimulated increase in Nox4 mRNA level but significantly inhibited total Nox4 expression, ROS generation, and apoptosis induced by TGF-β1. Moreover, double knockdown of Smad2 and 3 or only Smad4 completely suppressed TGF-β1-induced ERK1/2-mTORactivation. Our data suggest that TGF-β1 increases translation of Nox4 through the Smad-ERK1/2-mTORC1 axis, which is independent of transcriptional regulation. Activation of this pathway plays a crucial role in ROS generation and mitochondrial dysfunction, leading to podocyte apoptosis. Therefore, inhibition of the ERK1/2-mTORC1 pathway could be a potential therapeutic and preventive target in proteinuric and chronic

  5. Labor Inhibits Placental Mechanistic Target of Rapamycin Complex 1 Signaling

    Science.gov (United States)

    LAGER, Susanne; AYE, Irving L.M.H.; GACCIOLI, Francesca; RAMIREZ, Vanessa I.; JANSSON, Thomas; POWELL, Theresa L.

    2014-01-01

    Introduction Labor induces a myriad of changes in placental gene expression. These changes may represent a physiological adaptation inhibiting placental cellular processes associated with a high demand for oxygen and energy (e.g., protein synthesis and active transport) thereby promoting oxygen and glucose transfer to the fetus. We hypothesized that mechanistic target of rapamycin complex 1 (mTORC1) signaling, a positive regulator of trophoblast protein synthesis and amino acid transport, is inhibited by labor. Methods Placental tissue was collected from healthy, term pregnancies (n=15 no-labor; n=12 labor). Activation of Caspase-1, IRS1/Akt, STAT, mTOR, and inflammatory signaling pathways was determined by Western blot. NFκB p65 and PPARγ DNA binding activity was measured in isolated nuclei. Results Labor increased Caspase-1 activation and mTOR complex 2 signaling, as measured by phosphorylation of Akt (S473). However, mTORC1 signaling was inhibited in response to labor as evidenced by decreased phosphorylation of mTOR (S2448) and 4EBP1 (T37/46 and T70). Labor also decreased NFκB and PPARγ DNA binding activity, while having no effect on IRS1 or STAT signaling pathway. Discussion and conclusion Several placental signaling pathways are affected by labor, which has implications for experimental design in studies of placental signaling. Inhibition of placental mTORC1 signaling in response to labor may serve to down-regulate protein synthesis and amino acid transport, processes that account for a large share of placental oxygen and glucose consumption. We speculate that this response preserves glucose and oxygen for transfer to the fetus during the stressful events of labor. PMID:25454472

  6. Akt inhibition promotes ABCA1-mediated cholesterol efflux to ApoA-I through suppressing mTORC1.

    Directory of Open Access Journals (Sweden)

    Fumin Dong

    Full Text Available ATP-binding cassette transporter A1 (ABCA1 plays an essential role in mediating cholesterol efflux to apolipoprotein A-I (apoA-I, a major housekeeping mechanism for cellular cholesterol homeostasis. After initial engagement with ABCA1, apoA-I directly interacts with the plasma membrane to acquire cholesterol. This apoA-I lipidation process is also known to require cellular signaling processes, presumably to support cholesterol trafficking to the plasma membrane. We report here that one of major signaling pathways in mammalian cells, Akt, is also involved. In several cell models that express ABCA1 including macrophages, pancreatic beta cells and hepatocytes, inhibition of Akt increases cholesterol efflux to apoA-I. Importantly, Akt inhibition has little effect on cells expressing non-functional mutant of ABCA1, implicating a specific role of Akt in ABCA1 function. Furthermore, we provide evidence that mTORC1, a major downstream target of Akt, is also a negative regulator of cholesterol efflux. In cells where mTORC1 is constitutively activated due to tuberous sclerosis complex 2 deletion, cholesterol efflux to apoA-I is no longer sensitive to Akt activity. This suggests that Akt suppresses cholesterol efflux through mTORC1 activation. Indeed, inhibition of mTORC1 by rapamycin or Torin-1 promotes cholesterol efflux. On the other hand, autophagy, one of the major pathways of cholesterol trafficking, is increased upon Akt inhibition. Furthermore, Akt inhibition disrupts lipid rafts, which is known to promote cholesterol efflux to apoA-I. We therefore conclude that Akt, through its downstream targets, mTORC1 and hence autophagy, negatively regulates cholesterol efflux to apoA-I.

  7. Platelet-derived growth factor regulates vascular smooth muscle phenotype via mammalian target of rapamycin complex 1

    International Nuclear Information System (INIS)

    Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun; Song, Sang Heon; Shin, Hwa Kyoung; Bae, Sun Sik

    2015-01-01

    Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38 MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs

  8. Platelet-derived growth factor regulates vascular smooth muscle phenotype via mammalian target of rapamycin complex 1

    Energy Technology Data Exchange (ETDEWEB)

    Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun [Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Song, Sang Heon [Department of Internal Medicine, Pusan National University Hospital, Busan (Korea, Republic of); Shin, Hwa Kyoung [Department of Anatomy, Pusan National University School of Korean Medicine, Yangsan (Korea, Republic of); Bae, Sun Sik, E-mail: sunsik@pusan.ac.kr [Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine, Yangsan (Korea, Republic of)

    2015-08-14

    Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38 MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs.

  9. Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion.

    Science.gov (United States)

    Zhou, Jing; Tan, Shi-Hao; Nicolas, Valérie; Bauvy, Chantal; Yang, Nai-Di; Zhang, Jianbin; Xue, Yuan; Codogno, Patrice; Shen, Han-Ming

    2013-04-01

    Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.

  10. Intrahippocampal Glutamine Administration Inhibits mTORC1 Signaling and Impairs Long-Term Memory

    Science.gov (United States)

    Rozas, Natalia S.; Redell, John B.; Pita-Almenar, Juan D.; McKenna, James, III.; Moore, Anthony N.; Gambello, Michael J.; Dash, Pramod K.

    2015-01-01

    The mechanistic Target of Rapamycin Complex 1 (mTORC1), a key regulator of protein synthesis and cellular growth, is also required for long-term memory formation. Stimulation of mTORC1 signaling is known to be dependent on the availability of energy and growth factors, as well as the presence of amino acids. In vitro studies using serum- and amino…

  11. GSK-3 directly regulates phospho-4EBP1 in renal cell carcinoma cell-line: an intrinsic subcellular mechanism for resistance to mTORC1 inhibition

    International Nuclear Information System (INIS)

    Ito, Hiromi; Ichiyanagi, Osamu; Naito, Sei; Bilim, Vladimir N.; Tomita, Yoshihiko; Kato, Tomoyuki; Nagaoka, Akira; Tsuchiya, Norihiko

    2016-01-01

    The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin 1 (mTORC1) signaling pathway is aberrantly activated in renal cell carcinoma (RCC). We previously demonstrated glycogen synthase kinase-3β (GSK-3β) positively regulated RCC proliferation. The aim of this study was to evaluate the role of GSK-3 in the PI3K/Akt/mTORC1 pathway and regulation of the downstream substrates, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), ribosomal protein S6 kinase (S6K), and ribosomal protein S6 (S6RP). We used human RCC cell lines (ACHN, Caki1, and A498) and, as normal controls, human renal proximal tubular epithelial cell (HRPTEpC) and non-tumorous kidney tissues that were obtained surgically for treatment of RCC patients. Rapamycin-resistant ACHN (ACHN/RR) cells were generated with chronic exposure of ACHN to rapamycin ranging from 1nM finally to 1 μM. Cell viability, cell cycling and direct interaction between GSK-3β and 4EBP1 were evaluated with MTS assay, flowcytometry and in vitro kinase assay with recombinant GSK-3β and 4EBP1products, respectively. Protein expression and phosphorylation of molecules associated with the PI3K/Akt/mTORC1 pathway were examined by immunoblotting. Effects of drug combination were determined as the combination index with CompuSyn software. Overexpression and phosphorylation of 4EBP1 and S6RP together with GSK-3 activation were observed in RCC cell lines, but not in human normal kidney cells and tissues. Cell proliferation, p4EBP1 and pS6RP were strongly suppressed by GSK-3 inhibition. Rapamycin and LY294002 sufficiently decreased pS6RP, but only moderately p4EBP1. In vitro kinase assays showed that recombinant GSK-3β phosphorylated recombinant 4EBP1, and the effect was blocked by GSK-3 inhibitors. Different from rapamycin, AR- A014418 remarkably inhibited cell proliferation, and rapidly suppressed p4EBP1 and pS6RP in ACHN and ACHN/RR (in 30 min to 1 h). AR- A014418 and rapamycin combination showed

  12. BMAL1-dependent regulation of the mTOR signaling pathway delays aging.

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    Khapre, Rohini V; Kondratova, Anna A; Patel, Sonal; Dubrovsky, Yuliya; Wrobel, Michelle; Antoch, Marina P; Kondratov, Roman V

    2014-01-01

    The circadian clock, an internal time-keeping system, has been linked with control of aging, but molecular mechanisms of regulation are not known. BMAL1 is a transcriptional factor and core component of the circadian clock; BMAL1 deficiency is associated with premature aging and reduced lifespan. Here we report that activity of mammalian Target of Rapamycin Complex 1 (mTORC1) is increased upon BMAL1 deficiency both in vivo and in cell culture. Increased mTOR signaling is associated with accelerated aging; in accordance with that, treatment with the mTORC1 inhibitor rapamycin increased lifespan of Bmal1-/- mice by 50%. Our data suggest that BMAL1 is a negative regulator of mTORC1 signaling. We propose that the circadian clock controls the activity of the mTOR pathway through BMAL1-dependent mechanisms and this regulation is important for control of aging and metabolism.

  13. Inflammation induced mTORC2-Akt-mTORC1 signaling promotes macrophage foam cell formation.

    Science.gov (United States)

    Banerjee, Dipanjan; Sinha, Archana; Saikia, Sudeshna; Gogoi, Bhaskarjyoti; Rathore, Arvind K; Das, Anindhya Sundar; Pal, Durba; Buragohain, Alak K; Dasgupta, Suman

    2018-06-05

    The transformation of macrophages into lipid loaded foam cells is a critical and early event in the pathogenesis of atherosclerosis. Several recent reports highlighted that induction of TLR4 signaling promotes macrophage foam cell formation; however, the underlying molecular mechanisms have not been clearly elucidated. Here, we found that the TLR4 mediated inflammatory signaling communicated with mTORC2-Akt-mTORC1 metabolic cascade in macrophage and thereby promoting lipid uptake and foam cell formation. Mechanistically, LPS treatment markedly upregulates TLR4 mediated inflammatory pathway which by activating mTORC2 induces Akt phosphorylation at serine 473 and that aggravate mTORC1 dependent scavenger receptors expression and consequent lipid accumulation in THP-1 macrophages. Inhibition of mTORC2 either by silencing Rictor expression or inhibiting its association with mTOR notably prevents LPS induced Akt activation, scavenger receptors expression and macrophage lipid accumulation. Although suppression of mTORC1 expression by genetic knockdown of Raptor did not produce any significant change in Akt S473 phosphorylation, however, incubation with Akt activator in Rictor silenced cells failed to promote scavenger receptors expression and macrophage foam cell formation. Thus, present research explored the signaling pathway involved in inflammation induced macrophage foam cells formation and therefore, targeting this pathway might be useful for preventing macrophage foam cell formation. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  14. Lysosomal Regulation of mTORC1 by Amino Acids in Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Yao Yao

    2017-07-01

    Full Text Available The mechanistic target of rapamycin complex 1 (mTORC1 is a master regulator of cell growth in eukaryotic cells. The active mTORC1 promotes cellular anabolic processes including protein, pyrimidine, and lipid biosynthesis, and inhibits catabolic processes such as autophagy. Consistent with its growth-promoting functions, hyper-activation of mTORC1 signaling is one of the important pathomechanisms underlying major human health problems including diabetes, neurodegenerative disorders, and cancer. The mTORC1 receives multiple upstream signals such as an abundance of amino acids and growth factors, thus it regulates a wide range of downstream events relevant to cell growth and proliferation control. The regulation of mTORC1 by amino acids is a fast-evolving field with its detailed mechanisms currently being revealed as the precise picture emerges. In this review, we summarize recent progress with respect to biochemical and biological findings in the regulation of mTORC1 signaling on the lysosomal membrane by amino acids.

  15. Genetic and epigenetic inactivation of SESTRIN1 controls mTORC1 and response to EZH2 inhibition in follicular lymphoma.

    Science.gov (United States)

    Oricchio, Elisa; Katanayeva, Natalya; Donaldson, Maria Christine; Sungalee, Stephanie; Pasion, Joyce P; Béguelin, Wendy; Battistello, Elena; Sanghvi, Viraj R; Jiang, Man; Jiang, Yanwen; Teater, Matt; Parmigiani, Anita; Budanov, Andrei V; Chan, Fong Chun; Shah, Sohrab P; Kridel, Robert; Melnick, Ari M; Ciriello, Giovanni; Wendel, Hans-Guido

    2017-06-28

    Follicular lymphoma (FL) is an incurable form of B cell lymphoma. Genomic studies have cataloged common genetic lesions in FL such as translocation t(14;18), frequent losses of chromosome 6q, and mutations in epigenetic regulators such as EZH2 Using a focused genetic screen, we identified SESTRIN1 as a relevant target of the 6q deletion and demonstrate tumor suppression by SESTRIN1 in vivo. Moreover, SESTRIN1 is a direct target of the lymphoma-specific EZH2 gain-of-function mutation ( EZH2 Y641X ). SESTRIN1 inactivation disrupts p53-mediated control of mammalian target of rapamycin complex 1 (mTORC1) and enables mRNA translation under genotoxic stress. SESTRIN1 loss represents an alternative to RRAGC mutations that maintain mTORC1 activity under nutrient starvation. The antitumor efficacy of pharmacological EZH2 inhibition depends on SESTRIN1, indicating that mTORC1 control is a critical function of EZH2 in lymphoma. Conversely, EZH2 Y641X mutant lymphomas show increased sensitivity to RapaLink-1, a bifunctional mTOR inhibitor. Hence, SESTRIN1 contributes to the genetic and epigenetic control of mTORC1 in lymphoma and influences responses to targeted therapies. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  16. Key Markers of mTORC1-Dependent and mTORC1-Independent Signaling Pathways Regulating Protein Synthesis in Rat Soleus Muscle During Early Stages of Hindlimb Unloading.

    Science.gov (United States)

    Mirzoev, Timur; Tyganov, Sergey; Vilchinskaya, Natalia; Lomonosova, Yulia; Shenkman, Boris

    2016-01-01

    The purpose of the study was to assess the amount of rRNA and phosphorylation status of the key markers of mTORC1-dependent (70s6k, 4E-BP1) and mTORC1-independent (GSK-3β, AMPK) signaling pathways controlling protein synthesis in rat soleus during early stages of mechanical unloading (hindlimb suspension (HS) for 1-, 3- and 7 days). The content of the key signaling molecules of various anabolic signaling pathways was determined by Western-blotting. The amount of 28S rRNA was evaluated by RT-PCR. The rate of protein synthesis was assessed using in-vivo SUnSET technique. HS for 3 and 7 days induced a significant (pprotein synthesis in soleus muscle in comparison with control. HS within 24 hours resulted in a significant (pprotein synthesis in rat soleus during early stages of simulated microgravity is associated with impaired ribosome biogenesis as well as reduced activity of mTORC1-independent signaling pathways. © 2016 The Author(s) Published by S. Karger AG, Basel.

  17. BRAFV600E negatively regulates the AKT pathway in melanoma cell lines.

    Science.gov (United States)

    Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F; Niu, Huifeng

    2012-01-01

    Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.

  18. BRAFV600E negatively regulates the AKT pathway in melanoma cell lines.

    Directory of Open Access Journals (Sweden)

    Brenden Chen

    Full Text Available Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167 or mTORC1 inhibitor (rapamycin induced AKT phosphorylation (pAKT and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2 and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.

  19. Mammalian target of rapamycin complex 1 activation is required for the stimulation of human skeletal muscle protein synthesis by essential amino acids.

    Science.gov (United States)

    Dickinson, Jared M; Fry, Christopher S; Drummond, Micah J; Gundermann, David M; Walker, Dillon K; Glynn, Erin L; Timmerman, Kyle L; Dhanani, Shaheen; Volpi, Elena; Rasmussen, Blake B

    2011-05-01

    The relationship between mammalian target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis during instances of amino acid surplus in humans is based solely on correlational data. Therefore, the goal of this study was to use a mechanistic approach specifically designed to determine whether increased mTORC1 activation is requisite for the stimulation of muscle protein synthesis following L-essential amino acid (EAA) ingestion in humans. Examination of muscle protein synthesis and signaling were performed on vastus lateralis muscle biopsies obtained from 8 young (25 ± 2 y) individuals who were studied prior to and following ingestion of 10 g of EAA during 2 separate trials in a randomized, counterbalanced design. The trials were identical except during 1 trial, participants were administered a single oral dose of a potent mTORC1 inhibitor (rapamycin) prior to EAA ingestion. In response to EAA ingestion, an ~60% increase in muscle protein synthesis was observed during the control trial, concomitant with increased phosphorylation of mTOR (Ser(2448)), ribosomal S6 kinase 1 (Thr(389)), and eukaryotic initiation factor 4E binding protein 1 (Thr(37/46)). In contrast, prior administration of rapamycin completely blocked the increase in muscle protein synthesis and blocked or attenuated activation of mTORC1-signaling proteins. The inhibition of muscle protein synthesis and signaling was not due to differences in either extracellular or intracellular amino acid availability, because these variables were similar between trials. These data support a fundamental role for mTORC1 activation as a key regulator of human muscle protein synthesis in response to increased EAA availability. This information will be useful in the development of evidence-based nutritional therapies targeting mTORC1 to counteract muscle wasting associated with numerous clinical conditions.

  20. Deficiency in mTORC1-controlled C/EBP beta-mRNA translation improves metabolic health in mice

    NARCIS (Netherlands)

    Zidek, Laura M.; Ackermann, Tobias; Hartleben, Goetz; Eichwald, Sabrina; Kortman, Gertrud; Kiehntopf, Michael; Leutz, Achim; Sonenberg, Nahum; Wang, Zhao-Qi; von Maltzahn, Julia; Mueller, Christine; Calkhoven, Cornelis F.

    The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of physiological adaptations in response to changes in nutrient supply. Major downstream targets of mTORC1 signalling are the mRNA translation regulators p70 ribosomal protein S6 kinase 1 (S6K1p70) and the 4E-binding

  1. Hepatic mTORC1 controls locomotor activity, body temperature, and lipid metabolism through FGF21

    Science.gov (United States)

    Cornu, Marion; Oppliger, Wolfgang; Albert, Verena; Robitaille, Aaron M.; Trapani, Francesca; Quagliata, Luca; Fuhrer, Tobias; Sauer, Uwe; Terracciano, Luigi; Hall, Michael N.

    2014-01-01

    The liver is a key metabolic organ that controls whole-body physiology in response to nutrient availability. Mammalian target of rapamycin (mTOR) is a nutrient-activated kinase and central controller of growth and metabolism that is negatively regulated by the tumor suppressor tuberous sclerosis complex 1 (TSC1). To investigate the role of hepatic mTOR complex 1 (mTORC1) in whole-body physiology, we generated liver-specific Tsc1 (L-Tsc1 KO) knockout mice. L-Tsc1 KO mice displayed reduced locomotor activity, body temperature, and hepatic triglyceride content in a rapamycin-sensitive manner. Ectopic activation of mTORC1 also caused depletion of hepatic and plasma glutamine, leading to peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α)–dependent fibroblast growth factor 21 (FGF21) expression in the liver. Injection of glutamine or knockdown of PGC-1α or FGF21 in the liver suppressed the behavioral and metabolic defects due to mTORC1 activation. Thus, mTORC1 in the liver controls whole-body physiology through PGC-1α and FGF21. Finally, mTORC1 signaling correlated with FGF21 expression in human liver tumors, suggesting that treatment of glutamine-addicted cancers with mTOR inhibitors might have beneficial effects at both the tumor and whole-body level. PMID:25082895

  2. Neuroprotective effect of rapamycin on spinal cord injury via activation of the Wnt/β-catenin signaling pathway

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    Kai Gao

    2015-01-01

    Full Text Available The Wnt/β-catenin signaling pathway plays a crucial role in neural development, axonal guidance, neuropathic pain remission and neuronal survival. In this study, we initially examined the effect of rapamycin on the Wnt/β-catenin signaling pathway after spinal cord injury, by intraperitoneally injecting spinal cord injured rats with rapamycin over 2 days. Western blot analysis and immunofluorescence staining were used to detect the expression levels of β-catenin protein, ca-spase-3 protein and brain-derived neurotrophic factor protein, components of the Wnt/β-catenin signaling pathway. Rapamycin increased the levels of β-catenin and brain-derived neurotrophic factor in the injured spinal cord, improved the pathological morphology at the injury site, reduced the loss of motor neurons, and promoted motor functional recovery in rats after spinal cord injury. Our experimental findings suggest that the neuroprotective effect of rapamycin intervention is mediated through activation of the Wnt/β-catenin signaling pathway after spinal cord injury.

  3. Milk—A Nutrient System of Mammalian Evolution Promoting mTORC1-Dependent Translation

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    Melnik, Bodo C.

    2015-01-01

    Based on own translational research of the biochemical and hormonal effects of cow’s milk consumption in humans, this review presents milk as a signaling system of mammalian evolution that activates the nutrient-sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1), the pivotal regulator of translation. Milk, a mammary gland-derived secretory product, is required for species-specific gene-nutrient interactions that promote appropriate growth and development of the newborn mammal. This signaling system is highly conserved and tightly controlled by the lactation genome. Milk is sufficient to activate mTORC1, the crucial regulator of protein, lipid, and nucleotide synthesis orchestrating anabolism, cell growth and proliferation. To fulfill its mTORC1-activating function, milk delivers four key metabolic messengers: (1) essential branched-chain amino acids (BCAAs); (2) glutamine; (3) palmitic acid; and (4) bioactive exosomal microRNAs, which in a synergistical fashion promote mTORC1-dependent translation. In all mammals except Neolithic humans, postnatal activation of mTORC1 by milk intake is restricted to the postnatal lactation period. It is of critical concern that persistent hyperactivation of mTORC1 is associated with aging and the development of age-related disorders such as obesity, type 2 diabetes mellitus, cancer, and neurodegenerative diseases. Persistent mTORC1 activation promotes endoplasmic reticulum (ER) stress and drives an aimless quasi-program, which promotes aging and age-related diseases. PMID:26225961

  4. Milk—A Nutrient System of Mammalian Evolution Promoting mTORC1-Dependent Translation

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    Bodo C. Melnik

    2015-07-01

    Full Text Available Based on own translational research of the biochemical and hormonal effects of cow’s milk consumption in humans, this review presents milk as a signaling system of mammalian evolution that activates the nutrient-sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1, the pivotal regulator of translation. Milk, a mammary gland-derived secretory product, is required for species-specific gene-nutrient interactions that promote appropriate growth and development of the newborn mammal. This signaling system is highly conserved and tightly controlled by the lactation genome. Milk is sufficient to activate mTORC1, the crucial regulator of protein, lipid, and nucleotide synthesis orchestrating anabolism, cell growth and proliferation. To fulfill its mTORC1-activating function, milk delivers four key metabolic messengers: (1 essential branched-chain amino acids (BCAAs; (2 glutamine; (3 palmitic acid; and (4 bioactive exosomal microRNAs, which in a synergistical fashion promote mTORC1-dependent translation. In all mammals except Neolithic humans, postnatal activation of mTORC1 by milk intake is restricted to the postnatal lactation period. It is of critical concern that persistent hyperactivation of mTORC1 is associated with aging and the development of age-related disorders such as obesity, type 2 diabetes mellitus, cancer, and neurodegenerative diseases. Persistent mTORC1 activation promotes endoplasmic reticulum (ER stress and drives an aimless quasi-program, which promotes aging and age-related diseases.

  5. Dual inhibition of mTORC1 and mTORC2 perturbs cytoskeletal organization and impairs endothelial cell elongation.

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    Tsuji-Tamura, Kiyomi; Ogawa, Minetaro

    2018-02-26

    Elongation of endothelial cells is an important process in vascular formation and is expected to be a therapeutic target for inhibiting tumor angiogenesis. We have previously demonstrated that inhibition of mTORC1 and mTORC2 impaired endothelial cell elongation, although the mechanism has not been well defined. In this study, we analyzed the effects of the mTORC1-specific inhibitor everolimus and the mTORC1/mTORC2 dual inhibitor KU0063794 on the cytoskeletal organization and morphology of endothelial cell lines. While both inhibitors equally inhibited cell proliferation, KU0063794 specifically caused abnormal accumulation of F-actin and disordered distribution of microtubules, thereby markedly impairing endothelial cell elongation and tube formation. The effects of KU0063794 were phenocopied by paclitaxel treatment, suggesting that KU0063794 might impair endothelial cell morphology through over-stabilization of microtubules. Although mTORC1 is a key signaling molecule in cell proliferation and has been considered a target for preventing angiogenesis, mTORC1 inhibitors have not been sufficient to suppress angiogenesis. Our results suggest that mTORC1/mTORC2 dual inhibition is more effective for anti-angiogenic therapy, as it impairs not only endothelial cell proliferation, but also endothelial cell elongation. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Branched-chain amino acids enhance premature senescence through mammalian target of rapamycin complex I-mediated upregulation of p21 protein.

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    Masayuki Nakano

    Full Text Available Branched-chain amino acids (BCAAs have been applied as an oral supplementation to patients with liver cirrhosis. BCAAs not only improve nutritional status of patients but also decrease the incidence of liver cancer. Mammalian target of rapamycin (mTOR links cellular metabolism with growth and proliferation in response to nutrients, energy, and growth factors. BCAAs, especially leucine, have been shown to regulate protein synthesis through mTOR activities. On the other hand, cellular senescence is suggested to function as tumor suppressor mechanisms, and induced by a variety of stimuli including DNA damage-inducing drugs. However, it is not clear how BCAA supplementation prevents the incidence of liver cancer in patients with cirrhosis. Here we showed that human cancer cells, HepG2 and U2OS, cultured in medium containing BCAAs with Fischer's ratio about 3, which was shown to have highest activities to synthesize and secrete of albumin, had higher activities to induce premature senescence and elevate mTORC1 activities. Furthermore, BCAAs themselves enhanced the execution of premature senescence induced by DNA damage-inducing drugs, which was effectively prevented by rapamycin. These results strongly suggested the contribution of the mTORC1 pathway to the regulation of premature senescence. Interestingly, the protein levels of p21, a p53 target and well-known gene essential for the execution of cellular senescence, were upregulated in the presence of BCAAs. These results suggested that BCAAs possibly contribute to tumor suppression by enhancing cellular senescence mediated through the mTOR signalling pathway.

  7. Impaired mTORC2 signaling in catecholaminergic neurons exaggerates high fat diet-induced hyperphagia

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    Olga I. Dadalko

    2015-09-01

    Conclusions: Our data support a model in which mTORC2 signaling within catecholaminergic neurons constrains consumption of a high-fat diet, while disruption causes high-fat diet-specific exaggerated hyperphagia. In parallel, impaired mTORC2 signaling leads to aberrant striatal DA neurotransmission, which has been associated with obesity in human and animal models, as well as with escalating substance abuse. These data suggest that defects localized to the catecholaminergic pathways are capable of overriding homeostatic circuits, leading to obesity, metabolic impairment, and aberrant DA-dependent behaviors.

  8. SAD-A kinase controls islet β-cell size and function as a mediator of mTORC1 signaling.

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    Nie, Jia; Liu, Xiaolei; Lilley, Brendan N; Zhang, Hai; Pan, Y Albert; Kimball, Scot R; Zhang, Jun; Zhang, Weiping; Wang, Li; Jefferson, Leonard S; Sanes, Joshua R; Han, Xiao; Shi, Yuguang

    2013-08-20

    The mammalian target of rapamycin (mTOR) plays an important role in controlling islet β-cell function. However, the underlying molecular mechanisms remain poorly elucidated. Synapses of amphids defective kinase-A (SAD-A) is a 5' adenosine monophosphate-activated protein kinase-related protein kinase that is exclusively expressed in pancreas and brain. In this study, we investigated a role of the kinase in regulating pancreatic β-cell morphology and function as a mediator of mTOR complex 1 (mTORC1) signaling. We show that global SAD-A deletion leads to defective glucose-stimulated insulin secretion and petite islets, which are reminiscent of the defects in mice with global deletion of ribosomal protein S6 kinase 1, a downstream target of mTORC1. Consistent with these findings, selective deletion of SAD-A in pancreas decreased islet β-cell size, whereas SAD-A overexpression significantly increased the size of mouse insulinomas cell lines β-cells. In direct support of SAD-A as a unique mediator of mTORC1 signaling in islet β-cells, we demonstrate that glucose dramatically stimulated SAD-A protein translation in isolated mouse islets, which was potently inhibited by rapamycin, an inhibitor of mTORC1. Moreover, the 5'-untranslated region of SAD-A mRNA is highly structured and requires mTORC1 signaling for its translation initiation. Together, these findings identified SAD-A as a unique pancreas-specific effector protein of mTORC1 signaling.

  9. mTORC1 Balances Cellular Amino Acid Supply with Demand for Protein Synthesis through Post-transcriptional Control of ATF4

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    Yeonwoo Park

    2017-05-01

    Full Text Available The mammalian target of rapamycin complex 1 (mTORC1 is a master regulator of cell growth that is commonly deregulated in human diseases. Here we find that mTORC1 controls a transcriptional program encoding amino acid transporters and metabolic enzymes through a mechanism also used to regulate protein synthesis. Bioinformatic analysis of mTORC1-responsive mRNAs identified a promoter element recognized by activating transcription factor 4 (ATF4, a key effector of the integrated stress response. ATF4 translation is normally induced by the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α through a mechanism that requires upstream open reading frames (uORFs in the ATF4 5′ UTR. mTORC1 also controls ATF4 translation through uORFs, but independently of changes in eIF2α phosphorylation. mTORC1 instead employs the 4E-binding protein (4E-BP family of translation repressors. These results link mTORC1-regulated demand for protein synthesis with an ATF4-regulated transcriptional program that controls the supply of amino acids to the translation machinery.

  10. Moderate Autophagy Inhibits Vascular Smooth Muscle Cell Senescence to Stabilize Progressed Atherosclerotic Plaque via the mTORC1/ULK1/ATG13 Signal Pathway

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    Zhenli Luo

    2017-01-01

    Full Text Available In order to investigate the effects of autophagy induced by rapamycin in the development of atherosclerosis plaque we established murine atherosclerosis model which was induced in ApoE−/− mice by high fat and cholesterol diet (HFD for 16 weeks. Rapamycin and 3-Methyladenine (MA were used as autophagy inducer and inhibitor respectively. The plaque areas in aortic artery were detected with HE and Oil Red O staining. Immunohistochemical staining were applied to investigate content of plaque respectively. In contrast to control and 3-MA groups, rapamycin could inhibit atherosclerosis progression. Rapamycin was able to increase collagen content and a-SMA distribution relatively, as well as decrease necrotic core area. Then we used MOVAS and culture with ox-LDL for 72 h to induce smooth muscle-derived foam cell model in vitro. Rapamycin and 3-MA were cultured together respectively. Flow cytometry assay and SA-β-Gal staining experiments were performed to detect survival and senescence of VSMCs. Western blot analysis were utilized to analyze the levels of protein expression. We found that rapamycin could promote ox-LDL-induced VSMCs autophagy survival and alleviate cellular senescence, in comparison to control and 3-MA groups. Western blot analysis showed that rapamycin could upregulate ULK1, ATG13 and downregulate mTORC1 and p53 protein expression.

  11. Rapamycin targeting mTOR and hedgehog signaling pathways blocks human rhabdomyosarcoma growth in xenograft murine model

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    Kaylani, Samer Z. [Division of Hematology and Oncology, Department of Pediatrics, University of Alabama at Birmingham, 1600 7th Avenue South, ACC 414, Birmingham, AL 35233 (United States); Xu, Jianmin; Srivastava, Ritesh K. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States); Kopelovich, Levy [Division of Cancer Prevention, National Cancer Institute, Bethesda (United States); Pressey, Joseph G. [Division of Hematology and Oncology, Department of Pediatrics, University of Alabama at Birmingham, 1600 7th Avenue South, ACC 414, Birmingham, AL 35233 (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States)

    2013-06-14

    Graphical abstract: Intervention of poorly differentiated RMS by rapamycin: In poorly differentiated RMS, rapamycin blocks mTOR and Hh signaling pathways concomitantly. This leads to dampening in cell cycle regulation and induction of apoptosis. This study provides a rationale for the therapeutic intervention of poorly differentiated RMS by treating patients with rapamycin alone or in combination with other chemotherapeutic agents. -- Highlights: •Rapamycin abrogates RMS tumor growth by modulating proliferation and apoptosis. •Co-targeting mTOR/Hh pathways underlie the molecular basis of effectiveness. •Reduction in mTOR/Hh pathways diminish EMT leading to reduced invasiveness. -- Abstract: Rhabdomyosarcomas (RMS) represent the most common childhood soft-tissue sarcoma. Over the past few decades outcomes for low and intermediate risk RMS patients have slowly improved while patients with metastatic or relapsed RMS still face a grim prognosis. New chemotherapeutic agents or combinations of chemotherapies have largely failed to improve the outcome. Based on the identification of novel molecular targets, potential therapeutic approaches in RMS may offer a decreased reliance on conventional chemotherapy. Thus, identification of effective therapeutic agents that specifically target relevant pathways may be particularly beneficial for patients with metastatic and refractory RMS. The PI3K/AKT/mTOR pathway has been found to be a potentially attractive target in RMS therapy. In this study, we provide evidence that rapamycin (sirolimus) abrogates growth of RMS development in a RMS xenograft mouse model. As compared to a vehicle-treated control group, more than 95% inhibition in tumor growth was observed in mice receiving parenteral administration of rapamycin. The residual tumors in rapamycin-treated group showed significant reduction in the expression of biomarkers indicative of proliferation and tumor invasiveness. These tumors also showed enhanced apoptosis

  12. Amino Acids Regulate mTORC1 by an Obligate Two-step Mechanism*

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    Dyachok, Julia; Earnest, Svetlana; Iturraran, Erica N.; Cobb, Melanie H.

    2016-01-01

    The mechanistic target of rapamycin complex 1 (mTORC1) coordinates cell growth with its nutritional, hormonal, energy, and stress status. Amino acids are critical regulators of mTORC1 that permit other inputs to mTORC1 activity. However, the roles of individual amino acids and their interactions in mTORC1 activation are not well understood. Here we demonstrate that activation of mTORC1 by amino acids includes two discrete and separable steps: priming and activation. Sensitizing mTORC1 activation by priming amino acids is a prerequisite for subsequent stimulation of mTORC1 by activating amino acids. Priming is achieved by a group of amino acids that includes l-asparagine, l-glutamine, l-threonine, l-arginine, l-glycine, l-proline, l-serine, l-alanine, and l-glutamic acid. The group of activating amino acids is dominated by l-leucine but also includes l-methionine, l-isoleucine, and l-valine. l-Cysteine predominantly inhibits priming but not the activating step. Priming and activating steps differ in their requirements for amino acid concentration and duration of treatment. Priming and activating amino acids use mechanisms that are distinct both from each other and from growth factor signaling. Neither step requires intact tuberous sclerosis complex of proteins to activate mTORC1. Concerted action of priming and activating amino acids is required to localize mTORC1 to lysosomes and achieve its activation. PMID:27587390

  13. Macrophage mTORC1 disruption reduces inflammation and insulin resistance in obese mice

    NARCIS (Netherlands)

    Jiang, Hongfeng; Westerterp, Marit; Wang, Chunjiong; Zhu, Yi; Ai, Ding

    2014-01-01

    Inflammatory factors secreted by macrophages play an important role in obesity-related insulin resistance. Being at the crossroads of a nutrient-hormonal signalling network, the mammalian target of rapamycin complex 1 (mTORC1) controls important functions in the regulation of energy balance and

  14. Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin

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    Nishitani, Shinobu; Horie, Mayumi; Ishizaki, Sonoko; Yano, Hirohisa

    2013-01-01

    Differentiation of cancer stem cells (CSCs) into cancer cells causes increased sensitivity to chemotherapeutic agents. Although inhibition of mammalian target of rapamycin (mTOR) leads to CSC survival, the effect of branched chain amino acids (BCAAs), an mTOR complex 1 (mTORC1) activator remains unknown. In this study, we examined the effects of BCAA on hepatocellular carcinoma (HCC) cells expressing a hepatic CSC marker, EpCAM. We examined the effects of BCAA and/or 5-fluorouracil (FU) on expression of EpCAM and other CSC-related markers, as well as cell proliferation in HCC cells and in a xenograft mouse model. We also characterized CSC-related and mTOR signal-related molecule expression and tumorigenicity in HCC cells with knockdown of Rictor or Raptor, or overexpression of constitutively active rheb (caRheb). mTOR signal-related molecule expression was also examined in BCAA-treated HCC cells. In-vitro BCAA reduced the frequency of EpCAM-positive cells and improved sensitivity to the anti-proliferative effect of 5-FU. Combined 5-FU and BCAA provided better antitumor efficacy than 5-FU alone in the xenograft model. Stimulation with high doses of BCAA activated mTORC1. Knockdown and overexpression experiments revealed that inhibition of mTOR complex 2 (mTORC2) or activation of mTORC1 led to decreased EpCAM expression and little or no tumorigenicity. BCAA may enhance the sensitivity to chemotherapy by reducing the population of cscs via the mTOR pathway. This result suggests the utility of BCAA in liver cancer therapy. PMID:24312415

  15. Branched chain amino acid suppresses hepatocellular cancer stem cells through the activation of mammalian target of rapamycin.

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    Shinobu Nishitani

    Full Text Available Differentiation of cancer stem cells (CSCs into cancer cells causes increased sensitivity to chemotherapeutic agents. Although inhibition of mammalian target of rapamycin (mTOR leads to CSC survival, the effect of branched chain amino acids (BCAAs, an mTOR complex 1 (mTORC1 activator remains unknown. In this study, we examined the effects of BCAA on hepatocellular carcinoma (HCC cells expressing a hepatic CSC marker, EpCAM. We examined the effects of BCAA and/or 5-fluorouracil (FU on expression of EpCAM and other CSC-related markers, as well as cell proliferation in HCC cells and in a xenograft mouse model. We also characterized CSC-related and mTOR signal-related molecule expression and tumorigenicity in HCC cells with knockdown of Rictor or Raptor, or overexpression of constitutively active rheb (caRheb. mTOR signal-related molecule expression was also examined in BCAA-treated HCC cells. In-vitro BCAA reduced the frequency of EpCAM-positive cells and improved sensitivity to the anti-proliferative effect of 5-FU. Combined 5-FU and BCAA provided better antitumor efficacy than 5-FU alone in the xenograft model. Stimulation with high doses of BCAA activated mTORC1. Knockdown and overexpression experiments revealed that inhibition of mTOR complex 2 (mTORC2 or activation of mTORC1 led to decreased EpCAM expression and little or no tumorigenicity. BCAA may enhance the sensitivity to chemotherapy by reducing the population of cscs via the mTOR pathway. This result suggests the utility of BCAA in liver cancer therapy.

  16. Role of mTORC1 Controlling Proteostasis after Brain Ischemia

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    Maria J. Perez-Alvarez

    2018-02-01

    Full Text Available Intense efforts are being undertaken to understand the pathophysiological mechanisms triggered after brain ischemia and to develop effective pharmacological treatments. However, the underlying molecular mechanisms are complex and not completely understood. One of the main problems is the fact that the ischemic damage is time-dependent and ranges from negligible to massive, involving different cell types such as neurons, astrocytes, microglia, endothelial cells, and some blood-derived cells (neutrophils, lymphocytes, etc.. Thus, approaching such a complicated cellular response generates a more complex combination of molecular mechanisms, in which cell death, cellular damage, stress and repair are intermixed. For this reason, animal and cellular model systems are needed in order to dissect and clarify which molecular mechanisms have to be promoted and/or blocked. Brain ischemia may be analyzed from two different perspectives: that of oxygen deprivation (hypoxic damage per se and that of deprivation of glucose/serum factors. For investigations of ischemic stroke, middle cerebral artery occlusion (MCAO is the preferred in vivo model, and uses two different approaches: transient (tMCAO, where reperfusion is permitted; or permanent (pMCAO. As a complement to this model, many laboratories expose different primary cortical neuron or neuronal cell lines to oxygen-glucose deprivation (OGD. This ex vivo model permits the analysis of the impact of hypoxic damage and the specific response of different cell types implicated in vivo, such as neurons, glia or endothelial cells. Using in vivo and neuronal OGD models, it was recently established that mTORC1 (mammalian Target of Rapamycin Complex-1, a protein complex downstream of PI3K-Akt pathway, is one of the players deregulated after ischemia and OGD. In addition, neuroprotective intervention either by estradiol or by specific AT2R agonists shows an important regulatory role for the mTORC1 activity, for

  17. Chronic innate immune activation of TBK1 suppresses mTORC1 activity and dysregulates cellular metabolism.

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    Hasan, Maroof; Gonugunta, Vijay K; Dobbs, Nicole; Ali, Aktar; Palchik, Guillermo; Calvaruso, Maria A; DeBerardinis, Ralph J; Yan, Nan

    2017-01-24

    Three-prime repair exonuclease 1 knockout (Trex1 -/- ) mice suffer from systemic inflammation caused largely by chronic activation of the cyclic GMP-AMP synthase-stimulator of interferon genes-TANK-binding kinase-interferon regulatory factor 3 (cGAS-STING-TBK1-IRF3) signaling pathway. We showed previously that Trex1-deficient cells have reduced mammalian target of rapamycin complex 1 (mTORC1) activity, although the underlying mechanism is unclear. Here, we performed detailed metabolic analysis in Trex1 -/- mice and cells that revealed both cellular and systemic metabolic defects, including reduced mitochondrial respiration and increased glycolysis, energy expenditure, and fat metabolism. We also genetically separated the inflammatory and metabolic phenotypes by showing that Sting deficiency rescued both inflammatory and metabolic phenotypes, whereas Irf3 deficiency only rescued inflammation on the Trex1 -/- background, and many metabolic defects persist in Trex1 -/- Irf3 -/- cells and mice. We also showed that Leptin deficiency (ob/ob) increased lipogenesis and prolonged survival of Trex1 -/- mice without dampening inflammation. Mechanistically, we identified TBK1 as a key regulator of mTORC1 activity in Trex1 -/- cells. Together, our data demonstrate that chronic innate immune activation of TBK1 suppresses mTORC1 activity, leading to dysregulated cellular metabolism.

  18. Computational analysis of an autophagy/translation switch based on mutual inhibition of MTORC1 and ULK1.

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    Paulina Szymańska

    Full Text Available We constructed a mechanistic, computational model for regulation of (macroautophagy and protein synthesis (at the level of translation. The model was formulated to study the system-level consequences of interactions among the following proteins: two key components of MTOR complex 1 (MTORC1, namely the protein kinase MTOR (mechanistic target of rapamycin and the scaffold protein RPTOR; the autophagy-initiating protein kinase ULK1; and the multimeric energy-sensing AMP-activated protein kinase (AMPK. Inputs of the model include intrinsic AMPK kinase activity, which is taken as an adjustable surrogate parameter for cellular energy level or AMP:ATP ratio, and rapamycin dose, which controls MTORC1 activity. Outputs of the model include the phosphorylation level of the translational repressor EIF4EBP1, a substrate of MTORC1, and the phosphorylation level of AMBRA1 (activating molecule in BECN1-regulated autophagy, a substrate of ULK1 critical for autophagosome formation. The model incorporates reciprocal regulation of mTORC1 and ULK1 by AMPK, mutual inhibition of MTORC1 and ULK1, and ULK1-mediated negative feedback regulation of AMPK. Through analysis of the model, we find that these processes may be responsible, depending on conditions, for graded responses to stress inputs, for bistable switching between autophagy and protein synthesis, or relaxation oscillations, comprising alternating periods of autophagy and protein synthesis. A sensitivity analysis indicates that the prediction of oscillatory behavior is robust to changes of the parameter values of the model. The model provides testable predictions about the behavior of the AMPK-MTORC1-ULK1 network, which plays a central role in maintaining cellular energy and nutrient homeostasis.

  19. Paradoxical effects of rapamycin on experimental house dust mite-induced asthma.

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    Karin Fredriksson

    Full Text Available The mammalian target of rapamycin (mTOR modulates immune responses and cellular proliferation. The objective of this study was to assess whether inhibition of mTOR with rapamycin modifies disease severity in two experimental murine models of house dust mite (HDM-induced asthma. In an induction model, rapamycin was administered to BALB/c mice coincident with nasal HDM challenges for 3 weeks. In a treatment model, nasal HDM challenges were performed for 6 weeks and rapamycin treatment was administered during weeks 4 through 6. In the induction model, rapamycin significantly attenuated airway inflammation, airway hyperreactivity (AHR and goblet cell hyperplasia. In contrast, treatment of established HDM-induced asthma with rapamycin exacerbated AHR and airway inflammation, whereas goblet cell hyperplasia was not modified. Phosphorylation of the S6 ribosomal protein, which is downstream of mTORC1, was increased after 3 weeks, but not 6 weeks of HDM-challenge. Rapamycin reduced S6 phosphorylation in HDM-challenged mice in both the induction and treatment models. Thus, the paradoxical effects of rapamycin on asthma severity paralleled the activation of mTOR signaling. Lastly, mediastinal lymph node re-stimulation experiments showed that treatment of rapamycin-naive T cells with ex vivo rapamycin decreased antigen-specific Th2 cytokine production, whereas prior exposure to in vivo rapamycin rendered T cells refractory to the suppressive effects of ex vivo rapamycin. We conclude that rapamycin had paradoxical effects on the pathogenesis of experimental HDM-induced asthma. Thus, consistent with the context-dependent effects of rapamycin on inflammation, the timing of mTOR inhibition may be an important determinant of efficacy and toxicity in HDM-induced asthma.

  20. The mTORC1-4E-BP-eIF4E axis controls de novo Bcl6 protein synthesis in T cells and systemic autoimmunity.

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    Yi, Woelsung; Gupta, Sanjay; Ricker, Edd; Manni, Michela; Jessberger, Rolf; Chinenov, Yurii; Molina, Henrik; Pernis, Alessandra B

    2017-08-15

    Post-transcriptional modifications can control protein abundance, but the extent to which these alterations contribute to the expression of T helper (T H ) lineage-defining factors is unknown. Tight regulation of Bcl6 expression, an essential transcription factor for T follicular helper (T FH ) cells, is critical as aberrant T FH cell expansion is associated with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here we show that lack of the SLE risk variant Def6 results in deregulation of Bcl6 protein synthesis in T cells as a result of enhanced activation of the mTORC1-4E-BP-eIF4E axis, secondary to aberrant assembly of a raptor-p62-TRAF6 complex. Proteomic analysis reveals that this pathway selectively controls the abundance of a subset of proteins. Rapamycin or raptor deletion ameliorates the aberrant T FH cell expansion in mice lacking Def6. Thus deregulation of mTORC1-dependent pathways controlling protein synthesis can result in T-cell dysfunction, indicating a mechanism by which mTORC1 can promote autoimmunity.Excessive expansion of the T follicular helper (T FH ) cell pool is associated with autoimmune disease and Def6 has been identified as an SLE risk variant. Here the authors show that Def6 limits proliferation of T FH cells in mice via alteration of mTORC1 signaling and inhibition of Bcl6 expression.

  1. Striatal Transcriptome and Interactome Analysis of Shank3-overexpressing Mice Reveals the Connectivity between Shank3 and mTORC1 Signaling

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    Yeunkum Lee

    2017-06-01

    Full Text Available Mania causes symptoms of hyperactivity, impulsivity, elevated mood, reduced anxiety and decreased need for sleep, which suggests that the dysfunction of the striatum, a critical component of the brain motor and reward system, can be causally associated with mania. However, detailed molecular pathophysiology underlying the striatal dysfunction in mania remains largely unknown. In this study, we aimed to identify the molecular pathways showing alterations in the striatum of SH3 and multiple ankyrin repeat domains 3 (Shank3-overexpressing transgenic (TG mice that display manic-like behaviors. The results of transcriptome analysis suggested that mammalian target of rapamycin complex 1 (mTORC1 signaling may be the primary molecular signature altered in the Shank3 TG striatum. Indeed, we found that striatal mTORC1 activity, as measured by mTOR S2448 phosphorylation, was significantly decreased in the Shank3 TG mice compared to wild-type (WT mice. To elucidate the potential underlying mechanism, we re-analyzed previously reported protein interactomes, and detected a high connectivity between Shank3 and several upstream regulators of mTORC1, such as tuberous sclerosis 1 (TSC1, TSC2 and Ras homolog enriched in striatum (Rhes, via 94 common interactors that we denominated “Shank3-mTORC1 interactome”. We noticed that, among the 94 common interactors, 11 proteins were related to actin filaments, the level of which was increased in the dorsal striatum of Shank3 TG mice. Furthermore, we could co-immunoprecipitate Shank3, Rhes and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1 proteins from the striatal lysate of Shank3 TG mice. By comparing with the gene sets of psychiatric disorders, we also observed that the 94 proteins of Shank3-mTORC1 interactome were significantly associated with bipolar disorder (BD. Altogether, our results suggest a protein interaction-mediated connectivity between Shank3 and certain upstream

  2. Dietary intervention in acne: Attenuation of increased mTORC1 signaling promoted by Western diet.

    Science.gov (United States)

    Melnik, Bodo

    2012-01-01

    The purpose of this paper is to highlight the endocrine signaling of Western diet, a fundamental environmental factor involved in the pathogenesis of epidemic acne. Western nutrition is characterized by high calorie uptake, high glycemic load, high fat and meat intake, as well as increased consumption of insulin- and IGF-1-level elevating dairy proteins. Metabolic signals of Western diet are sensed by the nutrient-sensitive kinase, mammalian target of rapamycin complex 1 (mTORC1), which integrates signals of cellular energy, growth factors (insulin, IGF-1) and protein-derived signals, predominantly leucine, provided in high amounts by milk proteins and meat. mTORC1 activates SREBP, the master transcription factor of lipogenesis. Leucine stimulates mTORC1-SREBP signaling and leucine is directly converted by sebocytes into fatty acids and sterols for sebaceous lipid synthesis. Over-activated mTORC1 increases androgen hormone secretion and most likely amplifies androgen-driven mTORC1 signaling of sebaceous follicles. Testosterone directly activates mTORC1. Future research should investigate the effects of isotretinoin on sebocyte mTORC1 activity. It is conceivable that isotretinoin may downregulate mTORC1 in sebocytes by upregulation of nuclear levels of FoxO1. The role of Western diet in acne can only be fully appreciated when all stimulatory inputs for maximal mTORC1 activation, i.e., glucose, insulin, IGF-1 and leucine, are adequately considered. Epidemic acne has to be recognized as an mTORC1-driven disease of civilization like obesity, type 2 diabetes, cancer and neurodegenerative diseases. These new insights into Western diet-mediated mTORC1-hyperactivity provide a rational basis for dietary intervention in acne by attenuating mTORC1 signaling by reducing (1) total energy intake, (2) hyperglycemic carbohydrates, (3) insulinotropic dairy proteins and (4) leucine-rich meat and dairy proteins. The necessary dietary changes are opposed to the evolution of

  3. Differential response of skeletal muscles to mTORC1 signaling during atrophy and hypertrophy

    Science.gov (United States)

    2013-01-01

    Background Skeletal muscle mass is determined by the balance between protein synthesis and degradation. Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of protein translation and has been implicated in the control of muscle mass. Inactivation of mTORC1 by skeletal muscle-specific deletion of its obligatory component raptor results in smaller muscles and a lethal dystrophy. Moreover, raptor-deficient muscles are less oxidative through changes in the expression PGC-1α, a critical determinant of mitochondrial biogenesis. These results suggest that activation of mTORC1 might be beneficial to skeletal muscle by providing resistance to muscle atrophy and increasing oxidative function. Here, we tested this hypothesis by deletion of the mTORC1 inhibitor tuberous sclerosis complex (TSC) in muscle fibers. Method Skeletal muscles of mice with an acute or a permanent deletion of raptor or TSC1 were examined using histological, biochemical and molecular biological methods. Response of the muscles to changes in mechanical load and nerve input was investigated by ablation of synergistic muscles or by denervation . Results Genetic deletion or knockdown of raptor, causing inactivation of mTORC1, was sufficient to prevent muscle growth and enhance muscle atrophy. Conversely, short-term activation of mTORC1 by knockdown of TSC induced muscle fiber hypertrophy and atrophy-resistance upon denervation, in both fast tibialis anterior (TA) and slow soleus muscles. Surprisingly, however, sustained activation of mTORC1 by genetic deletion of Tsc1 caused muscle atrophy in all but soleus muscles. In contrast, oxidative capacity was increased in all muscles examined. Consistently, TSC1-deficient soleus muscle was atrophy-resistant whereas TA underwent normal atrophy upon denervation. Moreover, upon overloading, plantaris muscle did not display enhanced hypertrophy compared to controls. Biochemical analysis indicated that the atrophy response of muscles was based on the

  4. Rictor/mammalian target of rapamycin complex 2 promotes macrophage activation and kidney fibrosis.

    Science.gov (United States)

    Ren, Jiafa; Li, Jianzhong; Feng, Ye; Shu, Bingyan; Gui, Yuan; Wei, Wei; He, Weichun; Yang, Junwei; Dai, Chunsun

    2017-08-01

    Mammalian target of rapamycin (mTOR) signalling controls many essential cellular functions. However, the role of Rictor/mTOR complex 2 (mTORC2) in regulating macrophage activation and kidney fibrosis remains largely unknown. We report here that Rictor/mTORC2 was activated in macrophages from the fibrotic kidneys of mice. Ablation of Rictor in macrophages reduced kidney fibrosis, inflammatory cell accumulation, macrophage proliferation and polarization after unilateral ureter obstruction or ischaemia/reperfusion injury. In bone marrow-derived macrophages (BMMs), deletion of Rictor or blockade of protein kinase Cα inhibited cell migration. Additionally, deletion of Rictor or blockade of Akt abolished interleukin-4-stimulated or transforming growth factor (TGF)-β1-stimulated macrophage M2 polarization. Furthermore, deletion of Rictor downregulated TGF-β1-stimulated upregulation of multiple profibrotic cytokines, including platelet-derived growth factor, vascular endothelial growth factor and connective tissue growth factor, in BMMs. Conditioned medium from TGF-β1-pretreated Rictor -/- macrophages stimulated fibroblast activation less efficiently than that from TGF-β1-pretreated Rictor +/+ macrophages. These results demonstrate that Rictor/mTORC2 signalling can promote macrophage activation and kidney fibrosis. Targeting this signalling pathway in macrophages may shine light on ways to protect against kidney fibrosis in patients with chronic kidney diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  5. mTORC1 directly phosphorylates and regulates human MAF1.

    Science.gov (United States)

    Michels, Annemieke A; Robitaille, Aaron M; Buczynski-Ruchonnet, Diane; Hodroj, Wassim; Reina, Jaime H; Hall, Michael N; Hernandez, Nouria

    2010-08-01

    mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations nor the kinase involved is known. We find that human MAF1 is absolutely required for pol III repression in response to serum starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75, and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. Our results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription, and add a new branch to the signal transduction cascade immediately downstream of TORC1.

  6. Sestrin2 inhibits mTORC1 through modulation of GATOR complexes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jeong Sig; Ro, Seung-Hyun; Kim, Myungjin; Park, Hwan-Woo; Semple, Ian A.; Park, Haeli; Cho, Uhn-Soo; Wang, Wei; Guan, Kun-Liang; Karin, Michael; Lee, Jun Hee (Michigan); (UCSD)

    2015-03-30

    Sestrins are stress-inducible metabolic regulators that suppress a wide range of age- and obesity-associated pathologies, many of which are due to mTORC1 overactivation. Upon various stresses, the Sestrins inhibit mTORC1 activity through an indirect mechanism that is still unclear. GATORs are recently identified protein complexes that regulate the activity of RagB, a small GTPase essential for mTORC1 activation. GATOR1 is a GTPase activating protein (GAP) for RagB whereas GATOR2 functions as an inhibitor of GATOR1. However, how the GATORs are physiologically regulated is unknown. Here we show that Sestrin2 binds to GATOR2, and liberates GATOR1 from GATOR2-mediated inhibition. Released GATOR1 subsequently binds to and inactivates RagB, ultimately resulting in mTORC1 suppression. Consistent with this biochemical mechanism, genetic ablation of GATOR1 nullifies the mTORC1-inhibiting effect of Sestrin2 in both cell culture and Drosophila models. Collectively, we elucidate a new signaling cascade composed of Sestrin2-GATOR2-GATOR1-RagB that mediates stress-dependent suppression of mTORC1 activity.

  7. ROLE OF PI3K-AKT-mTOR AND Wnt SIGNALING PATHWAYS IN G1-S TRANSITION OF CELL CYCLE IN CANCER CELLS

    Directory of Open Access Journals (Sweden)

    LAKSHMIPATHI eVADLAKONDA

    2013-04-01

    Full Text Available The PI3K–Akt pathway together with one of its downstream targets, the mechanistic target of rapamycin (mTOR is a highly deregulated pathway in cancers. There is a reciprocal relation between the Akt phosphorylation and mTOR complexes. Akt phosphorylated at T308 activates mTORC1 by inhibition of the tuberous sclerosis complex (TSC1/2, where as mTORC2 is recognized as the kinase that phosphorylates Akt at S473. Recent developments in the research on regulatory mechanisms of autophagy places mTORC1 mediated inhibition of autophagy at the central position in activation of proliferation and survival pathways in cells. Autophagy is a negative regulator of Wnt signaling pathway and the downstream effectors of Wnt signaling pathway, cyclin D1 and the c-Myc, are the key players in initiation of cell cycle and regulation of the G1-S transition in cancer cells. Production of reaction oxygen species (ROS, a common feature of a cancer cell metabolism, activates several downstream targets like the transcription factors FoxO, which play key roles in promoting the progression of cell cycle. A model is presented on the role of PI3K -Akt - mTOR and Wnt pathways in regulation of the progression of cell cycle through Go-G1-and S phases.

  8. Effects of ketamine administration on mTOR and reticulum stress signaling pathways in the brain after the infusion of rapamycin into prefrontal cortex.

    Science.gov (United States)

    Abelaira, Helena M; Réus, Gislaine Z; Ignácio, Zuleide M; Dos Santos, Maria Augusta B; de Moura, Airam B; Matos, Danyela; Demo, Júlia P; da Silva, Júlia B I; Michels, Monique; Abatti, Mariane; Sonai, Beatriz; Dal Pizzol, Felipe; Carvalho, André F; Quevedo, João

    2017-04-01

    Recent studies show that activation of the mTOR signaling pathway is required for the rapid antidepressant actions of glutamate N-methyl-D-aspartate (NMDA) receptor antagonists. A relationship between mTOR kinase and the endoplasmic reticulum (ER) stress pathway, also known as the unfolded protein response (UPR) has been shown. We evaluate the effects of ketamine administration on the mTOR signaling pathway and proteins of UPR in the prefrontal cortex (PFC), hippocampus, amygdala and nucleus accumbens, after the inhibiton of mTOR signaling in the PFC. Male adult Wistar rats received pharmacological mTOR inhibitor, rapamycin (0.2 nmol), or vehicle into the PFC and then a single dose of ketamine (15 mg/kg, i.p.). The immunocontent of mTOR, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), eukaryotic elongation factor 2 kinase (eEF2K) homologous protein (CHOP), PKR-like ER kinase (PERK) and inositol-requiring enzyme 1 (IRE1) - alpha were determined in the brain. The mTOR levels were reduced in the rapamycin group treated with saline and ketamine in the PFC; p4EBP1 levels were reduced in the rapamycin group treated with ketamine in the PFC and nucleus accumbens; the levels of peEF2K were increased in the PFC in the vehicle group treated with ketamine and reduced in the rapamycin group treated with ketamine. The PERK and IRE1-alpha levels were decreased in the PFC in the rapamycin group treated with ketamine. Our results suggest that mTOR signaling inhibition by rapamycin could be involved, at least in part, with the mechanism of action of ketamine; and the ketamine antidepressant on ER stress pathway could be also mediated by mTOR signaling pathway in certain brain structures. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Saccharomyces cerevisiae FKBP12 binds Arabidopsis thaliana TOR and its expression in plants leads to rapamycin susceptibility.

    Science.gov (United States)

    Sormani, Rodnay; Yao, Lei; Menand, Benoît; Ennar, Najla; Lecampion, Cécile; Meyer, Christian; Robaglia, Christophe

    2007-06-01

    The eukaryotic TOR pathway controls translation, growth and the cell cycle in response to environmental signals such as nutrients or growth-stimulating factors. The TOR protein kinase can be inactivated by the antibiotic rapamycin following the formation of a ternary complex between TOR, rapamycin and FKBP12 proteins. The TOR protein is also found in higher plants despite the fact that they are rapamycin insensitive. Previous findings using the yeast two hybrid system suggest that the FKBP12 plant homolog is unable to form a complex with rapamycin and TOR, while the FRB domain of plant TOR is still able to bind to heterologous FKBP12 in the presence of rapamycin. The resistance to rapamycin is therefore limiting the molecular dissection of the TOR pathway in higher plants. Here we show that none of the FKBPs from the model plant Arabidopsis (AtFKBPs) is able to form a ternary complex with the FRB domain of AtTOR in the presence of rapamycin in a two hybrid system. An antibody has been raised against the AtTOR protein and binding of recombinant yeast ScFKBP12 to native Arabidopsis TOR in the presence of rapamycin was demonstrated in pull-down experiments. Transgenic lines expressing ScFKBP12 were produced and were found to display a rapamycin-dependent reduction of the primary root growth and a lowered accumulation of high molecular weight polysomes. These results further strengthen the idea that plant resistance to rapamycin evolved as a consequence of mutations in plant FKBP proteins. The production of rapamycin-sensitive plants through the expression of the ScFKBP12 protein illustrates the conservation of the TOR pathway in eukaryotes. Since AtTOR null mutants were found to be embryo lethal 1, transgenic ScFKBP12 plants will provide an useful tool for the post-embryonic study of plant TOR functions. This work also establish for the first time a link between TOR activity and translation in plant cells.

  10. Saccharomyces cerevisiae FKBP12 binds Arabidopsis thaliana TOR and its expression in plants leads to rapamycin susceptibility

    Directory of Open Access Journals (Sweden)

    Meyer Christian

    2007-06-01

    Full Text Available Abstract Background The eukaryotic TOR pathway controls translation, growth and the cell cycle in response to environmental signals such as nutrients or growth-stimulating factors. The TOR protein kinase can be inactivated by the antibiotic rapamycin following the formation of a ternary complex between TOR, rapamycin and FKBP12 proteins. The TOR protein is also found in higher plants despite the fact that they are rapamycin insensitive. Previous findings using the yeast two hybrid system suggest that the FKBP12 plant homolog is unable to form a complex with rapamycin and TOR, while the FRB domain of plant TOR is still able to bind to heterologous FKBP12 in the presence of rapamycin. The resistance to rapamycin is therefore limiting the molecular dissection of the TOR pathway in higher plants. Results Here we show that none of the FKBPs from the model plant Arabidopsis (AtFKBPs is able to form a ternary complex with the FRB domain of AtTOR in the presence of rapamycin in a two hybrid system. An antibody has been raised against the AtTOR protein and binding of recombinant yeast ScFKBP12 to native Arabidopsis TOR in the presence of rapamycin was demonstrated in pull-down experiments. Transgenic lines expressing ScFKBP12 were produced and were found to display a rapamycin-dependent reduction of the primary root growth and a lowered accumulation of high molecular weight polysomes. Conclusion These results further strengthen the idea that plant resistance to rapamycin evolved as a consequence of mutations in plant FKBP proteins. The production of rapamycin-sensitive plants through the expression of the ScFKBP12 protein illustrates the conservation of the TOR pathway in eukaryotes. Since AtTOR null mutants were found to be embryo lethal 1, transgenic ScFKBP12 plants will provide an useful tool for the post-embryonic study of plant TOR functions. This work also establish for the first time a link between TOR activity and translation in plant cells

  11. mTORC1 Directly Phosphorylates and Regulates Human MAF1▿

    Science.gov (United States)

    Michels, Annemieke A.; Robitaille, Aaron M.; Buczynski-Ruchonnet, Diane; Hodroj, Wassim; Reina, Jaime H.; Hall, Michael N.; Hernandez, Nouria

    2010-01-01

    mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations nor the kinase involved is known. We find that human MAF1 is absolutely required for pol III repression in response to serum starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75, and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. Our results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription, and add a new branch to the signal transduction cascade immediately downstream of TORC1. PMID:20516213

  12. Sleep deprivation impairs memory by attenuating mTORC1-dependent protein synthesis.

    Science.gov (United States)

    Tudor, Jennifer C; Davis, Emily J; Peixoto, Lucia; Wimmer, Mathieu E; van Tilborg, Erik; Park, Alan J; Poplawski, Shane G; Chung, Caroline W; Havekes, Robbert; Huang, Jiayan; Gatti, Evelina; Pierre, Philippe; Abel, Ted

    2016-04-26

    Sleep deprivation is a public health epidemic that causes wide-ranging deleterious consequences, including impaired memory and cognition. Protein synthesis in hippocampal neurons promotes memory and cognition. The kinase complex mammalian target of rapamycin complex 1 (mTORC1) stimulates protein synthesis by phosphorylating and inhibiting the eukaryotic translation initiation factor 4E-binding protein 2 (4EBP2). We investigated the involvement of the mTORC1-4EBP2 axis in the molecular mechanisms mediating the cognitive deficits caused by sleep deprivation in mice. Using an in vivo protein translation assay, we found that loss of sleep impaired protein synthesis in the hippocampus. Five hours of sleep loss attenuated both mTORC1-mediated phosphorylation of 4EBP2 and the interaction between eukaryotic initiation factor 4E (eIF4E) and eIF4G in the hippocampi of sleep-deprived mice. Increasing the abundance of 4EBP2 in hippocampal excitatory neurons before sleep deprivation increased the abundance of phosphorylated 4EBP2, restored the amount of eIF4E-eIF4G interaction and hippocampal protein synthesis to that seen in mice that were not sleep-deprived, and prevented the hippocampus-dependent memory deficits associated with sleep loss. These findings collectively demonstrate that 4EBP2-regulated protein synthesis is a critical mediator of the memory deficits caused by sleep deprivation. Copyright © 2016, American Association for the Advancement of Science.

  13. Mechanisms of amino acid sensing in mTOR signaling pathway

    OpenAIRE

    Kim, Eunjung

    2009-01-01

    Amino acids are fundamental nutrients for protein synthesis and cell growth (increase in cell size). Recently, many compelling evidences have shown that the level of amino acids is sensed by extra- or intra-cellular amino acids sensor(s) and regulates protein synthesis/degradation. Mammalian target of rapamycin complex 1 (mTORC1) is placed in a central position in cell growth regulation and dysregulation of mTOR signaling pathway has been implicated in many serious human diseases including ca...

  14. PDMP, a ceramide analogue, acts as an inhibitor of mTORC1 by inducing its translocation from lysosome to endoplasmic reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Ode, Takashi [Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan); Research Fellow of the Japan Society for the Promotion of Science (JSPS), 5-3-1 Kojimachi, Chiyoda-ku, Tokyo 102-0083 (Japan); Podyma-Inoue, Katarzyna A.; Terasawa, Kazue [Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan); Inokuchi, Jin-ichi [Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558 (Japan); Kobayashi, Toshihide [Lipid Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); CNRS, UMR 7213, University of Strasbourg, 67401 Illkirch (France); Watabe, Tetsuro [Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan); Izumi, Yuichi [Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan); Hara-Yokoyama, Miki, E-mail: m.yokoyama.bch@tmd.ac.jp [Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan)

    2017-01-01

    Mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism, and cell differentiation. Recent studies have revealed that the recruitment of mTORC1 to lysosomes is essential for its activation. The ceramide analogue 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a well known glycosphingolipid synthesis inhibitor, also affects the structures and functions of various organelles, including lysosomes and endoplasmic reticulum (ER). We investigated whether PDMP regulates the mTORC1 activity through its effects on organellar behavior. PDMP induced the translocation of mTORC1 from late endosomes/lysosomes, leading to the dissociation of mTORC1 from its activator Rheb in MC3T3-E1 cells. Surprisingly, we found mTORC1 translocation to the ER upon PDMP treatment. This effect of PDMP was independent of its action as the inhibitor, since two stereoisomers of PDMP, with and without the inhibitor activity, showed essentially the same effect. We confirmed that PDMP inhibits the mTORC1 activity based on the decrease in the phosphorylation of ribosomal S6 kinase, a downstream target of mTORC1, and the increase in LC3 puncta, reflecting autophagosome formation. Furthermore, PDMP inhibited the mTORC1-dependent osteoblastic cell proliferation and differentiation of MC3T3-E1 cells. Accordingly, the present results reveal a novel mechanism of PDMP, which inhibits the mTORC1 activity by inducing the translocation of mTOR from lysosomes to the ER. - Highlights: • The ceramide analogue, PDMP, suppressed the activation of mTORC1. • PDMP induced the translocation of mTOR from lysosomes to ER. • PDMP led to the dissociation of mTOR from its activator Rheb. • PDMP inhibited the mTORC1-dependent osteoblastic cell proliferation.

  15. mTORC1 activity as a determinant of cancer risk--rationalizing the cancer-preventive effects of adiponectin, metformin, rapamycin, and low-protein vegan diets.

    Science.gov (United States)

    McCarty, Mark F

    2011-10-01

    Increased plasma levels of adiponectin, metformin therapy of diabetes, rapamycin administration in transplant patients, and lifelong consumption of low-protein plant-based diets have all been linked to decreased risk for various cancers. These benefits may be mediated, at least in part, by down-regulated activity of the mTORC1 complex, a key regulator of protein translation. By boosting the effective availability of the translation initiator eIF4E, mTORC1 activity promotes the translation of a number of "weak" mRNAs that code for proteins, often up-regulated in cancer, that promote cellular proliferation, invasiveness, and angiogenesis, and that abet cancer promotion and chemoresistance by opposing apoptosis. Measures which inhibit eIF4E activity, either directly or indirectly, may have utility not only for cancer prevention, but also for the treatment of many cancers in which eIF4E drives malignancy. Since eIF4E is overexpressed in many cancers, strategies which target eIF4E directly--some of which are now being assessed clinically--may have the broadest efficacy in this regard. Many of the "weak" mRNAs coding for proteins that promote malignant behavior or chemoresistance are regulated transcriptionally by NF-kappaB and/or Stat3, which are active in a high proportion of cancers; thus, regimens concurrently targeting eIF4E, NF-kappaB, and Stat3 may suppress these proteins at both the transcriptional and translational levels, potentially achieving a very marked reduction in their expression. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Arctigenin functions as a selective agonist of estrogen receptor ? to restrict mTORC1 activation and consequent Th17 differentiation

    OpenAIRE

    Wu, Xin; Tong, Bei; Yang, Yan; Luo, Jinque; Yuan, Xusheng; Wei, Zhifeng; Yue, Mengfan; Xia, Yufeng; Dai, Yue

    2016-01-01

    Arctigenin was previously proven to inhibit Th17 cell differentiation and thereby attenuate colitis in mice by down-regulating the activation of mechanistic target of rapamycin complex 1 (mTORC1). The present study was performed to address its underlying mechanism in view of estrogen receptor (ER). The specific antagonist PHTPP or siRNA of ER? largely diminished the inhibitory effect of arctigenin on the mTORC1 activation in T cell lines and primary CD4+ T cells under Th17-polarization condit...

  17. Resolvin D1 and D2 Reverse Lipopolysaccharide-Induced Depression-Like Behaviors Through the mTORC1 Signaling Pathway.

    Science.gov (United States)

    Deyama, Satoshi; Ishikawa, Yuka; Yoshikawa, Kotomi; Shimoda, Kento; Ide, Soichiro; Satoh, Masamichi; Minami, Masabumi

    2017-07-01

    Resolvin D1 and D2 are bioactive lipid mediators that are generated from docosahexaenoic acid. Although recent preclinical studies suggest that these compounds have antidepressant effects, their mechanisms of action remain unclear. We investigated mechanisms underlying the antidepressant effects of resolvin D1 and resolvin D2 in lipopolysaccharide (0.8 mg/kg, i.p.)-induced depression model mice using a tail suspension test. I.c.v. infusion of resolvin D1 (10 ng) and resolvin D2 (10 ng) produced antidepressant effects; these effects were significantly blocked by a resolvin D1 receptor antagonist WRW4 (10 µg, i.c.v.) and a resolvin D2 receptor antagonist O-1918 (10 µg, i.c.v.), respectively. The mammalian target of rapamycin complex 1 inhibitor rapamycin (10 mg/kg, i.p.) and a mitogen-activated protein kinase kinase inhibitor U0126 (5 µg, i.c.v.) significantly blocked the antidepressant effects of resolvin D1 and resolvin D2. An AMPA receptor antagonist NBQX (10 mg/kg, i.p.) and a phosphoinositide 3-kinase inhibitor LY294002 (3 µg, i.c.v.) blocked the antidepressant effects of resolvin D1 significantly, but not of resolvin D2. Bilateral infusions of resolvin D1 (0.3 ng/side) or resolvin D2 (0.3 ng/side) into the medial prefrontal cortex or dentate gyrus of the hippocampus produced antidepressant effects. These findings demonstrate that resolvin D1 and resolvin D2 produce antidepressant effects via the mammalian target of rapamycin complex 1 signaling pathway, and that the medial prefrontal cortex and dentate gyrus are important brain regions for these antidepressant effects. These compounds and their receptors may be promising targets for the development of novel rapid-acting antidepressants, like ketamine and scopolamine. © The Author 2017. Published by Oxford University Press on behalf of CINP.

  18. The cochaperone BAG3 coordinates protein synthesis and autophagy under mechanical strain through spatial regulation of mTORC1.

    Science.gov (United States)

    Kathage, Barbara; Gehlert, Sebastian; Ulbricht, Anna; Lüdecke, Laura; Tapia, Victor E; Orfanos, Zacharias; Wenzel, Daniela; Bloch, Wilhelm; Volkmer, Rudolf; Fleischmann, Bernd K; Fürst, Dieter O; Höhfeld, Jörg

    2017-01-01

    The cochaperone BAG3 is a central protein homeostasis factor in mechanically strained mammalian cells. It mediates the degradation of unfolded and damaged forms of the actin-crosslinker filamin through chaperone-assisted selective autophagy (CASA). In addition, BAG3 stimulates filamin transcription in order to compensate autophagic disposal and to maintain the actin cytoskeleton under strain. Here we demonstrate that BAG3 coordinates protein synthesis and autophagy through spatial regulation of the mammalian target of rapamycin complex 1 (mTORC1). The cochaperone utilizes its WW domain to contact a proline-rich motif in the tuberous sclerosis protein TSC1 that functions as an mTORC1 inhibitor in association with TSC2. Interaction with BAG3 results in a recruitment of TSC complexes to actin stress fibers, where the complexes act on a subpopulation of mTOR-positive vesicles associated with the cytoskeleton. Local inhibition of mTORC1 is essential to initiate autophagy at sites of filamin unfolding and damage. At the same time, BAG3-mediated sequestration of TSC1/TSC2 relieves mTORC1 inhibition in the remaining cytoplasm, which stimulates protein translation. In human muscle, an exercise-induced association of TSC1 with the cytoskeleton coincides with mTORC1 activation in the cytoplasm. The spatial regulation of mTORC1 exerted by BAG3 apparently provides the basis for a simultaneous induction of autophagy and protein synthesis to maintain the proteome under mechanical strain. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Descending serotonergic facilitation mediated by spinal 5-HT3 receptors engages spinal rapamycin-sensitive pathways in the rat

    Science.gov (United States)

    Asante, Curtis O.; Dickenson, Anthony H.

    2010-01-01

    We have recently reported the importance of spinal rapamycin-sensitive pathways in maintaining persistent pain-like states. A descending facilitatory drive mediated through spinal 5-HT3 receptors (5-HT3Rs) originating from superficial dorsal horn NK1-expressing neurons and that relays through the parabrachial nucleus and the rostroventral medial medulla to act on deep dorsal horn neurons is known be important in maintaining these pain-like states. To determine if spinal rapamycin-sensitive pathways are activated by a descending serotonergic drive, we investigated the effects of spinally administered rapamycin on responses of deep dorsal horn neurons that had been pre-treated with the selective 5-HT3R antagonist ondansetron. We also investigated the effects of spinally administered cell cycle inhibitor (CCI)-779 (a rapamycin ester analogue) on deep dorsal horn neurons from rats with carrageenan-induced inflammation of the hind paw. Unlike some other models of persistent pain, this model does not involve an altered 5-HT3R-mediated descending serotonergic drive. We found that the inhibitory effects of rapamycin were significantly reduced for neuronal responses to mechanical and thermal stimuli when the spinal cord was pre-treated with ondansetron. Furthermore, CCI-779 was found to be ineffective in attenuating spinal neuronal responses to peripheral stimuli in carrageenan-treated rats. Therefore, we conclude that 5-HT3R-mediated descending facilitation is one requirement for activation of rapamycin-sensitive pathways that contribute to persistent pain-like states. PMID:20709148

  20. Eukaryotic initiation factor 2α--a downstream effector of mammalian target of rapamycin--modulates DNA repair and cancer response to treatment.

    Directory of Open Access Journals (Sweden)

    Liron Tuval-Kochen

    Full Text Available In an effort to circumvent resistance to rapamycin--an mTOR inhibitor--we searched for novel rapamycin-downstream-targets that may be key players in the response of cancer cells to therapy. We found that rapamycin, at nM concentrations, increased phosphorylation of eukaryotic initiation factor (eIF 2α in rapamycin-sensitive and estrogen-dependent MCF-7 cells, but had only a minimal effect on eIF2α phosphorylation in the rapamycin-insensitive triple-negative MDA-MB-231 cells. Addition of salubrinal--an inhibitor of eIF2α dephosphorylation--decreased expression of a surface marker associated with capacity for self renewal, increased senescence and induced clonogenic cell death, suggesting that excessive phosphorylation of eIF2α is detrimental to the cells' survival. Treating cells with salubrinal enhanced radiation-induced increase in eIF2α phosphorylation and clonogenic death and showed that irradiated cells are more sensitive to increased eIF2α phosphorylation than non-irradiated ones. Similar to salubrinal--the phosphomimetic eIF2α variant--S51D--increased sensitivity to radiation, and both abrogated radiation-induced increase in breast cancer type 1 susceptibility gene, thus implicating enhanced phosphorylation of eIF2α in modulation of DNA repair. Indeed, salubrinal inhibited non-homologous end joining as well as homologous recombination repair of double strand breaks that were induced by I-SceI in green fluorescent protein reporter plasmids. In addition to its effect on radiation, salubrinal enhanced eIF2α phosphorylation and clonogenic death in response to the histone deacetylase inhibitor--vorinostat. Finally, the catalytic competitive inhibitor of mTOR--Ku-0063794--increased phosphorylation of eIF2α demonstrating further the involvement of mTOR activity in modulating eIF2α phosphorylation. These experiments suggest that excessive phosphorylation of eIF2α decreases survival of cancer cells; making eIF2α a worthy target for

  1. Potential role of mTORC2 as a therapeutic target in clear cell carcinoma of the ovary.

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    Hisamatsu, Takeshi; Mabuchi, Seiji; Matsumoto, Yuri; Kawano, Mahiru; Sasano, Tomoyuki; Takahashi, Ryoko; Sawada, Kenjiro; Ito, Kimihiko; Kurachi, Hirohisa; Schilder, Russell J; Testa, Joseph R; Kimura, Tadashi

    2013-07-01

    The goal of this study was to examine the role of mTOR complex 2 (mTORC2) as a therapeutic target in ovarian clear cell carcinoma (CCC), which is regarded as an aggressive, chemoresistant histologic subtype. Using tissue microarrays of 98 primary ovarian cancers [52 CCCs and 46 serous adenocarcinomas (SAC)], activation of mTORC2 was assessed by immunohistochemistry. Then, the growth-inhibitory effect of mTORC2-targeting therapy, as well as the role of mTORC2 signaling as a mechanism for acquired resistance to the mTOR complex 1 (mTORC1) inhibitor RAD001 in ovarian CCC, were examined using two pairs of RAD001-sensitive parental (RMG2 and HAC2) and RAD001-resistant CCC cell lines (RMG2-RR and HAC2-RR). mTORC2 was more frequently activated in CCCs than in SACs (71.2% vs. 45.7%). Simultaneous inhibition of mTORC1 and mTORC2 by AZD8055 markedly inhibited the proliferation of both RAD001-sensitive and -resistant cells in vitro. Treatment with RAD001 induced mTORC2-mediated AKT activation in RAD001-sensitive CCC cells. Moreover, increased activation of mTORC2-AKT signaling was observed in RAD001-resistant CCC cells compared with the respective parental cells. Inhibition of mTORC2 during RAD001 treatment enhanced the antitumor effect of RAD001 and prevented CCC cells from acquiring resistance to RAD001. In conclusion, mTORC2 is frequently activated, and can be a promising therapeutic target, in ovarian CCCs. Moreover, mTORC2-targeted therapy may be efficacious in a first-line setting as well as for second-line treatment of recurrent disease developing after RAD001-treatment.

  2. A hepatic amino acid/mTOR/S6K-dependent signalling pathway modulates systemic lipid metabolism via neuronal signals.

    Science.gov (United States)

    Uno, Kenji; Yamada, Tetsuya; Ishigaki, Yasushi; Imai, Junta; Hasegawa, Yutaka; Sawada, Shojiro; Kaneko, Keizo; Ono, Hiraku; Asano, Tomoichiro; Oka, Yoshitomo; Katagiri, Hideki

    2015-08-13

    Metabolism is coordinated among tissues and organs via neuronal signals. Levels of circulating amino acids (AAs), which are elevated in obesity, activate the intracellular target of rapamycin complex-1 (mTORC1)/S6kinase (S6K) pathway in the liver. Here we demonstrate that hepatic AA/mTORC1/S6K signalling modulates systemic lipid metabolism via a mechanism involving neuronal inter-tissue communication. Hepatic expression of an AA transporter, SNAT2, activates the mTORC1/S6K pathway, and markedly elevates serum triglycerides (TGs), while downregulating adipose lipoprotein lipase (LPL). Hepatic Rheb or active-S6K expression have similar metabolic effects, whereas hepatic expression of dominant-negative-S6K inhibits TG elevation in SNAT2 mice. Denervation, pharmacological deafferentation and β-blocker administration suppress obesity-related hypertriglyceridemia with adipose LPL upregulation, suggesting that signals are transduced between liver and adipose tissue via a neuronal pathway consisting of afferent vagal and efferent sympathetic nerves. Thus, the neuronal mechanism uncovered here serves to coordinate amino acid and lipid levels and contributes to the development of obesity-related hypertriglyceridemia.

  3. Co-activation of AMPK and mTORC1 Induces Cytotoxicity in Acute Myeloid Leukemia

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    Pierre Sujobert

    2015-06-01

    Full Text Available AMPK is a master regulator of cellular metabolism that exerts either oncogenic or tumor suppressor activity depending on context. Here, we report that the specific AMPK agonist GSK621 selectively kills acute myeloid leukemia (AML cells but spares normal hematopoietic progenitors. This differential sensitivity results from a unique synthetic lethal interaction involving concurrent activation of AMPK and mTORC1. Strikingly, the lethality of GSK621 in primary AML cells and AML cell lines is abrogated by chemical or genetic ablation of mTORC1 signaling. The same synthetic lethality between AMPK and mTORC1 activation is established in CD34-positive hematopoietic progenitors by constitutive activation of AKT or enhanced in AML cells by deletion of TSC2. Finally, cytotoxicity in AML cells from GSK621 involves the eIF2α/ATF4 signaling pathway that specifically results from mTORC1 activation. AMPK activation may represent a therapeutic opportunity in mTORC1-overactivated cancers.

  4. Dual mTORC1/C2 inhibitors suppress cellular geroconversion (a senescence program).

    Science.gov (United States)

    Leontieva, Olga V; Demidenko, Zoya N; Blagosklonny, Mikhail V

    2015-09-15

    In proliferating cells, mTOR is active and promotes cell growth. When the cell cycle is arrested, then mTOR converts reversible arrest to senescence (geroconversion). Rapamycin and other rapalogs suppress geroconversion, maintaining quiescence instead. Here we showed that ATP-competitive kinase inhibitors (Torin1 and PP242), which inhibit both mTORC1 and TORC2, also suppressed geroconversion. Despite inhibition of proliferation (in proliferating cells), mTOR inhibitors preserved re-proliferative potential (RP) in arrested cells. In p21-arrested cells, Torin 1 and PP242 detectably suppressed geroconversion at concentrations as low as 1-3 nM and 10-30 nM, reaching maximal gerosuppression at 30 nM and 300 nM, respectively. Near-maximal gerosuppression coincided with inhibition of p-S6K(T389) and p-S6(S235/236). Dual mTOR inhibitors prevented senescent morphology and hypertrophy. Our study warrants investigation into whether low doses of dual mTOR inhibitors will prolong animal life span and delay age-related diseases. A new class of potential anti-aging drugs can be envisioned.

  5. mTORC1 Inhibition Corrects Neurodevelopmental and Synaptic Alterations in a Human Stem Cell Model of Tuberous Sclerosis

    Directory of Open Access Journals (Sweden)

    Veronica Costa

    2016-04-01

    Full Text Available Hyperfunction of the mTORC1 pathway has been associated with idiopathic and syndromic forms of autism spectrum disorder (ASD, including tuberous sclerosis, caused by loss of either TSC1 or TSC2. It remains largely unknown how developmental processes and biochemical signaling affected by mTORC1 dysregulation contribute to human neuronal dysfunction. Here, we have characterized multiple stages of neurogenesis and synapse formation in human neurons derived from TSC2-deleted pluripotent stem cells. Homozygous TSC2 deletion causes severe developmental abnormalities that recapitulate pathological hallmarks of cortical malformations in patients. Both TSC2+/− and TSC2−/− neurons display altered synaptic transmission paralleled by molecular changes in pathways associated with autism, suggesting the convergence of pathological mechanisms in ASD. Pharmacological inhibition of mTORC1 corrects developmental abnormalities and synaptic dysfunction during independent developmental stages. Our results uncouple stage-specific roles of mTORC1 in human neuronal development and contribute to a better understanding of the onset of neuronal pathophysiology in tuberous sclerosis.

  6. Morphoproteomic profiling of the mammalian target of rapamycin (mTOR) signaling pathway in desmoplastic small round cell tumor (EWS/WT1), Ewing's sarcoma (EWS/FLI1) and Wilms' tumor(WT1).

    Science.gov (United States)

    Subbiah, Vivek; Brown, Robert E; Jiang, Yunyun; Buryanek, Jamie; Hayes-Jordan, Andrea; Kurzrock, Razelle; Anderson, Pete M

    2013-01-01

    Desmoplastic small round cell tumor (DSRCT) is a rare sarcoma in adolescents and young adults. The hallmark of this disease is a EWS-WT1 translocation resulting from apposition of the Ewing's sarcoma (EWS) gene with the Wilms' tumor (WT1) gene. We performed morphoproteomic profiling of DSRCT (EWS-WT1), Ewing's sarcoma (EWS-FLI1) and Wilms' tumor (WT1) to better understand the signaling pathways for selecting future targeted therapies. This pilot study assessed patients with DSRCT, Wilms' tumor and Ewing's sarcoma. Morphoproteomics and immunohistochemical probes were applied to detect: p-mTOR (Ser2448); p-Akt (Ser473); p-ERK1/2 (Thr202/Tyr204); p-STAT3 (Tyr 705); and cell cycle-related analytes along with their negative controls. In DSRCT the PI3K/Akt/mTOR pathway is constitutively activated by p-Akt (Ser 473) expression in the nuclear compartment of the tumor cells and p-mTOR phosphorylated on Ser 2448, suggesting mTORC2 (rictor+mTOR) as the dominant form. Ewing's sarcoma had upregulated p-Akt and p-mTOR, predominantly mTORC2. In Wilm's tumor, the mTOR pathway is also activated with most tumor cells moderately expressing p-mTOR (Ser 2448) in plasmalemmal and cytoplasmic compartments. This coincides with the constitutive activation of one of the downstream effectors of the mTORC1 signaling pathway, namely p-p70S6K (Thr 389). There was constitutive activation of the Ras/Raf/ERK pathway p-ERK 1/2 (Thr202/Tyr204) expression in the Wilms tumor and metastatic Ewing's sarcoma, but not in the DSRCT. MORPHOPROTEOMIC TUMOR ANALYSES REVEALED CONSTITUTIVE ACTIVATION OF THE MTOR PATHWAY AS EVIDENCED BY: (a) expression of phosphorylated (p)-mTOR, p-p70S6K; (b) mTORC 2 in EWS and DSRCT; (c) ERK signaling was seen in the advanced setting indicating these as resistance pathways to IGF1R related therapies. This is the first morphoproteomic study of such pathways in these rare malignancies and may have potential therapeutic implications. Further study using morphoproteomic

  7. Rapamycin causes activation of protein phosphatase-2A1 and nuclear translocation of PCNA in CD4+ T cells

    International Nuclear Information System (INIS)

    Morrow, Peter W.; Tung, H.Y. Lim; Hemmings, Hugh C.

    2004-01-01

    Rapamycin is a powerful immunosuppressant that causes cell cycle arrest in T cells and several other cell types. Despite its important clinical role, the mechanism of action of rapamycin is not fully understood. Here, we show that rapamycin causes the activation of protein phosphatase-2A 1 which forms a complex with proliferation cell nuclear antigen (PCNA) in a CD 4+ T cell line. Rapamycin also induces PCNA translocation from the cytoplasm to the nucleus, an effect which is antagonized by okadaic acid, an inhibitor of type 2A protein phosphatases. These findings provide evidence for the existence of a signal transduction pathway that links a rapamycin-activated type 2A protein phosphatase to the control of DNA synthesis, DNA repair, cell cycle, and cell death via PCNA

  8. Mutations in the HECT domain of NEDD4L lead to AKT-mTOR pathway deregulation and cause periventricular nodular heterotopia.

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    Broix, Loïc; Jagline, Hélène; Ivanova, Ekaterina; Schmucker, Stéphane; Drouot, Nathalie; Clayton-Smith, Jill; Pagnamenta, Alistair T; Metcalfe, Kay A; Isidor, Bertrand; Louvier, Ulrike Walther; Poduri, Annapurna; Taylor, Jenny C; Tilly, Peggy; Poirier, Karine; Saillour, Yoann; Lebrun, Nicolas; Stemmelen, Tristan; Rudolf, Gabrielle; Muraca, Giuseppe; Saintpierre, Benjamin; Elmorjani, Adrienne; Moïse, Martin; Weirauch, Nathalie Bednarek; Guerrini, Renzo; Boland, Anne; Olaso, Robert; Masson, Cecile; Tripathy, Ratna; Keays, David; Beldjord, Cherif; Nguyen, Laurent; Godin, Juliette; Kini, Usha; Nischké, Patrick; Deleuze, Jean-François; Bahi-Buisson, Nadia; Sumara, Izabela; Hinckelmann, Maria-Victoria; Chelly, Jamel

    2016-11-01

    Neurodevelopmental disorders with periventricular nodular heterotopia (PNH) are etiologically heterogeneous, and their genetic causes remain in many cases unknown. Here we show that missense mutations in NEDD4L mapping to the HECT domain of the encoded E3 ubiquitin ligase lead to PNH associated with toe syndactyly, cleft palate and neurodevelopmental delay. Cellular and expression data showed sensitivity of PNH-associated mutants to proteasome degradation. Moreover, an in utero electroporation approach showed that PNH-related mutants and excess wild-type NEDD4L affect neurogenesis, neuronal positioning and terminal translocation. Further investigations, including rapamycin-based experiments, found differential deregulation of pathways involved. Excess wild-type NEDD4L leads to disruption of Dab1 and mTORC1 pathways, while PNH-related mutations are associated with deregulation of mTORC1 and AKT activities. Altogether, these data provide insights into the critical role of NEDD4L in the regulation of mTOR pathways and their contributions in cortical development.

  9. Rapamycin and chloroquine: the in vitro and in vivo effects of autophagy-modifying drugs show promising results in valosin containing protein multisystem proteinopathy.

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    Angèle Nalbandian

    Full Text Available Mutations in the valosin containing protein (VCP gene cause hereditary Inclusion body myopathy (hIBM associated with Paget disease of bone (PDB, frontotemporal dementia (FTD, more recently termed multisystem proteinopathy (MSP. Affected individuals exhibit scapular winging and die from progressive muscle weakness, and cardiac and respiratory failure, typically in their 40s to 50s. Histologically, patients show the presence of rimmed vacuoles and TAR DNA-binding protein 43 (TDP-43-positive large ubiquitinated inclusion bodies in the muscles. We have generated a VCPR155H/+ mouse model which recapitulates the disease phenotype and impaired autophagy typically observed in patients with VCP disease. Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux. Herein, we report results of administration of rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR signaling pathway, and chloroquine, a lysosomal inhibitor which reverses autophagy by accumulating in lysosomes, responsible for blocking autophagy in 20-month old VCPR155H/+ mice. Rapamycin-treated mice demonstrated significant improvement in muscle performance, quadriceps histological analysis, and rescue of ubiquitin, and TDP-43 pathology and defective autophagy as indicated by decreased protein expression levels of LC3-I/II, p62/SQSTM1, optineurin and inhibiting the mTORC1 substrates. Conversely, chloroquine-treated VCPR155H/+ mice revealed progressive muscle weakness, cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-I/II, p62/SQSTM1, and optineurin expression levels. Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers. Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem

  10. Drug Modulators of B Cell Signaling Pathways and Epstein-Barr Virus Lytic Activation.

    Science.gov (United States)

    Kosowicz, John G; Lee, Jaeyeun; Peiffer, Brandon; Guo, Zufeng; Chen, Jianmeng; Liao, Gangling; Hayward, S Diane; Liu, Jun O; Ambinder, Richard F

    2017-08-15

    Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that establishes a latency reservoir in B cells. In this work, we show that ibrutinib, idelalisib, and dasatinib, drugs that block B cell receptor (BCR) signaling and are used in the treatment of hematologic malignancies, block BCR-mediated lytic induction at clinically relevant doses. We confirm that the immunosuppressive drugs cyclosporine and tacrolimus also inhibit BCR-mediated lytic induction but find that rapamycin does not inhibit BCR-mediated lytic induction. Further investigation shows that mammalian target of rapamycin complex 2 (mTORC2) contributes to BCR-mediated lytic induction and that FK506-binding protein 12 (FKBP12) binding alone is not adequate to block activation. Finally, we show that BCR signaling can activate EBV lytic induction in freshly isolated B cells from peripheral blood mononuclear cells (PBMCs) and that activation can be inhibited by ibrutinib or idelalisib. IMPORTANCE EBV establishes viral latency in B cells. Activation of the B cell receptor pathway activates lytic viral expression in cell lines. Here we show that drugs that inhibit important kinases in the BCR signaling pathway inhibit activation of lytic viral expression but do not inhibit several other lytic activation pathways. Immunosuppressant drugs such as cyclosporine and tacrolimus but not rapamycin also inhibit BCR-mediated EBV activation. Finally, we show that BCR activation of lytic infection occurs not only in tumor cell lines but also in freshly isolated B cells from patients and that this activation can be blocked by BCR inhibitors. Copyright © 2017 American Society for Microbiology.

  11. mTORC2 and AMPK differentially regulate muscle triglyceride content via Perilipin 3

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    Maximilian Kleinert

    2016-08-01

    Conclusions: We identified a novel link between mTORC2 and PLIN3, which regulates lipid storage in muscle. While mTORC2 is a negative regulator, we further identified AMPK as a positive regulator of PLIN3, which impacts whole-body substrate utilization and nutrient partitioning.

  12. mTOR pathway is activated in endothelial cells from patients with Takayasu arteritis and is modulated by serum immunoglobulin G.

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    Hadjadj, Jérôme; Canaud, Guillaume; Mirault, Tristan; Samson, Maxime; Bruneval, Patrick; Régent, Alexis; Goulvestre, Claire; Witko-Sarsat, Véronique; Costedoat-Chalumeau, Nathalie; Guillevin, Loïc; Mouthon, Luc; Terrier, Benjamin

    2018-06-01

    Takayasu arteritis (TA) and GCA are large-vessel vasculitides characterized by vascular remodelling involving endothelial cells (ECs) and vascular smooth muscle cells. Mammalian target of rapamycin (mTOR) pathway has been involved in vascular remodelling. We hypothesized that the mTOR pathway was involved in the pathogenesis of large-vessel vasculitis. We used IF analysis on aortic and temporal artery biopsies from patients with TA and GCA to assess the involvement of the mTOR pathway and searched for antibodies targeting ECs in serum by IIF and cellular ELISA. We evaluated in vitro the effect of purified IgG from patients on mTOR pathway activation and cell proliferation. IF analyses on tissues revealed that both mTORC1 and mTORC2 are activated specifically in ECs from TA patients but not in ECs from GCA patients and healthy controls (HCs). Using IIF and ELISA, we observed higher levels of antibodies binding to ECs in TA patients compared with GCA patients and HCs. Using western blot, we demonstrated that purified IgG from TA patients caused mTORC1 activation in ECs, whereas this effect was not observed with purified IgG from GCA patients or HCs. Purified IgG from TA patients induced a significant EC proliferation compared with to GCA and HC IgG, and this effect was decreased after EC exposure with sirolimus, a specific mTOR inhibitor and PI3K inhibitor. Our results suggest that antibodies targeting ECs drive endothelial remodelling in TA through activation of the mTOR pathway, but not in GCA. Inhibition of the mTOR pathway could represent a therapeutic option in TA.

  13. Restoration of Corticosteroid Sensitivity in Chronic Obstructive Pulmonary Disease by Inhibition of Mammalian Target of Rapamycin.

    Science.gov (United States)

    Mitani, Akihisa; Ito, Kazuhiro; Vuppusetty, Chaitanya; Barnes, Peter J; Mercado, Nicolas

    2016-01-15

    Corticosteroid resistance is a major barrier to the effective treatment of chronic obstructive pulmonary disease (COPD). Several molecular mechanisms have been proposed, such as activations of the phosphoinositide-3-kinase/Akt pathway and p38 mitogen-activated protein kinase. However, the mechanism for corticosteroid resistance is still not fully elucidated. To investigate the role of mammalian target of rapamycin (mTOR) in corticosteroid sensitivity in COPD. The corticosteroid sensitivity of peripheral blood mononuclear cells collected from patients with COPD, smokers, and nonsmoking control subjects, or of human monocytic U937 cells exposed to cigarette smoke extract (CSE), was quantified as the dexamethasone concentration required to achieve 30% inhibition of tumor necrosis factor-α-induced CXCL8 production in the presence or absence of the mTOR inhibitor rapamycin. mTOR activity was determined as the phosphorylation of p70 S6 kinase, using Western blotting. mTOR activity was increased in peripheral blood mononuclear cells from patients with COPD, and treatment with rapamycin inhibited this as well as restoring corticosteroid sensitivity. In U937 cells, CSE stimulated mTOR activity and c-Jun expression, but pretreatment with rapamycin inhibited both and also reversed CSE-induced corticosteroid insensitivity. mTOR inhibition by rapamycin restores corticosteroid sensitivity via inhibition of c-Jun expression, and thus mTOR is a potential novel therapeutic target for COPD.

  14. In vitro antiglioma action of indomethacin is mediated via AMP-activated protein kinase/mTOR complex 1 signalling pathway.

    Science.gov (United States)

    Pantovic, Aleksandar; Bosnjak, Mihajlo; Arsikin, Katarina; Kosic, Milica; Mandic, Milos; Ristic, Biljana; Tosic, Jelena; Grujicic, Danica; Isakovic, Aleksandra; Micic, Nikola; Trajkovic, Vladimir; Harhaji-Trajkovic, Ljubica

    2017-02-01

    We investigated the role of the intracellular energy-sensing AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the in vitro antiglioma effect of the cyclooxygenase (COX) inhibitor indomethacin. Indomethacin was more potent than COX inhibitors diclofenac, naproxen, and ketoprofen in reducing the viability of U251 human glioma cells. Antiglioma effect of the drug was associated with p21 increase and G 2 M cell cycle arrest, as well as with oxidative stress, mitochondrial depolarization, caspase activation, and the induction of apoptosis. Indomethacin increased the phosphorylation of AMPK and its targets Raptor and acetyl-CoA carboxylase (ACC), and reduced the phosphorylation of mTOR and mTOR complex 1 (mTORC1) substrates p70S6 kinase and PRAS40 (Ser183). AMPK knockdown by RNA interference, as well as the treatment with the mTORC1 activator leucine, prevented indomethacin-mediated mTORC1 inhibition and cytotoxic action, while AMPK activators metformin and AICAR mimicked the effects of the drug. AMPK activation by indomethacin correlated with intracellular ATP depletion and increase in AMP/ATP ratio, and was apparently independent of COX inhibition or the increase in intracellular calcium. Finally, the toxicity of indomethacin towards primary human glioma cells was associated with the activation of AMPK/Raptor/ACC and subsequent suppression of mTORC1/S6K. By demonstrating the involvement of AMPK/mTORC1 pathway in the antiglioma action of indomethacin, our results support its further exploration in glioma therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. mTORC2 and AMPK differentially regulate muscle triglyceride content via Perilipin 3

    DEFF Research Database (Denmark)

    Kleinert, Maximilian; Parker, Benjamin L; Chaudhuri, Rima

    2016-01-01

    culture. RESULTS: Ric mKO mice exhibited a greater reliance on fat as an energy substrate, a re-partitioning of lean to fat mass and an increase in intramyocellular triglyceride (IMTG) content, along with increases in several lipid metabolites in muscle. Unbiased proteomics revealed an increase......OBJECTIVE: We have recently shown that acute inhibition of both mTOR complexes (mTORC1 and mTORC2) increases whole-body lipid utilization, while mTORC1 inhibition had no effect. Therefore, we tested the hypothesis that mTORC2 regulates lipid metabolism in skeletal muscle. METHODS: Body composition...... in the expression of the lipid droplet binding protein Perilipin 3 (PLIN3) in muscle from Ric mKO mice. This was associated with increased AMPK activity in Ric mKO muscle. Reducing AMPK kinase activity decreased muscle PLIN3 expression and IMTG content. AMPK agonism, in turn, increased PLIN3 expression in a FoxO1...

  16. Anorexigenic and Orexigenic Hormone Modulation of Mammalian Target of Rapamycin Complex 1 Activity and the Regulation of Hypothalamic Agouti-Related Protein mRNA Expression

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    Kenneth R. Watterson

    2012-03-01

    Full Text Available Activation of mammalian target of rapamycin 1 (mTORC1 by nutrients, insulin and leptin leads to appetite suppression (anorexia. Contrastingly, increased AMP-activated protein kinase (AMPK activity by ghrelin promotes appetite (orexia. However, the interplay between these mechanisms remains poorly defined. The relationship between the anorexigenic hormones, insulin and leptin, and the orexigenic hormone, ghrelin, on mTORC1 signalling was examined using S6 kinase phosphorylation as a marker for changes in mTORC1 activity in mouse hypothalamic GT1-7 cells. Additionally, the contribution of AMPK and mTORC1 signalling in relation to insulin-, leptin- and ghrelin-driven alterations to mouse hypothalamic agouti-related protein (AgRP mRNA levels was examined. Insulin and leptin increase mTORC1 activity in a phosphoinositide-3-kinase (PI3K- and protein kinase B (PKB-dependent manner, compared to vehicle controls, whereas increasing AMPK activity inhibits mTORC1 activity and blocks the actions of the anorexigenic hormones. Ghrelin mediates an AMPK-dependent decrease in mTORC1 activity and increases hypothalamic AgRP mRNA levels, the latter effect being prevented by insulin in an mTORC1-dependent manner. In conclusion, mTORC1 acts as an integration node in hypothalamic neurons for hormone-derived PI3K and AMPK signalling and mediates at least part of the assimilated output of anorexigenic and orexigenic hormone actions in the hypothalamus.

  17. Rapamycin down-regulates LDL-receptor expression independently of SREBP-2

    International Nuclear Information System (INIS)

    Sharpe, Laura J.; Brown, Andrew J.

    2008-01-01

    As a key regulator of cholesterol homeostasis, sterol-regulatory element binding protein-2 (SREBP-2) up-regulates expression of genes involved in cholesterol synthesis (e.g., 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) Reductase) and uptake (the low density lipoprotein (LDL)-receptor). Previously, we showed that Akt, a critical kinase in cell growth and proliferation, contributes to SREBP-2 activation. However, the specific Akt target involved is unknown. A potential candidate is the mammalian target of rapamycin, mTOR. Rapamycin can cause hyperlipidaemia clinically, and we hypothesised that this may be mediated via an effect of mTOR on SREBP-2. Herein, we found that SREBP-2 activation and HMG-CoA Reductase gene expression were unaffected by rapamycin treatment. However, LDL-receptor gene expression was decreased by rapamycin, suggesting that this may contribute to the hyperlipidaemia observed in rapamycin-treated patients. Rapamycin did not affect mRNA stability, so the decrease in LDL-receptor gene expression is likely to be occurring at the transcriptional level, although independently of SREBP-2

  18. Arctigenin functions as a selective agonist of estrogen receptor β to restrict mTORC1 activation and consequent Th17 differentiation.

    Science.gov (United States)

    Wu, Xin; Tong, Bei; Yang, Yan; Luo, Jinque; Yuan, Xusheng; Wei, Zhifeng; Yue, Mengfan; Xia, Yufeng; Dai, Yue

    2016-12-20

    Arctigenin was previously proven to inhibit Th17 cell differentiation and thereby attenuate colitis in mice by down-regulating the activation of mechanistic target of rapamycin complex 1 (mTORC1). The present study was performed to address its underlying mechanism in view of estrogen receptor (ER). The specific antagonist PHTPP or siRNA of ERβ largely diminished the inhibitory effect of arctigenin on the mTORC1 activation in T cell lines and primary CD4+ T cells under Th17-polarization condition, suggesting that arctigenin functioned in an ERβ-dependent manner. Moreover, arctigenin was recognized to be an agonist of ERβ, which could bind to ERβ with a moderate affinity, promote dissociation of ERβ/HSP90 complex and nuclear translocation and phosphorylation of ERβ, and increase the transcription activity. Following activation of ERβ, arctigenin inhibited the activity of mTORC1 by disruption of ERβ-raptor-mTOR complex assembly. Deficiency of ERβ markedly abolished arctigenin-mediated inhibition of Th17 cell differentiation. In colitis mice, the activation of ERβ, inhibition of mTORC1 activation and Th17 response by arctigenin were abolished by PHTPP treatment. In conclusion, ERβ might be the target protein of arctigenin responsible for inhibition of mTORC1 activation and resultant prevention of Th17 cell differentiation and colitis development.

  19. Contraction mode itself does not determine the level of mTORC1 activity in rat skeletal muscle.

    Science.gov (United States)

    Ato, Satoru; Makanae, Yuhei; Kido, Kohei; Fujita, Satoshi

    2016-10-01

    Resistance training with eccentric contraction has been shown to augment muscle hypertrophy more than other contraction modes do (i.e., concentric and isometric contraction). However, the molecular mechanisms involved remain unclear. The purpose of this study was to investigate the effect of muscle contraction mode on mammalian target of rapamycin complex 1 (mTORC1) signaling using a standardized force-time integral (load (weight) × contraction time). Male Sprague-Dawley rats were randomly assigned to three groups: eccentric contraction, concentric contraction, and isometric contraction. The right gastrocnemius muscle was exercised via percutaneous electrical stimulation-induced maximal contraction. In experiment 1, different modes of muscle contraction were exerted using the same number of reps in all groups, while in experiment 2, muscle contractions were exerted using a standardized force-time integral. Muscle samples were obtained immediately and 3 h after exercise. Phosphorylation of molecules associated with mTORC1 activity was assessed using western blot analysis. In experiment 1, the force-time integral was significantly different among contraction modes with a higher force-time integral for eccentric contraction compared to that for other contraction modes (P contraction compared to that for isometric contraction (P contraction than for other modes of contraction (P contraction was higher than isometric contraction (P contraction modes 3 h after exercise. Our results suggest that mTORC1 activity is not determined by differences in muscle contraction mode itself. Instead, mTORC1 activity is determined by differences in the force-time integral during muscle contraction. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  20. FK506 Binding Protein Mediates Glioma Cell Growth and Sensitivity to Rapamycin Treatment by Regulating NF-κB Signaling Pathway

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    Wei Jiang

    2008-03-01

    Full Text Available FK506 binding protein 5 (FKBP5 belongs to a family of immunophilins named for their ability to bind immunosuppressive drugs, also known as peptidyl-prolyl cis-trans isomerases, and also with chaperones to help protein folding. Using glioma cDNA microarray analysis, we found that FKBP5 was overexpressed in glioma tumors. This finding was further validated by real-time reverse transcription-polymerase chain reaction and Western blot analysis. The roles of FKBP5 in glioma cells were then examined. We found that cell growth was suppressed after FKBP5 expression was inhibited by short interfering RNA transfection and enhanced by FKBP5 overexpression. Electrophoretic mobility shift assay showed that nuclear factor-kappa B (NF-κB and DNA binding was enhanced by FKBP5 overexpression. The expression level of I-kappa B alpha and phosphorylated NF-κB was regulated by the expression of FKBP5. These data suggest that FKBP5 is involved in NF-κB pathway activation in glioma cells. In addition, FKBP5 overexpression in rapamycin-sensitive U87 cells blocked the cells' response to rapamycin treatment, whereas rapamycin-resistant glioma cells, both PTEN-positive and -negative, were synergistically sensitive to rapamycin after FKBP5 was knocked down, suggesting that the FKBP5 regulates glioma cell response to rapamycin treatment. In conclusion, our study demonstrates that FKBP5 plays an important role in glioma growth and chemoresistance through regulating signal transduction of the NF-κB pathway.

  1. Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway

    Science.gov (United States)

    Joubert, Pierre-Emmanuel; Stapleford, Kenneth; Guivel-Benhassine, Florence; Vignuzzi, Marco; Schwartz, Olivier; Albert, Matthew L.

    2015-01-01

    Chikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell’s cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition. PMID:26317997

  2. Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway.

    Directory of Open Access Journals (Sweden)

    Pierre-Emmanuel Joubert

    2015-08-01

    Full Text Available Chikungunya virus (CHIKV, the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell's cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K and MAP kinase-activated protein kinase (MnKs activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition.

  3. Four-week rapamycin treatment improves muscular dystrophy in a fukutin-deficient mouse model of dystroglycanopathy.

    Science.gov (United States)

    Foltz, Steven J; Luan, Junna; Call, Jarrod A; Patel, Ankit; Peissig, Kristen B; Fortunato, Marisa J; Beedle, Aaron M

    2016-01-01

    Secondary dystroglycanopathies are a subset of muscular dystrophy caused by abnormal glycosylation of α-dystroglycan (αDG). Loss of αDG functional glycosylation prevents it from binding to laminin and other extracellular matrix receptors, causing muscular dystrophy. Mutations in a number of genes, including FKTN (fukutin), disrupt αDG glycosylation. We analyzed conditional Fktn knockout (Fktn KO) muscle for levels of mTOR signaling pathway proteins by Western blot. Two cohorts of Myf5-cre/Fktn KO mice were treated with the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) for 4 weeks and evaluated for changes in functional and histopathological features. Muscle from 17- to 25-week-old fukutin-deficient mice has activated mTOR signaling. However, in tamoxifen-inducible Fktn KO mice, factors related to Akt/mTOR signaling were unchanged before the onset of dystrophic pathology, suggesting that Akt/mTOR signaling pathway abnormalities occur after the onset of disease pathology and are not causative in early dystroglycanopathy development. To determine any pharmacological benefit of targeting mTOR signaling, we administered RAPA daily for 4 weeks to Myf5/Fktn KO mice to inhibit mTORC1. RAPA treatment reduced fibrosis, inflammation, activity-induced damage, and central nucleation, and increased muscle fiber size in Myf5/Fktn KO mice compared to controls. RAPA-treated KO mice also produced significantly higher torque at the conclusion of dosing. These findings validate a misregulation of mTOR signaling in dystrophic dystroglycanopathy skeletal muscle and suggest that such signaling molecules may be relevant targets to delay and/or reduce disease burden in dystrophic patients.

  4. A complex interplay between PGC-1 co-activators and mTORC1 regulates hematopoietic recovery following 5-fluorouracil treatment

    Directory of Open Access Journals (Sweden)

    Sunanda Basu

    2014-01-01

    Full Text Available In vitro stimulation of HSCs with growth factors generally leads to their depletion. Understanding the molecular mechanisms underlying expansion of HSCs in vivo following myeloablation could lead to successful expansion of HSCs ex vivo for therapeutic purposes. Current findings show that mTORC1 is activated in HSPCs following 5-fluorouracil treatment and that mTORC1 activation is dependent on mitochondrial ETC capacity of HSPCs. Moreover, expression of PGC-1 family members, proteins that regulate mitochondrial biogenesis, in HSPCs following 5-fluorouracil treatment changes; also, these proteins play a stage specific role in hematopoietic recovery. While PRC regulates HSCs' expansion during early recovery phase, PGC-1α regulates progenitor cell proliferation and recovery of hematopoiesis during later phase. During early recovery phase, PRC expression, mitochondrial activity and mTORC1 activation are relatively higher in PGC-1α−/− HSCs compared to WT HSCs, and PGC-1α−/− HSCs show greater expansion. Administration of rapamycin, but not NAC, during early recovery phase improves WT HSC numbers but decreases PGC-1α−/− HSC numbers. The current findings demonstrate that mTOR activation can increase HSC numbers provided that the energy demand created by mTOR activation is successfully met. Thus, critical tuning between mTORC1 activation and mitochondrial ETC capacity is crucial for HSC maintenance/expansion in response to mitogenic stimulation.

  5. The novel mTORC1/2 dual inhibitor INK-128 suppresses survival and proliferation of primary and transformed human pancreatic cancer cells

    International Nuclear Information System (INIS)

    Lou, Hai-zhou; Weng, Xiao-chuan; Pan, Hong-ming; Pan, Qin; Sun, Peng; Liu, Li-li; Chen, Bin

    2014-01-01

    Highlights: • INK-128 inhibits the survival and growth of human pancreatic cancer cells. • INK-128 induced pancreatic cancer cell apoptosis and necrosis simultaneously. • INK-128 blocks mTORC1/2 activation simultaneously in pancreatic cancer cells. • INK-128 down-regulates cyclin D1 and causes pancreatic cancer cell cycle arrest. • INK-128 significantly increases sensitivity of pancreatic cancer cells to gemcitabine. - Abstract: Pancreatic cancer has one of worst prognosis among all human malignancies around the world, the development of novel and more efficient anti-cancer agents against this disease is urgent. In the current study, we tested the potential effect of INK-128, a novel mammalian target of rapamycin (mTOR) complex 1 and 2 (mTORC1/2) dual inhibitor, against pancreatic cancer cells in vitro. Our results demonstrated that INK-128 concentration- and time-dependently inhibited the survival and growth of pancreatic cancer cells (both primary cells and transformed cells). INK-128 induced pancreatic cancer cell apoptosis and necrosis simultaneously. Further, INK-128 dramatically inhibited phosphorylation of 4E-binding protein 1 (4E-BP1), ribosomal S6 kinase 1 (S6K1) and Akt at Ser 473 in pancreatic cancer cells. Meanwhile, it downregulated cyclin D1 expression and caused cell cycle arrest. Finally, we found that a low concentration of INK-128 significantly increased the sensitivity of pancreatic cancer cells to gemcitabine. Together, our in vitro results suggest that INK-128 might be further investigated as a novel anti-cancer agent or chemo-adjuvant for pancreatic cancer treatment

  6. The novel mTORC1/2 dual inhibitor INK-128 suppresses survival and proliferation of primary and transformed human pancreatic cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Lou, Hai-zhou [Department of Medical Oncology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou 310016 (China); Weng, Xiao-chuan [Department of Anesthesiology, Hangzhou Xia-sha Hospital, Hangzhou 310018 (China); Pan, Hong-ming; Pan, Qin [Department of Medical Oncology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou 310016 (China); Sun, Peng [Department of Medical Oncology, Sun Yat-Sen University Cancer Center, Guangzhou 510060 (China); Liu, Li-li [Department of Medical Oncology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou 310016 (China); Chen, Bin, E-mail: chenbinhangzhou126@126.com [Department of Hepatopancreatobiliary Surgery, First People’s Hospital of Hangzhou, Hangzhou 310006 (China)

    2014-07-25

    Highlights: • INK-128 inhibits the survival and growth of human pancreatic cancer cells. • INK-128 induced pancreatic cancer cell apoptosis and necrosis simultaneously. • INK-128 blocks mTORC1/2 activation simultaneously in pancreatic cancer cells. • INK-128 down-regulates cyclin D1 and causes pancreatic cancer cell cycle arrest. • INK-128 significantly increases sensitivity of pancreatic cancer cells to gemcitabine. - Abstract: Pancreatic cancer has one of worst prognosis among all human malignancies around the world, the development of novel and more efficient anti-cancer agents against this disease is urgent. In the current study, we tested the potential effect of INK-128, a novel mammalian target of rapamycin (mTOR) complex 1 and 2 (mTORC1/2) dual inhibitor, against pancreatic cancer cells in vitro. Our results demonstrated that INK-128 concentration- and time-dependently inhibited the survival and growth of pancreatic cancer cells (both primary cells and transformed cells). INK-128 induced pancreatic cancer cell apoptosis and necrosis simultaneously. Further, INK-128 dramatically inhibited phosphorylation of 4E-binding protein 1 (4E-BP1), ribosomal S6 kinase 1 (S6K1) and Akt at Ser 473 in pancreatic cancer cells. Meanwhile, it downregulated cyclin D1 expression and caused cell cycle arrest. Finally, we found that a low concentration of INK-128 significantly increased the sensitivity of pancreatic cancer cells to gemcitabine. Together, our in vitro results suggest that INK-128 might be further investigated as a novel anti-cancer agent or chemo-adjuvant for pancreatic cancer treatment.

  7. Leucine signaling in the pathogenesis of type 2 diabetes and obesity

    OpenAIRE

    Melnik, Bodo C

    2012-01-01

    Epidemiological evidence points to increased dairy and meat consumption, staples of the Western diet, as major risk factors for the development of type 2 diabetes (T2D). This paper presents a new concept and comprehensive review of leucine-mediated cell signaling explaining the pathogenesis of T2D and obesity by leucine-induced over-stimulation of mammalian target of rapamycin complex 1 (mTORC1). mTORC1, a pivotal nutrient-sensitive kinase, promotes growth and cell proliferation in response t...

  8. Divergent tissue and sex effects of rapamycin on the proteasome-chaperone network of old mice

    Directory of Open Access Journals (Sweden)

    Karl Andrew Rodriguez

    2014-11-01

    Full Text Available Rapamycin, an allosteric inhibitor of the mTOR kinase, increases longevity in mice in a sex-specific manner. In contrast to the widely accepted theory that a loss of proteasome activity is detrimental to both life- and healthspan, biochemical studies in vitro reveal that rapamycin inhibits 20S proteasome peptidase activity. We tested if this unexpected finding is also evident after chronic rapamycin treatment in vivo by measuring peptidase activities for both the 26S and 20S proteasome in liver, fat, and brain tissues of old, male and female mice fed encapsulated chow containing 2.24mg/kg (14 ppm rapamycin for 6 months. Further we assessed if rapamycin altered expression of the chaperone proteins known to interact with the proteasome-mediated degradation system (PMDS, heat shock factor 1 (HSF1, and the levels of key mTOR pathway proteins. Rapamycin had little effect on liver proteasome activity in either gender, but increased proteasome activity in female brain lysates and lowered its activity in female fat tissue. Rapamycin-induced changes in molecular chaperone levels were also more substantial in tissues from female animals. Furthermore, mTOR pathway proteins showed more significant changes in female tissues compared to those from males. These data show collectively that there are divergent tissue and sex effects of rapamycin on the proteasome-chaperone network and that these may be linked to the disparate effects of rapamycin on males and females. Further our findings suggest that rapamycin induces indirect regulation of the PMDS/heat-shock response through its modulation of the mTOR pathway rather than via direct interactions between rapamycin and the proteasome.

  9. The nuclear import of ribosomal proteins is regulated by mTOR

    Science.gov (United States)

    Kazyken, Dubek; Kaz, Yelimbek; Kiyan, Vladimir; Zhylkibayev, Assylbek A.; Chen, Chien-Hung; Agarwal, Nitin K.; Sarbassov, Dos D.

    2014-01-01

    Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins. PMID:25294810

  10. GSK3 is required for rapalogs to induce degradation of some oncogenic proteins and to suppress cancer cell growth.

    Science.gov (United States)

    Koo, Junghui; Wang, Xuerong; Owonikoko, Taofeek K; Ramalingam, Suresh S; Khuri, Fadlo R; Sun, Shi-Yong

    2015-04-20

    The single-agent activity of rapalogs (rapamycin and its analogues) in most tumor types has been modest at best. The underlying mechanisms are largely unclear. In this report, we have uncovered a critical role of GSK3 in regulating degradation of some oncogenic proteins induced by rapalogs and cell sensitivity to rapalogs. The basal level of GSK3 activity was positively correlated with cell sensitivity of lung cancer cell lines to rapalogs. GSK3 inhibition antagonized rapamycin's growth inhibitory effects both in vitro and in vivo, while enforced activation of GSK3β sensitized cells to rapamycin. GSK3 inhibition rescued rapamcyin-induced reduction of several oncogenic proteins such as cyclin D1, Mcl-1 and c-Myc, without interfering with the ability of rapamycin to suppress mTORC1 signaling and cap binding. Interestingly, rapamycin induces proteasomal degradation of these oncogenic proteins, as evidenced by their decreased stabilities induced by rapamcyin and rescue of their reduction by proteasomal inhibition. Moreover, acute or short-time rapamycin treatment dissociated not only raptor, but also rictor from mTOR in several tested cell lines, suggesting inhibition of both mTORC1 and mTORC2. Thus, induction of GSK3-dependent degradation of these oncogenic proteins is likely secondary to mTORC2 inhibition; this effect should be critical for rapamycin to exert its anticancer activity.

  11. Cystogenesis and elongated primary cilia in Tsc1-deficient distal convoluted tubules.

    Science.gov (United States)

    Armour, Eric A; Carson, Robert P; Ess, Kevin C

    2012-08-15

    Tuberous sclerosis complex (TSC) is a multiorgan hamartomatous disease caused by loss of function mutations of either the TSC1 or TSC2 genes. Neurological symptoms of TSC predominate in younger patients, but renal pathologies are a serious aspect of the disease in older children and adults. To study TSC pathogenesis in the kidney, we inactivated the mouse Tsc1 gene in the distal convoluted tubules (DCT). At young ages, Tsc1 conditional knockout (CKO) mice have enlarged kidneys and mild cystogenesis with increased mammalian target of rapamycin complex (mTORC)1 but decreased mTORC2 signaling. Treatment with the mTORC1 inhibitor rapamycin reduces kidney size and cystogenesis. Rapamycin withdrawal led to massive cystogenesis involving both distal as well as proximal tubules. To assess the contribution of decreased mTORC2 signaling in kidney pathogenesis, we also generated Rictor CKO mice. These animals did not have any detectable kidney pathology. Finally, we examined primary cilia in the DCT. Cilia were longer in Tsc1 CKO mice, and rapamycin treatment returned cilia length to normal. Rictor CKO mice had normal cilia in the DCT. Overall, our findings suggest that loss of the Tsc1 gene in the DCT is sufficient for renal cystogenesis. This cytogenesis appears to be mTORC1 but not mTORC2 dependent. Intriguingly, the mechanism may be cell autonomous as well as non-cell autonomous and possibly involves the length and function of primary cilia.

  12. Cystogenesis and elongated primary cilia in Tsc1-deficient distal convoluted tubules

    Science.gov (United States)

    Armour, Eric A.; Carson, Robert P.

    2012-01-01

    Tuberous sclerosis complex (TSC) is a multiorgan hamartomatous disease caused by loss of function mutations of either the TSC1 or TSC2 genes. Neurological symptoms of TSC predominate in younger patients, but renal pathologies are a serious aspect of the disease in older children and adults. To study TSC pathogenesis in the kidney, we inactivated the mouse Tsc1 gene in the distal convoluted tubules (DCT). At young ages, Tsc1 conditional knockout (CKO) mice have enlarged kidneys and mild cystogenesis with increased mammalian target of rapamycin complex (mTORC)1 but decreased mTORC2 signaling. Treatment with the mTORC1 inhibitor rapamycin reduces kidney size and cystogenesis. Rapamycin withdrawal led to massive cystogenesis involving both distal as well as proximal tubules. To assess the contribution of decreased mTORC2 signaling in kidney pathogenesis, we also generated Rictor CKO mice. These animals did not have any detectable kidney pathology. Finally, we examined primary cilia in the DCT. Cilia were longer in Tsc1 CKO mice, and rapamycin treatment returned cilia length to normal. Rictor CKO mice had normal cilia in the DCT. Overall, our findings suggest that loss of the Tsc1 gene in the DCT is sufficient for renal cystogenesis. This cytogenesis appears to be mTORC1 but not mTORC2 dependent. Intriguingly, the mechanism may be cell autonomous as well as non-cell autonomous and possibly involves the length and function of primary cilia. PMID:22674026

  13. Sarcopaenia: Where do we stand after two decades of research?

    African Journals Online (AJOL)

    muscle mass as sarcopaenia (Greek sarx=flesh and paenia=loss).2 The estimated relative annual rate of loss of muscle mass is 1% in men and 0.84% in women.1 ... over a prolonged period.1 The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in muscle plays a key role in regulating exercise-.

  14. RNA interference targeting raptor inhibits proliferation of gastric cancer cells

    International Nuclear Information System (INIS)

    Wu, William Ka Kei; Lee, Chung Wa; Cho, Chi Hin; Chan, Francis Ka Leung; Yu, Jun; Sung, Joseph Jao Yiu

    2011-01-01

    Mammalian target of rapamycin complex 1 (mTORC1) is dysregulated in gastric cancer. The biologic function of mTORC1 in gastric carcinogenesis is unclear. Here, we demonstrate that disruption of mTORC1 function by RNA interference-mediated downregulation of raptor substantially inhibited gastric cancer cell proliferation through induction of G 0 /G 1 -phase cell cycle arrest. The anti-proliferative effect was accompanied by concomitant downregulation of activator protein-1 and upregulation of Smad2/3 transcriptional activities. In addition, the expression of cyclin D 3 and p21 Waf1 , which stabilizes cyclin D/cdk4 complex for G 1 -S transition, was reduced by raptor knockdown. In conclusion, disruption of mTORC1 inhibits gastric cancer cell proliferation through multiple pathways. This discovery may have an implication in the application of mTORC1-directed therapy for the treatment of gastric cancer.

  15. TOR-inhibitor insensitive-1 (TRIN1) regulates cotyledons greening in Arabidopsis.

    Science.gov (United States)

    Li, Linxuan; Song, Yun; Wang, Kai; Dong, Pan; Zhang, Xueyan; Li, Fuguang; Li, Zhengguo; Ren, Maozhi

    2015-01-01

    Target of Rapamycin (TOR) is an eukaryotic protein kinase and evolutionally conserved from the last eukaryotic common ancestor (LECA) to humans. The growing evidences have shown that TOR signaling acts as a central controller of cell growth and development. The downstream effectors of TOR have been well-identified in yeast and animals by using the immunosuppression agent rapamycin. However, less is known about TOR in plants. This is largely due to the fact that plants are insensitive to rapamycin. In this study, AZD8055 (AZD), the novel ATP-competitive inhibitor of TOR, was employed to decipher the downstream effectors of TOR in Arabidopsis. One AZD insensitive mutant, T O R - i nhibitor i n sensitive- 1 (trin1), was screened from 10,000 EMS-induced mutation seeds. The cotyledons of trin1 can turn green when its seeds were germinated on ½ MS medium supplemented with 2 μM AZD, whereas the cotyledons greening of wild-type (WT) can be completely blocked at this concentration. Through genetic mapping, TRIN1 was mapped onto the long arm of chromosome 2, between markers SGCSNP26 and MI277. Positional cloning revealed that TRIN1 was an allele of ABI4, which encoded an ABA-regulated AP2 domain transcription factor. Plants containing P35S::TRIN1 or P35S::TRIN1-GUS were hypersensitive to AZD treatment and displayed the opposite phenotype observed in trin1. Importantly, GUS signaling was significantly enhanced in P35S::TRIN1-GUS transgenic plants in response to AZD treatment, indicating that suppression of TOR resulted in the accumulation of TRIN1. These observations revealed that TOR controlled seed-to-seedling transition by negatively regulating the stability of TRIN1 in Arabidopsis. For the first time, TRIN1, the downstream effector of TOR signaling, was identified through a chemical genetics approach.

  16. Multi-organ abnormalities and mTORC1 activation in zebrafish model of multiple acyl-CoA dehydrogenase deficiency.

    Directory of Open Access Journals (Sweden)

    Seok-Hyung Kim

    2013-06-01

    Full Text Available Multiple Acyl-CoA Dehydrogenase Deficiency (MADD is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxa(vu463 that has an inactivating mutation in the etfa gene. dxa(vu463 recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxa(vu463 zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxa(vu463 zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1 with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.

  17. The impact of cow's milk-mediated mTORC1-signaling in the initiation and progression of prostate cancer

    Science.gov (United States)

    2012-01-01

    Prostate cancer (PCa) is dependent on androgen receptor signaling and aberrations of the PI3K-Akt-mTORC1 pathway mediating excessive and sustained growth signaling. The nutrient-sensitive kinase mTORC1 is upregulated in nearly 100% of advanced human PCas. Oncogenic mTORC1 signaling activates key subsets of mRNAs that cooperate in distinct steps of PCa initiation and progression. Epidemiological evidence points to increased dairy protein consumption as a major dietary risk factor for the development of PCa. mTORC1 is a master regulator of protein synthesis, lipid synthesis and autophagy pathways that couple nutrient sensing to cell growth and cancer. This review provides evidence that PCa initiation and progression are promoted by cow´s milk, but not human milk, stimulation of mTORC1 signaling. Mammalian milk is presented as an endocrine signaling system, which activates mTORC1, promotes cell growth and proliferation and suppresses autophagy. Naturally, milk-mediated mTORC1 signaling is restricted only to the postnatal growth phase of mammals. However, persistent consumption of cow´s milk proteins in humans provide highly insulinotropic branched-chain amino acids (BCAAs) provided by milk´s fast hydrolysable whey proteins, which elevate postprandial plasma insulin levels, and increase hepatic IGF-1 plasma concentrations by casein-derived amino acids. BCAAs, insulin and IGF-1 are pivotal activating signals of mTORC1. Increased cow´s milk protein-mediated mTORC1 signaling along with constant exposure to commercial cow´s milk estrogens derived from pregnant cows may explain the observed association between high dairy consumption and increased risk of PCa in Westernized societies. As well-balanced mTORC1-signaling plays an important role in appropriate prostate morphogenesis and differentiation, exaggerated mTORC1-signaling by high cow´s milk consumption predominantly during critical growth phases of prostate development and differentiation may exert long

  18. The impact of cow's milk-mediated mTORC1-signaling in the initiation and progression of prostate cancer

    Directory of Open Access Journals (Sweden)

    Melnik Bodo C

    2012-08-01

    Full Text Available Abstract Prostate cancer (PCa is dependent on androgen receptor signaling and aberrations of the PI3K-Akt-mTORC1 pathway mediating excessive and sustained growth signaling. The nutrient-sensitive kinase mTORC1 is upregulated in nearly 100% of advanced human PCas. Oncogenic mTORC1 signaling activates key subsets of mRNAs that cooperate in distinct steps of PCa initiation and progression. Epidemiological evidence points to increased dairy protein consumption as a major dietary risk factor for the development of PCa. mTORC1 is a master regulator of protein synthesis, lipid synthesis and autophagy pathways that couple nutrient sensing to cell growth and cancer. This review provides evidence that PCa initiation and progression are promoted by cow´s milk, but not human milk, stimulation of mTORC1 signaling. Mammalian milk is presented as an endocrine signaling system, which activates mTORC1, promotes cell growth and proliferation and suppresses autophagy. Naturally, milk-mediated mTORC1 signaling is restricted only to the postnatal growth phase of mammals. However, persistent consumption of cow´s milk proteins in humans provide highly insulinotropic branched-chain amino acids (BCAAs provided by milk´s fast hydrolysable whey proteins, which elevate postprandial plasma insulin levels, and increase hepatic IGF-1 plasma concentrations by casein-derived amino acids. BCAAs, insulin and IGF-1 are pivotal activating signals of mTORC1. Increased cow´s milk protein-mediated mTORC1 signaling along with constant exposure to commercial cow´s milk estrogens derived from pregnant cows may explain the observed association between high dairy consumption and increased risk of PCa in Westernized societies. As well-balanced mTORC1-signaling plays an important role in appropriate prostate morphogenesis and differentiation, exaggerated mTORC1-signaling by high cow´s milk consumption predominantly during critical growth phases of prostate development and

  19. The crosstalk between Target of Rapamycin (TOR) and Jasmonic Acid (JA) signaling existing in Arabidopsis and cotton.

    Science.gov (United States)

    Song, Yun; Zhao, Ge; Zhang, Xueyan; Li, Linxuan; Xiong, Fangjie; Zhuo, Fengping; Zhang, Chaojun; Yang, Zuoren; Datla, Raju; Ren, Maozhi; Li, Fuguang

    2017-04-04

    Target of rapamycin (TOR) acts as an important regulator of cell growth, development and stress responses in most examined diploid eukaryotes. However, little is known about TOR in tetraploid species such as cotton. Here, we show that TORC1-S6K-RPS6, the major signaling components, are conserved and further expanded in cotton genome. Though the cotton seedlings are insensitive to rapamycin, AZD8055, the second-generation inhibitor of TOR, can significantly suppress the growth in cotton. Global transcriptome analysis revealed that genes associated with jasmonic acid (JA) biosynthesis and transduction were significantly altered in AZD8055 treated cotton seedlings, suggesting the potential crosstalk between TOR and JA signaling. Pharmacological and genetic approaches have been employed to get further insights into the molecular mechanism of the crosstalk between TOR and JA. Combination of AZD8055 with methyl jasmonate can synergistically inhibit cotton growth, and additionally JA levels were significantly increased when cotton seedlings were subjected to AZD8055. JA biosynthetic and signaling mutants including jar1, coi1-2 and myc2-2 displayed TOR inhibitor-resistant phenotypes, whereas COI1 overexpression transgenic lines and jaz10 exhibited sensitivity to AZD8055. Consistently, cotton JAZ can partially rescue TOR-suppressed phenotypes in Arabidopsis. These evidences revealed that the crosstalk between TOR and JA pathway operates in cotton and Arabidopsis.

  20. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    International Nuclear Information System (INIS)

    Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

    2009-01-01

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  1. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    Energy Technology Data Exchange (ETDEWEB)

    Fahrer, Joerg, E-mail: joerg.fahrer@uni-ulm.de [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Wagner, Silvia [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany); Buerkle, Alexander [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Koenigsrainer, Alfred [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany)

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  2. Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration

    Energy Technology Data Exchange (ETDEWEB)

    Montes, Daniela K.; Brenet, Marianne; Muñoz, Vanessa C.; Burgos, Patricia V.; Villanueva, Carolina I. [Department of Physiology, Universidad Austral de Chile, Valdivia 509-9200 (Chile); Figueroa, Carlos D. [Department of Anatomy, Histology and Pathology, Universidad Austral de Chile, Valdivia 509-9200 (Chile); González, Carlos B., E-mail: cbgonzal@uach.cl [Department of Physiology, Universidad Austral de Chile, Valdivia 509-9200 (Chile); Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555 (United States)

    2013-11-29

    Highlights: •AVP induces mTOR phosphorylation in A-10 cells cultured in high glucose concentration. •The mTOR phosphorylation is mediated by the PI3K/Akt pathway activation. •The AVP-induced mTOR phosphorylation inhibited autophagy and stimulated cell proliferation. -- Abstract: Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. The AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration.

  3. Combination of rapamycin, CI-1040, and 17-AAG inhibits metastatic capacity of prostate cancer via Slug inhibition.

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    Guanxiong Ding

    Full Text Available Though prostate cancer (PCa has slow progression, the hormone refractory (HRCP and metastatic entities are substantially lethal and lack effective treatments. Transcription factor Slug is critical in regulating metastases of various tumors including PCa. Here we studied targeted therapy against Slug using combination of 3 drugs targeting 3 pathways respectively converging via Slug and further regulating PCa metastasis. Using in vitro assays we confirmed that Slug up-regulation incurred inhibition of E-cadherin that was anti-metastatic, and inhibited Bim-regulated cell apoptosis in PCa. Upstream PTEN/Akt, mTOR, Erk, and AR/Hsp90 pathways were responsible for Slug up-regulation and each of these could be targeted by rapamycin, CI-1040, and 17-AAG respectively. In 4 PCa cell lines with different traits in terms of PTEN loss and androgen sensitivity we tested the efficacy of mono- and combined therapy with the drugs. We found that metastatic capacity of the cells was maximally inhibited only when all 3 drugs were combined, due to the crosstalk between the pathways. 17-AAG decreases Slug expression via blockade of HSP90-dependent AR stability. Combination of rapamycin and CI-1040 diminishes invasiveness more potently in PCa cells that are androgen insensitive and with PTEN loss. Slug inhibited Bim-mediated apoptosis that could be rescued by mTOR/Erk/HSP90 inhibitors. Using mouse models for circulating PCa DNA quantification, we found that combination of mTOR/Erk/HSP90 inhibitors reduced circulating PCa cells in vivo significantly more potently than combination of 2 or monotherapy. Conclusively, combination of mTOR/Erk/Hsp90 inhibits metastatic capacity of prostate cancer via Slug inhibition.

  4. Rapamycin sensitizes T-ALL cells to dexamethasone-induced apoptosis

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    Mu Dezhi

    2010-11-01

    Full Text Available Abstract Background Glucocorticoid (GC resistance is frequently seen in acute lymphoblastic leukemia of T-cell lineage (T-ALL. In this study we investigate the potential and mechanism of using rapamycin to restore the sensitivity of GC-resistant T-ALL cells to dexamethasone (Dex treatment. Methods Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl- 2,5-diphenyltetrazolium bromide (MTT assay. Fluorescence-activated cell sorting (FACS analysis was used to analyze apoptosis and cell cycles. Western blot analysis was performed to test the expression of the downstream effector proteins of mammalian target of rapamycin (mTOR, the cell cycle regulatory proteins, and apoptosis associated proteins. Results 10 nM rapamycin markedly increased GC sensitivity in GC-resistant T-ALL cells and this effect was mediated, at least in part, by inhibition of mTOR signaling pathway. Cell cycle arrest was associated with modulation of G1-S phase regulators. Both rapamycin and Dex can induce up-regulation of cyclin-dependent kinase (CDK inhibitors of p21 and p27 and co-treatment of rapamycin with Dex resulted in a synergistic induction of their expressions. Rapamycin did not obviously affect the expression of cyclin A, whereas Dex induced cyclin A expression. Rapamycin prevented Dex-induced expression of cyclin A. Rapamycin had a stronger inhibition of cyclin D1 expression than Dex. Rapamycin enhanced GC-induced apoptosis and this was not achieved by modulation of glucocorticoid receptor (GR expression, but synergistically up-regulation of pro-apoptotic proteins like caspase-3, Bax, and Bim, and down-regulation of anti-apoptotic protein of Mcl-1. Conclusion Our data suggests that rapamycin can effectively reverse GC resistance in T-ALL and this effect is achieved by inducing cell cycles arrested at G0/G1 phase and activating the intrinsic apoptotic program. Therefore, combination of mTOR inhibitor rapamycin with GC containing protocol might be an attracting

  5. Rapamycin suppresses brain aging in senescence-accelerated OXYS rats.

    Science.gov (United States)

    Kolosova, Nataliya G; Vitovtov, Anton O; Muraleva, Natalia A; Akulov, Andrey E; Stefanova, Natalia A; Blagosklonny, Mikhail V

    2013-06-01

    Cellular and organismal aging are driven in part by the MTOR (mechanistic target of rapamycin) pathway and rapamycin extends life span inC elegans, Drosophila and mice. Herein, we investigated effects of rapamycin on brain aging in OXYS rats. Previously we found, in OXYS rats, an early development of age-associated pathological phenotypes similar to several geriatric disorders in humans, including cerebral dysfunctions. Behavioral alterations as well as learning and memory deficits develop by 3 months. Here we show that rapamycin treatment (0.1 or 0.5 mg/kg as a food mixture daily from the age of 1.5 to 3.5 months) decreased anxiety and improved locomotor and exploratory behavior in OXYS rats. In untreated OXYS rats, MRI revealed an increase of the area of hippocampus, substantial hydrocephalus and 2-fold increased area of the lateral ventricles. Rapamycin treatment prevented these abnormalities, erasing the difference between OXYS and Wister rats (used as control). All untreated OXYS rats showed signs of neurodegeneration, manifested by loci of demyelination. Rapamycin decreased the percentage of animals with demyelination and the number of loci. Levels of Tau and phospho-Tau (T181) were increased in OXYS rats (compared with Wistar). Rapamycin significantly decreased Tau and inhibited its phosphorylation in the hippocampus of OXYS and Wistar rats. Importantly, rapamycin treatment caused a compensatory increase in levels of S6 and correspondingly levels of phospo-S6 in the frontal cortex, indicating that some downstream events were compensatory preserved, explaining the lack of toxicity. We conclude that rapamycin in low chronic doses can suppress brain aging.

  6. Differential Reponses of Hematopoietic Stem and Progenitor Cells to mTOR Inhibition

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    Aimin Yang

    2015-01-01

    Full Text Available Abnormal activation of the mammalian target of rapamycin (mTOR signaling pathway has been observed in a variety of human cancers. Therefore, targeting of the mTOR pathway is an attractive strategy for cancer treatment and several mTOR inhibitors, including AZD8055 (AZD, a novel dual mTORC1/2 inhibitor, are currently in clinical trials. Although bone marrow (BM suppression is one of the primary side effects of anticancer drugs, it is not known if pharmacological inhibition of dual mTORC1/2 affects BM hematopoietic stem and progenitor cells (HSPCs function and plasticity. Here we report that dual inhibition of mTORC1/2 by AZD or its analogue (KU-63794 depletes mouse BM Lin−Sca-1+c-Kit+ cells in cultures via the induction of apoptotic cell death. Subsequent colony-forming unit (CFU assays revealed that inhibition of mTORC1/2 suppresses the clonogenic function of hematopoietic progenitor cells (HPCs in a dose-dependent manner. Surprisingly, we found that dual inhibition of mTORC1/2 markedly inhibits the growth of day-14 cobblestone area-forming cells (CAFCs but enhances the generation of day-35 CAFCs. Given the fact that day-14 and day-35 CAFCs are functional surrogates of HPCs and hematopoietic stem cells (HSCs, respectively, these results suggest that dual inhibition of mTORC1/2 may have distinct effects on HPCs versus HSCs.

  7. Destabilization of Akt Promotes the Death of Myeloma Cell Lines

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    Yanan Zhang

    2014-01-01

    Full Text Available Constitutive activation of Akt is believed to be an oncogenic signal in multiple myeloma and is associated with poor patient prognosis and resistance to available treatment. The stability of Akt proteins is regulated by phosphorylating the highly conserved turn motif (TM of these proteins and the chaperone protein HSP90. In this study we investigate the antitumor effects of inhibiting mTORC2 plus HSP90 in myeloma cell lines. We show that chronic exposure of cells to rapamycin can inhibit mTORC2 pathway, and AKT will be destabilized by administration of the HSP90 inhibitor 17-allylamino-geldanamycin (17-AAG. Finally, we show that the rapamycin synergizes with 17-AAG and inhibits myeloma cells growth and promotes cell death to a greater extent than either drug alone. Our studies provide a clinical rationale of use mTOR inhibitors and chaperone protein inhibitors in combination regimens for the treatment of human blood cancers.

  8. Prolonged calorie restriction downregulates skeletal muscle mTORC1 signaling independent of dietary protein intake and associated microRNA expression

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    Lee M Margolis

    2016-10-01

    Full Text Available Short-term (5-10 days calorie restriction (CR downregulates muscle protein synthesis, with consumption of a high protein-based diet attenuating this decline. Benefit of increase protein intake is believed to be due to maintenance of amino acid-mediated anabolic signaling through the mechanistic target of rapamycin complex 1 (mTORC1, however, there is limited evidence to support this contention. The purpose of this investigation was to determine the effects of prolonged CR and high protein diets on skeletal muscle mTORC1 signaling and expression of associated microRNA (miR. 12-wk old male Sprague Dawley rats consumed ad libitum (AL or calorie restricted (CR; 40% adequate (10%, AIN-93M or high (32% protein milk-based diets for 16 weeks. Body composition was determined using dual energy X-ray absorptiometry and muscle protein content was calculated from muscle homogenate protein concentrations expressed relative to fat-free mass to estimate protein content. Western blot and RT-qPCR were used to determine mTORC1 signaling and mRNA and miR expression in fasted mixed gastrocnemius. Independent of dietary protein intake, muscle protein content was 38% lower (P < 0.05 in CR compared to AL. Phosphorylation and total Akt, mTOR, rpS6 and p70S6K were lower (P < 0.05 in CR versus AL, and total rpS6 was associated with muscle protein content (r = 0.64, r2 = 0.36. Skeletal muscle miR expression was not altered by either energy or protein intake. This study provides evidence that chronic CR attenuates muscle protein content by downregulating mTORC1 signaling. This response is independent of skeletal muscle miR and dietary protein.

  9. Heterozygous inactivation of tsc2 enhances tumorigenesis in p53 mutant zebrafish

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    Seok-Hyung Kim

    2013-07-01

    Tuberous sclerosis complex (TSC is a multi-organ disorder caused by mutations of the TSC1 or TSC2 genes. A key function of these genes is to inhibit mTORC1 (mechanistic target of rapamycin complex 1 kinase signaling. Cells deficient for TSC1 or TSC2 have increased mTORC1 signaling and give rise to benign tumors, although, as a rule, true malignancies are rarely seen. In contrast, other disorders with increased mTOR signaling typically have overt malignancies. A better understanding of genetic mechanisms that govern the transformation of benign cells to malignant ones is crucial to understand cancer pathogenesis. We generated a zebrafish model of TSC and cancer progression by placing a heterozygous mutation of the tsc2 gene in a p53 mutant background. Unlike tsc2 heterozygous mutant zebrafish, which never exhibited cancers, compound tsc2;p53 mutants had malignant tumors in multiple organs. Tumorigenesis was enhanced compared with p53 mutant zebrafish. p53 mutants also had increased mTORC1 signaling that was further enhanced in tsc2;p53 compound mutants. We found increased expression of Hif1-α, Hif2-α and Vegf-c in tsc2;p53 compound mutant zebrafish compared with p53 mutant zebrafish. Expression of these proteins probably underlies the increased angiogenesis seen in compound mutant zebrafish compared with p53 mutants and might further drive cancer progression. Treatment of p53 and compound mutant zebrafish with the mTORC1 inhibitor rapamycin caused rapid shrinkage of tumor size and decreased caliber of tumor-associated blood vessels. This is the first report using an animal model to show interactions between tsc2, mTORC1 and p53 during tumorigenesis. These results might explain why individuals with TSC rarely have malignant tumors, but also suggest that cancer arising in individuals without TSC might be influenced by the status of TSC1 and/or TSC2 mutations and be potentially treatable with mTORC1 inhibitors.

  10. mTORC1 Is a Major Regulatory Node in the FGF21 Signaling Network in Adipocytes

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    Annabel Y. Minard

    2016-09-01

    Full Text Available FGF21 improves the metabolic profile of obese animals through its actions on adipocytes. To elucidate the signaling network responsible for mediating these effects, we quantified dynamic changes in the adipocyte phosphoproteome following acute exposure to FGF21. FGF21 regulated a network of 821 phosphosites on 542 proteins. A major FGF21-regulated signaling node was mTORC1/S6K. In contrast to insulin, FGF21 activated mTORC1 via MAPK rather than through the canonical PI3K/AKT pathway. Activation of mTORC1/S6K by FGF21 was surprising because this is thought to contribute to deleterious metabolic effects such as obesity and insulin resistance. Rather, mTORC1 mediated many of the beneficial actions of FGF21 in vitro, including UCP1 and FGF21 induction, increased adiponectin secretion, and enhanced glucose uptake without any adverse effects on insulin action. This study provides a global view of FGF21 signaling and suggests that mTORC1 may act to facilitate FGF21-mediated health benefits in vivo.

  11. Are invertebrates relevant models in ageing research? Focus on the effects of rapamycin on TOR.

    Science.gov (United States)

    Erdogan, Cihan Suleyman; Hansen, Benni Winding; Vang, Ole

    2016-01-01

    Ageing is the organisms increased susceptibility to death, which is linked to accumulated damage in the cells and tissues. Ageing is a complex process regulated by crosstalk of various pathways in the cells. Ageing is highly regulated by the Target of Rapamycin (TOR) pathway activity. TOR is an evolutionary conserved key protein kinase in the TOR pathway that regulates growth, proliferation and cell metabolism in response to nutrients, growth factors and stress. Comparing the ageing process in invertebrate model organisms with relatively short lifespan with mammals provides valuable information about the molecular mechanisms underlying the ageing process faster than mammal systems. Inhibition of the TOR pathway activity via either genetic manipulation or rapamycin increases lifespan profoundly in most invertebrate model organisms. This contribution will review the recent findings in invertebrates concerning the TOR pathway and effects of TOR inhibition by rapamycin on lifespan. Besides some contradictory results, the majority points out that rapamycin induces longevity. This suggests that administration of rapamycin in invertebrates is a promising tool for pursuing the scientific puzzle of lifespan prolongation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. Role of mTOR Inhibitors in Kidney Disease

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    Moto Kajiwara

    2016-06-01

    Full Text Available The first compound that inhibited the mammalian target of rapamycin (mTOR, sirolimus (rapamycin was discovered in the 1970s as a soil bacterium metabolite collected on Easter Island (Rapa Nui. Because sirolimus showed antiproliferative activity, researchers investigated its molecular target and identified the TOR1 and TOR2. The mTOR consists of mTOR complex 1 (mTORC1 and mTORC2. Rapalogues including sirolimus, everolimus, and temsirolimus exert their effect mainly on mTORC1, whereas their inhibitory effect on mTORC2 is mild. To obtain compounds with more potent antiproliferative effects, ATP-competitive inhibitors of mTOR targeting both mTORC1 and mTORC2 have been developed and tested in clinical trials as anticancer drugs. Currently, mTOR inhibitors are used as anticancer drugs against several solid tumors, and immunosuppressive agents for transplantation of various organs. This review discusses the role of mTOR inhibitors in renal disease with a particular focus on renal cancer, diabetic nephropathy, and kidney transplantation.

  13. Dynamics of mTORC1 activation in response to amino acids

    Science.gov (United States)

    Manifava, Maria; Smith, Matthew; Rotondo, Sergio; Walker, Simon; Niewczas, Izabella; Zoncu, Roberto; Clark, Jonathan; Ktistakis, Nicholas T

    2016-01-01

    Amino acids are essential activators of mTORC1 via a complex containing RAG GTPases, RAGULATOR and the vacuolar ATPase. Sensing of amino acids causes translocation of mTORC1 to lysosomes, an obligate step for activation. To examine the spatial and temporal dynamics of this translocation, we used live imaging of the mTORC1 component RAPTOR and a cell permeant fluorescent analogue of di-leucine methyl ester. Translocation to lysosomes is a transient event, occurring within 2 min of aa addition and peaking within 5 min. It is temporally coupled with fluorescent leucine appearance in lysosomes and is sustained in comparison to aa stimulation. Sestrin2 and the vacuolar ATPase are negative and positive regulators of mTORC1 activity in our experimental system. Of note, phosphorylation of canonical mTORC1 targets is delayed compared to lysosomal translocation suggesting a dynamic and transient passage of mTORC1 from the lysosomal surface before targetting its substrates elsewhere. DOI: http://dx.doi.org/10.7554/eLife.19960.001 PMID:27725083

  14. Calcium pathways such as cAMP modulate clothianidin action through activation of α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors.

    Science.gov (United States)

    Calas-List, Delphine; List, Olivier; Quinchard, Sophie; Thany, Steeve H

    2013-07-01

    Clothianidin is a neonicotinoid insecticide developed in the early 2000s. We have recently demonstrated that it was a full agonist of α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors expressed in the cockroach dorsal unpaired median neurons. Clothianidin was able to act as an agonist of imidacloprid-insensitive nAChR2 receptor and internal regulation of cAMP concentration modulated nAChR2 sensitivity to clothianidin. In the present study, we demonstrated that cAMP modulated the agonist action of clothianidin via α-bungarotoxin-sensitive and insensitive receptors. Clothianidin-induced current-voltage curves were dependent to clothianidin concentrations. At 10 μM clothianidin, increasing cAMP concentration induced a linear current-voltage curve. Clothianidin effects were blocked by 0.5 μM α-bungarotoxin suggesting that cAMP modulation occurred through α-bungarotoxin-sensitive receptors. At 1 mM clothianidin, cAMP effects were associated to α-bungarotoxin-insensitive receptors because clothianidin-induced currents were blocked by 5 μM mecamylamine and 20 μM d-tubocurarine. In addition, we found that application of 1mM clothianidin induced a strong increase of intracellular calcium concentration. These data reinforced the finding that calcium pathways including cAMP modulated clothianidin action on insect nicotinic acetylcholine receptors. We proposed that intracellular calcium pathways such as cAMP could be a target to modulate the mode of action of neonicotinoid insecticides. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. S6K1 Is Required for Increasing Skeletal Muscle Force during Hypertrophy.

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    Marabita, Manuela; Baraldo, Martina; Solagna, Francesca; Ceelen, Judith Johanna Maria; Sartori, Roberta; Nolte, Hendrik; Nemazanyy, Ivan; Pyronnet, Stéphane; Kruger, Marcus; Pende, Mario; Blaauw, Bert

    2016-10-04

    Loss of skeletal muscle mass and force aggravates age-related sarcopenia and numerous pathologies, such as cancer and diabetes. The AKT-mTORC1 pathway plays a major role in stimulating adult muscle growth; however, the functional role of its downstream mediators in vivo is unknown. Here, we show that simultaneous inhibition of mTOR signaling to both S6K1 and 4E-BP1 is sufficient to reduce AKT-induced muscle growth and render it insensitive to the mTORC1-inhibitor rapamycin. Surprisingly, lack of mTOR signaling to 4E-BP1 only, or deletion of S6K1 alone, is not sufficient to reduce muscle hypertrophy or alter its sensitivity to rapamycin. However, we report that, while not required for muscle growth, S6K1 is essential for maintaining muscle structure and force production. Hypertrophy in the absence of S6K1 is characterized by compromised ribosome biogenesis and the formation of p62-positive protein aggregates. These findings identify S6K1 as a crucial player for maintaining muscle function during hypertrophy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Autophagy response: manipulating the mTOR-controlled machinery by amino acids and pathogens.

    Science.gov (United States)

    Fader, Claudio Marcelo; Aguilera, Milton Osmar; Colombo, María Isabel

    2015-10-01

    Macroautophagy is a self-degradative process that normally maintains cellular homeostasis via a lysosomal pathway. It is induced by different stress signals, including nutrients and growth factors' restriction as well as pathogen invasions. These stimuli are modulated by the serine/threonine protein kinase mammalian target of rapamycin (mTOR) which control not only autophagy but also protein translation and gene expression. This review focuses on the important role of mTOR as a master regulator of cell growth and the autophagy pathway. Here, we have discussed the role of intracellular amino acid availability and intracellular pH in the redistribution of autophagic structures, which may contribute to mammalian target of rapamycin complex 1 (mTORC1) activity regulation. We have also discussed that mTORC1 complex and components of the autophagy machinery are localized at the lysosomal surface, representing a fascinating mechanism to control the metabolism, cellular clearance and also to restrain invading intracellular pathogens.

  17. Inhibition of repopulation is not a determining factor for the radiosensitizing effects of rapamycin

    International Nuclear Information System (INIS)

    Sarkaria, J.N.; Carlson, B.L.; Mladek, A.C.

    2003-01-01

    The mammalian target of rapamycin (mTOR) is a key downstream effector of the PI3K-Akt signaling pathway, and we have previously shown that inhibition of mTOR by rapamycin significantly enhances the efficacy of prolonged fractionated radiation in U87 glioma cells grown as xenografts or spheroids. To test whether inhibition of repopulation between radiation fractions contributes to the sensitizing effects of rapamycin, the efficacy of our previous protracted radiation schedule was compared with an accelerated regimen in U87 spheroids. Regrowth of individual spheroids was tracked over time following treatment with either accelerated or protracted radiation in the presence or absence of rapamycin. As in our previous studies, treatment with 10 nM rapamycin significantly increased the time required for U87 spheroids to regrow to 10 times their original volume (22 ± 2 days [mean ± 95% CI]) compared to control (7 ± 1 days). Regrowth after protracted radiation (2 Gy every 3 days x 4; 9 ± 2 days)did not significantly differ from control treatment, while accelerated radiation (2 Gy every 4 hours x 4) modestly delayed spheroid regrowth (12 ± 2 days). Specific to our model, the relatively small difference in regrowth time between the two radiation fractionation schedules suggests that repopulation is not a major detrimental factor in the protracted radiation schedule. Interestingly, the combination of rapamycin with either protracted or accelerated RT significantly enhanced the efficacy of the radiation with regrowth times of 31 ± 4 days and 29 ± 4 days, respectively. Consistent with this in vitro data, preliminary results from an animal study suggest that treatment with a rapamycin analog and daily radiation is as effective as protracted radiation/ rapamycin schedules. Thus, any effects of rapamycin on repopulation in our model systems do not contribute significantly to the sensitizing effects of rapamycin

  18. Phosphorylation of Ribosomal Protein S6 Mediates Mammalian Target of Rapamycin Complex 1-Induced Parathyroid Cell Proliferation in Secondary Hyperparathyroidism.

    Science.gov (United States)

    Volovelsky, Oded; Cohen, Gili; Kenig, Ariel; Wasserman, Gilad; Dreazen, Avigail; Meyuhas, Oded; Silver, Justin; Naveh-Many, Tally

    2016-04-01

    Secondary hyperparathyroidism is characterized by increased serum parathyroid hormone (PTH) level and parathyroid cell proliferation. However, the molecular pathways mediating the increased parathyroid cell proliferation remain undefined. Here, we found that the mTOR pathway was activated in the parathyroid of rats with secondary hyperparathyroidism induced by either chronic hypocalcemia or uremia, which was measured by increased phosphorylation of ribosomal protein S6 (rpS6), a downstream target of the mTOR pathway. This activation correlated with increased parathyroid cell proliferation. Inhibition of mTOR complex 1 by rapamycin decreased or prevented parathyroid cell proliferation in secondary hyperparathyroidism rats and in vitro in uremic rat parathyroid glands in organ culture. Knockin rpS6(p-/-) mice, in which rpS6 cannot be phosphorylated because of substitution of all five phosphorylatable serines with alanines, had impaired PTH secretion after experimental uremia- or folic acid-induced AKI. Uremic rpS6(p-/-) mice had no increase in parathyroid cell proliferation compared with a marked increase in uremic wild-type mice. These results underscore the importance of mTOR activation and rpS6 phosphorylation for the pathogenesis of secondary hyperparathyroidism and indicate that mTORC1 is a significant regulator of parathyroid cell proliferation through rpS6. Copyright © 2016 by the American Society of Nephrology.

  19. Carbon monoxide protects against hepatic steatosis in mice by inducing sestrin-2 via the PERK-eIF2α-ATF4 pathway.

    Science.gov (United States)

    Kim, Hyo Jeong; Joe, Yeonsoo; Kim, Seul-Ki; Park, Se-Ung; Park, Jeongmin; Chen, Yingqing; Kim, Jin; Ryu, Jinhyun; Cho, Gyeong Jae; Surh, Young-Joon; Ryter, Stefan W; Kim, Uh-Hyun; Chung, Hun-Taeg

    2017-09-01

    Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, has emerged as one of the most common causes of chronic liver disease in developed countries over the last decade. NAFLD comprises a spectrum of pathological hepatic changes, including steatosis, steatohepatitis, advanced fibrosis, and cirrhosis. Autophagy, a homeostatic process for protein and organelle turnover, is decreased in the liver during the development of NAFLD. Previously, we have shown that carbon monoxide (CO), a reaction product of heme oxygenase (HO) activity, can confer protection in NAFLD, though the molecular mechanisms remain unclear. We therefore investigated the mechanisms underlying the protective effect of CO on methionine/choline-deficient (MCD) diet-induced hepatic steatosis. We found that CO induced sestrin-2 (SESN2) expression through enhanced mitochondrial ROS production and protected against MCD-induced NAFLD progression through activation of autophagy. SESN2 expression was increased by CO or CO-releasing molecule (CORM2), in a manner dependent on signaling through the protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor-2 alpha (eIF2α)/ activating transcription factor-4 (ATF4)-dependent pathway. CO-induced SESN2 upregulation in hepatocytes contributed to autophagy induction through activation of 5'-AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR) complex I (mTORC1). Furthermore, we demonstrate that CO significantly induced the expression of SESN2 and enhanced autophagy in the livers of MCD-fed mice or in MCD-media treated hepatocytes. Conversely, knockdown of SESN2 abrogated autophagy activation and mTOR inhibition in response to CO. We conclude that CO ameliorates hepatic steatosis through the autophagy pathway induced by SESN2 upregulation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Nutrient-induced stimulation of protein synthesis in mouse skeletal muscle is limited by the mTORC1 repressor REDD1.

    Science.gov (United States)

    Gordon, Bradley S; Williamson, David L; Lang, Charles H; Jefferson, Leonard S; Kimball, Scot R

    2015-04-01

    In skeletal muscle, the nutrient-induced stimulation of protein synthesis requires signaling through the mechanistic target of rapamycin complex 1 (mTORC1). Expression of the repressor of mTORC1 signaling, regulated in development and DNA damage 1 (REDD1), is elevated in muscle during various atrophic conditions and diminished under hypertrophic conditions. The question arises as to what extent REDD1 limits the nutrient-induced stimulation of protein synthesis. The objective was to examine the role of REDD1 in limiting the response of muscle protein synthesis and mTORC1 signaling to a nutrient stimulus. Wild type REDD1 gene (REDD1(+/+)) and disruption in the REDD1 gene (REDD1(-/-)) mice were feed deprived for 16 h and randomized to remain feed deprived or refed for 15 or 60 min. The tibialis anterior was then removed for analysis of protein synthesis and mTORC1 signaling. In feed-deprived mice, protein synthesis and mTORC1 signaling were significantly lower in REDD1(+/+) than in REDD1(-/-) mice. Thirty minutes after the start of refeeding, protein synthesis in REDD1(+/+) mice was stimulated by 28%, reaching a value similar to that observed in feed-deprived REDD1(-/-) mice, and was accompanied by increased phosphorylation of mTOR (Ser2448), p70S6K1 (Thr389), and 4E-BP1 (Ser65) by 81%, 167%, and 207%, respectively. In refed REDD1(-/-) mice, phosphorylation of mTOR (Ser2448), p70S6K1 (Thr389), and 4E-BP1 (Ser65) were significantly augmented above the values observed in refed REDD1(+/+) mice by 258%, 405%, and 401%, respectively, although protein synthesis was not coordinately increased. Seventy-five minutes after refeeding, REDD1 expression in REDD1(+/+) mice was reduced (∼15% of feed-deprived REDD1(+/+) values), and protein synthesis and mTORC1 signaling were not different between refed REDD1(+/+) mice and REDD1(-/-) mice. The results show that REDD1 expression limits protein synthesis in mouse skeletal muscle by inhibiting mTORC1 signaling during periods of feed

  1. GSK-3/Rb12 Pathway as a Novel Target of Rapamycin in Prostate Cancer

    National Research Council Canada - National Science Library

    Litovchick, Larissa

    2005-01-01

    .... Rapamycin exerts its effects through inhibition of mammalian Target of Rapamycin (mTOR) protein kinase resulting in a decreased expression of a subset of proteins essential for cell cycle progression...

  2. Rapamycin enhances the anti-angiogenesis and anti-proliferation ability of YM155 in oral squamous cell carcinoma.

    Science.gov (United States)

    Li, Kong-Liang; Wang, Yu-Fan; Qin, Jia-Ruo; Wang, Feng; Yang, Yong-Tao; Zheng, Li-Wu; Li, Ming-Hua; Kong, Jie; Zhang, Wei; Yang, Hong-Yu

    2017-06-01

    YM155, a small molecule inhibitor of survivin, has been studied in many tumors. It has been shown that YM155 inhibited oral squamous cell carcinoma through promoting apoptosis and autophagy and inhibiting proliferation. It was found that YM155 also inhibited the oral squamous cell carcinoma-mediated angiogenesis through the inactivation of the mammalian target of rapamycin pathway. Rapamycin, a mammalian target of rapamycin inhibitor, played an important role in the proliferation and angiogenesis of oral squamous cell carcinoma cell lines. In our study, cell proliferation assay, transwell assay, tube formation assay, and western blot assay were used to investigate the synergistic effect of rapamycin on YM155 in oral squamous cell carcinoma. Either in vitro or in vivo, rapamycin and YM155 exerted a synergistic effect on the inhibition of survivin and vascular endothelial growth factor through mammalian target of rapamycin pathway. Overall, our results revealed that low-dose rapamycin strongly promoted the sensitivity of oral squamous cell carcinoma cell lines to YM155.

  3. The TOR Pathway Is Involved in Adventitious Root Formation in Arabidopsis and Potato.

    Science.gov (United States)

    Deng, Kexuan; Dong, Pan; Wang, Wanjing; Feng, Li; Xiong, Fangjie; Wang, Kai; Zhang, Shumin; Feng, Shun; Wang, Bangjun; Zhang, Jiankui; Ren, Maozhi

    2017-01-01

    In the agriculture industry, adventitious root formation is a core issue of plants asexual propagation. However, the underlying molecular mechanism of adventitious root formation is far beyond understanding. In present study we found that target of rapamycin (TOR) signaling plays a key role in adventitious root formation in potato and Arabidopsis . The core components of TOR complex including TOR, RAPTOR, and LST8 are highly conserved in potato, but the seedlings of potato are insensitive to rapamycin, implying FK506 Binding Protein 12 KD (FKBP12) lost the function to bridge the interaction of rapamycin and TOR in potato. To dissect TOR signaling in potato, the rapamycin hypersensitive potato plants (BP12-OE) were engineered by introducing yeast FKBP12 ( ScFKBP12 ) into potato. We found that rapamycin can significantly attenuate the capability of adventitious root formation in BP12-OE potatoes. KU63794 (KU, an active-site TOR inhibitor) combined with rapamycin can more significantly suppress adventitious root formation of BP12-OE potato than the single treatments, such as KU63794 or rapamycin, indicating its synergistic inhibitory effects on potato adventitious root formation. Furthermore, RNA-seq data showed that many genes associated with auxin signaling pathway were altered when BP12-OE potato seedlings were treated with rapamycin + KU, suggesting that TOR may play a major role in adventitious root formation via auxin signaling. The auxin receptor mutant tir1 was sensitive to TOR inhibitors and the double and quadruple mutants including tir1afb2, tir1afb3 , and tir1afb1afb2afb3 displayed more sensitive to asTORis than single mutant tir1 . Consistently, overexpression of AtTIR1 in Arabidopsis and potato can partially overcome the inhibitory effect of asTORis and promote adventitious root formation under asTORis treatments. These observations suggest that TOR signaling regulates adventitious root formation by mediating auxin signaling in Arabidopsis and potato.

  4. Activation of mTORC1 by leucine is potentiated by branched-chain amino acids and even more so by essential amino acids following resistance exercise

    DEFF Research Database (Denmark)

    Moberg, Marcus; Apró, William; Ekblom, Björn

    2016-01-01

    Protein synthesis is stimulated by resistance exercise and intake of amino acids, in particular leucine. Moreover, activation of mammalian target of rapamycin complex 1 (mTORC1) signaling by leucine is potentiated by the presence of other essential amino acids (EAA). However, the contribution...... of the branched-chain amino acids (BCAA) to this effect is yet unknown. Here we compare the stimulatory role of leucine, BCAA, and EAA ingestion on anabolic signaling following exercise. Accordingly, eight trained volunteers completed four sessions of resistance exercise during which they ingested either placebo...

  5. Biochemical Basis of Sestrin Physiological Activities

    Energy Technology Data Exchange (ETDEWEB)

    Ho, Allison; Cho, Chun-Seok; Namkoong, Sim; Cho, Uhn-Soo; Lee, Jun Hee (Michigan)

    2016-05-10

    Excessive accumulation of reactive oxygen species (ROS) and chronic activation of mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) are well-characterized promoters of aging and age-associated degenerative pathologies. Sestrins, a family of highly conserved stress-inducible proteins, are important negative regulators of both ROS and mTORC1 signaling pathways; however, the mechanistic basis of how Sestrins suppress these pathways remains elusive. In the past couple of years, breakthrough discoveries about Sestrin signaling and its molecular nature have markedly increased our biochemical understanding of Sestrin function. These discoveries have also uncovered new potential therapeutic strategies that may eventually enable us to attenuate aging and age-associated diseases.

  6. Reactivation of cocaine reward memory engages the Akt/GSK3/mTOR signaling pathway and can be disrupted by GSK3 inhibition.

    Science.gov (United States)

    Shi, Xiangdang; Miller, Jonathan S; Harper, Lauren J; Poole, Rachel L; Gould, Thomas J; Unterwald, Ellen M

    2014-08-01

    Memories return to a labile state following their retrieval and must undergo a process of reconsolidation to be maintained. Thus, disruption of cocaine reward memories by interference with reconsolidation may be therapeutically beneficial in the treatment of cocaine addiction. The objectives were to elucidate the signaling pathway involved in reconsolidation of cocaine reward memory and to test whether targeting this pathway could disrupt cocaine-associated contextual memory. Using a mouse model of conditioned place preference, regulation of the activity of glycogen synthase kinase-3 (GSK3), mammalian target of Rapamycin complex 1 (mTORC1), P70S6K, β-catenin, and the upstream signaling molecule Akt, was studied in cortico-limbic-striatal circuitry after re-exposure to an environment previously paired with cocaine. Levels of phosporylated Akt-Thr308, GSK3α-Ser21, GSK3β-Ser9, mTORC1, and P70S6K were reduced in the nucleus accumbens and hippocampus 10 min after the reactivation of cocaine cue memories. Levels of pAkt and pGSK3 were also reduced in the prefrontal cortex. Since reduced phosphorylation of GSK3 indicates heightened enzyme activity, the effect of a selective GSK3 inhibitor, SB216763, on reconsolidation was tested. Administration of SB216763 immediately after exposure to an environment previously paired with cocaine abrogated a previously established place preference, suggesting that GSK3 inhibition interfered with reconsolidation of cocaine-associated reward memories. These findings suggest that the Akt/GSK3/mTORC1 signaling pathway in the nucleus accumbens, hippocampus, and/or prefrontal cortex is critically involved in the reconsolidation of cocaine contextual reward memory. Inhibition of GSK3 activity during memory retrieval can erase an established cocaine place preference.

  7. The anti-hepatocellular carcinoma cell activity by a novel mTOR kinase inhibitor CZ415

    International Nuclear Information System (INIS)

    Zhang, Wei; Chen, Bingyu; Zhang, Yu; Li, Kaiqiang; Hao, Ke; Jiang, Luxi; Wang, Ying; Mou, Xiaozhou; Xu, Xiaodong; Wang, Zhen

    2017-01-01

    Dysregulation of mammalian target of rapamycin (mTOR) in hepatocellular carcinoma (HCC) represents a valuable treatment target. Recent studies have developed a highly-selective and potent mTOR kinase inhibitor, CZ415. Here, we showed that nM concentrations of CZ415 efficiently inhibited survival and induced apoptosis in HCC cell lines (HepG2 and Huh-7) and primary-cultured human HCC cells. Meanwhile, CZ415 inhibited proliferation of HCC cells, more potently than mTORC1 inhibitors (rapamycin and RAD001). CZ415 was yet non-cytotoxic to the L02 human hepatocytes. Mechanistic studies showed that CZ415 disrupted assembly of mTOR complex 1 (mTORC1) and mTORC2 in HepG2 cells. Meanwhile, activation of mTORC1 (p-S6K1) and mTORC2 (p-AKT, Ser-473) was almost blocked by CZ415. In vivo studies revealed that oral administration of CZ415 significantly suppressed HepG2 xenograft tumor growth in severe combined immuno-deficient (SCID) mice. Activation of mTORC1/2 was also largely inhibited in CZ415-treated HepG2 tumor tissue. Together, these results show that CZ415 blocks mTORC1/2 activation and efficiently inhibits HCC cell growth in vitro and in vivo. - Highlights: • CZ415 is anti-survival and pro-apoptotic to hepatocellular carcinoma (HCC) cells. • CZ415 inhibits HCC cell proliferation, more efficiently than mTORC1 inhibitors. • CZ415 blocks assembly and activation of both mTORC1 and mTORC2 in HCC cells. • CZ415 oral administration inhibits HepG2 tumor growth in SCID mice. • mTORC1/2 activation in HepG2 tumor is inhibited with CZ415 administration.

  8. Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Karen K Y Lam

    Full Text Available Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

  9. Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis.

    Science.gov (United States)

    Lam, Karen K Y; Zheng, Xingji; Forestieri, Roberto; Balgi, Aruna D; Nodwell, Matt; Vollett, Sarah; Anderson, Hilary J; Andersen, Raymond J; Av-Gay, Yossef; Roberge, Michel

    2012-01-01

    Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

  10. Dysregulation of Nutrient Sensing and CLEARance in Presenilin Deficiency

    Directory of Open Access Journals (Sweden)

    Kavya Reddy

    2016-03-01

    Full Text Available Attenuated auto-lysosomal system has been associated with Alzheimer disease (AD, yet all underlying molecular mechanisms leading to this impairment are unknown. We show that the amino acid sensing of mechanistic target of rapamycin complex 1 (mTORC1 is dysregulated in cells deficient in presenilin, a protein associated with AD. In these cells, mTORC1 is constitutively tethered to lysosomal membranes, unresponsive to starvation, and inhibitory to TFEB-mediated clearance due to a reduction in Sestrin2 expression. Normalization of Sestrin2 levels through overexpression or elevation of nuclear calcium rescued mTORC1 tethering and initiated clearance. While CLEAR network attenuation in vivo results in buildup of amyloid, phospho-Tau, and neurodegeneration, presenilin-knockout fibroblasts and iPSC-derived AD human neurons fail to effectively initiate autophagy. These results propose an altered mechanism for nutrient sensing in presenilin deficiency and underline an importance of clearance pathways in the onset of AD.

  11. High-throughput drug screen identifies chelerythrine as a selective inducer of death in a TSC2-null setting.

    Science.gov (United States)

    Medvetz, Doug; Sun, Yang; Li, Chenggang; Khabibullin, Damir; Balan, Murugabaskar; Parkhitko, Andrey; Priolo, Carmen; Asara, John M; Pal, Soumitro; Yu, Jane; Henske, Elizabeth P

    2015-01-01

    Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome associated with tumors of the brain, heart, kidney, and lung. The TSC protein complex inhibits the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Inhibitors of mTORC1, including rapamycin, induce a cytostatic response in TSC tumors, resulting in temporary disease stabilization and prompt regrowth when treatment is stopped. The lack of TSC-specific cytotoxic therapies represents an important unmet clinical need. Using a high-throughput chemical screen in TSC2-deficient, patient-derived cells, we identified a series of molecules antagonized by rapamycin and therefore selective for cells with mTORC1 hyperactivity. In particular, the cell-permeable alkaloid chelerythrine induced reactive oxygen species (ROS) and depleted glutathione (GSH) selectively in TSC2-null cells based on metabolic profiling. N-acetylcysteine or GSH cotreatment protected TSC2-null cells from chelerythrine's effects, indicating that chelerythrine-induced cell death is ROS dependent. Induction of heme-oxygenase-1 (HMOX1/HO-1) with hemin also blocked chelerythrine-induced cell death. In vivo, chelerythrine inhibited the growth of TSC2-null xenograft tumors with no evidence of systemic toxicity with daily treatment over an extended period of time. This study reports the results of a bioactive compound screen and the identification of a potential lead candidate that acts via a novel oxidative stress-dependent mechanism to selectively induce necroptosis in TSC2-deficient tumors. This study demonstrates that TSC2-deficient tumor cells are hypersensitive to oxidative stress-dependent cell death, and provide critical proof of concept that TSC2-deficient cells can be therapeutically targeted without the use of a rapalog to induce a cell death response. ©2014 American Association for Cancer Research.

  12. The mTOR inhibitor rapamycin has limited acute anticonvulsant effects in mice.

    Directory of Open Access Journals (Sweden)

    Adam L Hartman

    Full Text Available The mammalian target of rapamycin (mTOR pathway integrates signals from different nutrient sources, including amino acids and glucose. Compounds that inhibit mTOR kinase activity such as rapamycin and everolimus can suppress seizures in some chronic animal models and in patients with tuberous sclerosis. However, it is not known whether mTOR inhibitors exert acute anticonvulsant effects in addition to their longer term antiepileptogenic effects. To gain insights into how rapamycin suppresses seizures, we investigated the anticonvulsant activity of rapamycin using acute seizure tests in mice.Following intraperitoneal injection of rapamycin, normal four-week-old male NIH Swiss mice were evaluated for susceptibility to a battery of acute seizure tests similar to those currently used to screen potential therapeutics by the US NIH Anticonvulsant Screening Program. To assess the short term effects of rapamycin, mice were seizure tested in ≤ 6 hours of a single dose of rapamycin, and for longer term effects of rapamycin, mice were tested after 3 or more daily doses of rapamycin.The only seizure test where short-term rapamycin treatment protected mice was against tonic hindlimb extension in the MES threshold test, though this protection waned with longer rapamycin treatment. Longer term rapamycin treatment protected against kainic acid-induced seizure activity, but only at late times after seizure onset. Rapamycin was not protective in the 6 Hz or PTZ seizure tests after short or longer rapamycin treatment times. In contrast to other metabolism-based therapies that protect in acute seizure tests, rapamycin has limited acute anticonvulsant effects in normal mice.The efficacy of rapamycin as an acute anticonvulsant agent may be limited. Furthermore, the combined pattern of acute seizure test results places rapamycin in a third category distinct from both fasting and the ketogenic diet, and which is more similar to drugs acting on sodium channels.

  13. Drosophila insulin and target of rapamycin (TOR pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo

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    Parisi Federica

    2011-09-01

    Full Text Available Abstract Background Genetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Results Our biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3β a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. Conclusions Our study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At

  14. Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo.

    Science.gov (United States)

    Parisi, Federica; Riccardo, Sara; Daniel, Margaret; Saqcena, Mahesh; Kundu, Nandini; Pession, Annalisa; Grifoni, Daniela; Stocker, Hugo; Tabak, Esteban; Bellosta, Paola

    2011-09-27

    Genetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Our biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3β ) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. Our study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level we found that both these pathways

  15. Amino Acid Activation of mTORC1 by a PB1-Domain-Driven Kinase Complex Cascade.

    Science.gov (United States)

    Linares, Juan F; Duran, Angeles; Reina-Campos, Miguel; Aza-Blanc, Pedro; Campos, Alex; Moscat, Jorge; Diaz-Meco, Maria T

    2015-08-25

    The mTORC1 complex is central to the cellular response to changes in nutrient availability. The signaling adaptor p62 contributes to mTORC1 activation in response to amino acids and interacts with TRAF6, which is required for the translocation of mTORC1 to the lysosome and the subsequent K63 polyubiquitination and activation of mTOR. However, the signal initiating these p62-driven processes was previously unknown. Here, we show that p62 is phosphorylated via a cascade that includes MEK3/6 and p38δ and is driven by the PB1-containing kinase MEKK3. This phosphorylation results in the recruitment of TRAF6 to p62, the ubiquitination and activation of mTOR, and the regulation of autophagy and cell proliferation. Genetic inactivation of MEKK3 or p38δ mimics that of p62 in that it leads to inhibited growth of PTEN-deficient prostate organoids. Analysis of human prostate cancer samples showed upregulation of these three components of the pathway, which correlated with enhanced mTORC1 activation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Amino Acid Activation of mTORC1 by a PB1-Domain-Driven Kinase Complex Cascade

    Directory of Open Access Journals (Sweden)

    Juan F. Linares

    2015-08-01

    Full Text Available The mTORC1 complex is central to the cellular response to changes in nutrient availability. The signaling adaptor p62 contributes to mTORC1 activation in response to amino acids and interacts with TRAF6, which is required for the translocation of mTORC1 to the lysosome and the subsequent K63 polyubiquitination and activation of mTOR. However, the signal initiating these p62-driven processes was previously unknown. Here, we show that p62 is phosphorylated via a cascade that includes MEK3/6 and p38δ and is driven by the PB1-containing kinase MEKK3. This phosphorylation results in the recruitment of TRAF6 to p62, the ubiquitination and activation of mTOR, and the regulation of autophagy and cell proliferation. Genetic inactivation of MEKK3 or p38δ mimics that of p62 in that it leads to inhibited growth of PTEN-deficient prostate organoids. Analysis of human prostate cancer samples showed upregulation of these three components of the pathway, which correlated with enhanced mTORC1 activation.

  17. The Molecular Basis for Load-Induced Skeletal Muscle Hypertrophy

    Science.gov (United States)

    Marcotte, George R.; West, Daniel W.D.; Baar, Keith

    2016-01-01

    In a mature (weight neutral) animal, an increase in muscle mass only occurs when the muscle is loaded sufficiently to cause an increase in myofibrillar protein balance. A tight relationship between muscle hypertrophy, acute increases in protein balance, and the activity of the mechanistic target of rapamycin complex 1 (mTORC1) was demonstrated 15 years ago. Since then, our understanding of the signals that regulate load-induced hypertrophy has evolved considerably. For example, we now know that mechanical load activates mTORC1 in the same way as growth factors, by moving TSC2 (a primary inhibitor of mTORC1) away from its target (the mTORC activator) Rheb. However, the kinase that phosphorylates and moves TSC2 is different in the two processes. Similarly, we have learned that a distinct pathway exists whereby amino acids activate mTORC1 by moving it to Rheb. While mTORC1 remains at the forefront of load-induced hypertrophy, the importance of other pathways that regulate muscle mass are becoming clearer. Myostatin, is best known for its control of developmental muscle size. However, new mechanisms to explain how loading regulates this process are suggesting that it could play an important role in hypertrophic muscle growth as well. Lastly, new mechanisms are highlighted for how β2 receptor agonists could be involved in load-induced muscle growth and why these agents are being developed as non-exercise-based therapies for muscle atrophy. Overall, the results highlight how studying the mechanism of load-induced skeletal muscle mass is leading the development of pharmaceutical interventions to promote muscle growth in those unwilling or unable to perform resistance exercise. PMID:25359125

  18. Rapamycin and Glucose-Target of Rapamycin (TOR) Protein Signaling in Plants*

    Science.gov (United States)

    Xiong, Yan; Sheen, Jen

    2012-01-01

    Target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, nutrients, growth factors, and stress signals to promote survival and growth in all eukaryotes. The reported land plant resistance to rapamycin and the embryo lethality of the Arabidopsis tor mutants have hindered functional dissection of TOR signaling in plants. We developed sensitive cellular and seedling assays to monitor endogenous Arabidopsis TOR activity based on its conserved S6 kinase (S6K) phosphorylation. Surprisingly, rapamycin effectively inhibits Arabidopsis TOR-S6K1 signaling and retards glucose-mediated root and leaf growth, mimicking estradiol-inducible tor mutants. Rapamycin inhibition is relieved in transgenic plants deficient in Arabidopsis FK506-binding protein 12 (FKP12), whereas FKP12 overexpression dramatically enhances rapamycin sensitivity. The role of Arabidopsis FKP12 is highly specific as overexpression of seven closely related FKP proteins fails to increase rapamycin sensitivity. Rapamycin exerts TOR inhibition by inducing direct interaction between the TOR-FRB (FKP-rapamycin binding) domain and FKP12 in plant cells. We suggest that variable endogenous FKP12 protein levels may underlie the molecular explanation for longstanding enigmatic observations on inconsistent rapamycin resistance in plants and in various mammalian cell lines or diverse animal cell types. Integrative analyses with rapamycin and conditional tor and fkp12 mutants also reveal a central role of glucose-TOR signaling in root hair formation. Our studies demonstrate the power of chemical genetic approaches in the discovery of previously unknown and pivotal functions of glucose-TOR signaling in governing the growth of cotyledons, true leaves, petioles, and primary and secondary roots and root hairs. PMID:22134914

  19. Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I.

    Science.gov (United States)

    Brockhoff, Marielle; Rion, Nathalie; Chojnowska, Kathrin; Wiktorowicz, Tatiana; Eickhorst, Christopher; Erne, Beat; Frank, Stephan; Angelini, Corrado; Furling, Denis; Rüegg, Markus A; Sinnreich, Michael; Castets, Perrine

    2017-02-01

    Myotonic dystrophy type I (DM1) is a disabling multisystemic disease that predominantly affects skeletal muscle. It is caused by expanded CTG repeats in the 3'-UTR of the dystrophia myotonica protein kinase (DMPK) gene. RNA hairpins formed by elongated DMPK transcripts sequester RNA-binding proteins, leading to mis-splicing of numerous pre-mRNAs. Here, we have investigated whether DM1-associated muscle pathology is related to deregulation of central metabolic pathways, which may identify potential therapeutic targets for the disease. In a well-characterized mouse model for DM1 (HSALR mice), activation of AMPK signaling in muscle was impaired under starved conditions, while mTORC1 signaling remained active. In parallel, autophagic flux was perturbed in HSALR muscle and in cultured human DM1 myotubes. Pharmacological approaches targeting AMPK/mTORC1 signaling greatly ameliorated muscle function in HSALR mice. AICAR, an AMPK activator, led to a strong reduction of myotonia, which was accompanied by partial correction of misregulated alternative splicing. Rapamycin, an mTORC1 inhibitor, improved muscle relaxation and increased muscle force in HSALR mice without affecting splicing. These findings highlight the involvement of AMPK/mTORC1 deregulation in DM1 muscle pathophysiology and may open potential avenues for the treatment of this disease.

  20. Mammalian Target of Rapamycin Inhibition With Rapamycin Mitigates Radiation-Induced Pulmonary Fibrosis in a Murine Model

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Eun Joo [Radiation Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); Sowers, Anastasia; Thetford, Angela [Radiation Biology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); McKay-Corkum, Grace; Chung, Su I. [Radiation Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); Mitchell, James B. [Radiation Biology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); Citrin, Deborah E., E-mail: citrind@mail.nih.gov [Radiation Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States)

    2016-11-15

    Purpose: Radiation-induced pulmonary fibrosis (RIPF) is a late toxicity of therapeutic radiation. Signaling of the mammalian target of rapamycin drives several processes implicated in RIPF, including inflammatory cytokine production, fibroblast proliferation, and epithelial senescence. We sought to determine if mammalian target of rapamycin inhibition with rapamycin would mitigate RIPF. Methods and Materials: C57BL/6NCr mice received a diet formulated with rapamycin (14 mg/kg food) or a control diet 2 days before and continuing for 16 weeks after exposure to 5 daily fractions of 6 Gy of thoracic irradiation. Fibrosis was assessed with Masson trichrome staining and hydroxyproline assay. Cytokine expression was evaluated by quantitative real-time polymerase chain reaction. Senescence was assessed by staining for β-galactosidase activity. Results: Administration of rapamycin extended the median survival of irradiated mice compared with the control diet from 116 days to 156 days (P=.006, log-rank test). Treatment with rapamycin reduced hydroxyproline content compared with the control diet (irradiation plus vehicle, 45.9 ± 11.8 μg per lung; irradiation plus rapamycin, 21.4 ± 6.0 μg per lung; P=.001) and reduced visible fibrotic foci. Rapamycin treatment attenuated interleukin 1β and transforming growth factor β induction in irradiated lungs compared with the control diet. Type II pneumocyte senescence after irradiation was reduced with rapamycin treatment at 16 weeks (3-fold reduction at 16 weeks, P<.001). Conclusions: Rapamycin protected against RIPF in a murine model. Rapamycin treatment reduced inflammatory cytokine expression, extracellular matrix production, and senescence in type II pneumocytes.

  1. Aspirin disrupts the mTOR-Raptor complex and potentiates the anti-cancer activities of sorafenib via mTORC1 inhibition.

    Science.gov (United States)

    Sun, Danni; Liu, Hongchun; Dai, Xiaoyang; Zheng, Xingling; Yan, Juan; Wei, Rongrui; Fu, Xuhong; Huang, Min; Shen, Aijun; Huang, Xun; Ding, Jian; Geng, Meiyu

    2017-10-10

    Aspirin is associated with a reduced risk of cancer and delayed progression of malignant disease. Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-mTOR signaling is believed to partially contribute to these anticancer effects, although the mechanism is unclear. In this study, we revealed the mechanism underlying the effects of aspirin on AMPK-mTOR signaling, and described a mechanism-based rationale for the use of aspirin in cancer therapy. We found that aspirin inhibited mTORC1 signaling through AMPK-dependent and -independent manners. Aspirin inhibited the AMPK-TSC pathway, thus resulting in the suppression of mTORC1 activity. In parallel, it directly disrupted the mTOR-raptor interaction. Additionally, the combination of aspirin and sorafenib showed synergetic effects via inhibiting mTORC1 signaling and the PI3K/AKT, MAPK/ERK pathways. Aspirin and sorafenib showed synergetic anticancer efficacy in the SMMC-7721 model. Our study provides mechanistic insights and a mechanism-based rationale for the roles of aspirin in cancer treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. mTOR up-regulation of PFKFB3 is essential for acute myeloid leukemia cell survival

    International Nuclear Information System (INIS)

    Feng, Yonghuai; Wu, Liusong

    2017-01-01

    Although mTOR (mammalian target of rapamycin) activation is frequently observed in acute myeloid leukemia (AML) patients, the precise function and the downstream targets of mTOR are poorly understood. Here we revealed that PFKFB3, but not PFKFB1, PFKFB2 nor PFKFB4 was a novel downstream substrate of mTOR signaling pathway as PFKFB3 level was augmented after knocking down TSC2 in THP1 and OCI-AML3 cells. Importantly, PFKFB3 silencing suppressed glycolysis and cell proliferation of TSC2 silencing OCI-AML3 cells and activated apoptosis pathway. These results suggested that mTOR up-regulation of PFKFB3 was essential for AML cells survival. Mechanistically, Rapamycin treatment or Raptor knockdown reduced the expression of PFKFB3 in TSC2 knockdown cells, while Rictor silencing did not have such effect. Furthermore, we also revealed that mTORC1 up-regulated PFKFB3 was dependent on hypoxia-inducible factor 1α (HIF1α), a positive regulator of glycolysis. Moreover, PFKFB3 inhibitor PFK15 and rapamycin synergistically blunted the AML cell proliferation. Taken together, PFKFB3 was a promising drug target in AML patients harboring mTOR hyper-activation.

  3. mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient.

    Science.gov (United States)

    Wyant, Gregory A; Abu-Remaileh, Monther; Wolfson, Rachel L; Chen, Walter W; Freinkman, Elizaveta; Danai, Laura V; Vander Heiden, Matthew G; Sabatini, David M

    2017-10-19

    The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Rapamycin delays growth of Wnt-1 tumors in spite of suppression of host immunity

    International Nuclear Information System (INIS)

    Svirshchevskaya, Elena V; Mariotti, Jacopo; Wright, Mollie H; Viskova, Natalia Y; Telford, William; Fowler, Daniel H; Varticovski, Lyuba

    2008-01-01

    Rapamycin, an inhibitor of mammalian target of Rapamycin (mTOR), is an immunosuppressive agent that has anti-proliferative effects on some tumors. However, the role of Rapamycin-induced immune suppression on tumor progression has not been examined. We developed a transplantation model for generation of mammary tumors in syngeneic recipients that can be used to address the role of the immune system on tumor progression. We examined the effect of Rapamycin on the immune system and growth of MMTV-driven Wnt-1 mammary tumors which were transplanted into irradiated and bone marrow-reconstituted, or naïve mice. Rapamycin induced severe immunosuppression and significantly delayed the growth of Wnt-1 tumors. T cell depletion in spleen and thymus and reduction in T cell cytokine secretion were evident within 7 days of therapy. By day 20, splenic but not thymic T cell counts, and cytokine secretion recovered. We determined whether adoptive T cell therapy enhances the anti-cancer effect using ex vivo generated Rapamycin-resistant T cells. However, T cell transfer during Rapamycin therapy did not improve the outcome relative to drug therapy alone. Thus, we could not confirm that suppression of T cell immunity contributes to tumor growth in this model. Consistent with suppression of the mTOR pathway, decreased 4E-BP1, p70 S6-kinase, and S6 protein phosphorylation correlated with a decrease in Wnt-1 tumor cell proliferation. Rapamycin has a direct anti-tumor effect on Wnt-1 breast cancer in vivo that involves inhibition of the mTOR pathway at doses that also suppress host immune responses

  5. Dysplasia and overgrowth. Magnetic resonance imaging of pediatric brain abnormalities secondary to alterations in the mechanistic target of rapamycin pathway

    International Nuclear Information System (INIS)

    Shrot, Shai; Hwang, Misun; Huisman, Thierry A.G.M.; Soares, Bruno P.; Stafstrom, Carl E.

    2018-01-01

    The current classification of malformations of cortical development is based on the type of disrupted embryological process (cell proliferation, migration, or cortical organization/post-migrational development) and the resulting morphological anomalous pattern of findings. An ideal classification would include knowledge of biological pathways. It has recently been demonstrated that alterations affecting the mechanistic target of rapamycin (mTOR) signaling pathway result in diverse abnormalities such as dysplastic megalencephaly, hemimegalencephaly, ganglioglioma, dysplastic cerebellar gangliocytoma, focal cortical dysplasia type IIb, and brain lesions associated with tuberous sclerosis. We review the neuroimaging findings in brain abnormalities related to alterations in the mTOR pathway, following the emerging trend from morphology towards genetics in the classification of malformations of cortical development. This approach improves the understanding of anomalous brain development and allows precise diagnosis and potentially targeted therapies that may regulate mTOR pathway function. (orig.)

  6. Dysplasia and overgrowth. Magnetic resonance imaging of pediatric brain abnormalities secondary to alterations in the mechanistic target of rapamycin pathway

    Energy Technology Data Exchange (ETDEWEB)

    Shrot, Shai [Johns Hopkins University School of Medicine, Division of Pediatric Radiology and Pediatric Neuroradiology, Russell H. Morgan Department of Radiology and Radiological Science, Baltimore, MD (United States); Sheba Medical Center, Department of Diagnostic Imaging, Ramat-Gan (Israel); Hwang, Misun; Huisman, Thierry A.G.M.; Soares, Bruno P. [Johns Hopkins University School of Medicine, Division of Pediatric Radiology and Pediatric Neuroradiology, Russell H. Morgan Department of Radiology and Radiological Science, Baltimore, MD (United States); Stafstrom, Carl E. [Johns Hopkins University School of Medicine, Division of Pediatric Neurology, Department of Neurology, Baltimore, MD (United States)

    2018-02-15

    The current classification of malformations of cortical development is based on the type of disrupted embryological process (cell proliferation, migration, or cortical organization/post-migrational development) and the resulting morphological anomalous pattern of findings. An ideal classification would include knowledge of biological pathways. It has recently been demonstrated that alterations affecting the mechanistic target of rapamycin (mTOR) signaling pathway result in diverse abnormalities such as dysplastic megalencephaly, hemimegalencephaly, ganglioglioma, dysplastic cerebellar gangliocytoma, focal cortical dysplasia type IIb, and brain lesions associated with tuberous sclerosis. We review the neuroimaging findings in brain abnormalities related to alterations in the mTOR pathway, following the emerging trend from morphology towards genetics in the classification of malformations of cortical development. This approach improves the understanding of anomalous brain development and allows precise diagnosis and potentially targeted therapies that may regulate mTOR pathway function. (orig.)

  7. Target of rapamycin complex 2 signals to downstream effector yeast protein kinase 2 (Ypk2) through adheres-voraciously-to-target-of-rapamycin-2 protein 1 (Avo1) in Saccharomyces cerevisiae.

    Science.gov (United States)

    Liao, Hsien-Ching; Chen, Mei-Yu

    2012-02-24

    The conserved Ser/Thr kinase target of rapamycin (TOR) serves as a central regulator in controlling cell growth-related functions. There exist two distinct TOR complexes, TORC1 and TORC2, each coupling to specific downstream effectors and signaling pathways. In Saccharomyces cerevisiae, TORC2 is involved in regulating actin organization and maintaining cell wall integrity. Ypk2 (yeast protein kinase 2), a member of the cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase family, is a TORC2 substrate known to participate in actin and cell wall regulation. Employing avo3(ts) mutants with defects in TORC2 functions that are suppressible by active Ypk2, we investigated the molecular interactions involved in mediating TORC2 signaling to Ypk2. GST pulldown assays in yeast lysates demonstrated physical interactions between Ypk2 and components of TORC2. In vitro binding assays revealed that Avo1 directly binds to Ypk2. In avo3(ts) mutants, the TORC2-Ypk2 interaction was reduced and could be restored by AVO1 overexpression, highlighting the important role of Avo1 in coupling TORC2 to Ypk2. The interaction was mapped to an internal region (amino acids 600-840) of Avo1 and a C-terminal region of Ypk2. Ypk2(334-677), a truncated form of Ypk2 containing the Avo1-interacting region, was able to interfere with Avo1-Ypk2 interaction in vitro. Overexpressing Ypk2(334-677) in yeast cells resulted in a perturbation of TORC2 functions, causing defective cell wall integrity, aberrant actin organization, and diminished TORC2-dependent Ypk2 phosphorylation evidenced by the loss of an electrophoretic mobility shift. Together, our data support the conclusion that the direct Avo1-Ypk2 interaction is crucial for TORC2 signaling to the downstream Ypk2 pathway.

  8. Leucine signaling in the pathogenesis of type 2 diabetes and obesity.

    Science.gov (United States)

    Melnik, Bodo C

    2012-03-15

    Epidemiological evidence points to increased dairy and meat consumption, staples of the Western diet, as major risk factors for the development of type 2 diabetes (T2D). This paper presents a new concept and comprehensive review of leucine-mediated cell signaling explaining the pathogenesis of T2D and obesity by leucine-induced over-stimulation of mammalian target of rapamycin complex 1 (mTORC1). mTORC1, a pivotal nutrient-sensitive kinase, promotes growth and cell proliferation in response to glucose, energy, growth factors and amino acids. Dairy proteins and meat stimulate insulin/insulin-like growth factor 1 signaling and provide high amounts of leucine, a primary and independent stimulator for mTORC1 activation. The downstream target of mTORC1, the kinase S6K1, induces insulin resistance by phosphorylation of insulin receptor substrate-1, thereby increasing the metabolic burden of β-cells. Moreover, leucine-mediated mTORC1-S6K1-signaling plays an important role in adipogenesis, thus increasing the risk of obesity-mediated insulin resistance. High consumption of leucine-rich proteins explains exaggerated mTORC1-dependent insulin secretion, increased β-cell growth and β-cell proliferation promoting an early onset of replicative β-cell senescence with subsequent β-cell apoptosis. Disturbances of β-cell mass regulation with increased β-cell proliferation and apoptosis as well as insulin resistance are hallmarks of T2D, which are all associated with hyperactivation of mTORC1. In contrast, the anti-diabetic drug metformin antagonizes leucine-mediated mTORC1 signaling. Plant-derived polyphenols and flavonoids are identified as natural inhibitors of mTORC1 and exert anti-diabetic and anti-obesity effects. Furthermore, bariatric surgery in obesity reduces increased plasma levels of leucine and other branched-chain amino acids. Attenuation of leucine-mediated mTORC1 signaling by defining appropriate upper limits of the daily intake of leucine-rich animal and dairy

  9. Enteroendocrine-derived glucagon-like peptide-2 controls intestinal amino acid transport.

    Science.gov (United States)

    Lee, Jennifer; Koehler, Jacqueline; Yusta, Bernardo; Bahrami, Jasmine; Matthews, Dianne; Rafii, Mahroukh; Pencharz, Paul B; Drucker, Daniel J

    2017-03-01

    Glucagon-like peptide-2 (GLP-2) is co-secreted with GLP-1 from gut endocrine cells, and both peptides act as growth factors to expand the surface area of the mucosal epithelium. Notably, GLP-2 also enhances glucose and lipid transport in enterocytes; however, its actions on control of amino acid (AA) transport remain unclear. Here we examined the mechanisms linking gain and loss of GLP-2 receptor (GLP-2R) signaling to control of intestinal amino acid absorption in mice. Absorption, transport, and clearance of essential AAs, specifically lysine, were measured in vivo by Liquid Chromatography triple quadrupole Mass Spectrometry (LC-MS/MS) and ex vivo with Ussing chambers using intestinal preparations from Glp2 r +/+ and Glp2r - / - mice. Immunoblotting determined jejunal levels of protein components of signaling pathways (PI3K-AKT, and mTORC1-pS6-p4E-BP1) following administration of GLP-2, protein gavage, and rapamycin to fasted Glp2 r +/+ and Glp2r - / - mice. Expression of AA transporters from full thickness jejunum and 4F2hc from brush border membrane vesicles (BBMVs) was measured by real-time PCR and immunoblotting, respectively. Acute administration of GLP-2 increased basal AA absorption in vivo and augmented basal lysine transport ex vivo . GLP-2-stimulated lysine transport was attenuated by co-incubation with wortmannin, rapamycin, or tetrodotoxin ex vivo . Phosphorylation of mTORC1 effector proteins S6 and 4E-BP1 was significantly increased in wild-type mice in response to GLP-2 alone, or when co-administered with protein gavage, and abolished following oral gavage of rapamycin. In contrast, activation of GLP-1R signaling did not enhance S6 phosphorylation. Disruption of GLP-2 action in Glp2r -/- mice reduced lysine transport ex vivo and attenuated the phosphorylation of S6 and 4E-BP1 in response to oral protein. Moreover, the expression of cationic AA transporter slc7a9 in response to refeeding, and the abundance of 4F2hc in BBMVs following protein

  10. Target of Rapamycin (TOR) Regulates Growth in Response to Nutritional Signals.

    Science.gov (United States)

    Weisman, Ronit

    2016-10-01

    All organisms can respond to the availability of nutrients by regulating their metabolism, growth, and cell division. Central to the regulation of growth in response to nutrient availability is the target of rapamycin (TOR) signaling that is composed of two structurally distinct complexes: TOR complex 1 (TORC1) and TOR complex 2 (TORC2). The TOR genes were first identified in yeast as target of rapamycin, a natural product of a soil bacterium, which proved beneficial as an immunosuppressive and anticancer drug and is currently being tested for a handful of other pathological conditions including diabetes, neurodegeneration, and age-related diseases. Studies of the TOR pathway unraveled a complex growth-regulating network. TOR regulates nutrient uptake, transcription, protein synthesis and degradation, as well as metabolic pathways, in a coordinated manner that ensures that cells grow or cease growth in response to nutrient availability. The identification of specific signals and mechanisms that stimulate TOR signaling is an active and exciting field of research that has already identified nitrogen and amino acids as key regulators of TORC1 activity. The signals, as well as the cellular functions of TORC2, are far less well understood. Additional open questions in the field concern the relationships between TORC1 and TORC2, as well as the links with other nutrient-responsive pathways. Here I review the main features of TORC1 and TORC2, with a particular focus on yeasts as model organisms.

  11. Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer.

    Science.gov (United States)

    Ribas, Ricardo; Pancholi, Sunil; Rani, Aradhana; Schuster, Eugene; Guest, Stephanie K; Nikitorowicz-Buniak, Joanna; Simigdala, Nikiana; Thornhill, Allan; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Dowsett, Mitch; Johnston, Stephen R; Martin, Lesley-Ann

    2018-06-08

    Endocrine therapies are the mainstay of treatment for oestrogen receptor (ER)-positive (ER + ) breast cancer (BC). However, resistance remains problematic largely due to enhanced cross-talk between ER and growth factor pathways, circumventing the need for steroid hormones. Previously, we reported the anti-proliferative effect of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models; however, potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB, and mTORC1) may provide enhanced long-term clinical utility. A panel of ER + BC cell lines adapted to long-term oestrogen deprivation (LTED) and expressing ESR1 wt or ESR1 Y537S , modelling acquired resistance to an aromatase-inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan-ERBB inhibitor) in the presence or absence of oestradiol (E2), tamoxifen (4-OHT), or fulvestrant (ICI182780). End points included proliferation, cell signalling, cell cycle, and effect on ER-mediated transactivation. An in-vivo model of AI resistance was treated with monotherapies and combinations to assess the efficacy in delaying tumour progression. RNA-seq analysis was performed to identify changes in global gene expression as a result of the indicated therapies. Here, we show RAD001 and neratinib (pan-ERBB inhibitor) caused a concentration-dependent decrease in proliferation, irrespective of the ESR1 mutation status. The combination of either agent with endocrine therapy further reduced proliferation but the maximum effect was observed with a triple combination of RAD001, neratinib, and endocrine therapy. In the absence of oestrogen, RAD001 caused a reduction in ER-mediated transcription in the majority of the cell lines, which associated with a decrease in recruitment of ER to an oestrogen-response element on the TFF1 promoter. Contrastingly, neratinib increased both ER

  12. The ABA-INSENSITIVE-4 (ABI4) transcription factor links redox, hormone and sugar signaling pathways.

    Science.gov (United States)

    Foyer, Christine H; Kerchev, Pavel I; Hancock, Robert D

    2012-02-01

    The cellular reduction-oxidation (redox) hub processes information from metabolism and the environment and so regulates plant growth and defense through integration with the hormone signaling network. One key pathway of redox control involves interactions with ABSCISIC ACID (ABA). Accumulating evidence suggests that the ABA-INSENSITIVE-4 (ABI4) transcription factor plays a key role in transmitting information concerning the abundance of ascorbate and hence the ability of cells to buffer oxidative challenges. ABI4 is required for the ascorbate-dependent control of growth, a process that involves enhancement of salicylic acid (SA) signaling and inhibition of jasmonic acid (JA) signaling pathways. Low redox buffering capacity reinforces SA- JA- interactions through the mediation of ABA and ABI4 to fine-tune plant growth and defense in relation to metabolic cues and environmental challenges. Moreover, ABI4-mediated pathways of sugar sensitivity are also responsive to the abundance of ascorbate, providing evidence of overlap between redox and sugar signaling pathways.

  13. Distinct roles of Rheb and Raptor in activating mTOR complex 1 for the self-renewal of hematopoietic stem cells.

    Science.gov (United States)

    Peng, Hui; Kasada, Atsuo; Ueno, Masaya; Hoshii, Takayuki; Tadokoro, Yuko; Nomura, Naho; Ito, Chiaki; Takase, Yusuke; Vu, Ha Thi; Kobayashi, Masahiko; Xiao, Bo; Worley, Paul F; Hirao, Atsushi

    2018-01-01

    The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) senses a cell's energy status and environmental levels of nutrients and growth factors. In response, mTORC1 mediates signaling that controls protein translation and cellular metabolism. Although mTORC1 plays a critical role in hematopoiesis, it remains unclear which upstream stimuli regulate mTORC1 activity in the context of hematopoietic stem cells (HSC) maintenance in vivo. In this study, we investigated the function of Rheb, a critical regulator of mTORC1 activity controlled by the PI3K-AKT-TSC axis, both in HSC maintenance in mice at steady-state and in HSC-derived hematopoiesis post-transplantation. In contrast to the severe hematopoietic dysfunction caused by Raptor deletion, which completely inactivates mTORC1, Rheb deficiency in adult mice did not show remarkable hematopoietic failure. Lack of Rheb caused abnormalities in myeloid cells but did not have impact on hematopoietic regeneration in mice subjected to injury by irradiation. As previously reported, Rheb deficiency resulted in defective HSC-derived hematopoiesis post-transplantation. However, while Raptor is essential for HSC competitiveness in vivo, Rheb is dispensable for HSC maintenance under physiological conditions, indicating that the PI3K-AKT-TSC pathway does not contribute to mTORC1 activity for sustaining HSC self-renewal activity at steady-state. Thus, the various regulatory elements that impinge upstream of mTORC1 activation pathways are differentially required for HSC homeostasis in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Effects of CD44 Ligation on Signaling and Metabolic Pathways in Acute Myeloid Leukemia

    KAUST Repository

    Madhoun, Nour Y.

    2017-04-01

    Acute myeloid leukemia (AML) is characterized by a blockage in the differentiation of myeloid cells at different stages. CD44-ligation using anti-CD44 monoclonal antibodies (mAbs) has been shown to reverse the blockage of differentiation and to inhibit the proliferation of blasts in most AML-subtypes. However, the molecular mechanisms underlying this property have not been fully elucidated. Here, we sought to I) analyze the effects of anti-CD44 mAbs on downstream signaling pathways, including the ERK1/2 (extracellular signal-regulated kinase 1 and 2) and mTOR (mammalian target of rapamycin) pathways and II) use state-of-the-art Nuclear Magnetic Resonance (NMR) technology to determine the global metabolic changes during differentiation induction of AML cells using anti-CD44 mAbs and other two previously reported differentiation agents. In the first objective (Chapter 4), our studies provide evidence that CD44-ligation with specific mAbs in AML cells induced an increase in ERK1/2 phosphorylation. The use of the MEK inhibitor (U0126) significantly inhibited the CD44-induced differentiation of HL60 cells, suggesting that ERK1/2 is critical for the CD44-triggered differentiation in AML. In addition, this was accompanied by a marked decrease in the phosphorylation of the mTORC1 and mTORC2 complexes, which are strongly correlated with the inhibition of the PI3K/Akt pathway. In the second objective (Chapter 5), 1H NMR experiments demonstrated that considerable changes in the metabolic profiles of HL60 cells were induced in response to each differentiation agent. These most notable metabolites that significantly changed upon CD44 ligation were involved in the tricarboxylic acid (TCA) cycle and glycolysis such as, succinate, fumarate and lactate. Therefore, we sought to analyze the mechanisms underlying their alterations. Our results revealed that anti-CD44 mAbs treatment induced upregulation in fumarate hydratase (FH) expression and its activity which was accompanied by a

  15. Phosphatidylcholine Transfer Protein Interacts with Thioesterase Superfamily Member 2 to Attenuate Insulin Signaling

    OpenAIRE

    Ersoy, Baran A.; Tarun, Akansha; D’Aquino, Katharine; Hancer, Nancy J.; Ukomadu, Chinweike; White, Morris F.; Michel, Thomas; Manning, Brendan D.; Cohen, David E.

    2013-01-01

    Phosphatidylcholine transfer protein (PC-TP) is a phospholipid-binding protein that is enriched in liver and that interacts with thioesterase superfamily member 2 (THEM2). Mice lacking either protein exhibit improved hepatic glucose homeostasis and are resistant to diet-induced diabetes. Insulin receptor substrate 2 (IRS2) and mammalian target of rapamycin complex 1 (mTORC1) are key effectors of insulin signaling, which is attenuated in diabetes. We found that PC-TP inhibited IRS2, as evidenc...

  16. Transmembrane Inhibitor of RICTOR/mTORC2 in Hematopoietic Progenitors

    Directory of Open Access Journals (Sweden)

    Dongjun Lee

    2014-11-01

    Full Text Available Central to cellular proliferative, survival, and metabolic responses is the serine/threonine kinase mTOR, which is activated in many human cancers. mTOR is present in distinct complexes that are either modulated by AKT (mTORC1 or are upstream and regulatory of it (mTORC2. Governance of mTORC2 activity is poorly understood. Here, we report a transmembrane molecule in hematopoietic progenitor cells that physically interacts with and inhibits RICTOR, an essential component of mTORC2. Upstream of mTORC2 (UT2 negatively regulates mTORC2 enzymatic activity, reducing AKTS473, PKCα, and NDRG1 phosphorylation and increasing FOXO transcriptional activity in an mTORC2-dependent manner. Modulating UT2 levels altered animal survival in a T cell acute lymphoid leukemia (T-ALL model that is known to be mTORC2 sensitive. These studies identify an inhibitory component upstream of mTORC2 in hematopoietic cells that can reduce mortality from NOTCH-induced T-ALL. A transmembrane inhibitor of mTORC2 may provide an attractive target to affect this critical cell regulatory pathway.

  17. Mammalian target of rapamycin complex 2 regulates muscle glucose uptake during exercise in mice

    DEFF Research Database (Denmark)

    Kleinert, Maximilian; Parker, Benjamin L; Fritzen, Andreas Mæchel

    2017-01-01

    Exercise increases glucose uptake into insulin-resistant muscle. Thus, elucidating the exercise signalling network in muscle may uncover new therapeutic targets. mTORC2, a regulator of insulin-controlled glucose uptake, has been reported to interact with Rac1, which plays a role in exercise-induc...

  18. Gβγ interacts with mTOR and promotes its activation

    Energy Technology Data Exchange (ETDEWEB)

    Robles-Molina, Evelyn [Department of Pharmacology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Apartado postal 14-740, México, D.F. 07360 (Mexico); Dionisio-Vicuña, Misael [Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Apartado postal 14-740, México, D.F. 07360 (Mexico); Guzmán-Hernández, María Luisa [Department of Pharmacology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Apartado postal 14-740, México, D.F. 07360 (Mexico); Reyes-Cruz, Guadalupe [Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Apartado postal 14-740, México, D.F. 07360 (Mexico); Vázquez-Prado, José, E-mail: jvazquez@cinvestav.mx [Department of Pharmacology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Apartado postal 14-740, México, D.F. 07360 (Mexico)

    2014-02-07

    Highlights: • Gβγ interacts with mTOR kinase domain via a mechanism sensitive to chronic treatment with rapamycin. • Gβγ interacts with mTORC1 and mTORC2 which correlates with its ability to promote mTORC1 and mTORC2 signaling. • Gβγ heterodimers containing different Gβ subunits, except Gβ{sub 4}, interact with mTOR. - Abstract: Diverse G protein-coupled receptors depend on Gβγ heterodimers to promote cell polarization and survival via direct activation of PI3Kγ and potentially other effectors. These events involve full activation of AKT via its phosphorylation at Ser473, suggesting that mTORC2, the kinase that phosphorylates AKT at Ser473, is activated downstream of Gβγ. Thus, we tested the hypothesis that Gβγ directly contributes to mTOR signaling. Here, we demonstrate that endogenous mTOR interacts with Gβγ. Cell stimulation with serum modulates Gβγ interaction with mTOR. The carboxyl terminal region of mTOR, expressed as a GST-fusion protein, including the serine/threonine kinase domain, binds Gβγ heterodimers containing different Gβ subunits, except Gβ{sub 4}. Both, mTORC1 and mTORC2 complexes interact with Gβ{sub 1}γ{sub 2} which promotes phosphorylation of their respective substrates, p70S6K and AKT. In addition, chronic treatment with rapamycin, a condition known to interfere with assembly of mTORC2, reduces the interaction between Gβγ and mTOR and the phosphorylation of AKT; whereas overexpression of Gαi interfered with the effect of Gβγ as promoter of p70S6K and AKT phosphorylation. Altogether, our results suggest that Gβγ positively regulates mTOR signaling via direct interactions and provide further support to emerging strategies based on the therapeutical potential of inhibiting different Gβγ signaling interfaces.

  19. Synthesis of I-125 labeled photoaffinity rapamycin analogs

    International Nuclear Information System (INIS)

    Shu, A.Y.L.; Yamashita, D.S.; Holt, D.A.; Heys, J.R.

    1996-01-01

    Two no-carrier-added 125 I-labelled photoaffinity rapamycin analogs were prepared: 7-demethoxy-7-(4-azido-3- 125 I-benzyloxy) rapamycin and its C 28 -C 29 seco analog. The key reactions of the synthesis were substitution of the C 7 methoxyl of rapamycin with 4-azido-3-tributylstannylbenzyloxy group, exchange of tributyltin with 125 I using Na 125 I and Chloramine-T, and a ZnCl 2 mediated retro-Aldol cleavage of the C 28 -C 29 bond of rapamycin. (author)

  20. TNF-α promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    International Nuclear Information System (INIS)

    Wang, Cheng-hu; Cao, Guo-Fan; Jiang, Qin; Yao, Jin

    2012-01-01

    Highlights: ► TNF-α induces MMP-9 expression and secretion to promote RPE cell migration. ► MAPK activation is not critical for TNF-α-induced MMP-9 expression. ► Akt and mTORC1 signaling mediate TNF-α-induced MMP-9 expression. ► SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-α. -- Abstract: Tumor necrosis factor-alpha (TNF-α) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-α promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-α-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-α-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-α promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  1. TNF-{alpha} promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Cheng-hu; Cao, Guo-Fan [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Jiang, Qin, E-mail: Jqin710@vip.sina.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Yao, Jin, E-mail: dryaojin@yahoo.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  2. TORC1 Inhibition by Rapamycin Promotes Antioxidant Defences in a Drosophila Model of Friedreich's Ataxia.

    Directory of Open Access Journals (Sweden)

    Pablo Calap-Quintana

    Full Text Available Friedreich's ataxia (FRDA, the most common inherited ataxia in the Caucasian population, is a multisystemic disease caused by a significant decrease in the frataxin level. To identify genes capable of modifying the severity of the symptoms of frataxin depletion, we performed a candidate genetic screen in a Drosophila RNAi-based model of FRDA. We found that genetic reduction in TOR Complex 1 (TORC1 signalling improves the impaired motor performance phenotype of FRDA model flies. Pharmacologic inhibition of TORC1 signalling by rapamycin also restored this phenotype and increased the lifespan and ATP levels. Furthermore, rapamycin reduced the altered levels of malondialdehyde + 4-hydroxyalkenals and total glutathione of the model flies. The rapamycin-mediated protection against oxidative stress is due in part to an increase in the transcription of antioxidant genes mediated by cap-n-collar (Drosophila ortholog of Nrf2. Our results suggest that autophagy is indeed necessary for the protective effect of rapamycin in hyperoxia. Rapamycin increased the survival and aconitase activity of model flies subjected to high oxidative insult, and this improvement was abolished by the autophagy inhibitor 3-methyladenine. These results point to the TORC1 pathway as a new potential therapeutic target for FRDA and as a guide to finding new promising molecules for disease treatment.

  3. Inhibition of Akt enhances the chemopreventive effects of topical rapamycin in mouse skin

    Science.gov (United States)

    Dickinson, Sally E; Janda, Jaroslav; Criswell, Jane; Blohm-Mangone, Karen; Olson, Erik R.; Liu, Zhonglin; Barber, Christie; Rusche, Jadrian J.; Petricoin, Emmanuel; Calvert, Valerie; Einspahr, Janine G.; Dickinson, Jesse; Stratton, Steven P.; Curiel-Lewandrowski, Clara; Saboda, Kathylynn; Hu, Chengcheng; Bode, Ann M.; Dong, Zigang; Alberts, David S.; Bowden, G. Timothy

    2016-01-01

    The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced non-melanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin, (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared to those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here we explored the use of topical rapamycin as a chemopreventive agent in the context of solar simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared to controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared to vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC.

  4. Biological constraints limit the use of rapamycin-inducible FKBP12-Inp54p for depleting PIP2 in dorsal root ganglia neurons.

    Science.gov (United States)

    Coutinho-Budd, Jaeda C; Snider, Samuel B; Fitzpatrick, Brendan J; Rittiner, Joseph E; Zylka, Mark J

    2013-09-08

    Rapamycin-induced translocation systems can be used to manipulate biological processes with precise temporal control. These systems are based on rapamycin-induced dimerization of FK506 Binding Protein 12 (FKBP12) with the FKBP Rapamycin Binding (FRB) domain of mammalian target of rapamycin (mTOR). Here, we sought to adapt a rapamycin-inducible phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphatase (Inp54p) system to deplete PIP2 in nociceptive dorsal root ganglia (DRG) neurons. We genetically targeted membrane-tethered CFP-FRBPLF (a destabilized FRB mutant) to the ubiquitously expressed Rosa26 locus, generating a Rosa26-FRBPLF knockin mouse. In a second knockin mouse line, we targeted Venus-FKBP12-Inp54p to the Calcitonin gene-related peptide-alpha (CGRPα) locus. We hypothesized that after intercrossing these mice, rapamycin treatment would induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in CGRP+ DRG neurons. In control experiments with cell lines, rapamycin induced translocation of Venus-FKBP12-Inp54p to the plasma membrane, and subsequent depletion of PIP2, as measured with a PIP2 biosensor. However, rapamycin did not induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in FRBPLF-expressing DRG neurons (in vitro or in vivo). Moreover, rapamycin treatment did not alter PIP2-dependent thermosensation in vivo. Instead, rapamycin treatment stabilized FRBPLF in cultured DRG neurons, suggesting that rapamycin promoted dimerization of FRBPLF with endogenous FKBP12. Taken together, our data indicate that these knockin mice cannot be used to inducibly deplete PIP2 in DRG neurons. Moreover, our data suggest that high levels of endogenous FKBP12 could compete for binding to FRBPLF, hence limiting the use of rapamycin-inducible systems to cells with low levels of endogenous FKBP12.

  5. Comment on "A dynamic network model of mTOR signaling reveals TSC-independent mTORC2 regulation": building a model of the mTOR signaling network with a potentially faulty tool.

    Science.gov (United States)

    Manning, Brendan D

    2012-07-10

    In their study published in Science Signaling (Research Article, 27 March 2012, DOI: 10.1126/scisignal.2002469), Dalle Pezze et al. tackle the dynamic and complex wiring of the signaling network involving the protein kinase mTOR, which exists within two distinct protein complexes (mTORC1 and mTORC2) that differ in their regulation and function. The authors use a combination of immunoblotting for specific phosphorylation events and computational modeling. The primary experimental tool employed is to monitor the autophosphorylation of mTOR on Ser(2481) in cell lysates as a surrogate for mTOR activity, which the authors conclude is a specific readout for mTORC2. However, Ser(2481) phosphorylation occurs on both mTORC1 and mTORC2 and will dynamically change as the network through which these two complexes are connected is manipulated. Therefore, models of mTOR network regulation built using this tool are inherently imperfect and open to alternative explanations. Specific issues with the main conclusion made in this study, involving the TSC1-TSC2 (tuberous sclerosis complex 1 and 2) complex and its potential regulation of mTORC2, are discussed here. A broader goal of this Letter is to clarify to other investigators the caveats of using mTOR Ser(2481) phosphorylation in cell lysates as a specific readout for either of the two mTOR complexes.

  6. The effect of the immunophilin ligands rapamycin and FK506 on proliferation of mast cells and other hematopoietic cell lines.

    Science.gov (United States)

    Hultsch, T; Martin, R; Hohman, R J

    1992-01-01

    The immunosuppressive drugs FK506 and cyclosporin A have an identical spectrum of activities with respect to IgE receptor (Fc epsilon RI)-mediated exocytosis from mast cells and T cell receptor-mediated transcription of IL-2. These findings suggest a common step in receptor-mediated signal transduction leading to exocytosis and transcription and imply that immunosuppressive drugs target specific signal transduction pathways, rather than specific cell types. This hypothesis is supported by studies on the effect of rapamycin on IL-3 dependent proliferation of the rodent mast cell line PT18. Rapamycin inhibits proliferation of PT18 cells, achieving a plateau of 80% inhibition at 1 nM. This inhibition is prevented in a competitive manner by FK506, a structural analogue of rapamycin. Proliferation of rat basophilic leukemia cells and WEHI-3 cells was also inhibited, at doses comparable to those shown previously to inhibit IL-2-dependent proliferation of cytotoxic T lymphocyte line (CTLL) cells. In contrast, proliferation of A-431 cells, a epidermoid cell line, was not affected by rapamycin. DNA histograms indicate that complexes formed between the rapamycin-FK506-binding protein (FKBP) and rapamycin arrest-proliferating PT18 cells in the G0/G1-phase. It is concluded that FKBP-rapamycin complexes may inhibit proliferative signals emanating from IL-3 receptors, resulting in growth arrest of cytokine-dependent, hematopoietic cells. PMID:1384815

  7. Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

    International Nuclear Information System (INIS)

    Zhang, Pengpeng; Shan, Tizhong; Liang, Xinrong; Deng, Changyan; Kuang, Shihuan

    2014-01-01

    Highlights: • mTOR is a critical regulator of many biological processes yet its function in heart is not well understood. • MCK-Cre/Mtor flox/flox mice were established to delete Mtor in cardiomyocytes. • The mTOR-mKO mice developed normally but die prematurely within 5 weeks after birth due to heart disease. • The mTOR-mKO mice had dilated myocardium and increased cell death. • mTOR-mKO hearts had reduced expression of metabolic genes and activation of mTOR target proteins. - Abstract: Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor flox/flox mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function

  8. Biphasic Rapamycin Effects in Lymphoma and Carcinoma Treatment.

    Science.gov (United States)

    Liu, Yang; Pandeswara, Srilakshmi; Dao, Vinh; Padrón, Álvaro; Drerup, Justin M; Lao, Shunhua; Liu, Aijie; Hurez, Vincent; Curiel, Tyler J

    2017-01-15

    mTOR drives tumor growth but also supports T-cell function, rendering the applications of mTOR inhibitors complex especially in T-cell malignancies. Here, we studied the effects of the mTOR inhibitor rapamycin in mouse EL4 T-cell lymphoma. Typical pharmacologic rapamycin (1-8 mg/kg) significantly reduced tumor burden via direct suppression of tumor cell proliferation and improved survival in EL4 challenge independent of antitumor immunity. Denileukin diftitox (DD)-mediated depletion of regulatory T cells significantly slowed EL4 growth in vivo in a T-cell-dependent fashion. However, typical rapamycin inhibited T-cell activation and tumor infiltration in vivo and failed to boost DD treatment effects. Low-dose (LD) rapamycin (75 μg/kg) increased potentially beneficial CD44hiCD62L + CD8 + central memory T cells in EL4 challenge, but without clinical benefit. LD rapamycin significantly enhanced DD treatment efficacy, but DD plus LD rapamycin treatment effects were independent of antitumor immunity. Instead, rapamycin upregulated EL4 IL2 receptor in vitro and in vivo, facilitating direct DD tumor cell killing. LD rapamycin augmented DD efficacy against B16 melanoma and a human B-cell lymphoma, but not against human Jurkat T-cell lymphoma or ID8agg ovarian cancer cells. Treatment effects correlated with IL2R expression, but mechanisms in some tumors were not fully defined. Overall, our data define a distinct, biphasic mechanisms of action of mTOR inhibition at doses that are clinically exploitable, including in T-cell lymphomas. Cancer Res; 77(2); 520-31. ©2016 AACR. ©2016 American Association for Cancer Research.

  9. Nuclear PIM1 confers resistance to rapamycin-impaired endothelial proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Walpen, Thomas; Kalus, Ina [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Schwaller, Juerg [Department of Biomedicine, University of Basel, 4031 Basel (Switzerland); Peier, Martin A. [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Battegay, Edouard J. [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), 8057 Zuerich (Switzerland); Humar, Rok, E-mail: Rok.Humar@usz.ch [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), 8057 Zuerich (Switzerland)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer Pim1{sup -/-} endothelial cell proliferation displays increased sensitivity to rapamycin. Black-Right-Pointing-Pointer mTOR inhibition by rapamycin enhances PIM1 cytosolic and nuclear protein levels. Black-Right-Pointing-Pointer Truncation of Pim1 beyond serine 276 results in nuclear localization of the kinase. Black-Right-Pointing-Pointer Nuclear PIM1 increases endothelial proliferation independent of rapamycin. -- Abstract: The PIM serine/threonine kinases and the mTOR/AKT pathway integrate growth factor signaling and promote cell proliferation and survival. They both share phosphorylation targets and have overlapping functions, which can partially substitute for each other. In cancer cells PIM kinases have been reported to produce resistance to mTOR inhibition by rapamycin. Tumor growth depends highly on blood vessel infiltration into the malignant tissue and therefore on endothelial cell proliferation. We therefore investigated how the PIM1 kinase modulates growth inhibitory effects of rapamycin in mouse aortic endothelial cells (MAEC). We found that proliferation of MAEC lacking Pim1 was significantly more sensitive to rapamycin inhibition, compared to wildtype cells. Inhibition of mTOR and AKT in normal MAEC resulted in significantly elevated PIM1 protein levels in the cytosol and in the nucleus. We observed that truncation of the C-terminal part of Pim1 beyond Ser 276 resulted in almost exclusive nuclear localization of the protein. Re-expression of this Pim1 deletion mutant significantly increased the proliferation of Pim1{sup -/-} cells when compared to expression of the wildtype Pim1 cDNA. Finally, overexpression of the nuclear localization mutant and the wildtype Pim1 resulted in complete resistance to growth inhibition by rapamycin. Thus, mTOR inhibition-induced nuclear accumulation of PIM1 or expression of a nuclear C-terminal PIM1 truncation mutant is sufficient to increase endothelial cell proliferation

  10. The rapamycin-binding domain of the protein kinase mammalian target of rapamycin is a destabilizing domain.

    Science.gov (United States)

    Edwards, Sarah R; Wandless, Thomas J

    2007-05-04

    Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to 10-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retained the ability to inhibit mTOR, although with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wild-type FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems.

  11. Beneficial role of rapamycin in experimental autoimmune myositis.

    Directory of Open Access Journals (Sweden)

    Nicolas Prevel

    Full Text Available We developed an experimental autoimmune myositis (EAM mouse model of polymyositis where we outlined the role of regulatory T (Treg cells. Rapamycin, this immunosuppressant drug used to prevent rejection in organ transplantation, is known to spare Treg. Our aim was to test the efficacy of rapamycin in vivo in this EAM model and to investigate the effects of the drug on different immune cell sub-populations.EAM is induced by 3 injections of myosin emulsified in CFA. Mice received rapamycin during 25 days starting one day before myosin immunization (preventive treatment, or during 10 days following the last myosin immunization (curative treatment.Under preventive or curative treatment, an increase of muscle strength was observed with a parallel decrease of muscle inflammation, both being well correlated (R(2 = -0.645, p<0.0001. Rapamycin induced a general decrease in muscle of CD4 and CD8 T cells in lymphoid tissues, but spared B cells. Among T cells, the frequency of Treg was increased in rapamycin treated mice in draining lymph nodes (16.9 ± 2.2% vs. 9.3 ± 1.4%, p<0.001, which were mostly activated regulatory T cells (CD62L(lowCD44(high: 58.1 ± 5.78% vs. 33.1 ± 7%, treated vs. untreated, p<0.001. In rapamycin treated mice, inhibition of proliferation (Ki-67(+ is more important in effector T cells compared to Tregs cells (p<0.05. Furthermore, during preventive treatment, rapamycin increased the levels of KLF2 transcript in CD44(low CD62L(high naive T cell and in CD62L(low CD44(high activated T cell.Rapamycin showed efficacy both as curative and preventive treatment in our murine model of experimental myositis, in which it induced an increase of muscle strength with a parallel decrease in muscle inflammation. Rapamycin administration was also associated with a decrease in the frequency of effector T cells, an increase in Tregs, and, when administered as preventive treatment, an upregulation of KFL2 in naive and activated T cells.

  12. mTORC1-independent reduction of retinal protein synthesis in type 1 diabetes.

    Science.gov (United States)

    Fort, Patrice E; Losiewicz, Mandy K; Pennathur, Subramaniam; Jefferson, Leonard S; Kimball, Scot R; Abcouwer, Steven F; Gardner, Thomas W

    2014-09-01

    Poorly controlled diabetes has long been known as a catabolic disorder with profound loss of muscle and fat body mass resulting from a simultaneous reduction in protein synthesis and enhanced protein degradation. By contrast, retinal structure is largely maintained during diabetes despite reduced Akt activity and increased rate of cell death. Therefore, we hypothesized that retinal protein turnover is regulated differently than in other insulin-sensitive tissues, such as skeletal muscle. Ins2(Akita) diabetic mice and streptozotocin-induced diabetic rats exhibited marked reductions in retinal protein synthesis matched by a concomitant reduction in retinal protein degradation associated with preserved retinal mass and protein content. The reduction in protein synthesis depended on both hyperglycemia and insulin deficiency, but protein degradation was only reversed by normalization of hyperglycemia. The reduction in protein synthesis was associated with diminished protein translation efficiency but, surprisingly, not with reduced activity of the mTORC1/S6K1/4E-BP1 pathway. Instead, diabetes induced a specific reduction of mTORC2 complex activity. These findings reveal distinctive responses of diabetes-induced retinal protein turnover compared with muscle and liver that may provide a new means to ameliorate diabetic retinopathy. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  13. Bone growth during rapamycin therapy in young rats

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    He Yu-Zhu

    2009-01-01

    Full Text Available Abstract Background Rapamycin is an effective immunosuppressant widely used to maintain the renal allograft in pediatric patients. Linear growth may be adversely affected in young children since rapamycin has potent anti-proliferative and anti-angiogenic properties. Methods Weanling three week old rats were given rapamycin at 2.5 mg/kg daily by gavage for 2 or 4 weeks and compared to a Control group given equivalent amount of saline. Morphometric measurements and biochemical determinations for serum calcium, phosphate, iPTH, urea nitrogen, creatinine and insulin-growth factor I (IGF-I were obtained. Histomorphometric analysis of the growth plate cartilage, in-situ hybridization experiments and immunohistochemical studies for various proteins were performed to evaluate for chondrocyte proliferation, chondrocyte differentiation and chondro/osteoclastic resorption. Results At the end of the 2 weeks, body and tibia length measurements were shorter after rapamycin therapy associated with an enlargement of the hypertrophic zone in the growth plate cartilage. There was a decrease in chondrocyte proliferation assessed by histone-4 and mammalian target of rapamycin (mTOR expression. A reduction in parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP and an increase in Indian hedgehog (Ihh expression may explain in part, the increase number of hypertrophic chondrocytes. The number of TRAP positive multinucleated chondro/osteoclasts declined in the chondro-osseous junction with a decrease in the receptor activator of nuclear factor kappa β ligand (RANKL and vascular endothelial growth factor (VEGF expression. Although body and tibial length remained short after 4 weeks of rapamycin, changes in the expression of chondrocyte proliferation, chondrocyte differentiation and chondro/osteoclastic resorption which were significant after 2 weeks of rapamycin improved at the end of 4 weeks. Conclusion When given to young rats, 2 weeks of rapamycin

  14. Suppression of AKT phosphorylation restores rapamycin-based synthetic lethality in SMAD4-defective pancreatic cancer cells.

    Science.gov (United States)

    Le Gendre, Onica; Sookdeo, Ayisha; Duliepre, Stephie-Anne; Utter, Matthew; Frias, Maria; Foster, David A

    2013-05-01

    mTOR has been implicated in survival signals for many human cancers. Rapamycin and TGF-β synergistically induce G1 cell-cycle arrest in several cell lines with intact TGF-β signaling pathway, which protects cells from the apoptotic effects of rapamycin during S-phase of the cell cycle. Thus, rapamycin is cytostatic in the presence of serum/TGF-β and cytotoxic in the absence of serum. However, if TGF-β signaling is defective, rapamycin induced apoptosis in both the presence and absence of serum/TGF-β in colon and breast cancer cell lines. Because genetic dysregulation of TGF-β signaling is commonly observed in pancreatic cancers-with defects in the Smad4 gene being most prevalent, we hypothesized that pancreatic cancers would display a synthetic lethality to rapamycin in the presence of serum/TGF-β. We report here that Smad4-deficient pancreatic cancer cells are killed by rapamycin in the absence of serum; however, in the presence of serum, we did not observe the predicted synthetic lethality with rapamycin. Rapamycin also induced elevated phosphorylation of the survival kinase Akt at Ser473. Suppression of rapamycin-induced Akt phosphorylation restored rapamycin sensitivity in Smad4-null, but not Smad4 wild-type pancreatic cancer cells. This study shows that the synthetic lethality to rapamycin in pancreatic cancers with defective TGF-β signaling is masked by rapamycin-induced increases in Akt phosphorylation. The implication is that a combination of approaches that suppress both Akt phosphorylation and mTOR could be effective in targeting pancreatic cancers with defective TGF-β signaling. ©2013 AACR.

  15. mTOR complex 1: a key player in neuroadaptations induced by drugs of abuse.

    Science.gov (United States)

    Neasta, Jeremie; Barak, Segev; Hamida, Sami Ben; Ron, Dorit

    2014-07-01

    The mammalian (or mechanistic) target of rapamycin (mTOR) complex 1 (mTORC1) is a serine and threonine kinase that regulates cell growth, survival, and proliferation. mTORC1 is a master controller of the translation of a subset of mRNAs. In the central nervous system mTORC1 plays a crucial role in mechanisms underlying learning and memory by controlling synaptic protein synthesis. Here, we review recent evidence suggesting that the mTORC1 signaling pathway promotes neuroadaptations following exposure to a diverse group of drugs of abuse including stimulants, cannabinoids, opiates, and alcohol. We further describe potential molecular mechanisms by which drug-induced mTORC1 activation may alter brain functions. Finally, we propose that mTORC1 is a focal point shared by drugs of abuse to mediate drug-related behaviors such as reward seeking and excessive drug intake, and offer future directions to decipher the contribution of the kinase to mechanisms underlying addiction. Recent studies suggesting that exposure to diverse classes of drugs of abuse as well as exposure to drug-associated memories lead to mTORC1 kinase activation in the limbic system. In turn, mTORC1 controls the onset and the maintenance of pathological neuroadaptions that underlie several features of drug addiction such as drug seeking and relapse. Therefore, we propose that targeting mTORC1 and its effectors is a promising strategy to treat drug disorders. © 2014 International Society for Neurochemistry.

  16. PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis.

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    Kathrin Thedieck

    Full Text Available TOR (Target of Rapamycin is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1 and TOR complex 2 (TORC2. In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS based proteomic strategy to identify new mammalian TOR (mTOR binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40 and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFalpha and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5 and was therefore named PRR5-Like (PRR5L. PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1 and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.

  17. Target of rapamycin (TOR) plays a critical role in triacylglycerol accumulation in microalgae.

    Science.gov (United States)

    Imamura, Sousuke; Kawase, Yasuko; Kobayashi, Ikki; Sone, Toshiyuki; Era, Atsuko; Miyagishima, Shin-Ya; Shimojima, Mie; Ohta, Hiroyuki; Tanaka, Kan

    2015-10-01

    Most microalgae produce triacylglycerol (TAG) under stress conditions such as nitrogen depletion, but the underlying molecular mechanism remains unclear. In this study, we focused on the role of target of rapamycin (TOR) in TAG accumulation. TOR is a serine/threonine protein kinase that is highly conserved and plays pivotal roles in nitrogen and other signaling pathways in eukaryotes. We previously constructed a rapamycin-susceptible Cyanidioschyzon merolae, a unicellular red alga, by expressing yeast FKBP12 protein to evaluate the results of TOR inhibition (Imamura et al. in Biochem Biophys Res Commun 439:264-269, 2013). By using this strain, we here report that rapamycin-induced TOR inhibition results in accumulation of cytoplasmic lipid droplets containing TAG. Transcripts for TAG synthesis-related genes, such as glycerol-3-phosphate acyltransferase and acyl-CoA:diacylglycerol acyltransferase (DGAT), were increased by rapamycin treatment. We also found that fatty acid synthase-dependent de novo fatty acid synthesis was required for the accumulation of lipid droplets. Induction of TAG and up-regulation of DGAT gene expression by rapamycin were similarly observed in the unicellular green alga, Chlamydomonas reinhardtii. These results suggest the general involvement of TOR signaling in TAG accumulation in divergent microalgae.

  18. Rapamycin up-regulates triglycerides in hepatocytes by down-regulating Prox1.

    Science.gov (United States)

    Kwon, Sora; Jeon, Ji-Sook; Kim, Su Bin; Hong, Young-Kwon; Ahn, Curie; Sung, Jung-Suk; Choi, Inho

    2016-02-27

    Although the prolonged use of rapamycin may cause unwanted side effects such as hyperlipidemia, the underlying mechanism remains unknown. Prox1 is a transcription factor responsible for the development of several tissues including lymphatics and liver. There is growing evidences that Prox1 participates in metabolism in addition to embryogenesis. However, whether Prox1 is directly related to lipid metabolism is currently unknown. HepG2 human hepatoma cells were treated with rapamycin and total lipids were analyzed by thin layer chromatography. The effect of rapamycin on the expression of Prox1 was determined by western blotting. To investigate the role of Prox1 in triglycerides regulation, siRNA and overexpression system were employed. Rapamycin was injected into mice for 2 weeks and total lipids and proteins in liver were measured by thin layer chromatography and western blot analysis, respectively. Rapamycin up-regulated the amount of triglyceride and down-regulated the expression of Prox1 in HepG2 cells by reducing protein half-life but did not affect its transcript. The loss-of-function of Prox1 was coincident with the increase of triglycerides in HepG2 cells treated with rapamycin. The up-regulation of triglycerides by rapamycin in HepG2 cells reverted to normal levels by the compensation of Prox1 using the overexpression system. Rapamycin also down-regulated Prox1 expression but increased triglycerides in mouse liver. This study suggests that rapamycin can increase the amount of triglycerides by down-regulating Prox1 expression in hepatocytes, which means that the mammalian target of rapamycin (mTOR) signaling is important for the regulation of triglycerides by maintaining Prox1 expression.

  19. Architecture of human mTOR complex 1.

    Science.gov (United States)

    Aylett, Christopher H S; Sauer, Evelyn; Imseng, Stefan; Boehringer, Daniel; Hall, Michael N; Ban, Nenad; Maier, Timm

    2016-01-01

    Target of rapamycin (TOR), a conserved protein kinase and central controller of cell growth, functions in two structurally and functionally distinct complexes: TORC1 and TORC2. Dysregulation of mammalian TOR (mTOR) signaling is implicated in pathologies that include diabetes, cancer, and neurodegeneration. We resolved the architecture of human mTORC1 (mTOR with subunits Raptor and mLST8) bound to FK506 binding protein (FKBP)-rapamycin, by combining cryo-electron microscopy at 5.9 angstrom resolution with crystallographic studies of Chaetomium thermophilum Raptor at 4.3 angstrom resolution. The structure explains how FKBP-rapamycin and architectural elements of mTORC1 limit access to the recessed active site. Consistent with a role in substrate recognition and delivery, the conserved amino-terminal domain of Raptor is juxtaposed to the kinase active site. Copyright © 2016, American Association for the Advancement of Science.

  20. The gene expression of the neuronal protein, SLC38A9, changes in mouse brain after in vivo starvation and high-fat diet.

    Directory of Open Access Journals (Sweden)

    Sofie V Hellsten

    Full Text Available SLC38A9 is characterized as a lysosomal component of the amino acid sensing Ragulator-RAG GTPase complex, controlling the mechanistic target of rapamycin complex 1 (mTORC1. Here, immunohistochemistry was used to map SLC38A9 in mouse brain and staining was detected throughout the brain, in cortex, hypothalamus, thalamus, hippocampus, brainstem and cerebellum. More specifically, immunostaining was found in areas known to be involved in amino acid sensing and signaling pathways e.g. piriform cortex and hypothalamus. SLC38A9 immunoreactivity co-localized with both GABAergic and glutamatergic neurons, but not with astrocytes. SLC38A9 play a key role in the mTORC1 pathway, and therefore we performed in vivo starvation and high-fat diet studies, to measure gene expression alterations in specific brain tissues and in larger brain regions. Following starvation, Slc38a9 was upregulated in brainstem and cortex, and in anterior parts of the brain (Bregma 3.2 to -2.1mm. After high-fat diet, Slc38a9 was specifically upregulated in hypothalamus, while overall downregulation was noticed throughout the brain (Bregma 3.2 to -8.6mm.

  1. Blood-brain barrier leakage after status epilepticus in rapamycin-treated rats I: Magnetic resonance imaging.

    Science.gov (United States)

    van Vliet, Erwin A; Otte, Willem M; Wadman, Wytse J; Aronica, Eleonora; Kooij, Gijs; de Vries, Helga E; Dijkhuizen, Rick M; Gorter, Jan A

    2016-01-01

    The mammalian target of rapamycin (mTOR) pathway has received increasing attention as a potential antiepileptogenic target. Treatment with the mTOR inhibitor rapamycin after status epilepticus reduces the development of epilepsy in a rat model. To study whether rapamycin mediates this effect via restoration of blood-brain barrier (BBB) dysfunction, contrast-enhanced magnetic resonance imaging (CE-MRI) was used to determine BBB permeability throughout epileptogenesis. Imaging was repeatedly performed until 6 weeks after kainic acid-induced status epilepticus in rapamycin (6 mg/kg for 6 weeks starting 4 h after SE) and vehicle-treated rats, using gadobutrol as contrast agent. Seizures were detected using video monitoring in the week following the last imaging session. Gadobutrol leakage was widespread and extensive in both rapamycin and vehicle-treated epileptic rats during the acute phase, with the piriform cortex and amygdala as the most affected regions. Gadobutrol leakage was higher in rapamycin-treated rats 4 and 8 days after status epilepticus compared to vehicle-treated rats. However, during the chronic epileptic phase, gadobutrol leakage was lower in rapamycin-treated epileptic rats along with a decreased seizure frequency. This was confirmed by local fluorescein staining in the brains of the same rats. Total brain volume was reduced by this rapamycin treatment regimen. The initial slow recovery of BBB function in rapamycin-treated epileptic rats indicates that rapamycin does not reduce seizure activity by a gradual recovery of BBB integrity. The reduced BBB leakage during the chronic phase, however, could contribute to the decreased seizure frequency in post-status epilepticus rats treated with rapamycin. Furthermore, the data show that CE-MRI (using step-down infusion with gadobutrol) can be used as biomarker for monitoring the effect of drug therapy in rats. Wiley Periodicals, Inc. © 2015 International League Against Epilepsy.

  2. β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells.

    Science.gov (United States)

    Salto, Rafael; Vílchez, Jose D; Girón, María D; Cabrera, Elena; Campos, Nefertiti; Manzano, Manuel; Rueda, Ricardo; López-Pedrosa, Jose M

    2015-01-01

    β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth.

  3. mTORC2 Regulation of Muscle Metabolism and Insulin Sensitivity

    DEFF Research Database (Denmark)

    Kleinert, Maximilian

    and skeletal muscle to take up blood glucose, ultimately lowering blood glucose levels. A hallmark of T2D is decreased organ sensitivity to the effects of the insulin. Therefore, an early event in the pathogenesis of T2D is an increase in insulin secretion in response to eating a meal, as more insulin....... In the absence of insulin, the majority of GLUT4 resides within the muscle. Conversely, insulin stimulation increases the muscle’s permeability to glucose, by triggering GLUT4 translocation to the plasma membrane. The effect of insulin on GLUT4 translocation is mediated by a chain of molecular signaling events...... that mTORC2 controls skeletal muscle glycolysis and lipid storage. In agreement, Ric mKO mice exhibited reduced muscle glycolytic flux, greater reliance on fat as an energy substrate, re-partitioning of lean to fat mass and higher intramyocellular triacylglycerol (IMTG) levels compared to Ric WT mice...

  4. The Effects of Glucagon-like Peptide-2 on the Tight Junction and Barrier Function in IPEC-J2 Cells through Phosphatidylinositol 3-kinase–Protein Kinase B–Mammalian Target of Rapamycin Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Changsong Yu

    2016-05-01

    Full Text Available Glucagon-like peptide-2 (GLP-2 is important for intestinal barrier function and regulation of tight junction (TJ proteins, but the intracellular mechanisms of action remain undefined. The purpose of this research was to determine the protective effect of GLP-2 mediated TJ and transepithelial electrical resistance (TER in lipopolysaccharide (LPS stressed IPEC-J2 cells and to test the hypothesis that GLP-2 regulate TJ and TER through the phosphatidylinositol 3-kinase (PI3K-protein kinase B (Akt-mammalian target of rapamycin (mTOR signaling pathway in IPEC-J2 cells. Wortmannin and LY294002 are specific inhibitors of PI3K. The results showed that 100 μg/mL LPS stress decreased TER and TJ proteins occludin, claudin-1 and zonula occludens protein 1 (ZO-1 mRNA, proteins expressions (p<0.01 respectively. GLP-2 (100 nmol/L promote TER and TJ proteins occludin, claudin-1, and zo-1 mRNA, proteins expressions in LPS stressed and normal IPEC-J2 cells (p<0.01 respectively. In normal cells, both wortmannin and LY294002, PI3K inhibitors, prevented the mRNA and protein expressions of Akt and mTOR increase induced by GLP-2 (p<0.01 following with the significant decreasing of occludin, claudin-1, ZO-1 mRNA and proteins expressions and TER (p<0.01. In conclusion, these results indicated that GLP-2 can promote TJ’s expression and TER in LPS stressed and normal IPEC-J2 cells and GLP-2 could regulate TJ and TER through the PI3K/Akt/mTOR pathway.

  5. The rapamycin-regulated gene expression signature determines prognosis for breast cancer

    Directory of Open Access Journals (Sweden)

    Tsavachidis Spiridon

    2009-09-01

    Full Text Available Abstract Background Mammalian target of rapamycin (mTOR is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes may also be used to simulate a biologic process or effects of a drug treatment. In this study, we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer. Results Colony formation and sulforhodamine B (IC50 in vitro and in vivo gene expression data identified a signature, termed rapamycin metagene index (RMI, of 31 genes upregulated by rapamycin treatment in vitro as well as in vivo (false discovery rate of 10%. In the Miller dataset, RMI did not correlate with tumor size or lymph node status. High (>75th percentile RMI was significantly associated with longer survival (P = 0.015. On multivariate analysis, RMI (P = 0.029, tumor size (P = 0.015 and lymph node status (P = 0.001 were prognostic. In van 't Veer study, RMI was not associated with the time to develop distant metastasis (P = 0.41. In the Wang dataset, RMI predicted time to disease relapse (P = 0.009. Conclusion Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment.

  6. MAOHUZI6/ETHYLENE INSENSITIVE3-LIKE1 and ETHYLENE INSENSITIVE3-LIKE2 Regulate Ethylene Response of Roots and Coleoptiles and Negatively Affect Salt Tolerance in Rice1[OPEN

    Science.gov (United States)

    Yang, Chao; Ma, Biao; He, Si-Jie; Xiong, Qing; Duan, Kai-Xuan; Yin, Cui-Cui; Chen, Hui; Lu, Xiang; Chen, Shou-Yi; Zhang, Jin-Song

    2015-01-01

    Ethylene plays important roles in plant growth, development, and stress responses. The ethylene signaling pathway has been studied extensively, mainly in Arabidopsis (Arabidopsis thaliana). However, the molecular mechanism of ethylene signaling is largely unknown in rice (Oryza sativa). Previously, we have isolated a set of rice ethylene-response mutants. Here, we characterized the mutant maohuzi6 (mhz6). Through map-based cloning, we found that MHZ6 encodes ETHYLENE INSENSITIVE3-LIKE1 (OsEIL1), a rice homolog of ETHYLENE INSENSITIVE3 (EIN3), which is the master transcriptional regulator of ethylene signaling in Arabidopsis. Disruption of MHZ6/OsEIL1 caused ethylene insensitivity mainly in roots, whereas silencing of the closely related OsEIL2 led to ethylene insensitivity mainly in coleoptiles of etiolated seedlings. This organ-specific functional divergence is different from the functional features of EIN3 and EIL1, both of which mediate the incomplete ethylene responses of Arabidopsis etiolated seedlings. In Arabidopsis, EIN3 and EIL1 play positive roles in plant salt tolerance. In rice, however, lack of MHZ6/OsEIL1 or OsEIL2 functions improves salt tolerance, whereas the overexpressing lines exhibit salt hypersensitivity at the seedling stage, indicating that MHZ6/OsEIL1 and OsEIL2 negatively regulate salt tolerance in rice. Furthermore, this negative regulation by MHZ6/OsEIL1 and OsEIL2 in salt tolerance is likely attributable in part to the direct regulation of HIGH-AFFINITY K+ TRANSPORTER2;1 expression and Na+ uptake in roots. Additionally, MHZ6/OsEIL1 overexpression promotes grain size and thousand-grain weight. Together, our study provides insights for the functional diversification of MHZ6/OsEIL1 and OsEIL2 in ethylene response and finds a novel mode of ethylene-regulated salt stress response that could be helpful for engineering salt-tolerant crops. PMID:25995326

  7. O2-insensitive photosynthesis in C3 plants: its occurrence and a possible explanation

    International Nuclear Information System (INIS)

    Sharkey, T.D.

    1985-01-01

    Leaves of C 3 plants which exhibit a normal O 2 inhibition of CO 2 fixation at less than saturating light intensity were found to exhibit O 2 -insensitive photosynthesis at high light. This behavior was observed in Phaseolus vulgaris L., Xanthium strumarium L., and Scrophularia desertorum (Shaw.) Munz. O 2 -insensitive photosynthesis has been reported in nine other C 3 species and usually occurred when the intercellular CO 2 pressure was about double the normal pressure. A lack of O 2 inhibition of photosynthesis was always accompanied by a failure of increased CO 2 pressure to stimulate photosynthesis to the expected degree. O 2 -insensitive photosynthesis also occurred after plants had been water stressed. Under such conditions, however, photosynthesis became O 2 and CO 2 insensitive at physiological CO 2 pressures. Postillumination CO 2 exchange kinetics showed that O 2 and CO 2 insensitivity was not the result of elimination of photorespiration. It is proposed that O 2 and CO 2 insensitivity occurs when the concentration of phosphate in the chloroplast stroma cannot be both high enough to allow photophosphorylation and low enough to allow starch and sucrose synthesis at the rates required by the rest of the photosynthetic component processes. Under these conditions, the energy diverted to photorespiration does not adversely affect the potential for CO 2 assimilation

  8. Go-6976 Reverses Hyperglycemia-Induced Insulin Resistance Independently of cPKC Inhibition in Adipocytes

    Science.gov (United States)

    Robinson, Katherine A.; Hegyi, Krisztina; Hannun, Yusuf A.; Buse, Maria G.; Sethi, Jaswinder K.

    2014-01-01

    Chronic hyperglycemia induces insulin resistance by mechanisms that are incompletely understood. One model of hyperglycemia-induced insulin resistance involves chronic preincubation of adipocytes in the presence of high glucose and low insulin concentrations. We have previously shown that the mTOR complex 1 (mTORC1) plays a partial role in the development of insulin resistance in this model. Here, we demonstrate that treatment with Go-6976, a widely used “specific” inhibitor of cPKCs, alleviates hyperglycemia-induced insulin resistance. However, the effects of mTOR inhibitor, rapamycin and Go-6976 were not additive and only rapamycin restored impaired insulin-stimulated AKT activation. Although, PKCα, (but not –β) was abundantly expressed in these adipocytes, our studies indicate cPKCs do not play a major role in causing insulin-resistance in this model. There was no evidence of changes in the expression or phosphorylation of PKCα, and PKCα knock-down did not prevent the reduction of insulin-stimulated glucose transport. This was also consistent with lack of IRS-1 phosphorylation on Ser-24 in hyperglycemia-induced insulin-resistant adipocytes. Treatment with Go-6976 did inhibit a component of the mTORC1 pathway, as evidenced by decreased phosphorylation of S6 ribosomal protein. Raptor knock-down enhanced the effect of insulin on glucose transport in insulin resistant adipocytes. Go-6976 had the same effect in control cells, but was ineffective in cells with Raptor knock-down. Taken together these findings suggest that Go-6976 exerts its effect in alleviating hyperglycemia-induced insulin-resistance independently of cPKC inhibition and may target components of the mTORC1 signaling pathway. PMID:25330241

  9. Nitrogen-responsive Regulation of GATA Protein Family Activators Gln3 and Gat1 Occurs by Two Distinct Pathways, One Inhibited by Rapamycin and the Other by Methionine Sulfoximine*

    Science.gov (United States)

    Georis, Isabelle; Tate, Jennifer J.; Cooper, Terrance G.; Dubois, Evelyne

    2011-01-01

    Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin. PMID:22039046

  10. Nitrogen-responsive regulation of GATA protein family activators Gln3 and Gat1 occurs by two distinct pathways, one inhibited by rapamycin and the other by methionine sulfoximine.

    Science.gov (United States)

    Georis, Isabelle; Tate, Jennifer J; Cooper, Terrance G; Dubois, Evelyne

    2011-12-30

    Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin.

  11. Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Pengpeng [Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Shan, Tizhong; Liang, Xinrong [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Deng, Changyan [Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Kuang, Shihuan, E-mail: skuang@purdue.edu [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States)

    2014-09-12

    Highlights: • mTOR is a critical regulator of many biological processes yet its function in heart is not well understood. • MCK-Cre/Mtor{sup flox/flox} mice were established to delete Mtor in cardiomyocytes. • The mTOR-mKO mice developed normally but die prematurely within 5 weeks after birth due to heart disease. • The mTOR-mKO mice had dilated myocardium and increased cell death. • mTOR-mKO hearts had reduced expression of metabolic genes and activation of mTOR target proteins. - Abstract: Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor{sup flox/flox} mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function.

  12. Ethylene signaling renders the jasmonate response of Arabidopsis insensitive to future suppression by salicylic Acid.

    Science.gov (United States)

    Leon-Reyes, Antonio; Du, Yujuan; Koornneef, Annemart; Proietti, Silvia; Körbes, Ana P; Memelink, Johan; Pieterse, Corné M J; Ritsema, Tita

    2010-02-01

    Cross-talk between jasmonate (JA), ethylene (ET), and Salicylic acid (SA) signaling is thought to operate as a mechanism to fine-tune induced defenses that are activated in response to multiple attackers. Here, 43 Arabidopsis genotypes impaired in hormone signaling or defense-related processes were screened for their ability to express SA-mediated suppression of JA-responsive gene expression. Mutant cev1, which displays constitutive expression of JA and ET responses, appeared to be insensitive to SA-mediated suppression of the JA-responsive marker genes PDF1.2 and VSP2. Accordingly, strong activation of JA and ET responses by the necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola prior to SA treatment counteracted the ability of SA to suppress the JA response. Pharmacological assays, mutant analysis, and studies with the ET-signaling inhibitor 1-methylcyclopropene revealed that ET signaling renders the JA response insensitive to subsequent suppression by SA. The APETALA2/ETHYLENE RESPONSE FACTOR transcription factor ORA59, which regulates JA/ET-responsive genes such as PDF1.2, emerged as a potential mediator in this process. Collectively, our results point to a model in which simultaneous induction of the JA and ET pathway renders the plant insensitive to future SA-mediated suppression of JA-dependent defenses, which may prioritize the JA/ET pathway over the SA pathway during multi-attacker interactions.

  13. Rapamycin Rescues the Poor Developmental Capacity of Aged Porcine Oocytes

    Directory of Open Access Journals (Sweden)

    Seung Eun Lee

    2014-05-01

    Full Text Available Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; 44 h+10 μM rapamycin/24 h, 47.52±5.68 or control oocytes (44 h IVM; 42.14±4.40 significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04±5.68 (p<0.05. Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK, and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1 compared with untreated, 24 h-aged IVM oocytes (p<0.05. Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS activity and DNA fragmentation (p<0.05, and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05 and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1, anti-apoptosis (BCL2L1 and BIRC5; p<0.05, and development (NANOG and SOX2; p<0.05 genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3 compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

  14. Phase II study of everolimus in children and adults with neurofibromatosis type 2 and progressive vestibular schwannomas

    Science.gov (United States)

    Karajannis, Matthias A.; Legault, Geneviève; Hagiwara, Mari; Giancotti, Filippo G.; Filatov, Alexander; Derman, Anna; Hochman, Tsivia; Goldberg, Judith D.; Vega, Emilio; Wisoff, Jeffrey H.; Golfinos, John G.; Merkelson, Amanda; Roland, J. Thomas; Allen, Jeffrey C.

    2014-01-01

    Background Activation of the mammalian target of rapamycin (mTOR) signaling pathway is thought to be a key driver of tumor growth in Merlin (NF2)-deficient tumors. Everolimus is an oral inhibitor of mTOR complex 1 (mTORC1) with antitumor activity in a variety of cancers. Methods We conducted a single-institution, prospective, 2-stage, open-label phase II study to estimate the response rate to everolimus in neurofibromatosis type 2 (NF2) patients with progressive vestibular schwannoma (VS). Ten eligible patients were enrolled, including 2 pediatric patients. Everolimus was administered at a daily dose of 10 mg (adults) or 5 mg/m2/day (children <18 y) orally in continuous 28-day courses, for up to 12 courses. Response was assessed every 3 months with MRI, using 3-dimensional volumetric tumor analysis, and audiograms. Nine patients were evaluable for the primary response, defined as ≥15% decrease in VS volume. Hearing response was evaluable as a secondary endpoint in 8 patients. Results None of the 9 patients with evaluable disease experienced a clinical or MRI response. No objective imaging or hearing responses were observed in stage 1 of the trial, and the study was closed according to predefined stopping rules. Conclusion Everolimus is ineffective for the treatment of progressive VS in NF2 patients. We are currently conducting a pharmacokinetic/pharmacodynamic (“phase 0”) study of everolimus in presurgical VS patients to elucidate the biological basis for apparent treatment resistance to mTORC1 inhibition in these tumors. PMID:24311643

  15. The antiaging activity and cerebral protection of rapamycin at micro-doses.

    Science.gov (United States)

    Qi, Haiyan; Su, Feng-Yun; Wan, Shan; Chen, Yongjie; Cheng, Yan-Qiong; Liu, Ai-Jun

    2014-11-01

    The immunosuppressant drug rapamycin was reported to have an antiaging activity, which was attributed to the TORC1 inhibition that inhibits cell proliferation and increases autophagy. However, rapamycin also exhibits a number of harmful adverse effects. Whether rapamycin can be developed into an antiaging agent remains unclear. We demonstrated that rapamycin at micro-doses (below the TORC1 inhibiting concentration) exhibits a cell-protective activity: (1) It protects cultured neurons against neurotoxin MPP(+) and H2O2. (2) It increases survival time of neuron in culture. (3) It maintains the nonproliferative state of cultured senescent human fibroblasts and prevents cell death induced by telomere dysfunction. (4) In animal models, it decreased the cerebral infarct sizes induced by acute ischemia and dramatically extended the life span of stroke prone spontaneously hypertensive rats (SHR-SPs). We propose that rapamycin at micro-dose can be developed into an antiaging agent with a novel mechanism. © 2014 John Wiley & Sons Ltd.

  16. Enhanced genetic modification of adult growth factor mobilized peripheral blood hematopoietic stem and progenitor cells with rapamycin.

    Science.gov (United States)

    Li, Lijing; Torres-Coronado, Mónica; Gu, Angel; Rao, Anitha; Gardner, Agnes M; Epps, Elizabeth W; Gonzalez, Nancy; Tran, Chy-Anh; Wu, Xiwei; Wang, Jin-Hui; DiGiusto, David L

    2014-10-01

    Genetic modification of adult human hematopoietic stem and progenitor cells (HSPCs) with lentiviral vectors leads to long-term gene expression in the progeny of the HSPCs and has been used to successfully treat several monogenic diseases. In some cases, the gene-modified cells have a selective growth advantage over nonmodified cells and eventually are the dominant engrafted population. However, in disease indications for which the gene-modified cells do not have a selective advantage, optimizing transduction of HSPC is paramount to successful stem cell-based gene therapy. We demonstrate here that transduction of adult CD34+ HSPCs with lentiviral vectors in the presence of rapamycin, a widely used mTORC1 inhibitor, results in an approximately threefold increase in stable gene marking with minimal effects on HSPC growth and differentiation. Using this approach, we have demonstrated that we can enhance the frequency of gene-modified HSPCs that give rise to clonogenic progeny in vitro without excessive increases in the number of vector copies per cell or changes in integration pattern. The genetic marking of HSPCs and expression of transgenes is durable, and transplantation of gene-modified HSPCs into immunodeficient mice results in high levels of gene marking of the lymphoid and myeloid progeny in vivo. The prior safe clinical history of rapamycin in other applications supports the use of this compound to generate gene-modified autologous HSPCs for our HIV gene therapy clinical trials. ©AlphaMed Press.

  17. Longitudinal imaging studies of tumor microenvironment in mice treated with the mTOR inhibitor rapamycin.

    Directory of Open Access Journals (Sweden)

    Keita Saito

    Full Text Available Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation. The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell carcinoma (SCCVII by electron paramagnetic resonance imaging (EPRI and magnetic resonance imaging (MRI to identify an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on SCCVII solid tumors 8 days after implantation (500-750 mm(3 and measurements of tumor pO(2 and blood volume were conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin treatment. Tumor pO(2 levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO(2<10 mm Hg in the tumor region decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization. Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal window for anti-cancer therapies to realize enhanced response to radiotherapy.

  18. FOXO3a reactivation mediates the synergistic cytotoxic effects of rapamycin and cisplatin in oral squamous cell carcinoma cells

    International Nuclear Information System (INIS)

    Fang Liang; Wang Huiming; Zhou Lin; Yu Da

    2011-01-01

    FOXO3a, a well-known transcriptional regulator, controls a wide spectrum of biological processes. The Phosphoinositide-3-kinase (PI3K)/Akt signaling pathway inactivates FOXO3a via phosphorylation-induced nuclear exclusion and degradation. A loss or gain of FOXO3a activity has been correlated with efficiency of chemotherapies in various cancers including oral squamous cell carcinoma (OSCC). Therefore, in the current study, we have investigated the FOXO3a activity modulating and antitumor effects of rapamycin and cisplatin in OSCC cells. Cisplatin inhibited proliferation and induced apoptosis in a dose-dependent way in OSCC Tca8113 cells. Rapamycin alone had no effect on cell proliferation and apoptosis. Rapamycin downregulated the expression of S-phase kinase associated protein-2 (Skp2) and increased the FOXO3a protein stability but induced the upregulation of feedback Akt activation-mediated FOXO3a phosphorylation. Cisplatin decreased the phosphorylation of FOXO3a via Akt inhibition. Rapamycin combined with cisplatin as its feedback Akt activation inhibitor revealed the most dramatic FOXO3a nuclear localization and reactivation with the prevention of its feedback loop and exposed significant synergistic effects of decreased cell proliferation and increased apoptosis in vitro and decreased tumor size in vivo. Furthermore, the downstream effects of FOXO3a reactivation were found to be accumulation of p27 and Bim. In conclusion, rapamycin/cisplatin combination therapy boosts synergistic antitumor effects through the significant FOXO3a reactivation in OSCC cells. These results may represent a novel mechanism by which rapamycin/cisplatin combination therapy proves to be a potent molecular-targeted strategy for OSCC.

  19. Pharmacokinetics of orally administered low-dose rapamycin in healthy dogs.

    Science.gov (United States)

    Larson, Jeanne C; Allstadt, Sara D; Fan, Timothy M; Khanna, Chand; Lunghofer, Paul J; Hansen, Ryan J; Gustafson, Daniel L; Legendre, Alfred M; Galyon, Gina D; LeBlanc, Amy K; Martin-Jimenez, Tomas

    2016-01-01

    To determine the pharmacokinetics of orally administered rapamycin in healthy dogs. 5 healthy purpose-bred hounds. The study consisted of 2 experiments. In experiment 1, each dog received rapamycin (0.1 mg/kg, PO) once; blood samples were obtained immediately before and at 0.5, 1, 2, 4, 6, 12, 24, 48, and 72 hours after administration. In experiment 2, each dog received rapamycin (0.1 mg/kg, PO) once daily for 5 days; blood samples were obtained immediately before and at 3, 6, 24, 27, 30, 48, 51, 54, 72, 75, 78, 96, 96.5, 97, 98, 100, 102, 108, 120, 144, and 168 hours after the first dose. Blood rapamycin concentration was determined by a validated liquid chromatography-tandem mass spectrometry assay. Pharmacokinetic parameters were determined by compartmental and noncompartmental analyses. Mean ± SD blood rapamycin terminal half-life, area under the concentration-time curve from 0 to 48 hours after dosing, and maximum concentration were 38.7 ± 12.7 h, 140 ± 23.9 ng•h/mL, and 8.39 ± 1.73 ng/mL, respectively, for experiment 1, and 99.5 ± 89.5 h, 126 ± 27.1 ng•h/mL, and 5.49 ± 1.99 ng/mL, respectively, for experiment 2. Pharmacokinetic parameters for rapamycin after administration of 5 daily doses differed significantly from those after administration of 1 dose. Results indicated that oral administration of low-dose (0.1 mg/kg) rapamycin to healthy dogs achieved blood concentrations measured in nanograms per milliliter. The optimal dose and administration frequency of rapamcyin required to achieve therapeutic effects in tumor-bearing dogs, as well as toxicity after chronic dosing, need to be determined.

  20. Novel nervous and multi-system regenerative therapeutic strategies for diabetes mellitus with mTOR

    Directory of Open Access Journals (Sweden)

    Kenneth Maiese

    2016-01-01

    Full Text Available Throughout the globe, diabetes mellitus (DM is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder. DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy. The mechanistic target of rapamycin (mTOR is a promising agent for the development of novel regenerative strategies for the treatment of DM. mTOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis, insulin resistance, insulin secretion, stem cell proliferation and differentiation, pancreatic β-cell function, and programmed cell death with apoptosis and autophagy. mTOR is central element for the protein complexes mTOR Complex 1 (mTORC1 and mTOR Complex 2 (mTORC2 and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase (PI 3-K, protein kinase B (Akt, AMP activated protein kinase (AMPK, silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae (SIRT1, Wnt1 inducible signaling pathway protein 1 (WISP1, and growth factors. As a result, mTOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease. Future studies directed to elucidate the delicate balance mTOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.

  1. Synergism between the mTOR inhibitor rapamycin and FAK down-regulation in the treatment of acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Pei-Jie Shi

    2016-02-01

    Full Text Available Abstract Background Acute lymphoblastic leukemia (ALL is an aggressive malignant disorder of lymphoid progenitor cells in both children and adults. Although improvements in contemporary therapy and development of new treatment strategies have led to dramatic increases in the cure rate in children with ALL, the relapse rate remains high and the prognosis of relapsed childhood ALL is poor. Molecularly targeted therapies have emerged as the leading treatments in cancer therapy. Multi-cytotoxic drug regimens have achieved success, yet many studies addressing targeted therapies have focused on only one single agent. In this study, we attempted to investigate whether the effect of the mammalian target of rapamycin (mTOR inhibitor rapamycin is synergistic with the effect of focal adhesion kinase (FAK down-regulation in the treatment of ALL. Methods The effect of rapamycin combined with FAK down-regulation on cell proliferation, the cell cycle, and apoptosis was investigated in the human precursor B acute lymphoblastic leukemia cells REH and on survival time and leukemia progression in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID mouse model. Results When combined with FAK down-regulation, rapamycin-induced suppression of cell proliferation, G0/G1 cell cycle arrest, and apoptosis were significantly enhanced. In addition, REH cell-injected NOD/SCID mice treated with rapamycin and a short-hairpin RNA (shRNA to down-regulate FAK had significantly longer survival times and slower leukemia progression compared with mice injected with REH-empty vector cells and treated with rapamycin. Moreover, the B-cell CLL/lymphoma-2 (BCL-2 gene family was shown to be involved in the enhancement, by combined treatment, of REH cell apoptosis. Conclusions FAK down-regulation enhanced the in vitro and in vivo inhibitory effects of rapamycin on REH cell growth, indicating that the simultaneous targeting of mTOR- and FAK-related pathways might offer a novel

  2. High-fat feeding impairs nutrient sensing and gut brain integration in the caudomedial nucleus of the solitary tract in mice.

    Directory of Open Access Journals (Sweden)

    Althea R Cavanaugh

    Full Text Available Hyperphagic obesity is characterized in part by a specific increase in meal size that contributes to increased daily energy intake, but the mechanisms underlying impaired activity of meal size regulatory circuits, particularly those converging at the caudomedial nucleus of the solitary tract in the hindbrain (cmNTS, remain poorly understood. In this paper, we assessed the consequences of high-fat (HF feeding and diet-induced obesity (DIO on cmNTS nutrient sensing and metabolic integration in the control of meal size. Mice maintained on a standard chow diet, low-fat (LF diet or HF diet for 2 weeks or 6 months were implanted with a bilateral brain cannula targeting the cmNTS. Feeding behavior was assessed using behavioral chambers and meal-pattern analysis following cmNTS L-leucine injections alone or together with ip CCK. Molecular mechanisms implicated in the feeding responses were assessed using western blot, immunofluorescence and pharmacological inhibition of the amino acid sensing mTORC1 pathway (mammalian target of rapamycin complex 1. We found that HF feeding blunts the anorectic consequences of cmNTS L-leucine administration. Increased baseline activity of the L-leucine sensor P70 S6 kinase 1 and impaired L-leucine-induced activation of this pathway in the cmNTS of HF-fed mice indicate that HF feeding is associated with an impairment in cmNTS mTOR nutritional and hormonal sensing. Interestingly, the acute orexigenic effect of the mTORC1 inhibitor rapamycin was preserved in HF-fed mice, supporting the assertion that HF-induced increase in baseline cmNTS mTORC1 activity underlies the defect in L-leucine sensing. Last, the synergistic feeding-suppressive effect of CCK and cmNTS L-leucine was abrogated in DIO mice. These results indicate that HF feeding leads to an impairment in cmNTS nutrient sensing and metabolic integration in the regulation of meal size.

  3. Cardiac mTORC1 Dysregulation Impacts Stress Adaptation and Survival in Huntington’s Disease

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    Daniel D. Child

    2018-04-01

    Full Text Available Summary: Huntington’s disease (HD is a dominantly inherited neurological disorder caused by CAG-repeat expansion in exon 1 of Huntingtin (HTT. But in addition to the neurological disease, mutant HTT (mHTT, which is ubiquitously expressed, impairs other organ systems. Indeed, epidemiological and animal model studies suggest higher incidence of and mortality from heart disease in HD. Here, we show that the protein complex mTORC1 is dysregulated in two HD mouse models through a mechanism that requires intrinsic mHTT expression. Moreover, restoring cardiac mTORC1 activity with constitutively active Rheb prevents mortality and relieves the mHTT-induced block to hypertrophic adaptation to cardiac stress. Finally, we show that chronic mTORC1 dysregulation is due in part to mislocalization of endogenous Rheb. These data provide insight into the increased cardiac-related mortality of HD patients, with cardiac mHTT expression inhibiting mTORC1 activity, limiting heart growth, and decreasing the heart’s ability to compensate to chronic stress. : Child et al. demonstrate that mTORC1 dysregulation is a key molecular mechanism in the Huntington’s disease (HD heart phenotype. Impaired cardiac mTORC1 activity in HD mouse models requires intrinsic mHTT expression and explains the limited adaptation to cardiac stress. Keywords: Huntington’s disease, heart, mTOR, Rheb

  4. Phosphatidylcholine transfer protein interacts with thioesterase superfamily member 2 to attenuate insulin signaling.

    Science.gov (United States)

    Ersoy, Baran A; Tarun, Akansha; D'Aquino, Katharine; Hancer, Nancy J; Ukomadu, Chinweike; White, Morris F; Michel, Thomas; Manning, Brendan D; Cohen, David E

    2013-07-30

    Phosphatidylcholine transfer protein (PC-TP) is a phospholipid-binding protein that is enriched in liver and that interacts with thioesterase superfamily member 2 (THEM2). Mice lacking either protein exhibit improved hepatic glucose homeostasis and are resistant to diet-induced diabetes. Insulin receptor substrate 2 (IRS2) and mammalian target of rapamycin complex 1 (mTORC1) are key effectors of insulin signaling, which is attenuated in diabetes. We found that PC-TP inhibited IRS2, as evidenced by insulin-independent IRS2 activation after knockdown, genetic ablation, or chemical inhibition of PC-TP. In addition, IRS2 was activated after knockdown of THEM2, providing support for a role for the interaction of PC-TP with THEM2 in suppressing insulin signaling. Additionally, we showed that PC-TP bound to tuberous sclerosis complex 2 (TSC2) and stabilized the components of the TSC1-TSC2 complex, which functions to inhibit mTORC1. Preventing phosphatidylcholine from binding to PC-TP disrupted interactions of PC-TP with THEM2 and TSC2, and disruption of the PC-TP-THEM2 complex was associated with increased activation of both IRS2 and mTORC1. In livers of mice with genetic ablation of PC-TP or that had been treated with a PC-TP inhibitor, steady-state amounts of IRS2 were increased, whereas those of TSC2 were decreased. These findings reveal a phospholipid-dependent mechanism that suppresses insulin signaling downstream of its receptor.

  5. Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development

    OpenAIRE

    Guan, Yingjie; Yang, Xu; Yang, Wentian; Charbonneau, Cherie; Chen, Qian

    2014-01-01

    Mechanical stress regulates development by modulating cell signaling and gene expression. However, the cytoplasmic components mediating mechanotransduction remain unclear. In this study, elimination of muscle contraction during chicken embryonic development resulted in a reduction in the activity of mammalian target of rapamycin (mTOR) in the cartilaginous growth plate. Inhibition of mTOR activity led to significant inhibition of chondrocyte proliferation, cartilage tissue growth, and express...

  6. TNF-alpha impairs the S-G2/M cell cycle checkpoint and cyclobutane pyrimidine dimer repair in premalignant skin cells: Role of the PI3K-Akt pathway

    DEFF Research Database (Denmark)

    Faurschou, A.; Gniadecki, R.; Calay, D.

    2008-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is induced by UVB radiation and has been implicated in the early stages of skin carcinogenesis. Here, we show that in normal keratinocytes and the transformed keratinocyte cell lines, HaCaT and A431, TNF-alpha stimulates protein kinase B/Akt, which results...... cycling. TNF-alpha enhanced apoptosis less potently and did not increase the level of CPD or stimulate cell cycle progression in normal keratinocytes. Our data suggest that TNF-alpha overrides the G2/M checkpoint in premalignant skin cells and allows for some cells containing unrepaired CPD to enter...... in activation of the survival complex mTORC1 (mammalian target of rapamycin complex 1) and inhibition of the proapoptotic proteins Bad and Fox03a. In UVB-irradiated HaCaT cells (10-20 mJ cm(-2)), TNF-alpha increased the proportion of cycling cells and enhanced the rate of apoptosis. A significantly higher...

  7. Rapamycin preconditioning attenuates transient focal cerebral ischemia/reperfusion injury in mice.

    Science.gov (United States)

    Yin, Lele; Ye, Shasha; Chen, Zhen; Zeng, Yaoying

    2012-12-01

    Rapamycin, an mTOR inhibitor and immunosuppressive agent in clinic, has protective effects on traumatic brain injury and neurodegenerative diseases. But, its effects on transient focal ischemia/reperfusion disease are not very clear. In this study, we examined the effects of rapamycin preconditioning on mice treated with middle cerebral artery occlusion/reperfusion operation (MCAO/R). We found that the rapamycin preconditioning by intrahippocampal injection 20 hr before MCAO/R significantly improved the survival rate and longevity of mice. It also decreased the neurological deficit score, infracted areas and brain edema. In addition, rapamycin preconditioning decreased the production of NF-κB, TNF-α, and Bax, but not Bcl-2, an antiapoptotic protein in the ischemic area. From these results, we may conclude that rapamycin preconditioning attenuate transient focal cerebral ischemia/reperfusion injury and inhibits apoptosis induced by MCAO/R in mice.

  8. Laser ignitibility of insensitive secondary explosive 1,1-diamino-2,2-dinitroethene (FOX-7)

    International Nuclear Information System (INIS)

    Fang, Xiao; McLuckie, Warren G

    2015-01-01

    Highlights: • Carbon black (CB) is an efficient optical sensitizer compatible with 1,1-diamino-2,2-dinitroethene (FOX-7). • A CB doped insensitive explosive FOX-7 can be ignited by a low-power diode laser. • Laser ignitibility of the optically sensitized FOX-7 is mainly affected by laser power among the other parameters. • Inhomogeneities in the explosive affect laser ignition reliability. - Abstract: An experimental investigation into laser ignitibility of insensitive secondary explosives, 1,1-diamino-2,2-dinitroethene (FOX-7) has been carried out, using a diode laser of continuous wave at the laser wavelength of 974 nm. The direct optical ignition of an insensitive explosive will add more safety features to insensitive munitions (IM) or explosive devices. In this study, effects of laser parameters on the ignitibility were analysed in terms of laser ignition threshold, the times to initiate the ignition and full combustion, and burning sustainability. The results have shown that carbon black (CB) as an optical sensitizer is compatible with FOX-7, and significantly enhances laser ignitibility of the explosive when a small amount of CB is uniformly doped in FOX-7. The delay times for ignition and subsequent development of sustainable burning of the material are mainly determined by ignition laser power, although the other laser parameters have effects. The minimum laser power required to ignite the optically sensitized FOX-7 was found below 10 W and a fast ignition was initiated in as short as 70 μs by a laser power of 40 W. Also the effect of the mixture uniformity of FOX-7/CB on laser ignition performance was evaluated in this study

  9. Rictor/mTORC2 Loss in the Myf5 Lineage Reprograms Brown Fat Metabolism and Protects Mice against Obesity and Metabolic Disease

    Directory of Open Access Journals (Sweden)

    Chien-Min Hung

    2014-07-01

    Full Text Available The in vivo functions of mechanistic target of rapamycin complex 2 (mTORC2 and the signaling mechanisms that control brown adipose tissue (BAT fuel utilization and activity are not well understood. Here, by conditionally deleting Rictor in the Myf5 lineage, we provide in vivo evidence that mTORC2 is dispensable for skeletal muscle development and regeneration but essential for BAT growth. Furthermore, deleting Rictor in Myf5 precursors shifts BAT metabolism to a more oxidative and less lipogenic state and protects mice from obesity and metabolic disease at thermoneutrality. We additionally find that Rictor is required for brown adipocyte differentiation in vitro and that the mechanism specifically requires AKT1 hydrophobic motif phosphorylation but is independent of pan-AKT signaling and is rescued with BMP7. Our findings provide insights into the signaling circuitry that regulates brown adipocytes and could have important implications for developing therapies aimed at increasing energy expenditure as a means to combat human obesity.

  10. mTOR Complex Signaling through the SEMA4A-Plexin B2 Axis Is Required for Optimal Activation and Differentiation of CD8+ T Cells.

    Science.gov (United States)

    Ito, Daisuke; Nojima, Satoshi; Nishide, Masayuki; Okuno, Tatsusada; Takamatsu, Hyota; Kang, Sujin; Kimura, Tetsuya; Yoshida, Yuji; Morimoto, Keiko; Maeda, Yohei; Hosokawa, Takashi; Toyofuku, Toshihiko; Ohshima, Jun; Kamimura, Daisuke; Yamamoto, Masahiro; Murakami, Masaaki; Morii, Eiichi; Rakugi, Hiromi; Isaka, Yoshitaka; Kumanogoh, Atsushi

    2015-08-01

    Mammalian target of rapamycin (mTOR) plays crucial roles in activation and differentiation of diverse types of immune cells. Although several lines of evidence have demonstrated the importance of mTOR-mediated signals in CD4(+) T cell responses, the involvement of mTOR in CD8(+) T cell responses is not fully understood. In this study, we show that a class IV semaphorin, SEMA4A, regulates CD8(+) T cell activation and differentiation through activation of mTOR complex (mTORC) 1. SEMA4A(-/-) CD8(+) T cells exhibited impairments in production of IFN-γ and TNF-α and induction of the effector molecules granzyme B, perforin, and FAS-L. Upon infection with OVA-expressing Listeria monocytogenes, pathogen-specific effector CD8(+) T cell responses were significantly impaired in SEMA4A(-/-) mice. Furthermore, SEMA4A(-/-) CD8(+) T cells exhibited reduced mTORC1 activity and elevated mTORC2 activity, suggesting that SEMA4A is required for optimal activation of mTORC1 in CD8(+) T cells. IFN-γ production and mTORC1 activity in SEMA4A(-/-) CD8(+) T cells were restored by administration of recombinant Sema4A protein. In addition, we show that plexin B2 is a functional receptor of SEMA4A in CD8(+) T cells. Collectively, these results not only demonstrate the role of SEMA4A in CD8(+) T cells, but also reveal a novel link between a semaphorin and mTOR signaling. Copyright © 2015 by The American Association of Immunologists, Inc.

  11. Genetic Deletion of Rheb1 in the Brain Reduces Food Intake and Causes Hypoglycemia with Altered Peripheral Metabolism

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    Wanchun Yang

    2014-01-01

    Full Text Available Excessive food/energy intake is linked to obesity and metabolic disorders, such as diabetes. The hypothalamus in the brain plays a critical role in the control of food intake and peripheral metabolism. The signaling pathways in hypothalamic neurons that regulate food intake and peripheral metabolism need to be better understood for developing pharmacological interventions to manage eating behavior and obesity. Mammalian target of rapamycin (mTOR, a serine/threonine kinase, is a master regulator of cellular metabolism in different cell types. Pharmacological manipulations of mTOR complex 1 (mTORC1 activity in hypothalamic neurons alter food intake and body weight. Our previous study identified Rheb1 (Ras homolog enriched in brain 1 as an essential activator of mTORC1 activity in the brain. Here we examine whether central Rheb1 regulates food intake and peripheral metabolism through mTORC1 signaling. We find that genetic deletion of Rheb1 in the brain causes a reduction in mTORC1 activity and impairs normal food intake. As a result, Rheb1 knockout mice exhibit hypoglycemia and increased lipid mobilization in adipose tissue and ketogenesis in the liver. Our work highlights the importance of central Rheb1 signaling in euglycemia and energy homeostasis in animals.

  12. Metastatic Group 3 Medulloblastoma in a Patient With Tuberous Sclerosis Complex: Case Description and Molecular Characterization of the Tumor.

    Science.gov (United States)

    Moavero, Romina; Folgiero, Valentina; Carai, Andrea; Miele, Evelina; Ferretti, Elisabetta; Po, Agnese; Diomedi Camassei, Francesca; Lepri, Francesca Romana; Vigevano, Federico; Curatolo, Paolo; Valeriani, Massimiliano; Colafati, Giovanna S; Locatelli, Franco; Tornesello, Assunta; Mastronuzzi, Angela

    2016-04-01

    Medulloblastoma is the most common pediatric brain tumor. We describe a child with tuberous sclerosis complex that developed a Group 3, myc overexpressed, metastatic medulloblastoma (MB). Considering the high risk of treatment-induced malignancies, a tailored therapy, omitting radiation, was given. Based on the evidence of mammalian target of rapamycin mTORC, mTOR Complex; RAS, Rat sarcoma; RAF, rapidly accelerated fibrosarcoma (mTOR) pathway activation in the tumor, targeted therapy was applied resulting in complete remission of disease. Although the PI3K/AKT/mTOR signaling pathway plays a role in MB, we did not find TSC1/TSC2 (TSC, tuberous sclerosis complex) mutation in our patient. We speculate that a different pathway resulting in mTOR activation is the basis of both TSC and MB in this child; H&E, haematoxilin and eosin; Gd, gadolinium. © 2015 Wiley Periodicals, Inc.

  13. Surface Engineering of Porous Silicon Microparticles for Intravitreal Sustained Delivery of Rapamycin

    Science.gov (United States)

    Nieto, Alejandra; Hou, Huiyuan; Moon, Sang Woong; Sailor, Michael J.; Freeman, William R.; Cheng, Lingyun

    2015-01-01

    Purpose. To understand the relationship between rapamycin loading/release and surface chemistries of porous silicon (pSi) to optimize pSi-based intravitreal delivery system. Methods. Three types of surface chemical modifications were studied: (1) pSi-COOH, containing 10-carbon aliphatic chains with terminal carboxyl groups grafted via hydrosilylation of undecylenic acid; (2) pSi-C12, containing 12-carbon aliphatic chains grafted via hydrosilylation of 1-dodecene; and (3) pSiO2-C8, prepared by mild oxidation of the pSi particles followed by grafting of 8-hydrocarbon chains to the resulting porous silica surface via a silanization. Results. The efficiency of rapamycin loading follows the order (micrograms of drug/milligrams of carrier): pSiO2-C8 (105 ± 18) > pSi-COOH (68 ± 8) > pSi-C12 (36 ± 6). Powder X-ray diffraction data showed that loaded rapamycin was amorphous and dynamic drug-release study showed that the availability of the free drug was increased by 6-fold (compared with crystalline rapamycin) by using pSiO2-C8 formulation (P = 0.0039). Of the three formulations in this study, pSiO2-C8-RAP showed optimal performance in terms of simultaneous release of the active drug and carrier degradation, and drug-loading capacity. Released rapamycin was confirmed with the fingerprints of the mass spectrometry and biologically functional as the control of commercial crystalline rapamycin. Single intravitreal injections of 2.9 ± 0.37 mg pSiO2-C8-RAP into rabbit eyes resulted in more than 8 weeks of residence in the vitreous while maintaining clear optical media and normal histology of the retina in comparison to the controls. Conclusions. Porous silicon–based rapamycin delivery system using the pSiO2-C8 formulation demonstrated good ocular compatibility and may provide sustained drug release for retina. PMID:25613937

  14. Surface engineering of porous silicon microparticles for intravitreal sustained delivery of rapamycin.

    Science.gov (United States)

    Nieto, Alejandra; Hou, Huiyuan; Moon, Sang Woong; Sailor, Michael J; Freeman, William R; Cheng, Lingyun

    2015-01-22

    To understand the relationship between rapamycin loading/release and surface chemistries of porous silicon (pSi) to optimize pSi-based intravitreal delivery system. Three types of surface chemical modifications were studied: (1) pSi-COOH, containing 10-carbon aliphatic chains with terminal carboxyl groups grafted via hydrosilylation of undecylenic acid; (2) pSi-C12, containing 12-carbon aliphatic chains grafted via hydrosilylation of 1-dodecene; and (3) pSiO2-C8, prepared by mild oxidation of the pSi particles followed by grafting of 8-hydrocarbon chains to the resulting porous silica surface via a silanization. The efficiency of rapamycin loading follows the order (micrograms of drug/milligrams of carrier): pSiO2-C8 (105 ± 18) > pSi-COOH (68 ± 8) > pSi-C12 (36 ± 6). Powder X-ray diffraction data showed that loaded rapamycin was amorphous and dynamic drug-release study showed that the availability of the free drug was increased by 6-fold (compared with crystalline rapamycin) by using pSiO2-C8 formulation (P = 0.0039). Of the three formulations in this study, pSiO2-C8-RAP showed optimal performance in terms of simultaneous release of the active drug and carrier degradation, and drug-loading capacity. Released rapamycin was confirmed with the fingerprints of the mass spectrometry and biologically functional as the control of commercial crystalline rapamycin. Single intravitreal injections of 2.9 ± 0.37 mg pSiO2-C8-RAP into rabbit eyes resulted in more than 8 weeks of residence in the vitreous while maintaining clear optical media and normal histology of the retina in comparison to the controls. Porous silicon-based rapamycin delivery system using the pSiO2-C8 formulation demonstrated good ocular compatibility and may provide sustained drug release for retina. Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.

  15. 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression.

    Science.gov (United States)

    Shives, Katherine D; Massey, Aaron R; May, Nicholas A; Morrison, Thomas E; Beckham, J David

    2016-10-18

    West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7 GpppN m 5' cap with 2'- O -methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

  16. 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression

    Directory of Open Access Journals (Sweden)

    Katherine D. Shives

    2016-10-01

    Full Text Available West Nile virus (WNV is a (+ sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1 for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K and eukaryotic translation initiation factor 4E-binding protein (4EBP pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E interaction and eukaryotic initiation factor 4F (eIF4F complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6 and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

  17. Acute mTOR inhibition induces insulin resistance and alters substrate utilization in vivo

    DEFF Research Database (Denmark)

    Kleinert, Maximilian; Sylow, Lykke; Fazakerley, Daniel J

    2014-01-01

    , but not rapamycin reduced insulin-stimulated glucose uptake into incubated muscles, despite normal GLUT4 translocation in muscle cells. AZD8055 inhibited glycolysis in MEF cells. Abrogation of mTORC2 activity by SIN1 deletion impaired glycolysis and AZD8055 had no effect in SIN1 KO MEFs. Re-expression of wildtype...... SIN1 rescued glycolysis. Glucose intolerance following AZD8055 administration was absent in mice lacking the mTORC2 subunit Rictor in muscle, and in vivo glucose uptake into Rictor-deficient muscle was reduced despite normal Akt activity. Taken together, acute mTOR inhibition is detrimental to glucose...

  18. Acute mTOR inhibition induces insulin resistance and alters substrate utilization in vivo

    DEFF Research Database (Denmark)

    Kleinert, Maximilian; Sylow, Lykke; Fazakerley, Daniel J.

    2014-01-01

    , but not rapamycin reduced insulin-stimulated glucose uptake into incubated muscles, despite normal GLUT4 translocation in muscle cells. AZD8055 inhibited glycolysis in MEF cells. Abrogation of mTORC2 activity by SIN1 deletion impaired glycolysis and AZD8055 had no effect in SIN1 KO MEFs. Re-expression of wildtype...... SIN1 rescued glycolysis. Glucose intolerance following AZD8055 administration was absent in mice lacking the mTORC2 subunit Rictor in muscle, and in vivo glucose uptake into Rictor-deficient muscle was reduced despite normal Akt activity. Taken together, acute mTOR inhibition is detrimental to glucose...

  19. Transient activation of mTOR following forced treadmill exercise in rats

    DEFF Research Database (Denmark)

    Elfving, Betina; Christensen, Tina; Ratner, Cecilia

    2013-01-01

    , while the induction of neurogenesis requires signaling through the VEGF receptor, Flk-1 (VEGFR-2). VEGF expression is believed to be regulated by two distinct mTOR (mammalian Target of Rapamycin)-containing multiprotein complexes mTORC1 and mTORC2, respectively. This study was initiated to investigate...... of mTOR was regulated after a single bout of exercise. In conclusion, the effect of treadmill exercise on the VEGF system is acute rather than chronic and there is a transient activation of mTOR. More studies are needed to understand whether this could be beneficial in the treatment of neuropsychiatric...

  20. Enhanced antitumor activity of 3-bromopyruvate in combination with rapamycin in vivo and in vitro.

    Science.gov (United States)

    Zhang, Qi; Pan, Jing; Lubet, Ronald A; Komas, Steven M; Kalyanaraman, Balaraman; Wang, Yian; You, Ming

    2015-04-01

    3-Bromopyruvate (3-BrPA) is an alkylating agent and a well-known inhibitor of energy metabolism. Rapamycin is an inhibitor of the serine/threonine protein kinase mTOR. Both 3-BrPA and rapamycin show chemopreventive efficacy in mouse models of lung cancer. Aerosol delivery of therapeutic drugs for lung cancer has been reported to be an effective route of delivery with little systemic distribution in humans. In this study, 3-BrPA and rapamycin were evaluated in combination for their preventive effects against lung cancer in mice by aerosol treatment, revealing a synergistic ability as measured by tumor multiplicity and tumor load compared treatment with either single-agent alone. No evidence of liver toxicity was detected by monitoring serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. To understand the mechanism in vitro experiments were performed using human non-small cell lung cancer (NSCLC) cell lines. 3-BrPA and rapamycin also synergistically inhibited cell proliferation. Rapamycin alone blocked the mTOR signaling pathway, whereas 3-BrPA did not potentiate this effect. Given the known role of 3-BrPA as an inhibitor of glycolysis, we investigated mitochondrial bioenergetics changes in vitro in 3-BrPA-treated NSCLC cells. 3-BrPA significantly decreased glycolytic activity, which may be due to adenosine triphosphate (ATP) depletion and decreased expression of GAPDH. Our results demonstrate that rapamycin enhanced the antitumor efficacy of 3-BrPA, and that dual inhibition of mTOR signaling and glycolysis may be an effective therapeutic strategy for lung cancer chemoprevention. ©2015 American Association for Cancer Research.

  1. Second-hit mosaic mutation in mTORC1 repressor DEPDC5 causes focal cortical dysplasia-associated epilepsy.

    Science.gov (United States)

    Ribierre, Théo; Deleuze, Charlotte; Bacq, Alexandre; Baldassari, Sara; Marsan, Elise; Chipaux, Mathilde; Muraca, Giuseppe; Roussel, Delphine; Navarro, Vincent; Leguern, Eric; Miles, Richard; Baulac, Stéphanie

    2018-04-30

    DEP domain-containing 5 protein (DEPDC5) is a repressor of the recently recognized amino acid-sensing branch of the mTORC1 pathway. So far, its function in the brain remains largely unknown. Germline loss-of-function mutations in DEPDC5 have emerged as a major cause of familial refractory focal epilepsies, with case reports of sudden unexpected death in epilepsy (SUDEP). Remarkably, a fraction of patients also develop focal cortical dysplasia (FCD), a neurodevelopmental cortical malformation. We therefore hypothesized that a somatic second-hit mutation arising during brain development may support the focal nature of the dysplasia. Here, using postoperative human tissue, we provide the proof of concept that a biallelic 2-hit - brain somatic and germline - mutational mechanism in DEPDC5 causes focal epilepsy with FCD. We discovered a mutation gradient with a higher rate of mosaicism in the seizure-onset zone than in the surrounding epileptogenic zone. Furthermore, we demonstrate the causality of a Depdc5 brain mosaic inactivation using CRISPR-Cas9 editing and in utero electroporation in a mouse model recapitulating focal epilepsy with FCD and SUDEP-like events. We further unveil a key role of Depdc5 in shaping dendrite and spine morphology of excitatory neurons. This study reveals promising therapeutic avenues for treating drug-resistant focal epilepsies with mTORC1-targeting molecules.

  2. mTORC1 Coordinates Protein Synthesis and Immunoproteasome Formation via PRAS40 to Prevent Accumulation of Protein Stress.

    Science.gov (United States)

    Yun, Young Sung; Kim, Kwan Hyun; Tschida, Barbara; Sachs, Zohar; Noble-Orcutt, Klara E; Moriarity, Branden S; Ai, Teng; Ding, Rui; Williams, Jessica; Chen, Liqiang; Largaespada, David; Kim, Do-Hyung

    2016-02-18

    Reduction of translational fidelity often occurs in cells with high rates of protein synthesis, generating defective ribosomal products. If not removed, such aberrant proteins can be a major source of cellular stress causing human diseases. Here, we demonstrate that mTORC1 promotes the formation of immunoproteasomes for efficient turnover of defective proteins and cell survival. mTORC1 sequesters precursors of immunoproteasome β subunits via PRAS40. When activated, mTORC1 phosphorylates PRAS40 to enhance protein synthesis and simultaneously to facilitate the assembly of the β subunits for forming immunoproteasomes. Consequently, the PRAS40 phosphorylations play crucial roles in clearing aberrant proteins that accumulate due to mTORC1 activation. Mutations of RAS, PTEN, and TSC1, which cause mTORC1 hyperactivation, enhance immunoproteasome formation in cells and tissues. Those mutations increase cellular dependence on immunoproteasomes for stress response and survival. These results define a mechanism by which mTORC1 couples elevated protein synthesis with immunoproteasome biogenesis to protect cells against protein stress. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. The Human Papillomavirus Type 16 E6 Oncoprotein Activates mTORC1 Signaling and Increases Protein Synthesis ▿ †

    OpenAIRE

    Spangle, Jennifer M.; Münger, Karl

    2010-01-01

    The mammalian target of rapamycin (mTOR) kinase acts as a cellular rheostat that integrates signals from a variety of cellular signal transduction pathways that sense growth factor and nutrient availability as well as intracellular energy status. It was previously reported that the human papillomavirus type 16 (HPV16) E6 oncoprotein may activate the S6 protein kinase (S6K) through binding and E6AP-mediated degradation of the mTOR inhibitor tuberous sclerosis complex 2 (TSC2) (Z. Lu, X. Hu, Y....

  4. GTPase ROP2 binds and promotes activation of target of rapamycin, TOR, in response to auxin.

    Science.gov (United States)

    Schepetilnikov, Mikhail; Makarian, Joelle; Srour, Ola; Geldreich, Angèle; Yang, Zhenbiao; Chicher, Johana; Hammann, Philippe; Ryabova, Lyubov A

    2017-04-03

    Target of rapamycin (TOR) promotes reinitiation at upstream ORFs (uORFs) in genes that play important roles in stem cell regulation and organogenesis in plants. Here, we report that the small GTPase ROP2, if activated by the phytohormone auxin, promotes activation of TOR, and thus translation reinitiation of uORF-containing mRNAs. Plants with high levels of active ROP2, including those expressing constitutively active ROP2 (CA-ROP2), contain high levels of active TOR ROP2 physically interacts with and, when GTP-bound, activates TOR in vitro TOR activation in response to auxin is abolished in ROP-deficient rop2 rop6 ROP4 RNAi plants. GFP-TOR can associate with endosome-like structures in ROP2-overexpressing plants, indicating that endosomes mediate ROP2 effects on TOR activation. CA-ROP2 is efficient in loading uORF-containing mRNAs onto polysomes and stimulates translation in protoplasts, and both processes are sensitive to TOR inhibitor AZD-8055. TOR inactivation abolishes ROP2 regulation of translation reinitiation, but not its effects on cytoskeleton or intracellular trafficking. These findings imply a mode of translation control whereby, as an upstream effector of TOR, ROP2 coordinates TOR function in translation reinitiation pathways in response to auxin. © 2017 The Authors.

  5. Large Intergenic Non-coding RNA-RoR Inhibits Aerobic Glycolysis of Glioblastoma Cells via Akt Pathway

    Science.gov (United States)

    Li, Yong; He, Zhi-Cheng; Liu, Qing; Zhou, Kai; Shi, Yu; Yao, Xiao-Hong; Zhang, Xia; Kung, Hsiang-Fu; Ping, Yi-Fang; Bian, Xiu-Wu

    2018-01-01

    Reprogramming energy metabolism is a hallmark of malignant tumors, including glioblastoma (GBM). Aerobic glycolysis is often utilized by tumor cells to maintain survival and proliferation. However, the underlying mechanisms of aerobic glycolysis in GBM remain elusive. Herein, we demonstrated that large intergenic non-coding RNA-RoR (LincRNA-RoR) functioned as a critical suppressor to inhibit the aerobic glycolysis and viability of GBM cells. We found that LincRNA-RoR was markedly reduced in GBM tissues compared with adjacent non-tumor tissues from 10 cases of GBM patients. Consistently, LincRNA-RoR expression in GBM cells was significantly lower than that in normal glial cells. The aerobic glycolysis of GBM cells, as determined by the measurement of glucose uptake and lactate production, was impaired by LincRNA-RoR overexpression. Mechanistically, LincRNA-RoR inhibited the expression of Rictor, the key component of mTORC2 (mammalian target of rapamycin complex 2), to suppress the activity of Akt pathway and impair the expression of glycolytic effectors, including Glut1, HK2, PKM2 and LDHA. Finally, enforced expression of LincRNA-RoR reduced the proliferation of GBM cells in vitro, restrained tumor growth in vivo, and repressed the expression of glycolytic molecules in GBM xenografts. Collectively, our results underscore LincRNA-RoR as a new suppressor of GBM aerobic glycolysis with therapeutic potential. PMID:29581766

  6. Inhibition of Bcl-2 potentiates AZD-2014-induced anti-head and neck squamous cell carcinoma cell activity

    International Nuclear Information System (INIS)

    Li, Yi; Cui, Jiang-Tao

    2016-01-01

    Mammalian target of rapamycin (mTOR) is a therapeutic target for head and neck squamous cell carcinoma (HNSCC). Here, we evaluated the activity of AZD-2014, a potent mTOR complex 1/2 (mTORC1/2) dual inhibitor, against HNSCC cells. We showed that AZD-2014 blocked mTORC1/2 activation in established and primary human HNSCC cells, where it was anti-proliferative and pro-apoptotic. Yet, AZD-2014 was non-cytotoxic to the human oral epithelial cells with low basal mTORC1/2 activation. In an effect to identify possible AZD-2014 resistance factors, we showed that the anti-apoptosis protein Bcl-2 was upregulated in AZD-2014-resistant SQ20B HNSCC cells. Inhibition of Bcl-2 by ABT-737 (a known Bcl-2 inhibitor) or Bcl-2 shRNA dramatically potentiated AZD-2014 lethality against HNSCC cells. On the other hand, exogenous overexpression of Bcl-2 largely attenuated AZD-2014’s activity against HNSCC cells. For the in vivo studies, we showed that oral gavage of AZD-2014 suppressed SQ20B xenograft growth in severe combined immunodeficient (SCID) mice. It also significantly improved mice survival. Importantly, AZD-2014’s anti-HNSCC activity in vivo was potentiated with co-administration of ABT-737. The preclinical results of this study suggest that AZD-2014 could be further tested as a valuable anti-HNSCC agent, either alone or in combination with Bcl-2 inhibitors. - Highlights: • AZD-2014 blocks mTORC1/2 activation in HNSCC cells. • AZD-2014 suppresses HNSCC cell proliferation. • AZD-2014 activates caspase-3 and apoptosis in HNSCC cells. • Bcl-2 is the key resistance factor of AZD-2014 in HNSCC cells. • ABT-737 sensitizes AZD-2014-induced anti-HNSCC activity in vivo.

  7. Inhibition of Bcl-2 potentiates AZD-2014-induced anti-head and neck squamous cell carcinoma cell activity

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yi; Cui, Jiang-Tao, E-mail: cuijingtaopaper@126.com

    2016-09-02

    Mammalian target of rapamycin (mTOR) is a therapeutic target for head and neck squamous cell carcinoma (HNSCC). Here, we evaluated the activity of AZD-2014, a potent mTOR complex 1/2 (mTORC1/2) dual inhibitor, against HNSCC cells. We showed that AZD-2014 blocked mTORC1/2 activation in established and primary human HNSCC cells, where it was anti-proliferative and pro-apoptotic. Yet, AZD-2014 was non-cytotoxic to the human oral epithelial cells with low basal mTORC1/2 activation. In an effect to identify possible AZD-2014 resistance factors, we showed that the anti-apoptosis protein Bcl-2 was upregulated in AZD-2014-resistant SQ20B HNSCC cells. Inhibition of Bcl-2 by ABT-737 (a known Bcl-2 inhibitor) or Bcl-2 shRNA dramatically potentiated AZD-2014 lethality against HNSCC cells. On the other hand, exogenous overexpression of Bcl-2 largely attenuated AZD-2014’s activity against HNSCC cells. For the in vivo studies, we showed that oral gavage of AZD-2014 suppressed SQ20B xenograft growth in severe combined immunodeficient (SCID) mice. It also significantly improved mice survival. Importantly, AZD-2014’s anti-HNSCC activity in vivo was potentiated with co-administration of ABT-737. The preclinical results of this study suggest that AZD-2014 could be further tested as a valuable anti-HNSCC agent, either alone or in combination with Bcl-2 inhibitors. - Highlights: • AZD-2014 blocks mTORC1/2 activation in HNSCC cells. • AZD-2014 suppresses HNSCC cell proliferation. • AZD-2014 activates caspase-3 and apoptosis in HNSCC cells. • Bcl-2 is the key resistance factor of AZD-2014 in HNSCC cells. • ABT-737 sensitizes AZD-2014-induced anti-HNSCC activity in vivo.

  8. Effects of chronic Akt/mTOR inhibition by rapamycin on mechanical overload-induced hypertrophy and myosin heavy chain transition in masseter muscle.

    Science.gov (United States)

    Umeki, Daisuke; Ohnuki, Yoshiki; Mototani, Yasumasa; Shiozawa, Kouichi; Fujita, Takayuki; Nakamura, Yoshiki; Saeki, Yasutake; Okumura, Satoshi

    2013-01-01

    To examine the effects of the Akt/mammalian target of rapamycin (mTOR) pathway on masseter muscle hypertrophy and myosin heavy chain (MHC) transition in response to mechanical overload, we analyzed the effects of bite-opening (BO) on the hypertrophy and MHC composition of masseter muscle of BO-rats treated or not treated with rapamycin (RAPA), a selective mTOR inhibitor. The masseter muscle weight in BO-rats was significantly greater than that in controls, and this increase was attenuated by RAPA treatment. Expression of slow-twitch MHC isoforms was significantly increased in BO-rats with/without RAPA treatment, compared with controls, but the magnitude of the increase was much smaller in RAPA-treated BO-rats. Phosphorylation of p44/42 MAPK (ERK1/2), which preserves fast-twitch MHC isoforms in skeletal muscle, was significantly decreased in BO-rats, but the decrease was abrogated by RAPA treatment. Calcineurin signaling is known to be important for masseter muscle hypertrophy and fast-to-slow MHC isoform transition, but expression of known calcineurin activity modulators was unaffected by RAPA treatment. Taken together, these results indicate that the Akt/mTOR pathway is involved in both development of masseter muscle hypertrophy and fast-to-slow MHC isoform transition in response to mechanical overload with inhibition of the ERK1/2 pathway and operates independently of the calcineurin pathway.

  9. Obesity challenges the hepatoprotective function of the integrated stress response to asparaginase exposure in mice.

    Science.gov (United States)

    Nikonorova, Inna A; Al-Baghdadi, Rana J T; Mirek, Emily T; Wang, Yongping; Goudie, Michael P; Wetstein, Berish B; Dixon, Joseph L; Hine, Christopher; Mitchell, James R; Adams, Christopher M; Wek, Ronald C; Anthony, Tracy G

    2017-04-21

    Obesity increases risk for liver toxicity by the anti-leukemic agent asparaginase, but the mechanism is unknown. Asparaginase activates the integrated stress response (ISR) via sensing amino acid depletion by the eukaryotic initiation factor 2 (eIF2) kinase GCN2. The goal of this work was to discern the impact of obesity, alone versus alongside genetic disruption of the ISR, on mechanisms of liver protection during chronic asparaginase exposure in mice. Following diet-induced obesity, biochemical analysis of livers revealed that asparaginase provoked hepatic steatosis that coincided with activation of another eIF2 kinase PKR-like endoplasmic reticulum kinase (PERK), a major ISR transducer to ER stress. Genetic loss of Gcn2 intensified hepatic PERK activation to asparaginase, yet surprisingly, mRNA levels of key ISR gene targets such as Atf5 and Trib3 failed to increase. Instead, mechanistic target of rapamycin complex 1 (mTORC1) signal transduction was unleashed, and this coincided with liver dysfunction reflected by a failure to maintain hydrogen sulfide production or apolipoprotein B100 (ApoB100) expression. In contrast, obese mice lacking hepatic activating transcription factor 4 ( Atf4 ) showed an exaggerated ISR and greater loss of endogenous hydrogen sulfide but normal inhibition of mTORC1 and maintenance of ApoB100 during asparaginase exposure. In both genetic mouse models, expression and phosphorylation of Sestrin2, an ATF4 gene target, was increased by asparaginase, suggesting mTORC1 inhibition during asparaginase exposure is not driven via eIF2-ATF4-Sestrin2. In conclusion, obesity promotes a maladaptive ISR during asparaginase exposure. GCN2 functions to repress mTORC1 activity and maintain ApoB100 protein levels independently of Atf4 expression, whereas hydrogen sulfide production is promoted via GCN2-ATF4 pathway. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. The antidepressant sertraline inhibits translation initiation by curtailing mammalian target of rapamycin signaling.

    Science.gov (United States)

    Lin, Chen-Ju; Robert, Francis; Sukarieh, Rami; Michnick, Stephen; Pelletier, Jerry

    2010-04-15

    Sertraline, a selective serotonin reuptake inhibitor, is a widely used antidepressant agent. Here, we show that sertraline also exhibits antiproliferative activity. Exposure to sertraline leads to a concentration-dependent decrease in protein synthesis. Moreover, polysome profile analysis of sertraline-treated cells shows a reduction in polysome content and a concomitant increase in 80S ribosomes. The inhibition in translation caused by sertraline is associated with decreased levels of the eukaryotic initiation factor (eIF) 4F complex, altered localization of eIF4E, and increased eIF2alpha phosphorylation. The latter event leads to increased REDD1 expression, which in turn impinges on the mammalian target of rapamycin (mTOR) pathway by affecting TSC1/2 signaling. Sertraline also independently targets the mTOR signaling pathway downstream of Rheb. In the Emu-myc murine lymphoma model where carcinogenesis is driven by phosphatase and tensin homologue (PTEN) inactivation, sertraline is able to enhance chemosensitivity to doxorubicin. Our results indicate that sertraline exerts antiproliferative activity by targeting the mTOR signaling pathway in a REDD1-dependent manner. (c) 2010 AACR.

  11. Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK, DNA Synthesis and Proliferation in Pancreatic Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Ming Ming

    Full Text Available Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC cells (PANC-1, MiaPaCa-2 with the isoquinoline alkaloid berberine (0.3-6 µM inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70% the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC at Ser79. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr389 and S6 at Ser240/244 and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α1 and α2 catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.

  12. Effects of rapamycin treatment after controlled cortical impact injury on neurogenesis and synaptic reorganization in the mouse dentate gyrus

    Directory of Open Access Journals (Sweden)

    Corwin R Butler

    2015-11-01

    Full Text Available Post-traumatic epilepsy (PTE is one consequence of traumatic brain injury (TBI. A prominent cell signaling pathway activated in animal models of both TBI and epilepsy is the mammalian target of rapamycin (mTOR. Inhibition of mTOR with rapamycin has shown promise as a potential modulator of epileptogenesis in several animal models of epilepsy, but cellular mechanisms linking mTOR expression and epileptogenesis are unclear. In this study, the role of mTOR in modifying functional hippocampal circuit reorganization after focal TBI induced by controlled cortical impact was investigated. Rapamycin (3 or 10 mg/kg, an inhibitor of mTOR signaling, was administered by intraperitoneal injection beginning on the day of injury and continued daily until tissue collection. Relative to controls, rapamycin treatment reduced dentate granule cell area in the hemisphere ipsilateral to the injury two weeks post-injury. Brain injury resulted in a significant increase in doublecortin immunolabeling in the dentate gyrus ipsilateral to the injury, indicating increased neurogenesis shortly after TBI. Rapamycin treatment prevented the increase in doublecortin labeling, with no overall effect on Fluoro-Jade B staining in the ipsilateral hemisphere, suggesting that rapamycin treatment reduced posttraumatic neurogenesis but did not prevent cell loss after injury. At later times post-injury (8-13 weeks, evidence of mossy fiber sprouting and increased recurrent excitation of dentate granule cells was detected, which were attenuated by rapamycin treatment. Rapamycin treatment also diminished seizure prevalence relative to vehicle-treated controls after TBI. Collectively, these results support a role for adult neurogenesis in PTE development and suggest that suppression of epileptogenesis by mTOR inhibition includes effects on post-injury neurogenesis.

  13. Photochemical transformation of the insensitive munitions compound 2,4-dinitroanisole

    Energy Technology Data Exchange (ETDEWEB)

    Rao, Balaji, E-mail: anandharaob@ornl.gov [Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN (United States); Wang, Wei [Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN (United States); Cai, Qingsong; Anderson, Todd [Department of Environmental Toxicology, The Institute of Environment and Human Health, Texas Tech University, Lubbock, TX (United States); Gu, Baohua, E-mail: gub1@ornl.gov [Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN (United States)

    2013-01-15

    The insensitive munitions compound 2,4-dinitroanisole (DNAN) is increasingly being used as a replacement for traditional, sensitive munitions compounds (e.g., trinitrotoluene [TNT]), but the environmental fate and photo-transformation of DNAN in natural water systems are currently unknown. In this study, we investigated the photo-transformation rates of DNAN with both ultraviolet (UV) and sunlight irradiation under different environmentally relevant conditions. Sunlight photo-transformation of DNAN in water was found to follow predominantly pseudo-first-order decay kinetics with an average half-life (t{sub 1/2}) of approximately 0.70 d and activation energy (E{sub a}) of 53 kJ mol{sup −1}. Photo-transformation rates of DNAN were dependent on the wavelength of the light source: irradiation with UV-B light (280–315 nm) resulted in a greater quantum yield of transformation (ϕ{sub UV-B} = 3.7 × 10{sup −4}) than rates obtained with UV-A light (ϕ{sub UV-A} = 2.9 × 10{sup −4} at 316–400 nm) and sunlight (ϕ{sub sun} = 1.1 × 10{sup −4}). Photo-oxidation was the dominant mechanism for DNAN photo-transformation, based on the formation of nitrite (NO{sub 2}{sup −}) and nitrate (NO{sub 3}{sup −}) as major N species and 2,4-dinitrophenol as the minor species. Environmental factors (e.g., temperature, pH, and the presence or absence of naturally dissolved organic matter) displayed modest to little effects on the rate of DNAN photo-transformation. These observations indicate that sunlight-induced photo-transformation of DNAN may represent a significant abiotic degradation pathway in surface water, which may have important implications in evaluating the potential impacts and risks of DNAN in the environment. - Highlights: ► DNAN photo-transformation kinetics was dependent on light source and temperature. ► Photolysis produced harmful by-products that included dinitrophenol and nitrate. ► Photo-oxidation was determined to be the likely pathway of DNAN

  14. C. elegans DAF-16/FOXO interacts with TGF-ß/BMP signaling to induce germline tumor formation via mTORC1 activation.

    Directory of Open Access Journals (Sweden)

    Wenjing Qi

    2017-05-01

    Full Text Available Activation of the FOXO transcription factor DAF-16 by reduced insulin/IGF signaling (IIS is considered to be beneficial in C. elegans due to its ability to extend lifespan and to enhance stress resistance. In the germline, cell-autonomous DAF-16 activity prevents stem cell proliferation, thus acting tumor-suppressive. In contrast, hypodermal DAF-16 causes a tumorous germline phenotype characterized by hyperproliferation of the germline stem cells and rupture of the adjacent basement membrane. Here we show that cross-talk between DAF-16 and the transforming growth factor ß (TGFß/bone morphogenic protein (BMP signaling pathway causes germline hyperplasia and results in disruption of the basement membrane. In addition to activating MADM/NRBP/hpo-11 gene alone, DAF-16 also directly interacts with both R-SMAD proteins SMA-2 and SMA-3 in the nucleus to regulate the expression of mTORC1 pathway. Knocking-down of BMP genes or each of the four target genes in the hypodermis was sufficient to inhibit germline proliferation, indicating a cell-non-autonomously controlled regulation of stem cell proliferation by somatic tissues. We propose the existence of two antagonistic DAF-16/FOXO functions, a cell-proliferative somatic and an anti-proliferative germline activity. Whereas germline hyperplasia under reduced IIS is inhibited by DAF-16 cell-autonomously, activation of somatic DAF-16 in the presence of active IIS promotes germline proliferation and eventually induces tumor-like germline growth. In summary, our results suggest a novel pathway crosstalk of DAF-16 and TGF-ß/BMP that can modulate mTORC1 at the transcriptional level to cause stem-cell hyperproliferation. Such cell-type specific differences may help explaining why human FOXO activity is considered to be tumor-suppressive in most contexts, but may become oncogenic, e.g. in chronic and acute myeloid leukemia.

  15. C. elegans DAF-16/FOXO interacts with TGF-ß/BMP signaling to induce germline tumor formation via mTORC1 activation.

    Science.gov (United States)

    Qi, Wenjing; Yan, Yijian; Pfeifer, Dietmar; Donner V Gromoff, Erika; Wang, Yimin; Maier, Wolfgang; Baumeister, Ralf

    2017-05-01

    Activation of the FOXO transcription factor DAF-16 by reduced insulin/IGF signaling (IIS) is considered to be beneficial in C. elegans due to its ability to extend lifespan and to enhance stress resistance. In the germline, cell-autonomous DAF-16 activity prevents stem cell proliferation, thus acting tumor-suppressive. In contrast, hypodermal DAF-16 causes a tumorous germline phenotype characterized by hyperproliferation of the germline stem cells and rupture of the adjacent basement membrane. Here we show that cross-talk between DAF-16 and the transforming growth factor ß (TGFß)/bone morphogenic protein (BMP) signaling pathway causes germline hyperplasia and results in disruption of the basement membrane. In addition to activating MADM/NRBP/hpo-11 gene alone, DAF-16 also directly interacts with both R-SMAD proteins SMA-2 and SMA-3 in the nucleus to regulate the expression of mTORC1 pathway. Knocking-down of BMP genes or each of the four target genes in the hypodermis was sufficient to inhibit germline proliferation, indicating a cell-non-autonomously controlled regulation of stem cell proliferation by somatic tissues. We propose the existence of two antagonistic DAF-16/FOXO functions, a cell-proliferative somatic and an anti-proliferative germline activity. Whereas germline hyperplasia under reduced IIS is inhibited by DAF-16 cell-autonomously, activation of somatic DAF-16 in the presence of active IIS promotes germline proliferation and eventually induces tumor-like germline growth. In summary, our results suggest a novel pathway crosstalk of DAF-16 and TGF-ß/BMP that can modulate mTORC1 at the transcriptional level to cause stem-cell hyperproliferation. Such cell-type specific differences may help explaining why human FOXO activity is considered to be tumor-suppressive in most contexts, but may become oncogenic, e.g. in chronic and acute myeloid leukemia.

  16. Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies.

    Science.gov (United States)

    Le Plénier, Servane; Goron, Arthur; Sotiropoulos, Athanassia; Archambault, Eliane; Guihenneuc, Chantal; Walrand, Stéphane; Salles, Jérome; Jourdan, Marion; Neveux, Nathalie; Cynober, Luc; Moinard, Christophe

    2017-01-01

    Citrulline (CIT) is an endogenous amino acid produced by the intestine. Recent literature has consistently shown CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both interorgan and intraorgan trafficking and metabolism, we combined three approaches: in vivo, ex vivo, and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated (P < 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, P < 0.05), and the CIT effect was abolished in the presence of wortmannin, rapamycin, and PD-98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD-98059 inhibited the CIT effect on S6K1, whereas only LY-294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control. Copyright © 2017 the American Physiological Society.

  17. Tomato FK506 Binding Protein 12KD (FKBP12 mediates the interaction between rapamycin and Target of Rapamycin (TOR

    Directory of Open Access Journals (Sweden)

    Fangjie Xiong

    2016-11-01

    Full Text Available Target of Rapamycin (TOR signaling is an important regulator in multiple organisms including yeast, plants and animals. However, the TOR signaling in plants is much less understood as compared to that in yeast and animals. TOR kinase can be efficiently suppressed by rapamycin in the presence of functional FK506 Binding Protein 12KD (FKBP12 in yeast and animals. In most examined higher plants rapamycin fails to inhibit TOR kinase due to the non-functional FKBP12. Here we find that tomato plants showed obvious growth inhibition when treated with rapamycin and the inhibitory phenotype is similar to suppression of TOR causing by active-site TOR inhibitors (asTORis such as KU63794, AZD8055 and Torin1. The chemical genetic assays using TOR inhibitors and heterologous expressing SlFKBP12 in Arabidopsis indicated that the TOR signaling is functional in tomato. The protein gel shifting and TOR inhibitors combination assays showed that SlFKBP12 can mediate the interaction between rapamycin and TOR. Furthermore, comparative expression profiling analysis between treatments with rapamycin and KU63794 identified highly overlapped Differentially Expressed Genes (DEGs which are involved in many anabolic and catabolic processes, such as photosynthesis, cell wall restructuring, and senescence in tomato. These observations suggest that SlFFBP12 is functional in tomato. The results provided basic information of TOR signaling in tomato, and also some new insights into how TOR controls plant growth and development through reprogramming the transcription profiles

  18. Tomato FK506 Binding Protein 12KD (FKBP12) Mediates the Interaction between Rapamycin and Target of Rapamycin (TOR).

    Science.gov (United States)

    Xiong, Fangjie; Dong, Pan; Liu, Mei; Xie, Gengxin; Wang, Kai; Zhuo, Fengping; Feng, Li; Yang, Lu; Li, Zhengguo; Ren, Maozhi

    2016-01-01

    Target of Rapamycin (TOR) signaling is an important regulator in multiple organisms including yeast, plants, and animals. However, the TOR signaling in plants is much less understood as compared to that in yeast and animals. TOR kinase can be efficiently suppressed by rapamycin in the presence of functional FK506 Binding Protein 12 KD (FKBP12) in yeast and animals. In most examined higher plants rapamycin fails to inhibit TOR kinase due to the non-functional FKBP12. Here we find that tomato plants showed obvious growth inhibition when treated with rapamycin and the inhibitory phenotype is similar to suppression of TOR causing by active-site TOR inhibitors (asTORis) such as KU63794, AZD8055, and Torin1. The chemical genetic assays using TOR inhibitors and heterologous expressing SlFKBP12 in Arabidopsis indicated that the TOR signaling is functional in tomato. The protein gel shifting and TOR inhibitors combination assays showed that SlFKBP12 can mediate the interaction between rapamycin and TOR. Furthermore, comparative expression profile analysis between treatments with rapamycin and KU63794 identified highly overlapped Differentially Expressed Genes (DEGs) which are involved in many anabolic and catabolic processes, such as photosynthesis, cell wall restructuring, and senescence in tomato. These observations suggest that SlFFBP12 is functional in tomato. The results provided basic information of TOR signaling in tomato, and also some new insights into how TOR controls plant growth and development through reprogramming the transcription profiles.

  19. Rapamycin protects kidney against ischemia reperfusion injury through recruitment of NKT cells.

    Science.gov (United States)

    Zhang, Chao; Zheng, Long; Li, Long; Wang, Lingyan; Li, Liping; Huang, Shang; Gu, Chenli; Zhang, Lexi; Yang, Cheng; Zhu, Tongyu; Rong, Ruiming

    2014-08-19

    NKT cells play a protective role in ischemia reperfusion (IR) injury, of which the trafficking in the body and recruitment in injured organs can be influenced by immunosuppressive therapy. Therefore, we investigated the effects of rapamycin on kidneys exposed to IR injury in early stage and on trafficking of NKT cells in a murine model. Balb/c mice were subjected to kidney 30 min ischemia followed by 24 h reperfusion. Rapamycin (2.5 ml/kg) was administered by gavage daily, starting 1 day before the operation. Renal function and histological changes were assessed. The proportion of NKT cells in peripheral blood, spleen and kidney was detected by flow cytometry. The chemokines and corresponding receptor involved in NKT cell trafficking were determined by RT-PCR and flow cytometry respectively. Rapamycin significantly improved renal function and ameliorated histological injury. In rapamycin-treated group, the proportion of NKT cells in spleen was significantly decreased but increased in peripheral blood and kidney. In addition, the CXCR3+ NKT cell in the kidney increased remarkably in the rapamycin-treated group. The chemokines, CXCL9 and CXCL10, as the ligands of CXCR3, were also increased in the rapamycin-treated kidney. Rapamycin may recruit NKT cells from spleen to the IR-induced kidney to ameliorate renal IR injury in the early stage.

  20. The Inhibitory Effect of Rapamycin on Toll Like Receptor 4 and Interleukin 17 in the Early Stage of Rat Diabetic Nephropathy

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    Ruichao Yu

    2016-02-01

    Full Text Available Background/Aims: There is increasing evidence showing that innate immune responses and inflammatory processes play an important role in the development and progression of diabetic nephropathy (DN. The potential effect of innate immunity in the early stage of DN is still unclear. Toll-Like-Receptor 4 (TLR4 is vigorously involved in the progress of kidney diseases in a sterile environment. The activation of the interleukin 17 (IL-17 pathway produces inflammatory cytokines, appearing in various kidney diseases. Unfortunately the relationship between TLR4 and IL-17 has not been investigated in diabetic nephropathy to date. The aim of this study is to investigate whether mammalian target of rapamycin (mTOR inhibition may be dependent on TLR4 signaling and the pro-inflammatory factor IL-17 to delay the progression of DN. Methods: Streptozotocin (STZ-induced diabetic rats were randomly assigned to 3 experimental groups: a diabetic nephropathy group (DN, n = 6; and a diabetic nephropathy treated with rapamycin group (Rapa, n = 6 and a control group (Control, n =6. Body weight, fasting blood sugar, and 24h urine albumin were assessed at week 2, week 4 and week 8. Renal tissues were harvested for H&E, PAS staining, as well as an immunohistochemistry assay for TLR4 and IL-17. TLR4 quantitative expression was measured by Western-Blot analysis and RT-PCR. Results: Our results demonstrated that the expression of both TLR4 and IL-17 were upregulated in early stage DN and reduced by rapamycin. TLR4 and IL-17 both increased and positively related to 24h urinary albumin and kidney/weight ratio. However, neither TLR4 nor IL-17 made a significant difference on fasting blood sugar. Conclusions: Taken together, our results confirm and extend previous studies identifying the significance of the TLR4 and Th17 pathways in development of early stage DN. Furthermore, we suggest this overexpression of TLR4 might be involved in the immunopathogenesis of DN through

  1. Prevention of age-related macular degeneration-like retinopathy by rapamycin in rats.

    Science.gov (United States)

    Kolosova, Nataliya G; Muraleva, Natalia A; Zhdankina, Anna A; Stefanova, Natalia A; Fursova, Anzhela Z; Blagosklonny, Mikhail V

    2012-08-01

    Age-related macular degeneration, a neurodegenerative and vascular retinal disease, is the most common cause of blindness in the Western countries. Evidence accumulates that target of rapamycin is involved in aging and age-related diseases, including neurodegeneration. The target of rapamycin inhibitor, rapamycin, suppresses the senescent cell phenotype and extends life span in diverse species, including mice. Rapamycin decreases senescence-associated phenotypes in retinal pigment epithelial cells in culture. Herein, we investigated the effect of rapamycin on spontaneous retinopathy in senescence-accelerated OXYS rats, an animal model of age-related macular degeneration. Rats were treated with either 0.1 or 0.5 mg/kg rapamycin, which was given orally as a food mixture. In a dose-dependent manner, rapamycin decreased the incidence and severity of retinopathy. Rapamycin improved some (but not all) histological abnormalities associated with retinopathy. Thus, in retinal pigment epithelial cell layers, rapamycin decreased nuclei heterogeneity and normalized intervals between nuclei. In photoreceptor cells, associated neurons, and radial glial cells, rapamycin prevented nuclear and cellular pyknosis. More important, rapamycin prevented destruction of ganglionar neurons in the retina. Rapamycin did not exert any adverse effects on the retina in control disease-free Wistar rats. Taken together, our data suggest the therapeutic potential of rapamycin for treatment and prevention of retinopathy. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  2. Novel synergistic antitumor effects of rapamycin with bortezomib on hepatocellular carcinoma cells and orthotopic tumor model

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    Wang Cun

    2012-05-01

    Full Text Available Abstract Background Despite recent advances in the treatment of hepatocellular carcinoma (HCC, the chemotherapy efficacy against HCC is still unsatisfactory. The mammalian target of rapamycin (mTOR has been emerged as an important cancer therapeutic target. However, HCC cells often resistant to rapamycin because of the paradoxical activation of Akt by rapamycin. In this study, we investigated whether bortezomib could enhance the antitumor effects of rapamycin. Methods The effects of rapamycin and bortezomib on HCC proliferation, apoptosis, migration, and invasiveness in vitro were assessed by CCK-8 analysis, flow cytometry, Hoechst 33342 staining and transwell assays, respectively. Total and phosphorylated protein levels of Akt were detected by Western blotting. The effects of rapamycin and/or bortezomib on the mRNA expression levels of p53, p27, p21 and Bcl-2 family in HCCLM3 cells were evaluated by RT-PCR. The roles of rapamycin and bortezomib on HCC growth and metastasis in xenograft models were evaluated by tumor volumes and fluorescent signals. The effects of rapamycin and bortezomib on cell proliferation and apoptosis in vivo were test by PCNA and TUNEL staining. Results Bortezomib synergized with rapamycin to reduce cell growth, induce apoptosis, and inhibit cell mobility in vitro. Further mechanistic studies showed that bortezomib inhibited rapamycin-induced phosphorylated Akt, which in turn enhanced apoptosis of HCC cell lines. The alteration of the mRNA expression of cell cycle inhibitors p53, p27, p21 and apoptosis associated genes Bcl-2, Bax were also involved in the synergistic antitumor effects of rapamycin and bortezomib. P53 inhibitor PFT-α significantly attenuate the effect of rapamycin and bortezomib on cell apoptosis, which indicated that the pro-apoptotic effect of rapamycin and bortezomib may be p53-dependent. Treatment of HCCLM3-R bearing nude mice with rapamycin and bortezomib significantly enhanced tumor growth

  3. Target of rapamycin signalling mediates the lifespan-extending effects of dietary restriction by essential amino acid alteration

    NARCIS (Netherlands)

    Emran, S.; Yang, M.Y.; He, X.L.; Zandveld, J.; Piper, M.D.W.

    2014-01-01

    Dietary restriction (DR), defined as a moderate reduction in food intake short of malnutrition, has been shown to extend healthy lifespan in a diverse range of organisms, from yeast to primates. Reduced signalling through the insulin/IGF-like (IIS) and Target of Rapamycin (TOR) signalling pathways

  4. Effects of rapamycin and curcumin treatment on the development of epilepsy after electrically induced status epilepticus in rats.

    NARCIS (Netherlands)

    Drion, C.M.; Borm, L.E.; Kooijman, L.; Aronica, E.; Wadman, W.J.; Hartog, A.F.; van Vliet, E.A.; Gorter, J.A.

    2016-01-01

    OBJECTIVE: Inhibition of the mammalian target of rapamycin (mTOR) pathway has been suggested as a possible antiepileptogenic strategy in temporal lobe epilepsy (TLE). Here we aim to elucidate whether mTOR inhibition has antiepileptogenic and/or antiseizure effects using different treatment

  5. A novel mouse model of tuberous sclerosis complex (TSC): eye-specific Tsc1-ablation disrupts visual-pathway development.

    Science.gov (United States)

    Jones, Iwan; Hägglund, Anna-Carin; Törnqvist, Gunilla; Nord, Christoffer; Ahlgren, Ulf; Carlsson, Leif

    2015-12-01

    Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome that is best characterised by neurodevelopmental deficits and the presence of benign tumours (called hamartomas) in affected organs. This multi-organ disorder results from inactivating point mutations in either the TSC1 or the TSC2 genes and consequent activation of the canonical mammalian target of rapamycin complex 1 signalling (mTORC1) pathway. Because lesions to the eye are central to TSC diagnosis, we report here the generation and characterisation of the first eye-specific TSC mouse model. We demonstrate that conditional ablation of Tsc1 in eye-committed progenitor cells leads to the accelerated differentiation and subsequent ectopic radial migration of retinal ganglion cells. This results in an increase in retinal ganglion cell apoptosis and consequent regionalised axonal loss within the optic nerve and topographical changes to the contra- and ipsilateral input within the dorsal lateral geniculate nucleus. Eyes from adult mice exhibit aberrant retinal architecture and display all the classic neuropathological hallmarks of TSC, including an increase in organ and cell size, ring heterotopias, hamartomas with retinal detachment, and lamination defects. Our results provide the first major insight into the molecular etiology of TSC within the developing eye and demonstrate a pivotal role for Tsc1 in regulating various aspects of visual-pathway development. Our novel mouse model therefore provides a valuable resource for future studies concerning the molecular mechanisms underlying TSC and also as a platform to evaluate new therapeutic approaches for the treatment of this multi-organ disorder. © 2015. Published by The Company of Biologists Ltd.

  6. Differential effects of rapamycin and dexamethasone in mouse models of established allergic asthma.

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    Elizabeth M Mushaben

    Full Text Available The mammalian target of rapamycin (mTOR plays an important role in cell growth/differentiation, integrating environmental cues, and regulating immune responses. Our lab previously demonstrated that inhibition of mTOR with rapamycin prevented house dust mite (HDM-induced allergic asthma in mice. Here, we utilized two treatment protocols to investigate whether rapamycin, compared to the steroid, dexamethasone, could inhibit allergic responses during the later stages of the disease process, namely allergen re-exposure and/or during progression of chronic allergic disease. In protocol 1, BALB/c mice were sensitized to HDM (three i.p. injections and administered two intranasal HDM exposures. After 6 weeks of rest/recovery, mice were re-exposed to HDM while being treated with rapamycin or dexamethasone. In protocol 2, mice were exposed to HDM for 3 or 6 weeks and treated with rapamycin or dexamethasone during weeks 4-6. Characteristic features of allergic asthma, including IgE, goblet cells, airway hyperreactivity (AHR, inflammatory cells, cytokines/chemokines, and T cell responses were assessed. In protocol 1, both rapamycin and dexamethasone suppressed goblet cells and total CD4(+ T cells including activated, effector, and regulatory T cells in the lung tissue, with no effect on AHR or total inflammatory cell numbers in the bronchoalveolar lavage fluid. Rapamycin also suppressed IgE, although IL-4 and eotaxin 1 levels were augmented. In protocol 2, both drugs suppressed total CD4(+ T cells, including activated, effector, and regulatory T cells and IgE levels. IL-4, eotaxin, and inflammatory cell numbers were increased after rapamycin and no effect on AHR was observed. Dexamethasone suppressed inflammatory cell numbers, especially eosinophils, but had limited effects on AHR. We conclude that while mTOR signaling is critical during the early phases of allergic asthma, its role is much more limited once disease is established.

  7. Finding a better drug for epilepsy: The mTOR pathway as an antiepileptogenic target

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    Galanopoulou, Aristea S.; Gorter, Jan A.; Cepeda, Carlos

    2012-01-01

    Summary The mTOR signaling pathway regulates cell growth, differentiation, proliferation and metabolism. Loss of function mutations in upstream regulators of mTOR have been highly associated with dysplasias, epilepsy and neurodevelopmental disorders. These include tuberous sclerosis, which is due to mutations in TSC1 or TSC2 genes, mutations in phosphatase and tensin homolog (PTEN) as in Cowden syndrome, polyhydramnios, megalencephaly, symptomatic epilepsy syndrome (PMSE) due to mutations in the STE20-related kinase adaptor alpha (STRADalpha), and neurofibromatosis type 1 attributed to neurofibromin 1 mutations. Inhibition of the mTOR pathway with rapamycin may prevent epilepsy and improve the underlying pathology in mouse models with disrupted mTOR signaling, due to PTEN or TSC mutations. However the timing and duration of its administration appear critical in defining the seizure and pathology-related outcomes. Rapamycin application in human cortical slices from patients with cortical dysplasias reduces the 4-aminopyridine induced oscillations. In the multiple-hit model of infantile spasms, pulse high dose rapamycin administration can reduce the cortical overactivation of the mTOR pathway, suppresses spasms and has disease-modifying effects by partially improving cognitive deficits. In post-status epilepticus models of temporal lobe epilepsy, rapamycin may ameliorate the development of epilepsy-related pathology and reduce the expression of spontaneous seizures, but its effects depend on the timing and duration of administration, and possibly the model used. The observed recurrence of seizures and epilepsy-related pathology after rapamycin discontinuation suggests the need for continuous administration to maintain the benefit. However, the use of pulse administration protocols may be useful in certain age-specific epilepsy syndromes, like infantile spasms, whereas repetitive pulse rapamycin protocols may suffice to sustain a long-term benefit in genetic disorders

  8. AMPK-mediated up-regulation of mTORC2 and MCL-1 compromises the anti-cancer effects of aspirin

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    Hua, Hui; Yin, Yancun; Wang, Jiao; Luo, Ting; Jiang, Yangfu

    2016-01-01

    AMP-activated protein kinase (AMPK) is an important energy sensor that may inhibit cell proliferation or promote cell survival during stresses. Besides cyclooxygenase, AMPK is another target of the nonsteroid anti-inflammatory agent aspirin. Preclinical and clinical investigations demonstrate that aspirin can inhibit several types of cancer such as colorectal adenomas and hepatocellular carcinoma (HCC). However, little is known about the cellular response to aspirin that may lead to aspirin resistance. Here, we show that aspirin induces the expression of MCL-1 in HepG2 and SW480 cells through AMPK-mTOR-Akt/ERK axis. Treatment of HepG2 and SW480 cells with aspirin leads to increased MCL-1 expression, Akt and ERK1/2 phosphorylation. Inhibition of Akt/MEK abrogates the induction of MCL-1 by aspirin. Aspirin activates AMPK, which in turn up-regulates mTORC2 activity, Akt, ERK1/2 phosphorylation and MCL-1 expression. MCL-1 knockdown sensitizes cancer cells to aspirin-induced apoptosis. Combination of aspirin and AMPK, Akt or MEK inhibitor results in more significant inhibition of cell proliferation and induction of apoptosis than single agent. Moreover, sorafenib blocks aspirin-induced MCL-1 up-regulation. Combination of aspirin and sorafenib leads to much more cell death and less cell proliferation than each drug alone. Treatment of HCC and colon cancer xenografts with both aspirin and sorafenib results in more significant tumor suppression than single agent. These data demonstrate that AMPK-mediated up-regulation of mTORC2 and MCL-1 may compromise the anticancer effects of aspirin. Combination of aspirin and sorafenib may be an effective regimen to treat HCC and colon cancer. PMID:26918349

  9. Severe energy deficit at high altitude inhibits skeletal muscle mTORC1-mediated anabolic signaling without increased ubiquitin proteasome activity.

    Science.gov (United States)

    Margolis, Lee M; Carbone, John W; Berryman, Claire E; Carrigan, Christopher T; Murphy, Nancy E; Ferrando, Arny A; Young, Andrew J; Pasiakos, Stefan M

    2018-06-07

    Muscle loss at high altitude (HA) is attributable to energy deficit and a potential dysregulation of anabolic signaling. Exercise and protein ingestion can attenuate the effects of energy deficit on muscle at sea level (SL). Whether these effects are observed when energy deficit occurs at HA is unknown. To address this, muscle obtained from lowlanders ( n = 8 males) at SL, acute HA (3 h, 4300 m), and chronic HA (21 d, -1766 kcal/d energy balance) before [baseline (Base)] and after 80 min of aerobic exercise followed by a 2-mile time trial [postexercise (Post)] and 3 h into recovery (Rec) after ingesting whey protein (25 g) were analyzed using standard molecular techniques. At SL, Post, and REC, p-mechanistic target of rapamycin (mTOR) Ser2448 , p-p70 ribosomal protein S6 kinase (p70S6K) Ser424/421 , and p-ribosomal protein S6 (rpS6) Ser235/236 were similar and higher ( P anabolic resistance that is exacerbated by energy deficit during acclimatization, with no change in proteolysis.-Margolis, L. M., Carbone, J. W., Berryman, C. E., Carrigan, C. T., Murphy, N. E., Ferrando, A. A., Young, A. J., Pasiakos, S. M. Severe energy deficit at high altitude inhibits skeletal muscle mTORC1-mediated anabolic signaling without increased ubiquitin proteasome activity.

  10. Effects of rapamycin and curcumin treatment on the development of epilepsy after electrically induced status epilepticus in rats

    NARCIS (Netherlands)

    Drion, Cato M.; Borm, Lars E.; Kooijman, Lieneke; Aronica, Eleonora; Wadman, Wytse J.; Hartog, Aloysius F.; van Vliet, Erwin A.; Gorter, Jan A.

    2016-01-01

    Inhibition of the mammalian target of rapamycin (mTOR) pathway has been suggested as a possible antiepileptogenic strategy in temporal lobe epilepsy (TLE). Here we aim to elucidate whether mTOR inhibition has antiepileptogenic and/or antiseizure effects using different treatment strategies in the

  11. Ketamine Exhibits Different Neuroanatomical Profile After Mammalian Target of Rapamycin Inhibition in the Prefrontal Cortex: the Role of Inflammation and Oxidative Stress.

    Science.gov (United States)

    Abelaira, Helena M; Réus, Gislaine Z; Ignácio, Zuleide M; Dos Santos, Maria Augusta B; de Moura, Airam B; Matos, Danyela; Demo, Júlia P; da Silva, Júlia B I; Danielski, Lucineia G; Petronilho, Fabricia; Carvalho, André F; Quevedo, João

    2017-09-01

    Studies indicated that mammalian target of rapamycin (mTOR), oxidative stress, and inflammation are involved in the pathophysiology of major depressive disorder (MDD). Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been identified as a novel MDD therapy; however, the antidepressant mechanism is not fully understood. In addition, the effects of ketamine after mTOR inhibition have not been fully investigated. In the present study, we examined the behavioral and biochemical effects of ketamine in the prefrontal cortex (PFC), hippocampus, amygdala, and nucleus accumbens after inhibition of mTOR signaling in the PFC. Male adult Wistar rats received pharmacological mTOR inhibitor, rapamycin (0.2 nmol) or vehicle into the PFC and then a single dose of ketamine (15 mg/kg, i.p.). Immobility was assessed in forced swimming tests, and then oxidative stress parameters and inflammatory markers were evaluated in the brain and periphery. mTOR activation in the PFC was essential to ketamine's antidepressant-like effects. Ketamine increased lipid damage in the PFC, hippocampus, and amygdala. Protein carbonyl was elevated in the PFC, amygdala, and NAc after ketamine administration. Ketamine also increased nitrite/nitrate in the PFC, hippocampus, amygdala, and NAc. Myeloperoxidase activity increased in the hippocampus and NAc after ketamine administration. The activities of superoxide dismutase and catalase were reduced after ketamine administration in all brain areas studied. Inhibition of mTOR signaling pathways by rapamycin in the PFC was required to protect against oxidative stress by reducing damage and increasing antioxidant enzymes. Finally, the TNF-α level was increased in serum by ketamine; however, the rapamycin plus treatment group was not able to block this increase. Activation of mTOR in the PFC is involved in the antidepressant-like effects of ketamine; however, the inhibition of this pathway was able to protect certain brain areas against

  12. LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs.

    Science.gov (United States)

    Hong, Sungki; Freeberg, Mallory A; Han, Ting; Kamath, Avani; Yao, Yao; Fukuda, Tomoko; Suzuki, Tsukasa; Kim, John K; Inoki, Ken

    2017-06-26

    The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5'TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5'TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5' and 3'UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5'UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3'UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis.

  13. Rapamycin causes growth arrest and inhibition of invasion in human chondrosarcoma cells.

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    Song, Jian; Wang, Xiaobo; Zhu, Jiaxue; Liu, Jun

    2016-01-01

    Chondrosarcoma is a highly malignant tumor that is characterized by a potent capacity to invade locally and cause distant metastasis and notable for its lack of response to conventional chemotherapy or radiotherapy. Rapamycin, the inhibitor of mammalian target of rapamycin (mTOR), is a valuable drug with diverse clinical applications and regulates many cellular processes. However, the effects of rapamycin on cell growth and invasion of human chondrosarcoma cells are not well known. We determined the effect of rapamycin on cell proliferation, cell cycle arrest and invasion by using MTS, flow cytometry and invasion assays in two human chondrosarcoma cell lines, SW1353 and JJ012. Cell cycle regulatory and invasion-related genes' expression analysis was performed by quantitative RT-PCR (qRT-PCR). We also evaluated the effect of rapamycin on tumor growth by using mice xenograph models. Rapamycin significantly inhibited the cell proliferation, induced cell cycle arrest and decreased the invasion ability of human chondrosarcoma cells. Meanwhile, rapamycin modulated the cell cycle regulatory and invasion-related genes' expression. Furthermore, the tumor growth of mice xenograph models with human chondrosarcoma cells was significantly inhibited by rapamycin. These results provided further insight into the role of rapamycin in chondrosarcoma. Therefore, rapamycin targeted therapy may be a potential treatment strategy for chondrosarcoma.

  14. Insensitive Enough Semantics

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    Richard Vallée

    2006-06-01

    Full Text Available According to some philosophers, sentences like (1 “It is raining” and (2 “John is ready” are context sensitive sentences even if they do not contain indexicals or demonstratives. That view initiated a context sensitivity frenzy. Cappelen and Lepore (2005 summarize the frenzy by the slogan “Every sentence is context sensitive” (Insensitive Semantics, p. 6, note 5. They suggest a view they call Minimalism according to which the truth conditions of utterances of sentences like (1/(2 are exactly what Convention T gives you. I will distinguish different propositions, and refocus semantics on sentences. As distinct from what the protagonists in the ongoing debate think, I argue that the content or truth conditions of utterances of both context sensitive sentences and sentences like (1/(2 are not interesting from a semantic point of view, and that the problem sentences like (1/(2 raises is not about context sensitivity or context insensitivity of sentences, but relevance of the content of utterances.

  15. Pharmacokinetics of orally administered low-dose rapamycin in healthy dogs: A pilot study

    Science.gov (United States)

    Larson, Jeanne C.; Allstadt, Sara D.; Fan, Timothy M.; Khanna, Chand; Lunghofer, Paul J.; Hansen, Ryan J.; Gustafson, Daniel L.; Legendre, Alfred M.; Galyon, Gina D.; LeBlanc, Amy K.; Martin-Jimenez, Tomas

    2017-01-01

    Objective To determine the pharmacokinetics of orally administered rapamycin in healthy dogs. Animals 5 healthy purpose-bred hounds. Procedures The study consisted of 2 experiments. In experiment 1, each dog received rapamycin (0.1 mg/kg, PO) once; blood samples were obtained immediately before and at 0.5, 1, 2, 4, 6, 12, 24, 48, and 72 hours after administration. In experiment 2, each dog received (0.1 mg/kg, PO) once daily for 5 days; blood samples were obtained immediately before and at 3, 6, 24, 27, 30, 48, 51, 54, 72, 75, 78, 96, 96.5, 97, 98, 100, 102, 108, 120, 144, and 168 hours after the first dose. Blood rapamycin concentration was determined by a validated liquid chromatography-tandem mass spectrometry assay. Pharmacokinetic parameters were determined by compartmental and non-compartmental analyses. Results Mean ± SD blood rapamycin terminal half-life, area under the concentration-time curve from 0 to 48 hours after dosing, and maximum concentration were 38.7 ± 12.7 h, 140 ± 23.9 ng•h/mL, and 8.39 ± 1.73 ng/mL, respectively, for experiment 1, and 99.5 ± 89.5 h, 126 ± 27.1 ng•h/mL, and 5.49 ± 1.99 ng/mL, respectively, for experiment 2. Pharmacokinetic parameters for rapamycin after administration of 5 daily doses differed significantly from those after administration of 1 dose. Conclusions and Clinical Relevance Results indicated that oral administration of low-dose (0.1 mg/kg) rapamycin to healthy dogs achieved blood concentrations measured in ng/mL. The optimal dose and administration frequency of rapamcyin required to achieve therapeutic effects in tumor-bearing dogs, as well as toxicity after chronic dosing, needs to be determined. PMID:26709938

  16. Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development.

    Science.gov (United States)

    Guan, Yingjie; Yang, Xu; Yang, Wentian; Charbonneau, Cherie; Chen, Qian

    2014-10-01

    Mechanical stress regulates development by modulating cell signaling and gene expression. However, the cytoplasmic components mediating mechanotransduction remain unclear. In this study, elimination of muscle contraction during chicken embryonic development resulted in a reduction in the activity of mammalian target of rapamycin (mTOR) in the cartilaginous growth plate. Inhibition of mTOR activity led to significant inhibition of chondrocyte proliferation, cartilage tissue growth, and expression of chondrogenic genes, including Indian hedgehog (Ihh), a critical mediator of mechanotransduction. Conversely, cyclic loading (1 Hz, 5% matrix deformation) of embryonic chicken growth plate chondrocytes in 3-dimensional (3D) collagen scaffolding induced sustained activation of mTOR. Mechanical activation of mTOR occurred in serum-free medium, indicating that it is independent of growth factor or nutrients. Treatment of chondrocytes with Rapa abolished mechanical activation of cell proliferation and Ihh gene expression. Cyclic loading of chondroprogenitor cells deficient in SH2-containing protein tyrosine phosphatase 2 (Shp2) further enhanced mechanical activation of mTOR, cell proliferation, and chondrogenic gene expression. This result suggests that Shp2 is an antagonist of mechanotransduction through inhibition of mTOR activity. Our data demonstrate that mechanical activation of mTOR is necessary for cell proliferation, chondrogenesis, and cartilage growth during bone development, and that mTOR is an essential mechanotransduction component modulated by Shp2 in the cytoplasm. © FASEB.

  17. Activation of mTORC1 by leucine is potentiated by branched-chain amino acids and even more so by essential amino acids following resistance exercise.

    Science.gov (United States)

    Moberg, Marcus; Apró, William; Ekblom, Björn; van Hall, Gerrit; Holmberg, Hans-Christer; Blomstrand, Eva

    2016-06-01

    Protein synthesis is stimulated by resistance exercise and intake of amino acids, in particular leucine. Moreover, activation of mammalian target of rapamycin complex 1 (mTORC1) signaling by leucine is potentiated by the presence of other essential amino acids (EAA). However, the contribution of the branched-chain amino acids (BCAA) to this effect is yet unknown. Here we compare the stimulatory role of leucine, BCAA, and EAA ingestion on anabolic signaling following exercise. Accordingly, eight trained volunteers completed four sessions of resistance exercise during which they ingested either placebo, leucine, BCAA, or EAA (including the BCAA) in random order. Muscle biopsies were taken at rest, immediately after exercise, and following 90 and 180 min of recovery. Following 90 min of recovery the activity of S6 kinase 1 (S6K1) was greater than at rest in all four trials (PlaceboBCAABCAA. However, after 180 min of recovery this difference between EAA and BCAA had disappeared, although with both these supplements the increases were still higher than with leucine (40%, P BCAA. Copyright © 2016 the American Physiological Society.

  18. Raptor is phosphorylated by cdc2 during mitosis.

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    Dana M Gwinn

    2010-02-01

    Full Text Available The appropriate control of mitotic entry and exit is reliant on a series of interlocking signaling events that coordinately drive the biological processes required for accurate cell division. Overlaid onto these signals that promote orchestrated cell division are checkpoints that ensure appropriate mitotic spindle formation, a lack of DNA damage, kinetochore attachment, and that each daughter cell has the appropriate complement of DNA. We recently discovered that AMP-activated protein kinase (AMPK modulates the G2/M phase of cell cycle progression in part through its suppression of mammalian target of rapamycin (mTOR signaling. AMPK directly phosphorylates the critical mTOR binding partner raptor inhibiting mTORC1 (mTOR-raptor rapamycin sensitive mTOR kinase complex 1. As mTOR has been previously tied to mitotic control, we examined further how raptor may contribute to this process.We have discovered that raptor becomes highly phosphorylated in cells in mitosis. Utilizing tandem mass spectrometry, we identified a number of novel phosphorylation sites in raptor, and using phospho-specific antibodies demonstrated that raptor becomes phosphorylated on phospho-serine/threonine-proline sites in mitosis. A combination of site-directed mutagenesis in a tagged raptor cDNA and analysis with a series of new phospho-specific antibodies generated against different sites in raptor revealed that Serine 696 and Threonine 706 represent two key sites in raptor phosphorylated in mitosis. We demonstrate that the mitotic cyclin-dependent kinase cdc2/CDK1 is the kinase responsible for phosphorylating these sites, and its mitotic partner Cyclin B efficiently coimmunoprecipitates with raptor in mitotic cells.This study demonstrates that the key mTOR binding partner raptor is directly phosphorylated during mitosis by cdc2. This reinforces previous studies suggesting that mTOR activity is highly regulated and important for mitotic progression, and points to a direct

  19. Rapamycin prevents drug seeking via disrupting reconsolidation of reward memory in rats.

    Science.gov (United States)

    Lin, Jue; Liu, Lingqi; Wen, Quan; Zheng, Chunming; Gao, Yang; Peng, Shuxian; Tan, Yalun; Li, Yanqin

    2014-01-01

    The maladaptive drug memory developed between the drug-rewarding effect and environmental cues contributes to difficulty in preventing drug relapse. Established reward memories can be disrupted by pharmacologic interventions following their reactivation. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) kinase, has been proved to be involved in various memory consolidation. However, it is less well characterized in drug memory reconsolidation. Using a conditioned place preference (CPP) procedure, we examined the effects of systemically administered rapamycin on reconsolidation of drug memory in rats. We found that systemically administered rapamycin (0.1 or 10 mg/kg, i.p.) after re-exposure to drug-paired environment, dose dependently decreased the expression of CPP 1 d later, and the effect lasted for up to 14 d and could not be reversed by a priming injection of morphine. The effect of rapamycin on morphine-associated memory was specific to drug-paired context, and rapamycin had no effect on subsequent CPP expression when rats were exposed to saline-paired context or homecage. These results indicated that systemic administration of rapamycin after memory reactivation can persistently inhibit the drug seeking behaviour via disruption of morphine memory reconsolidation in rats. Additionally, the effect of rapamycin on memory reconsolidation was reproduced in cocaine CPP and alcohol CPP. Furthermore, rapamycin did not induce conditioned place aversion and had no effect on locomotor activity and anxiety behaviour. These findings suggest that rapamycin could erase the acquired drug CPP in rats, and that mTOR activity plays an important role in drug reconsolidation and is required for drug relapse.

  20. La-related protein 1 (LARP1) represses terminal oligopyrimidine (TOP) mRNA translation downstream of mTOR complex 1 (mTORC1)

    DEFF Research Database (Denmark)

    Fonseca, Bruno; Zakaria, Chadi; Jia, J J

    2015-01-01

    is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5′TOP motif...

  1. TDP-43 loss of function increases TFEB activity and blocks autophagosome-lysosome fusion.

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    Xia, Qin; Wang, Hongfeng; Hao, Zongbing; Fu, Cheng; Hu, Qingsong; Gao, Feng; Ren, Haigang; Chen, Dong; Han, Junhai; Ying, Zheng; Wang, Guanghui

    2016-01-18

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA-binding protein 43 (TDP-43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP-43 is a multi-functional protein involved in RNA processing and a large number of TDP-43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP-43-linked neurodegeneration remain elusive. In this study, we found that loss of TDP-43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy-lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP-43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP-43-depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP-43-mediated neurodegeneration. © 2015 The Authors.

  2. Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

    International Nuclear Information System (INIS)

    Varma, Shailly; Shrivastav, Anuraag; Changela, Sheena; Khandelwal, Ramji L.

    2008-01-01

    Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3β (GSK-3β) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-α (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity

  3. γ-Oryzanol Enhances Adipocyte Differentiation and Glucose Uptake

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    Chang Hwa Jung

    2015-06-01

    Full Text Available Recent studies show that brown rice improves glucose intolerance and potentially the risk of diabetes, although the underlying molecular mechanisms remain unclear. One of the phytochemicals found in high concentration in brown rice is γ-oryzanol (Orz, a group of ferulic acid esters of phytosterols and triterpene alcohols. Here, we found that Orz stimulated differentiation of 3T3-L1 preadipocytes and increased the protein expression of adipogenic marker genes such as peroxisome proliferator-activated receptor gamma (PPAR-γ and CCAAT/enhanced binding protein alpha (C/EBPα. Moreover, Orz significantly increased the glucose uptake in insulin-resistant cells and translocation of glucose transporter type 4 (GLUT4 from the cytosol to the cell surface. To investigate the mechanism by which Orz stimulated cell differentiation, we examined its effects on cellular signaling of the mammalian target of rapamycin complex 1 (mTORC1, a central mediator of cellular growth and proliferation. The Orz treatment increased mTORC1 kinase activity based on phosphorylation of 70-kDa ribosomal S6 kinase 1 (S6K1. The effect of Orz on adipocyte differentiation was dependent on mTORC1 activity because rapamycin blocks cell differentiation in Orz-treated cells. Collectively, our results indicate that Orz stimulates adipocyte differentiation, enhances glucose uptake, and may be associated with cellular signaling mediated by PPAR-γ and mTORC1.

  4. γ-Oryzanol Enhances Adipocyte Differentiation and Glucose Uptake.

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    Jung, Chang Hwa; Lee, Da-Hye; Ahn, Jiyun; Lee, Hyunjung; Choi, Won Hee; Jang, Young Jin; Ha, Tae-Youl

    2015-06-15

    Recent studies show that brown rice improves glucose intolerance and potentially the risk of diabetes, although the underlying molecular mechanisms remain unclear. One of the phytochemicals found in high concentration in brown rice is γ-oryzanol (Orz), a group of ferulic acid esters of phytosterols and triterpene alcohols. Here, we found that Orz stimulated differentiation of 3T3-L1 preadipocytes and increased the protein expression of adipogenic marker genes such as peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT/enhanced binding protein alpha (C/EBPα). Moreover, Orz significantly increased the glucose uptake in insulin-resistant cells and translocation of glucose transporter type 4 (GLUT4) from the cytosol to the cell surface. To investigate the mechanism by which Orz stimulated cell differentiation, we examined its effects on cellular signaling of the mammalian target of rapamycin complex 1 (mTORC1), a central mediator of cellular growth and proliferation. The Orz treatment increased mTORC1 kinase activity based on phosphorylation of 70-kDa ribosomal S6 kinase 1 (S6K1). The effect of Orz on adipocyte differentiation was dependent on mTORC1 activity because rapamycin blocks cell differentiation in Orz-treated cells. Collectively, our results indicate that Orz stimulates adipocyte differentiation, enhances glucose uptake, and may be associated with cellular signaling mediated by PPAR-γ and mTORC1.

  5. Comparison of three rapamycin dosing schedules in A/J Tsc2+/- mice and improved survival with angiogenesis inhibitor or asparaginase treatment in mice with subcutaneous tuberous sclerosis related tumors

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    Dabora Sandra L

    2010-02-01

    Full Text Available Abstract Background Tuberous Sclerosis Complex (TSC is an autosomal dominant tumor disorder characterized by the growth of hamartomas in various organs including the kidney, brain, skin, lungs, and heart. Rapamycin has been shown to reduce the size of kidney angiomyolipomas associated with TSC; however, tumor regression is incomplete and kidney angiomyolipomas regrow after cessation of treatment. Mouse models of TSC2 related tumors are useful for evaluating new approaches to drug therapy for TSC. Methods In cohorts of Tsc2+/- mice, we compared kidney cystadenoma severity in A/J and C57BL/6 mouse strains at both 9 and 12 months of age. We also investigated age related kidney tumor progression and compared three different rapamycin treatment schedules in cohorts of A/J Tsc2+/- mice. In addition, we used nude mice bearing Tsc2-/- subcutaneous tumors to evaluate the therapeutic utility of sunitinib, bevacizumab, vincristine, and asparaginase. Results TSC related kidney disease severity is 5-10 fold higher in A/J Tsc2+/- mice compared with C57BL/6 Tsc2+/- mice. Similar to kidney angiomyolipomas associated with TSC, the severity of kidney cystadenomas increases with age in A/J Tsc2+/- mice. When rapamycin dosing schedules were compared in A/J Tsc2+/- cohorts, we observed a 66% reduction in kidney tumor burden in mice treated daily for 4 weeks, an 82% reduction in mice treated daily for 4 weeks followed by weekly for 8 weeks, and an 81% reduction in mice treated weekly for 12 weeks. In the Tsc2-/- subcutaneous tumor mouse model, vincristine is not effective, but angiogenesis inhibitors (sunitinib and bevacizumab and asparaginase are effective as single agents. However, these drugs are not as effective as rapamycin in that they increased median survival only by 24-27%, while rapamycin increased median survival by 173%. Conclusions Our results indicate that the A/J Tsc2+/- mouse model is an improved, higher through-put mouse model for future TSC

  6. A natural frameshift mutation in Campanula EIL2 correlates with ethylene insensitivity in flowers

    DEFF Research Database (Denmark)

    Jensen, Line; Hegelund, Josefine Nymark; Olsen, Andreas

    2016-01-01

    BACKGROUND: The phytohormone ethylene plays a central role in development and senescence of climacteric flowers. In ornamental plant production, ethylene sensitive plants are usually protected against negative effects of ethylene by application of chemical inhibitors. In Campanula, flowers...... are sensitive to even minute concentrations of ethylene. RESULTS: Monitoring flower longevity in three Campanula species revealed C. portenschlagiana (Cp) as ethylene sensitive, C. formanekiana (Cf) with intermediate sensitivity and C. medium (Cm) as ethylene insensitive. We identified key elements in ethylene...... signal transduction, specifically in Ethylene Response Sensor 2 (ERS2), Constitutive Triple Response 1 (CTR1) and Ethylene Insensitive 3- Like 1 and 2 (EIL1 and EIL2) homologous. Transcripts of ERS2, CTR1 and EIL1 were constitutively expressed in all species both throughout flower development...

  7. LMW-E/CDK2 Deregulates Acinar Morphogenesis, Induces Tumorigenesis, and Associates with the Activated b-Raf-ERK1/2-mTOR Pathway in Breast Cancer Patients

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    Duong, MyLinh T.; Akli, Said; Wei, Caimiao; Wingate, Hannah F.; Liu, Wenbin; Lu, Yiling; Yi, Min; Mills, Gordon B.; Hunt, Kelly K.; Keyomarsi, Khandan

    2012-01-01

    Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2–associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E–expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E–expressing cells by inducing G1/S cell cycle arrest. LMW

  8. Enteral delivery of proteins stimulates protein synthesis in human duodenal mucosa in the fed state through a mammalian target of rapamycin-independent pathway.

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    Coëffier, Moïse; Claeyssens, Sophie; Bôle-Feysot, Christine; Guérin, Charlène; Maurer, Brigitte; Lecleire, Stéphane; Lavoinne, Alain; Donnadieu, Nathalie; Cailleux, Anne-Françoise; Déchelotte, Pierre

    2013-02-01

    Glutamine modulates duodenal protein metabolism in fasted healthy humans, but its effects in a fed state remain unknown. We aimed to assess the effects of either glutamine or an isonitrogenous protein mixture on duodenal protein metabolism in humans in the fed state. Twenty-four healthy volunteers were randomly included in 2 groups. Each volunteer was studied on 2 occasions in a random order and received, during 5 h, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹; both groups) that mimicked a carbohydrate fed state or maltodextrins with glutamine (group 1) or an isonitrogenous (22.4 mg N · kg⁻¹ · h⁻¹) protein powder (group 2). Simultaneously, a continuous intravenous infusion of ¹³C-leucine and ²H₅-phenylalanine (both 9 μmol · kg⁻¹ · h⁻¹) was performed. Endoscopic duodenal biopsies were taken. Leucine and phenylalanine enrichments were assessed by using gas chromatography-mass spectrometry in duodenal proteins and the intracellular free amino acids pool to calculate the mucosal fractional synthesis rate (FSR). Proteasome proteolytic activities and phosphokinase expression were assessed by using specific fluorogenic substrates and macroarrays, respectively. The FSR and proteasome activity were not different after the glutamine supply compared with after maltodextrins alone. In contrast, the FSR increased (1.7-fold increase; P protein-powder delivery without modification of total proteasome activity. The protein powder increased insulinemia, PI3 kinase, and erk phosphorylation but did not affect the mammalian target of rapamycin (mTOR) pathway and mitogen-activated protein kinase signal-integrating kinase 1 phosphorylation. A trend for an increase of eukaryotic translation initiation factor 4E phosphorylation was observed (P = 0.07). In the carbohydrate fed state, enteral proteins but not glutamine increased duodenal protein synthesis through an mTOR independent pathway in humans.

  9. Blocking mammalian target of rapamycin (mTOR) improves neuropathic pain evoked by spinal cord injury.

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    Wang, Xiaoping; Li, Xiaojia; Huang, Bin; Ma, Shuai

    2016-01-01

    Spinal cord injury (SCI) is an extremely serious type of physical trauma observed in clinics. Neuropathic pain resulting from SCI has a lasting and significant impact on most aspects of daily life. Thus, a better understanding of the molecular pathways responsible for the cause of neuropathic pain observed in SCI is important to develop effective therapeutic agents and treatment strategies. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that is well known for its critical roles in regulating protein synthesis and growth. Furthermore, compelling evidence supports the notion that widespread dysregulation of mTOR and its downstream pathways are involved in neuropathic pain. Thus, in this study we specifically examined the underlying mechanisms by which mTOR and its signaling pathways are involved in SCI-evoked neuropathic pain in a rat model. Overall, we demonstrated that SCI increased the protein expression of p-mTOR, and mTORmediated- phosphorylation of 4E-binding protein 4 (4E-BP1) and p70 ribosomal S6 protein kinase 1 (S6K1) in the superficial dorsal horn of the spinal cord. Also, we showed that blocking spinal mTOR by intrathecal injection of rapamycin significantly inhibited pain responses induced by mechanical and thermal stimulation. In addition, blocking spinal phosphatidylinositide 3-kinase (p-PI3K) pathway significantly attenuated activities of p-mTOR pathways as well as mechanical and thermal hyperalgesia in SCI rats. Moreover, blocking mTOR and PI3K decreased the enhanced levels of substance P and calcitonin gene-related peptide (CGRP) in the dorsal horn of SCI rats. We revealed specific signaling pathways leading to SCI-evoked neuropathic pain, including the activation of PI3K, mTOR and its downstream signaling pathways. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of neuropathic pain often observed in patients with SCI.

  10. Blocking mammalian target of rapamycin (mTOR improves neuropathic pain evoked by spinal cord injury

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    Wang Xiaoping

    2016-01-01

    Full Text Available Spinal cord injury (SCI is an extremely serious type of physical trauma observed in clinics. Neuropathic pain resulting from SCI has a lasting and significant impact on most aspects of daily life. Thus, a better understanding of the molecular pathways responsible for the cause of neuropathic pain observed in SCI is important to develop effective therapeutic agents and treatment strategies. Mammalian target of rapamycin (mTOR is a serine/threonine protein kinase that is well known for its critical roles in regulating protein synthesis and growth. Furthermore, compelling evidence supports the notion that widespread dysregulation of mTOR and its downstream pathways are involved in neuropathic pain. Thus, in this study we specifically examined the underlying mechanisms by which mTOR and its signaling pathways are involved in SCI-evoked neuropathic pain in a rat model. Overall, we demonstrated that SCI increased the protein expression of p-mTOR, and mTORmediated- phosphorylation of 4E–binding protein 4 (4E-BP1 and p70 ribosomal S6 protein kinase 1 (S6K1 in the superficial dorsal horn of the spinal cord. Also, we showed that blocking spinal mTOR by intrathecal injection of rapamycin significantly inhibited pain responses induced by mechanical and thermal stimulation. In addition, blocking spinal phosphatidylinositide 3-kinase (p-PI3K pathway significantly attenuated activities of p-mTOR pathways as well as mechanical and thermal hyperalgesia in SCI rats. Moreover, blocking mTOR and PI3K decreased the enhanced levels of substance P and calcitonin gene-related peptide (CGRP in the dorsal horn of SCI rats. We revealed specific signaling pathways leading to SCI-evoked neuropathic pain, including the activation of PI3K, mTOR and its downstream signaling pathways. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of neuropathic pain often observed in patients with SCI.

  11. PF-4708671, a specific inhibitor of p70 ribosomal S6 kinase 1, activates Nrf2 by promoting p62-dependent autophagic degradation of Keap1

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    Park, Jeong Su [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Kang, Dong Hoon [Department of Life Science and Ewha Research Center for Systems Biology (Korea, Republic of); The Research Center for Cell Homeostasis, Ewha Womans University, Seoul 127-750 (Korea, Republic of); Lee, Da Hyun [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Bae, Soo Han, E-mail: soohanbae@yuhs.ac [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of)

    2015-10-23

    p70 ribosomal S6 kinase 1 (S6K1) is an important serine/threonine kinase and downstream target of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. PF-4708671 is a specific inhibitor of S6K1, and prevents S6K1-mediated phosphorylation of the S6 protein. PF-4708671 treatment often leads to apoptotic cell death. However, the protective mechanism against PF-4708671-induced cell death has not been elucidated. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is essential for protecting cells against oxidative stress. p62, an adaptor protein in the autophagic process, enhances Nrf2 activation through the impairment of Keap1 activity. In this study, we showed that PF-4708671 induces autophagic Keap1 degradation-mediated Nrf2 activation in p62-dependent manner. Furthermore, p62-dependent Nrf2 activation plays a crucial role in protecting cells from PF-4708671-mediated apoptosis. - Highlights: • PF-4708671, a S6K1-specific inhibitor, prevents S6K1-mediated S6 phosphorylation. • However, PF-4708671 treatment often leads to apoptotic cell death. • Protective mechanism against PF-4708671-induced cell death remains to be elucidated. • PF-4708671 induced p62-dependent, autophagic Keap1 degradation-mediated Nrf2 activation. • p62-dependent Nrf2 activation protects cells from PF-4708671-mediated apoptosis.

  12. A pseudo-response regulator is misexpressed in the photoperiod insensitive Ppd-D1a mutant of wheat (Triticum aestivum L.).

    Science.gov (United States)

    Beales, James; Turner, Adrian; Griffiths, Simon; Snape, John W; Laurie, David A

    2007-09-01

    Ppd-D1 on chromosome 2D is the major photoperiod response locus in hexaploid wheat (Triticum aestivum). A semi-dominant mutation widely used in the "green revolution" converts wheat from a long day (LD) to a photoperiod insensitive (day neutral) plant, providing adaptation to a broad range of environments. Comparative mapping shows Ppd-D1 to be colinear with the Ppd-H1 gene of barley (Hordeum vulgare) which is a member of the pseudo-response regulator (PRR) gene family. To investigate the relationship between wheat and barley photoperiod genes we isolated homologues of Ppd-H1 from a 'Chinese Spring' wheat BAC library and compared them to sequences from other wheat varieties with known Ppd alleles. Varieties with the photoperiod insensitive Ppd-D1a allele which causes early flowering in short (SD) or LDs had a 2 kb deletion upstream of the coding region. This was associated with misexpression of the 2D PRR gene and expression of the key floral regulator FT in SDs, showing that photoperiod insensitivity is due to activation of a known photoperiod pathway irrespective of day length. Five Ppd-D1 alleles were found but only the 2 kb deletion was associated with photoperiod insensitivity. Photoperiod insensitivity can also be conferred by mutation at a homoeologous locus on chromosome 2B (Ppd-B1). No candidate mutation was found in the 2B PRR gene but polymorphism within the 2B PRR gene cosegregated with the Ppd-B1 locus in a doubled haploid population, suggesting that insensitivity on 2B is due to a mutation outside the sequenced region or to a closely linked gene.

  13. A central role for the mammalian target of rapamycin in LPS-induced anorexia in mice.

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    Yue, Yunshuang; Wang, Yi; Li, Dan; Song, Zhigang; Jiao, Hongchao; Lin, Hai

    2015-01-01

    Bacterial lipopolysaccharide (LPS), also known as endotoxin, induces profound anorexia. However, the LPS-provoked pro-inflammatory signaling cascades and the neural mechanisms underlying the development of anorexia are not clear. Mammalian target of rapamycin (mTOR) is a key regulator of metabolism, cell growth, and protein synthesis. This study aimed to determine whether the mTOR pathway is involved in LPS-induced anorexia. Effects of LPS on hypothalamic gene/protein expression in mice were measured by RT-PCR or western blotting analysis. To determine whether inhibition of mTOR signaling could attenuate LPS-induced anorexia, we administered an i.c.v. injection of rapamycin, an mTOR inhibitor, on LPS-treated male mice. In this study, we showed that LPS stimulates the mTOR signaling pathway through the enhanced phosphorylation of mTOR(Ser2448) and p70S6K(Thr389). We also showed that LPS administration increased the phosphorylation of FOXO1(Ser256), the p65 subunit of nuclear factor kappa B (Panorexia by decreasing the phosphorylation of p70S6K(Thr389), FOXO1(Ser256), and FOXO1/3a(Thr) (24) (/) (32). These results suggest promising approaches for the prevention and treatment of LPS-induced anorexia. © 2015 Society for Endocrinology.

  14. Role of nutrients and mTOR signaling in the regulation of pancreatic progenitors development

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    Lynda Elghazi

    2017-06-01

    Full Text Available Objective: Poor fetal nutrition increases the risk of type 2 diabetes in the offspring at least in part by reduced embryonic β-cell growth and impaired function. However, it is not entirely clear how fetal nutrients and growth factors impact β-cells during development to alter glucose homeostasis and metabolism later in life. The current experiments aimed to test the impact of fetal nutrients and growth factors on endocrine development and how these signals acting on mTOR signaling regulate β-cell mass and glucose homeostasis. Method: Pancreatic rudiments in culture were used to study the role of glucose, growth factors, and amino acids on β-cell development. The number and proliferation of pancreatic and endocrine progenitor were assessed in the presence or absence of rapamycin. The impact of mTOR signaling in vivo on pancreas development and glucose homeostasis was assessed in models deficient for mTOR or Raptor in Pdx1 expressing pancreatic progenitors. Results: We found that amino acid concentrations, and leucine in particular, enhance the number of pancreatic and endocrine progenitors and are essential for growth factor induced proliferation. Rapamycin, an mTORC1 complex inhibitor, reduced the number and proliferation of pancreatic and endocrine progenitors. Mice lacking mTOR in pancreatic progenitors exhibited hyperglycemia in neonates, hypoinsulinemia and pancreatic agenesis/hypoplasia with pancreas rudiments containing ductal structures lacking differentiated acinar and endocrine cells. In addition, loss of mTORC1 by deletion of raptor in pancreatic progenitors reduced pancreas size with reduced number of β-cells. Conclusion: Together, these results suggest that amino acids concentrations and in particular leucine modulates growth responses of pancreatic and endocrine progenitors and that mTOR signaling is critical for these responses. Inactivation of mTOR and raptor in pancreatic progenitors suggested that alterations in some of

  15. The Immunosuppressive drug – Rapamycin – Electroanalytical Sensing Using Boron- Doped Diamond electrode

    International Nuclear Information System (INIS)

    Stanković, Dalibor M.; Kalcher, Kurt

    2015-01-01

    Graphical abstract: Display Omitted -- Abstract: This paper presents for the first time the study of electrochemical behavior of well known immunosuppressant drug – rapamycin (sirolimus) using boron-doped diamond electrode. Rapamycin provided single and oval-shaped oxidation peak at +1.1 V vs. Ag/AgCl electrode in Britton–Robinson buffer solution at pH 3 confirming highly irreversible behavior of analyte at boron-doped diamond electrode. A differential pulse voltammetry was used for quantification of tested drug under the optimum experimental conditions. The calibration curve was linear over the range from 0.5 to 19.5 μM (R 2 = 0.9976) with detection limit of 0.22 μM. Repeatability of ten successfully measurements of three different concentrations (5, 10 and 15 μM) was 2.5, 1.9 and 1,7 %, respectively. Influence of most common biomolecules presented in urine samples was evaluated. The suggested analytical methodology was successfully applied for determination of rapamycin in four urine samples with excellent recoveries. The developed approach could be beneficial in analysis of rapamycin in biological samples using boron-doped diamond electrode as up-to-date electrochemical sensor and could represent inexpensive analytical alternative to separation methods

  16. Hyperosmotic stress inhibits insulin receptor substrate-1 function by distinct mechanisms in 3T3-L1 adipocytes

    DEFF Research Database (Denmark)

    Gual, Philippe; Gonzalez, Teresa; Grémeaux, Thierry

    2003-01-01

    . Furthermore, the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented the osmotic shock-induced phosphorylation of IRS-1 on Ser307. The inhibition of mTOR completely reversed the inhibitory effect of hyperosmotic stress on insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase activation......In 3T3-L1 adipocytes, hyperosmotic stress was found to inhibit insulin signaling, leading to an insulin-resistant state. We show here that, despite normal activation of insulin receptor, hyperosmotic stress inhibits both tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-1....... In addition, prolonged osmotic stress enhanced the degradation of IRS proteins through a rapamycin-insensitive pathway and a proteasome-independent process. These data support evidence of new mechanisms involved in osmotic stress-induced cellular insulin resistance. Short-term osmotic stress induces...

  17. Identification, cloning and characterization of sis7 and sis10 sugar-insensitive mutants of Arabidopsis

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    Biddle Kelly D

    2008-10-01

    Full Text Available Abstract Background The levels of soluble sugars, such as glucose and sucrose, help regulate many plant metabolic, physiological and developmental processes. Genetic screens are helping identify some of the loci involved in plant sugar response and reveal extensive cross-talk between sugar and phytohormone response pathways. Results A forward genetic screen was performed to identify mutants with increased resistance to the inhibitory effects of high levels of exogenous sugars on early Arabidopsis seedling development. The positional cloning and characterization of two of these sugar insensitive (sis mutants, both of which are also involved in abscisic acid (ABA biosynthesis or response, are reported. Plants carrying mutations in SIS7/NCED3/STO1 or SIS10/ABI3 are resistant to the inhibitory effects of high levels of exogenous Glc and Suc. Quantitative RT-PCR analyses indicate transcriptional upregulation of ABA biosynthesis genes by high concentrations of Glc in wild-type germinating seeds. Gene expression profiling revealed that a significant number of genes that are expressed at lower levels in germinating sis7-1/nced3-4/sto1-4 seeds than in wild-type seeds are implicated in auxin biosynthesis or transport, suggesting cross-talk between ABA and auxin response pathways. The degree of sugar insensitivity of different sis10/abi3 mutant seedlings shows a strong positive correlation with their level of ABA insensitivity during seed germination. Conclusion Mutations in the SIS7/NCED3/STO1 gene, which is primarily required for ABA biosynthesis under drought conditions, confer a sugar-insensitive phenotype, indicating that a constitutive role in ABA biosynthesis is not necessary to confer sugar insensitivity. Findings presented here clearly demonstrate that mutations in ABI3 can confer a sugar-insensitive phenotype and help explain previous, mixed reports on this topic by showing that ABA and sugar insensitivity exhibit a strong positive correlation in

  18. Rapamycin-ameliorated diabetic symptoms involved in increasing adiponectin expression in diabetic mice on a high-fat diet.

    Science.gov (United States)

    Gong, Fang-Hua; Ye, Yan-Na; Li, Jin-Meng; Zhao, Hai-Yang; Li, Xiao-Kun

    2017-07-01

    Recent studies showed that rapamycin improved diabetic complications. Here, we investigated the metabolic effects of rapamycin in type 2 diabetes model (T2DM) mice. Mice were treated with a daily intraperitoneal injection of rapamycin at 2 mg/kg or vehicle only for 3 weeks and were maintained on a high-fat diet. The treated diabetic mice exhibited decreased body weight, blood glucose levels, and fat mass. FGF21 expression was suppressed in C57B/L6 mice, but adiponectin expression increased both in FGF21 KO and C57B/L6 mice. These results suggest that rapamycin may alleviate FGF21 resistance in mice on a high-fat diet. The reduction of adipose tissue mass of the diabetic mice may be due to the increased adiponectin. Copyright © 2017. Published by Elsevier Taiwan.

  19. The association of four common polymorphisms from four candidate genes (COX-1, COX-2, ITGA2B, ITGA2 with aspirin insensitivity: a meta-analysis.

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    Zhiyuan Weng

    Full Text Available OBJECTIVE: Evidence is mounting suggesting that a strong genetic component underlies aspirin insensitivity. To generate more information, we aimed to evaluate the association of four common polymorphisms (rs3842787, rs20417, rs201184269, rs1126643 from four candidate genes (COX-1, COX-2, ITGA2B, ITGA2 with aspirin insensitivity via a meta-analysis. METHODS AND RESULTS: In total, there were 4 (353/595, 6 (344/698, 10 (588/878 and 7 (209/676 articles (patients/controls qualified for rs3842787, rs20417, rs20118426 and rs1126643, respectively. The data were extracted in duplicate and analyzed by STATA software (Version 11.2. The risk estimate was expressed as odds ratio (OR and 95% confidence interval (95% CI. Analyses of the full data set indicated significant associations of rs20417 (OR; 95% CI; P: 1.86; 1.44-2.41; <0.0005 and rs1126643 (2.37; 1.44-3.89; 0.001 with aspirin insensitivity under allelic model. In subgroup analyses, the risk estimate for rs1126643 was greatly potentiated among patients with aspirin semi-resistance relative to those with aspirin resistance, especially under dominant model (aspirin semi-resistance: 5.44; 1.42-20.83; 0.013 versus aspirin resistance: 1.96; 1.07-3.6; 0.03. Further grouping articles by ethnicity observed a stronger prediction of all, but rs20417, examined polymorphisms for aspirin insensitivity in Chinese than in Caucasians. Finally, meta-regression analyses observed that the differences in percentage of coronary artery disease (P = 0.034 and averaged platelet numbers (P = 0.012 between two groups explained a large part of heterogeneity for rs20417 and rs1126643, respectively. CONCLUSION: Our findings provide strong evidence that COX-2 and ITGA2 genetic defects might increase the risk of having aspirin insensitivity, especially for aspirin semi-resistance and in Chinese populations.

  20. mTOR Inhibition Induces EGFR Feedback Activation in Association with Its Resistance to Human Pancreatic Cancer

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    Feng Wei

    2015-02-01

    Full Text Available The mammalian target of rapamycin (mTOR is dysregulated in diverse cancers and contributes to tumor progression and drug resistance. The first generation of mTOR inhibitors have failed to show clinical efficiency in treating pancreatic cancers due in part to the feedback relief of the insulin-like growth factor-1 receptor (IGF-1R-AKT signaling pathway. The second generation of mTOR inhibitors, such as AZD8055, could inhibit AKT activation upon mTOR complex 2 (mTORC2 inhibition. However, whether this generation of mTOR inhibitors can obtain satisfactory activities in pancreatic cancer therapy remains unclear. In this study, we found AZD8055 did not show great improvement compared with everolimus, AZD8055 induced a temporal inhibition of AKT kinase activities and AKT was then rephosphorylated. Additionally, we found that AZD8055-induced transient AKT inhibition increased the expression and activation of epidermal growth factor receptor (EGFR by releasing its transcriptional factors Fork-head box O 1/3a (FoxO1/3a, which might contribute to cell resistance to AZD8055. The in vitro and in vivo experiments further indicated the combination of AZD8055 and erlotinib synergistically inhibited the mTORC1/C2 signaling pathway, EGFR/AKT feedback activation, and cell growth, as well as suppressed the progression of pancreatic cancer in a xenograft model. This study provides a rationale and strategy for overcoming AZD8055 resistance by a combined treatment with the EGFR inhibitor erlotinib in pancreatic cancer therapy.

  1. The Rapamycin-Binding Domain of the Protein Kinase mTOR is a Destabilizing Domain*

    Science.gov (United States)

    Edwards, Sarah R.; Wandless, Thomas J.

    2013-01-01

    Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding domain (FRB) of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to ten-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retain the ability to inhibit mTOR, albeit with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wildtype FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems. PMID:17350953

  2. Mechanosensitive molecular networks involved in transducing resistance exercise-signals into muscle protein accretion

    Directory of Open Access Journals (Sweden)

    Emil Rindom

    2016-11-01

    Full Text Available Loss of skeletal muscle myofibrillar protein with disease and/or inactivity can severely deteriorate muscle strength and function. Strategies to counteract wasting of muscle myofibrillar protein are therefore desirable and invite for considerations on the potential superiority of specific modes of resistance exercise and/or the adequacy of low load resistance exercise regimens as well as underlying mechanisms. In this regard, delineation of the potentially mechanosensitive molecular mechanisms underlying muscle protein synthesis (MPS, may contribute to understanding on how differentiated resistance exercise can transduce a mechanical signal into stimulation of muscle accretion. Recent findings suggest specific upstream exercise-induced mechano-sensitive myocellular signaling pathways to converge on mammalian target of rapamycin complex 1 (mTORC1, to influence MPS. This may e.g. implicate mechanical activation of signaling through a diacylglycerol kinase (DGKζ-phosphatidic acid (PA axis or implicate integrin deformation to signal through a Focal adhesion kinase (FAK-Tuberous Sclerosis Complex 2TSC2-Ras homolog enriched in brain (Rheb axis. Moreover, since initiation of translation is reliant on mRNA, it is also relevant to consider potentially mechanosensitive signaling pathways involved in muscle myofibrillar gene transcription and whether some of these pathways converge with those affecting mTORC1 activation for MPS. In this regard, recent findings suggest how mechanical stress may implicate integrin deformation and/or actin dynamics to signal through a Ras homolog gene family member A protein (RhoA-striated muscle activator of Rho signaling (STARS axis or how it may implicate deformation of Notch to affect Bone Morphogenetic Protein (BMP signaling through a small mother of decapentaplegic (Smad axis.

  3. Gremlin promotes retinal pigmentation epithelial (RPE) cell proliferation, migration and VEGF production via activating VEGFR2-Akt-mTORC2 signaling.

    Science.gov (United States)

    Liu, Yuan; Chen, Zhijun; Cheng, Haixia; Chen, Juan; Qian, Jing

    2017-01-03

    Retinopathy of prematurity (ROP) is characterized by late-phase pathologic retinal vasoproliferation. Gremlin is a novel vascular endothelial growth factors (VEGF) receptor 2 (VEGFR2) agonist and promotes angiogenic response. We demonstrated that gremlin expression was significantly increased in retinas of ROP model mice, which was correlated with VEGF upregulation. In retinal pigmentation epithelial (RPE) cells, gremlin activated VEGFR2-Akt-mTORC2 (mammalian target of rapamycin complex 2) signaling, and promoted cell proliferation, migration and VEGF production. VEGFR inhibition (by SU5416) or shRNA knockdown almost abolished gremlin-mediated pleiotropic functions in RPE cells. Further, pharmacological inhibition of Akt-mTOR, or shRNA knockdown of key mTORC2 component (Rictor or Sin1) also attenuated gremlin-exerted activities in RPE cells. We conclude that gremlin promotes RPE cell proliferation, migration and VEGF production possibly via activating VEGFR2-Akt-mTORC2 signaling. Gremlin could be a novel therapeutic target of ROP or other retinal vasoproliferation diseases.

  4. Joint inhibition of TOR and JNK pathways interacts to extend the lifespan of Brachionus manjavacas (Rotifera).

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    Snell, Terry W; Johnston, Rachel K; Rabeneck, Brett; Zipperer, Cody; Teat, Stephanie

    2014-04-01

    The TOR kinase pathway is central in modulating aging in a variety of animal models. The target of rapamycin (TOR) integrates a complex network of signals from growth conditions, nutrient availability, energy status, and physiological stresses and matches an organism's growth rate to the resource environment. Important remaining problems are the identification of the pathways that interact with TOR and their characterization as additive or synergistic. One of the most versatile stress sensors in metazoans is the Jun-N-terminal kinase (JNK) signaling pathway. JNK is an evolutionarily conserved stress-activated protein kinase that is induced by a range of stressors, including UV irradiation, reactive oxygen species, DNA damage, heat, and bacterial antigens. JNK is thought to interact with the TOR pathway, but its effects on TOR are poorly understood. We used the rotifer Brachionus manjavacas as a model animal to probe the regulation of TOR and JNK pathways and explore their interaction. The effect of various chemical inhibitors was examined in life table and stressor challenge experiments. A survey of 12 inhibitors revealed two, rapamycin and JNK inhibitor, that significantly extended lifespan of B. manjavacas. At 1 μM concentration, exposure to rapamycin or JNK inhibitor extended mean rotifer lifespan by 35% and maximum lifespan by 37%. Exposure to both rapamycin and JNK inhibitor simultaneously extended mean rotifer lifespan by 65% more than either alone. Exposure to a combination of rapamycin and JNK inhibitors conveyed greater protection to starvation, UV and osmotic stress than either inhibitor alone. RNAi knockdown of TOR and JNK gene expression was investigated for its ability to extend rotifer lifespan. RNAi knockdown of the TOR gene resulted in 29% extension of the mean lifespan compared to control and knockdown of the JNK gene resulted in 51% mean lifespan extension. In addition to the lifespan, we quantified mitochondria activity using the fluorescent

  5. TOR Pathway-Mediated Juvenile Hormone Synthesis Regulates Nutrient-Dependent Female Reproduction in Nilaparvata lugens (Stål).

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    Lu, Kai; Chen, Xia; Liu, Wen-Ting; Zhou, Qiang

    2016-03-28

    The "target of rapamycin" (TOR) nutritional signaling pathway and juvenile hormone (JH) regulation of vitellogenesis has been known for a long time. However, the interplay between these two pathways regulating vitellogenin (Vg) expression remains obscure. Here, we first demonstrated the key role of amino acids (AAs) in activation of Vg synthesis and egg development in Nilaparvata lugens using chemically defined artificial diets. AAs induced the expression of TOR and S6K (S6 kinase), whereas RNAi-mediated silencing of these two TOR pathway genes and rapamycin application strongly inhibited the AAs-induced Vg synthesis. Furthermore, knockdown of Rheb (Ras homologue enriched in brain), TOR, S6K and application of rapamycin resulted in a dramatic reduction in the mRNA levels of jmtN (juvenile hormone acid methyltransferase, JHAMT). Application of JH III on the RNAi (Rheb and TOR) and rapamycin-treated females partially rescued the Vg expression. Conversely, knockdown of either jmtN or met (methoprene-tolerant, JH receptor) and application of JH III had no effects on mRNA levels of Rheb, TOR and S6K and phosphorylation of S6K. In summary, our results demonstrate that the TOR pathway induces JH biosynthesis that in turn regulates AAs-mediated Vg synthesis in N. lugens.

  6. Mechanistic Target of Rapamycin-Independent Antidepressant Effects of (R)-Ketamine in a Social Defeat Stress Model.

    Science.gov (United States)

    Yang, Chun; Ren, Qian; Qu, Youge; Zhang, Ji-Chun; Ma, Min; Dong, Chao; Hashimoto, Kenji

    2018-01-01

    The role of the mechanistic target of rapamycin (mTOR) signaling in the antidepressant effects of ketamine is controversial. In addition to mTOR, extracellular signal-regulated kinase (ERK) is a key signaling molecule in prominent pathways that regulate protein synthesis. (R)-Ketamine has a greater potency and longer-lasting antidepressant effects than (S)-ketamine. Here we investigated whether mTOR signaling and ERK signaling play a role in the antidepressant effects of two enantiomers. The effects of mTOR inhibitors (rapamycin and AZD8055) and an ERK inhibitor (SL327) on the antidepressant effects of ketamine enantiomers in the chronic social defeat stress (CSDS) model (n = 7 or 8) and on those of ketamine enantiomers in these signaling pathways in mouse brain regions were examined. The intracerebroventricular infusion of rapamycin or AZD8055 blocked the antidepressant effects of (S)-ketamine, but not (R)-ketamine, in the CSDS model. Furthermore, (S)-ketamine, but not (R)-ketamine, significantly attenuated the decreased phosphorylation of mTOR and its downstream effector, ribosomal protein S6 kinase, in the prefrontal cortex of susceptible mice after CSDS. Pretreatment with SL327 blocked the antidepressant effects of (R)-ketamine but not (S)-ketamine. Moreover, (R)-ketamine, but not (S)-ketamine, significantly attenuated the decreased phosphorylation of ERK and its upstream effector, mitogen-activated protein kinase/ERK kinase, in the prefrontal cortex and hippocampal dentate gyrus of susceptible mice after CSDS. This study suggests that mTOR plays a role in the antidepressant effects of (S)-ketamine, but not (R)-ketamine, and that ERK plays a role in (R)-ketamine's antidepressant effects. Thus, it is unlikely that the activation of mTOR signaling is necessary for antidepressant actions of (R)-ketamine. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  7. Pirarubicin induces an autophagic cytoprotective response through suppression of the mammalian target of rapamycin signaling pathway in human bladder cancer cells

    International Nuclear Information System (INIS)

    Li, Kuiqing; Chen, Xu; Liu, Cheng; Gu, Peng; Li, Zhuohang; Wu, Shaoxu; Xu, Kewei; Lin, Tianxin; Huang, Jian

    2015-01-01

    Pirarubicin is widely used in intravesical chemotherapy for bladder cancer, but its efficacy is limited due to drug resistance; the mechanism has not been well studied. Emerging evidence shows that autophagy can be a novel target for cancer therapy. This study aimed to investigate the role of autophagy in pirarubicin-treated bladder cancer cells. Bladder cancer cells EJ and J82 were treated with pirarubicin, siRNA, 3-methyladenine or hydroxychloroquine. Cell proliferation and apoptosis were tested by cell survival assay and flow cytometric analysis, respectively. Autophagy was evaluated by immunoblotting before and after the treatments. The phosphorylated mammalian target of rapamycin, serine/threonine kinase p70 S6 kinase, and eukaryotic translation initiation factor 4E binding protein 1 were also investigated by immunoblotting. We found that pirarubicin could induce autophagy in bladder cancer cells. Inhibition of autophagy by 3-methyladenine, hydroxychloroquine or knockdown of autophagy related gene 3 significantly increased apoptosis in pirarubicin-treated bladder cancer cells. Pirarubicin-induced autophagy was mediated via the mTOR/p70S6K/4E-BP1 signaling pathway. In conclusion, autophagy induced by pirarubicin plays a cytoprotective role in bladder cancer cells, suggesting that inhibition of autophagy may improve efficacy over traditional pirarubicin chemotherapy in bladder cancer patients. - Highlights: • Pirarubicin induced autophagy in bladder cancer cells. • Inhibition of autophagy enhanced pirarubicin-induced apoptosis. • Pirarubicin induced autophagy through inhibition of mTOR signaling pathway

  8. Pirarubicin induces an autophagic cytoprotective response through suppression of the mammalian target of rapamycin signaling pathway in human bladder cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Kuiqing; Chen, Xu [Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Liu, Cheng [Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Gu, Peng; Li, Zhuohang; Wu, Shaoxu [Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Xu, Kewei [Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Lin, Tianxin, E-mail: tianxinl@sina.com [Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Huang, Jian, E-mail: urolhj@sina.com [Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China)

    2015-05-01

    Pirarubicin is widely used in intravesical chemotherapy for bladder cancer, but its efficacy is limited due to drug resistance; the mechanism has not been well studied. Emerging evidence shows that autophagy can be a novel target for cancer therapy. This study aimed to investigate the role of autophagy in pirarubicin-treated bladder cancer cells. Bladder cancer cells EJ and J82 were treated with pirarubicin, siRNA, 3-methyladenine or hydroxychloroquine. Cell proliferation and apoptosis were tested by cell survival assay and flow cytometric analysis, respectively. Autophagy was evaluated by immunoblotting before and after the treatments. The phosphorylated mammalian target of rapamycin, serine/threonine kinase p70 S6 kinase, and eukaryotic translation initiation factor 4E binding protein 1 were also investigated by immunoblotting. We found that pirarubicin could induce autophagy in bladder cancer cells. Inhibition of autophagy by 3-methyladenine, hydroxychloroquine or knockdown of autophagy related gene 3 significantly increased apoptosis in pirarubicin-treated bladder cancer cells. Pirarubicin-induced autophagy was mediated via the mTOR/p70S6K/4E-BP1 signaling pathway. In conclusion, autophagy induced by pirarubicin plays a cytoprotective role in bladder cancer cells, suggesting that inhibition of autophagy may improve efficacy over traditional pirarubicin chemotherapy in bladder cancer patients. - Highlights: • Pirarubicin induced autophagy in bladder cancer cells. • Inhibition of autophagy enhanced pirarubicin-induced apoptosis. • Pirarubicin induced autophagy through inhibition of mTOR signaling pathway.

  9. Evolutionary Conservation of the Components in the TOR Signaling Pathways.

    Science.gov (United States)

    Tatebe, Hisashi; Shiozaki, Kazuhiro

    2017-11-01

    Target of rapamycin (TOR) is an evolutionarily conserved protein kinase that controls multiple cellular processes upon various intracellular and extracellular stimuli. Since its first discovery, extensive studies have been conducted both in yeast and animal species including humans. Those studies have revealed that TOR forms two structurally and physiologically distinct protein complexes; TOR complex 1 (TORC1) is ubiquitous among eukaryotes including animals, yeast, protozoa, and plants, while TOR complex 2 (TORC2) is conserved in diverse eukaryotic species other than plants. The studies have also identified two crucial regulators of mammalian TORC1 (mTORC1), Ras homolog enriched in brain (RHEB) and RAG GTPases. Of these, RAG regulates TORC1 in yeast as well and is conserved among eukaryotes with the green algae and land plants as apparent exceptions. RHEB is present in various eukaryotes but sporadically missing in multiple taxa. RHEB, in the budding yeast Saccharomyces cerevisiae , appears to be extremely divergent with concomitant loss of its function as a TORC1 regulator. In this review, we summarize the evolutionarily conserved functions of the key regulatory subunits of TORC1 and TORC2, namely RAPTOR, RICTOR, and SIN1. We also delve into the evolutionary conservation of RHEB and RAG and discuss the conserved roles of these GTPases in regulating TORC1.

  10. Drug synergy drives conserved pathways to increase fission yeast lifespan.

    Directory of Open Access Journals (Sweden)

    Xinhe Huang

    Full Text Available Aging occurs over time with gradual and progressive loss of physiological function. Strategies to reduce the rate of functional loss and mitigate the subsequent onset of deadly age-related diseases are being sought. We demonstrated previously that a combination of rapamycin and myriocin reduces age-related functional loss in the Baker's yeast Saccharomyces cerevisiae and produces a synergistic increase in lifespan. Here we show that the same drug combination also produces a synergistic increase in the lifespan of the fission yeast Schizosaccharomyces pombe and does so by controlling signal transduction pathways conserved across a wide evolutionary time span ranging from yeasts to mammals. Pathways include the target of rapamycin complex 1 (TORC1 protein kinase, the protein kinase A (PKA and a stress response pathway, which in fission yeasts contains the Sty1 protein kinase, an ortholog of the mammalian p38 MAP kinase, a type of Stress Activated Protein Kinase (SAPK. These results along with previous studies in S. cerevisiae support the premise that the combination of rapamycin and myriocin enhances lifespan by regulating signaling pathways that couple nutrient and environmental conditions to cellular processes that fine-tune growth and stress protection in ways that foster long term survival. The molecular mechanisms for fine-tuning are probably species-specific, but since they are driven by conserved nutrient and stress sensing pathways, the drug combination may enhance survival in other organisms.

  11. The Role of the Mammalian Target of Rapamycin (mTOR) in Pulmonary Fibrosis

    Science.gov (United States)

    Nho, Richard

    2018-01-01

    The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR)-dependent pathway is one of the most integral pathways linked to cell metabolism, proliferation, differentiation, and survival. This pathway is dysregulated in a variety of diseases, including neoplasia, immune-mediated diseases, and fibroproliferative diseases such as pulmonary fibrosis. The mTOR kinase is frequently referred to as the master regulator of this pathway. Alterations in mTOR signaling are closely associated with dysregulation of autophagy, inflammation, and cell growth and survival, leading to the development of lung fibrosis. Inhibitors of mTOR have been widely studied in cancer therapy, as they may sensitize cancer cells to radiation therapy. Studies also suggest that mTOR inhibitors are promising modulators of fibroproliferative diseases such as idiopathic pulmonary fibrosis (IPF) and radiation-induced pulmonary fibrosis (RIPF). Therefore, mTOR represents an attractive and unique therapeutic target in pulmonary fibrosis. In this review, we discuss the pathological role of mTOR kinase in pulmonary fibrosis and examine how mTOR inhibitors may mitigate fibrotic progression. PMID:29518028

  12. Repletion of branched chain amino acids reverses mTORC1 signaling but not improved metabolism during dietary protein dilution

    Directory of Open Access Journals (Sweden)

    Adriano Maida

    2017-08-01

    Conclusions: Repletion of BCAAs in dietary PD is sufficient to oppose changes in somatic mTORC1 signaling but does not reverse the hepatic ISR nor induce insulin resistance in type 2 diabetes during dietary PD.

  13. Paradigmenwechsel in der Anti-Aging-Medizin: Hormesis, Target-of-Rapamycin-Komplex und erste Anti-Aging-Pillen // Paradigm Shift in Anti-Aging Medicine: Hormesis, Target of Rapamycin Complex and First Human Anti-Aging-Pills

    Directory of Open Access Journals (Sweden)

    Römmler A

    2016-01-01

    Full Text Available Studies in model organisms have shown that some drugs and lifestyle practices (calorie-restricted diets, regular exercise, e.g. can extend life and health span and protect against the onset of age-related chronic diseases by targeting physiological pathways.brA common mode of action was found via mTOR (mechanistic Target Of Rapamycin pathway signalling. This intracellular protein kinase complex plays a key role in stimulating anabolic and cell growth promoting processes, while inhibiting autophagy. On the other hand, downregulation results in antiproliferative, anticancer and intensive cell-repairing effects leading to life and health span extension and stress resistance. The mTOR complex regulates such basic cell activities and integrates signals from nutrition sensing, energy metabolism, insulin and growth factors, stress and hypoxia.brImportantly, mTOR can be inhibited by some molecules and their analogs (rapamycin, resveratrol, metformin, e.g., which are released naturally from plants, yeast or bacteria to protect against natural enemies. Its dosage resembles an adaptive hormetic response relationship, as high concentrations are toxic and mild doses are associated with anticancer and antiaging effects. This opens up new avenues for their use as „anti-aging pills“ in humans.brRecent human data suggest that metformin, rapamycin and other mTOR-inhibitors could delay heart disease, cancer, cognitive decline and improve survival time in people with diabetes mellitus. In addition, response to influenca vaccine was enhanced by rapamycin in adults with immunosenescence, indicating beneficial anti-aging effects in the elderly.br“Treat aging” is an actual call to recognize aging as an indication appropriate for clinical trials and treatments, as it was recently approved by the Federal Drug Administration (FDA USA. p bKurzfassung/b: Die ansteigende Morbidität und Invalidität in alternden Industrienationen stößt an die Grenzen der Ressourcen

  14. The nutrient transceptor/PKA pathway functions independently of TOR and responds to leucine and Gcn2 in a TOR-independent manner.

    Science.gov (United States)

    Conrad, Michaela; Kankipati, Harish Nag; Kimpe, Marlies; Van Zeebroeck, Griet; Zhang, Zhiqiang; Thevelein, Johan M

    2017-08-01

    Two nutrient-controlled signalling pathways, the PKA and TOR pathway, play a major role in nutrient regulation of growth as well as growth-correlated properties in yeast. The relationship between the two pathways is not well understood. We have used Gap1 and Pho84 transceptor-mediated activation of trehalase and phosphorylation of fragmented Sch9 as a read-out for rapid nutrient activation of PKA or TORC1, respectively. We have identified conditions in which L-citrulline-induced activation of Sch9 phosphorylation is compromised, but not activation of trehalase: addition of the TORC1 inhibitor, rapamycin and low levels of L-citrulline. The same disconnection was observed for phosphate activation in phosphate-starved cells. The leu2 auxotrophic mutation reduces amino acid activation of trehalase, which is counteracted by deletion of GCN2. Both effects were also independent of TORC1. Our results show that rapid activation of the TOR pathway by amino acids is not involved in rapid activation of the PKA pathway and that effects of Gcn2 inactivation as well as leu2 auxotrophy all act independently of the TOR pathway. Hence, rapid nutrient signalling to PKA and TOR in cells arrested by nutrient starvation acts through parallel pathways. © FEMS 2017.

  15. Rapamycin potentiates cytotoxicity by docetaxel possibly through downregulation of Survivin in lung cancer cells

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    Li Hui

    2011-03-01

    Full Text Available Abstract Background To elucidate whether rapamycin, the inhibitor of mTOR (mammalian target of rapamycin, can potentiate the cytotoxic effect of docetaxel in lung cancer cells and to probe the mechanism underlying such enhancement. Methods Lung cancer cells were treated with docetaxel and rapamycin. The effect on the proliferation of lung cancer cells was evaluated using the MTT method, and cell apoptosis was measured by flow cytometry. Protein expression and level of phosphorylation were assayed using Western Blot method. Results Co-treatment of rapamycin and docetaxel was found to favorably enhance the cytotoxic effect of docetaxel in four lung cancer cell lines. This tumoricidal boost is associated with a reduction in the expression and phosphorylation levels of Survivin and ERK1/2, respectively. Conclusion The combined application of mTOR inhibitor and docetaxel led to a greater degree of cancer cell killing than that by either compound used alone. Therefore, this combination warrants further investigation in its suitability of serving as a novel therapeutic scheme for treating advanced and recurrent lung cancer patients.

  16. OSI-027 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine both in vitro and in vivo.

    Science.gov (United States)

    Zhi, Xiao; Chen, Wei; Xue, Fei; Liang, Chao; Chen, Bryan Wei; Zhou, Yue; Wen, Liang; Hu, Liqiang; Shen, Jian; Bai, Xueli; Liang, Tingbo

    2015-09-22

    Despite its relative rarity, pancreatic ductal adenocarcinoma (PDAC) accounts for a large percentage of cancer deaths. In this study, we investigated the in vitro efficacy of OSI-027, a selective inhibitor of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, to treat PDAC cell lines alone, and in combination with gemcitabine (GEM). Similarly, we tested the efficacy of these two compounds in a xenograft mouse model of PDAC. OSI-027 significantly arrested cell cycle in G0/G1 phase, inhibited the proliferation of Panc-1, BxPC-3, and CFPAC-1 cells, and downregulated mTORC1, mTORC2, phospho-Akt, phospho-p70S6K, phospho-4E-BP1, cyclin D1, and cyclin-dependent kinase 4 (CDK4) in these cells. Moreover, OSI-027 also downregulated multidrug resistance (MDR)-1, which has been implicated in chemotherapy resistance in PDAC cells and enhanced apoptosis induced by GEM in the three PDAC cell lines. When combined, OSI-027 with GEM showed synergistic cytotoxic effects both in vitro and in vivo. This is the first evidence of the efficacy of OSI-027 in PDAC and may provide the groundwork for a new clinical PDAC therapy.

  17. Anti-bombing insensitivity life of molybdenum cathode doped with La2O3 and Y2O3

    International Nuclear Information System (INIS)

    Wang Jinshu; Wang Yiman; Zhou Meiling

    2006-01-01

    Anti-bombing insensitivity of La 2 O 3 -Y 2 O 3 -Mo secondary emitter has been studied in this paper. The variation of maximum secondary emission coefficient δ max with time was measured. The cathode after life experiment was analyzed by means of HRM, SEM, EDS and XRD. The results showed that δ max of La 2 O 3 -Y 2 O 3 -Mo cathode operating at 1100 deg. C under continuous electron bombardment of 300 W/cm 2 was still about 2.5 after 1000 h operation, indicating that this kind of cathode had good anti-bombing insensitivity. In the internal part of the cathode, RE 2 O 3 (rare earth oxide) and molybdenum grains distributed alternately and there existed a certain relationship between crystallographic orientation of RE 2 O 3 and that of molybdenum. It was found that a RE 2 O 3 layer was formed on the surface after operation. The high δ max of La 2 O 3 -Y 2 O 3 -Mo cathode was related to the RE 2 O 3 layer on the surface and the amount of nanosized La 2 O 3 particles on the Y 2 O 3 layer

  18. Dietary intervention in acne

    Science.gov (United States)

    Melnik, Bodo

    2012-01-01

    The purpose of this paper is to highlight the endocrine signaling of Western diet, a fundamental environmental factor involved in the pathogenesis of epidemic acne. Western nutrition is characterized by high calorie uptake, high glycemic load, high fat and meat intake, as well as increased consumption of insulin- and IGF-1-level elevating dairy proteins. Metabolic signals of Western diet are sensed by the nutrient-sensitive kinase, mammalian target of rapamycin complex 1 (mTORC1), which integrates signals of cellular energy, growth factors (insulin, IGF-1) and protein-derived signals, predominantly leucine, provided in high amounts by milk proteins and meat. mTORC1 activates SREBP, the master transcription factor of lipogenesis. Leucine stimulates mTORC1-SREBP signaling and leucine is directly converted by sebocytes into fatty acids and sterols for sebaceous lipid synthesis. Over-activated mTORC1 increases androgen hormone secretion and most likely amplifies androgen-driven mTORC1 signaling of sebaceous follicles. Testosterone directly activates mTORC1. Future research should investigate the effects of isotretinoin on sebocyte mTORC1 activity. It is conceivable that isotretinoin may downregulate mTORC1 in sebocytes by upregulation of nuclear levels of FoxO1. The role of Western diet in acne can only be fully appreciated when all stimulatory inputs for maximal mTORC1 activation, i.e., glucose, insulin, IGF-1 and leucine, are adequately considered. Epidemic acne has to be recognized as an mTORC1-driven disease of civilization like obesity, type 2 diabetes, cancer and neurodegenerative diseases. These new insights into Western diet-mediated mTORC1-hyperactivity provide a rational basis for dietary intervention in acne by attenuating mTORC1 signaling by reducing (1) total energy intake, (2) hyperglycemic carbohydrates, (3) insulinotropic dairy proteins and (4) leucine-rich meat and dairy proteins. The necessary dietary changes are opposed to the evolution of

  19. Regulated Assembly of Vacuolar ATPase Is Increased during Cluster Disruption-induced Maturation of Dendritic Cells through a Phosphatidylinositol 3-Kinase/mTOR-dependent Pathway*

    Science.gov (United States)

    Liberman, Rachel; Bond, Sarah; Shainheit, Mara G.; Stadecker, Miguel J.; Forgac, Michael

    2014-01-01

    The vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation. PMID:24273170

  20. Rapamycin-based inducible translocation systems for studying phagocytosis.

    Science.gov (United States)

    Bohdanowicz, Michal; Fairn, Gregory D

    2011-01-01

    Phagocytosis is an immune receptor-mediated process whereby cells engulf large particles. The process is dynamic and requires several localized factors acting in concert with and sequentially after the engagement of immune receptors to envelope the particle. Once the particle is internalized, the nascent -phagosome undergoes a series of events leading to its maturation to the microbicidal phagolysosome. Investigating these dynamic and temporally controlled series of events in live cells requires noninvasive methods. The ability to rapidly recruit the proteins of interest to the sites of phagocytosis or to nascent phagosomes would help dissect the regulatory mechanisms involved during phagocytosis. Here, we describe a general approach to express in RAW264.7 murine macrophages, a genetically encoded rapamycin--induced heterodimerization system. In the presence of rapamycin, tight association between FK506-binding protein (FKBP) and FKBP rapamycin-binding protein (FRB) is observed. Based on this principle, a synthetic system consisting of a targeting domain attached to FKBP can recruit a protein of interest fused to FRB upon the addition of rapamycin. Previously, this technique has been used to target lipid-modifying enzymes and small GTPases to the phagosome or plasma membrane. The recruitment of the FRB module can be monitored by fluorescent microscopy if a fluorescent protein is fused to the FRB sequence. While the focus of this chapter is on phagocytic events, this method can be employed to study any organelle of interest when the appropriate targeting sequence is used.

  1. The TOR signaling pathway regulates vegetative development and virulence in Fusarium graminearum.

    Science.gov (United States)

    Yu, Fangwei; Gu, Qin; Yun, Yingzi; Yin, Yanni; Xu, Jin-Rong; Shim, Won-Bo; Ma, Zhonghua

    2014-07-01

    The target of rapamycin (TOR) signaling pathway plays critical roles in controlling cell growth in a variety of eukaryotes. However, the contribution of this pathway in regulating virulence of plant pathogenic fungi is unknown. We identified and characterized nine genes encoding components of the TOR pathway in Fusarium graminearum. Biological, genetic and biochemical functions of each component were investigated. The FgFkbp12-rapamycin complex binds to the FgTor kinase. The type 2A phosphatases FgPp2A, FgSit4 and FgPpg1 were found to interact with FgTap42, a downstream component of FgTor. Among these, we determined that FgPp2A is likely to be essential for F. graminearum survival, and FgSit4 and FgPpg1 play important roles in cell wall integrity by positively regulating the phosphorylation of FgMgv1, a key MAP kinase in the cell wall integrity pathway. In addition, the FgPpg1 interacting protein, FgTip41, is involved in regulating mycelial growth and virulence. Notably, FgTip41 does not interact with FgTap42 but with FgPpg1, suggesting the existence of FgTap42:FgPpg1:FgTip41 heterotrimer in F. graminearum, a complex not observed in the yeast model. Collectively, we defined a genetic regulatory framework that elucidates how the TOR pathway regulates virulence and vegetative development in F. graminearum. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  2. A Signal-On Fluorosensor Based on Quench-Release Principle for Sensitive Detection of Antibiotic Rapamycin

    Directory of Open Access Journals (Sweden)

    Hee-Jin Jeong

    2015-03-01

    Full Text Available An antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose “Q’-body”, which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body technology. We constructed rapamycin Q’-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q’-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.

  3. A signal-on fluorosensor based on quench-release principle for sensitive detection of antibiotic rapamycin.

    Science.gov (United States)

    Jeong, Hee-Jin; Itayama, Shuya; Ueda, Hiroshi

    2015-03-26

    An antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose "Q'-body", which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body) technology. We constructed rapamycin Q'-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q'-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.

  4. Rapamycin treatment is associated with an increased apoptosis rate in experimental vein grafts.

    Science.gov (United States)

    Schachner, Thomas; Oberhuber, Alexander; Zou, Yping; Tzankov, Alexandar; Ott, Harald; Laufer, Günther; Bonatti, Johannes

    2005-02-01

    Rapamycin is an immunosuppressive agent with marked antiproliferative properties and is effective in reducing in stent restenosis and vein graft neointimal hyperplasia. Apoptosis is one mechanism counterbalancing cellular proliferation. We therefore investigated the role of apoptosis in rapamycin treated vein grafts in a mouse model. C57BL6J mice underwent interposition of the inferior vena cava from isogenic donor mice into the common carotid artery using a cuff technique. In the treatment group 200 microg of rapamycin were applied locally in pluronic gel. The control group did not receive local treatment. Vein grafts were harvested at 4 weeks postoperatively and underwent morphometric analysis as well as immunohistochemical analysis for apoptosis (TUNEL). In grafted veins without treatment (controls) neointimal thickness was 50 (12-58) microm at 4 weeks postoperatively. In 200 microg rapamycin treated grafts the neointimal thickness was 17 (5-55) microm. Rapamycin treated vein grafts showed a significantly increased rate of apoptosis in the adventitia as compared with controls (P=0.032). In the neointima the apoptosis rate was lower in both groups with no significant difference between rapamycin treated grafts and controls. We conclude that treatment of experimental vein grafts with rapamycin is associated with an increased apoptosis rate in the vascular wall and a trend towards reduction of neointimal hyperplasia. These results suggest that apoptosis may be a beneficial antiproliferative component for the treatment of vein graft disease.

  5. Crosstalk between mTORC1 and cAMP Signaling

    Science.gov (United States)

    2016-09-01

    whether bidirectional inhibition of trafficking be- tween the endoplasmic reticulum (ER) and Golgi would affect Gln-induced activation of mTORC1 (23). We...Shimizu N, Matsumoto K, Itoh M, Ishitani T. 2012. NLK positively regulates Wnt/β-catenin signalling by phosphorylating LEF1 in neural progenitor...L, Pan D, Edgar BA. 2003. Rheb promotes cell growth as a component of the insulin/ TOR signalling network . Nat Cell Biol 5: 566–571. Sengupta S

  6. Regulation of mTORC1 Signaling by Src Kinase Activity Is Akt1-Independent in RSV-Transformed Cells

    Directory of Open Access Journals (Sweden)

    Martina Vojtěchová

    2008-02-01

    Full Text Available Increased activity of the Src tyrosine protein kinase that has been observed in a large number of human malignancies appears to be a promising target for drug therapy. In the present study, a critical role of the Src activity in the deregulation of mTOR signaling pathway in Rous sarcoma virus (RSV-transformed hamster fibroblasts, H19 cells, was shown using these cells treated with the Src-specific inhibitor, SU6656, and clones of fibroblasts expressing either the active Src or the dominant-negative Src kinase-dead mutant. Disruption of the Src kinase activity results in substantial reduction of the phosphorylation and activity of the Akt/protein kinase B (PKB, phosphorylation of tuberin (TSC2, mammalian target of rapamycin (mTOR, S6K1, ribosomal protein S6, and eukaryotic initiation factor 4E-binding protein 4E-BP1. The ectopic, active Akt1 that was expressed in Src-deficient cells significantly enhanced phosphorylation of TSC2 in these cells, but it failed to activate the inhibited components of the mTOR pathway that are downstream of TSC2. The data indicate that the Src kinase activity is essential for the activity of mTOR-dependent signaling pathway and suggest that mTOR targets may be controlled by Src independently of Akt1/TSC2 cascade in cells expressing hyperactive Src protein. These observations might have an implication in drug resistance to mTOR inhibitor-based cancer therapy in certain cell types.

  7. DEP domain-containing mTOR-interacting protein suppresses lipogenesis and ameliorates hepatic steatosis and acute-on-chronic liver injury in alcoholic liver disease.

    Science.gov (United States)

    Chen, Hanqing; Shen, Feng; Sherban, Alex; Nocon, Allison; Li, Yu; Wang, Hua; Xu, Ming-Jiang; Rui, Xianliang; Han, Jinyan; Jiang, Bingbing; Lee, Donghwan; Li, Na; Keyhani-Nejad, Farnaz; Fan, Jian-Gao; Liu, Feng; Kamat, Amrita; Musi, Nicolas; Guarente, Leonard; Pacher, Pal; Gao, Bin; Zang, Mengwei

    2018-02-19

    Alcoholic liver disease (ALD) is characterized by lipid accumulation and liver injury. However, how chronic alcohol consumption causes hepatic lipid accumulation remains elusive. The present study demonstrates that activation of the mechanistic target of rapamycin complex 1 (mTORC1) plays a causal role in alcoholic steatosis, inflammation, and liver injury. Chronic-plus-binge ethanol feeding led to hyperactivation of mTORC1, as evidenced by increased phosphorylation of mTOR and its downstream kinase S6 kinase 1 (S6K1) in hepatocytes. Aberrant activation of mTORC1 was likely attributed to the defects of the DEP domain-containing mTOR-interacting protein (DEPTOR) and the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1) in the liver of chronic-plus-binge ethanol-fed mice and in the liver of patients with ALD. Conversely, adenoviral overexpression of hepatic DEPTOR suppressed mTORC1 signaling and ameliorated alcoholic hepatosteatosis, inflammation, and acute-on-chronic liver injury. Mechanistically, the lipid-lowering effect of hepatic DEPTOR was attributable to decreased proteolytic processing, nuclear translocation, and transcriptional activity of the lipogenic transcription factor sterol regulatory element-binding protein-1 (SREBP-1). DEPTOR-dependent inhibition of mTORC1 also attenuated alcohol-induced cytoplasmic accumulation of the lipogenic regulator lipin 1 and prevented alcohol-mediated inhibition of fatty acid oxidation. Pharmacological intervention with rapamycin alleviated the ability of alcohol to up-regulate lipogenesis, to down-regulate fatty acid oxidation, and to induce steatogenic phenotypes. Chronic-plus-binge ethanol feeding led to activation of SREBP-1 and lipin 1 through S6K1-dependent and independent mechanisms. Furthermore, hepatocyte-specific deletion of SIRT1 disrupted DEPTOR function, enhanced mTORC1 activity, and exacerbated alcoholic fatty liver, inflammation, and liver injury in mice. The dysregulation of SIRT1

  8. The Drosophila FoxA ortholog Fork head regulates growth and gene expression downstream of Target of rapamycin.

    Directory of Open Access Journals (Sweden)

    Margret H Bülow

    2010-12-01

    Full Text Available Forkhead transcription factors of the FoxO subfamily regulate gene expression programs downstream of the insulin signaling network. It is less clear which proteins mediate transcriptional control exerted by Target of rapamycin (TOR signaling, but recent studies in nematodes suggest a role for FoxA transcription factors downstream of TOR. In this study we present evidence that outlines a similar connection in Drosophila, in which the FoxA protein Fork head (FKH regulates cellular and organismal size downstream of TOR. We find that ectopic expression and targeted knockdown of FKH in larval tissues elicits different size phenotypes depending on nutrient state and TOR signaling levels. FKH overexpression has a negative effect on growth under fed conditions, and this phenotype is not further exacerbated by inhibition of TOR via rapamycin feeding. Under conditions of starvation or low TOR signaling levels, knockdown of FKH attenuates the size reduction associated with these conditions. Subcellular localization of endogenous FKH protein is shifted from predominantly cytoplasmic on a high-protein diet to a pronounced nuclear accumulation in animals with reduced levels of TOR or fed with rapamycin. Two putative FKH target genes, CG6770 and cabut, are transcriptionally induced by rapamycin or FKH expression, and silenced by FKH knockdown. Induction of both target genes in heterozygous TOR mutant animals is suppressed by mutations in fkh. Furthermore, TOR signaling levels and FKH impact on transcription of the dFOXO target gene d4E-BP, implying a point of crosstalk with the insulin pathway. In summary, our observations show that an alteration of FKH levels has an effect on cellular and organismal size, and that FKH function is required for the growth inhibition and target gene induction caused by low TOR signaling levels.

  9. Brain Injury-Induced Synaptic Reorganization in Hilar Inhibitory Neurons Is Differentially Suppressed by Rapamycin.

    Science.gov (United States)

    Butler, Corwin R; Boychuk, Jeffery A; Smith, Bret N

    2017-01-01

    Following traumatic brain injury (TBI), treatment with rapamycin suppresses mammalian (mechanistic) target of rapamycin (mTOR) activity and specific components of hippocampal synaptic reorganization associated with altered cortical excitability and seizure susceptibility. Reemergence of seizures after cessation of rapamycin treatment suggests, however, an incomplete suppression of epileptogenesis. Hilar inhibitory interneurons regulate dentate granule cell (DGC) activity, and de novo synaptic input from both DGCs and CA3 pyramidal cells after TBI increases their excitability but effects of rapamycin treatment on the injury-induced plasticity of interneurons is only partially described. Using transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed in the somatostatinergic subset of hilar inhibitory interneurons, we tested the effect of daily systemic rapamycin treatment (3 mg/kg) on the excitability of hilar inhibitory interneurons after controlled cortical impact (CCI)-induced focal brain injury. Rapamycin treatment reduced, but did not normalize, the injury-induced increase in excitability of surviving eGFP+ hilar interneurons. The injury-induced increase in response to selective glutamate photostimulation of DGCs was reduced to normal levels after mTOR inhibition, but the postinjury increase in synaptic excitation arising from CA3 pyramidal cell activity was unaffected by rapamycin treatment. The incomplete suppression of synaptic reorganization in inhibitory circuits after brain injury could contribute to hippocampal hyperexcitability and the eventual reemergence of the epileptogenic process upon cessation of mTOR inhibition. Further, the cell-selective effect of mTOR inhibition on synaptic reorganization after CCI suggests possible mechanisms by which rapamycin treatment modifies epileptogenesis in some models but not others.

  10. Suppression of Th17-polarized airway inflammation by rapamycin.

    Science.gov (United States)

    Joean, Oana; Hueber, Anja; Feller, Felix; Jirmo, Adan Chari; Lochner, Matthias; Dittrich, Anna-Maria; Albrecht, Melanie

    2017-11-10

    Because Th17-polarized airway inflammation correlates with poor control in bronchial asthma and is a feature of numerous other difficult-to-treat inflammatory lung diseases, new therapeutic approaches for this type of airway inflammation are necessary. We assessed different licensed anti-inflammatory agents with known or expected efficacy against Th17-polarization in mouse models of Th17-dependent airway inflammation. Upon intravenous transfer of in vitro derived Th17 cells and intranasal challenge with the corresponding antigen, we established acute and chronic murine models of Th17-polarised airway inflammation. Consecutively, we assessed the efficacy of methylprednisolone, roflumilast, azithromycin, AM80 and rapamycin against acute or chronic Th17-dependent airway inflammation. Quantifiers for Th17-associated inflammation comprised: bronchoalveolar lavage (BAL) differential cell counts, allergen-specific cytokine and immunoglobulin secretion, as well as flow cytometric phenotyping of pulmonary inflammatory cells. Only rapamycin proved effective against acute Th17-dependent airway inflammation, accompanied by increased plasmacytoid dendritic cells (pDCs) and reduced neutrophils as well as reduced CXCL-1 levels in BAL. Chronic Th17-dependent airway inflammation was unaltered by rapamycin treatment. None of the other agents showed efficacy in our models. Our results demonstrate that Th17-dependent airway inflammation is difficult to treat with known agents. However, we identify rapamycin as an agent with inhibitory potential against acute Th17-polarized airway inflammation.

  11. Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Zheng [Markey Cancer Center, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Dadao Bei, Guangzhou 510515 (China); Zhou, Yuning [Markey Cancer Center, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Evers, B. Mark [Markey Cancer Center, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Department of Surgery, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Wang, Qingding, E-mail: qingding.wang@uky.edu [Markey Cancer Center, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Department of Surgery, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer Rictor associates with FBXW7 to form an E3 complex. Black-Right-Pointing-Pointer Knockdown of rictor decreases ubiquitination of c-Myc and cylin E. Black-Right-Pointing-Pointer Knockdown of rictor increases protein levels of c-Myc and cylin E. Black-Right-Pointing-Pointer Overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Black-Right-Pointing-Pointer Rictor regulation of c-Myc and cyclin E requires FBXW7. -- Abstract: Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor-FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.

  12. Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells

    International Nuclear Information System (INIS)

    Guo, Zheng; Zhou, Yuning; Evers, B. Mark; Wang, Qingding

    2012-01-01

    Highlights: ► Rictor associates with FBXW7 to form an E3 complex. ► Knockdown of rictor decreases ubiquitination of c-Myc and cylin E. ► Knockdown of rictor increases protein levels of c-Myc and cylin E. ► Overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. ► Rictor regulation of c-Myc and cyclin E requires FBXW7. -- Abstract: Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor–FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.

  13. Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase

    DEFF Research Database (Denmark)

    Brinch-Pedersen, H.; Galili, G.; Sørensen, K.

    1996-01-01

    In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance...... the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T0) exhibited a 14-fold increase of free lysine and an 8......, no differences were observed in the composition of total amino acids. The introduced genes were inherited in the T1 generation where enzymic activities revealed a 2.3-fold increase of AK activity and a 4.0-9.5-fold increase for DHPS. T1 seeds of DHPS transformants showed the same changes in free amino acids...

  14. AZD2014 Radiosensitizes Oral Squamous Cell Carcinoma by Inhibiting AKT/mTOR Axis and Inducing G1/G2/M Cell Cycle Arrest.

    Directory of Open Access Journals (Sweden)

    Chih-Chia Yu

    Full Text Available Oral squamous cell carcinoma (OSCC is one of the most common malignant neoplasms in Taiwan. Activation of the mTOR signaling pathway has been linked to decreased radiation responsiveness in human oral cancer, thus it limits efficacy of radiotherapy. To address this question, we investigated the effect of AZD2014, a novel small molecular ATP-competitive inhibitor of mTORC1 and mTORC2 kinase, as a radiosensitizer in primary OSCC and OSCC-derived cell line models.We isolated primary tumor cells from OSCC tissues and cell lines. AZD2014 was administered with and without ionizing radiation. The radiosensitizing effect of AZD2014 were then assessed using cell viability assays, clonogenic survival assays, and cell cycle analyses. Western blotting was used to detect protein expression.Combination treatment with AZD2014 and irradiation resulted in significant reduction in OSCC cell line and primary OSCC cell colony formation due to the enhanced inhibition of AKT and both mTORC1 and mTORC2 activity. Pre-treatment with AZD2014 in irradiated oral cancer cells induced tumor cell cycle arrest at the G1 and G2/M phases, which led to disruption of cyclin D1-CDK4 and cyclin B1-CDC2 complexes. Moreover, AZD2014 synergized with radiation to promote both apoptosis and autophagy by increasing caspase-3 and LC3 in primary OSCC cells.These findings suggest that in irradiated OSCC cells, co-treatment with AZD2014, which targets mTORC1 and mTORC2 blockade, is an effective radiosensitizing strategy for oral squamous cell carcinoma.

  15. The role of the TOR pathway in mediating the link between nutrition and longevity.

    Science.gov (United States)

    Lushchak, Oleh; Strilbytska, Olha; Piskovatska, Veronika; Storey, Kenneth B; Koliada, Alexander; Vaiserman, Alexander

    2017-06-01

    The target of rapamycin (TOR) pathway integrates signals from extracellular and intracellular agents, such as growth factors, nutrients, mediators of energy balance, oxygen availability and other environmental cues. It allows the regulation of multiple cellular processes including protein and lipid synthesis, ribosome biogenesis, autophagy and metabolic processes. Being conserved across different phyla, TOR regulates longevity of various organisms in response to dietary conditions. In this review we described the main components of the TOR pathway and its upstream effectors and downstream processes in relation to aging. The potential contribution of the TOR pathway in lifespan-extending effects of varied dietary interventions, and the anti-aging drugs rapamycin and metformin direct or indirect regulation of TOR activity in yeasts, worms, flies and mammals are also discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Protein kinase FgSch9 serves as a mediator of the target of rapamycin and high osmolarity glycerol pathways and regulates multiple stress responses and secondary metabolism in Fusarium graminearum.

    Science.gov (United States)

    Gu, Qin; Zhang, Chengqi; Yu, Fangwei; Yin, Yanni; Shim, Won-Bo; Ma, Zhonghua

    2015-08-01

    Saccharomyces cerevisiae protein kinase Sch9 is one of the downstream effectors of the target of rapamycin (TOR) complex 1 and plays multiple roles in stress resistance, longevity and nutrient sensing. However, the functions of Sch9 orthologs in filamentous fungi, particularly in pathogenic species, have not been characterized to date. Here, we investigated biological and genetic functions of FgSch9 in Fusarium graminearum. The FgSCH9 deletion mutant (ΔFgSch9) was defective in aerial hyphal growth, hyphal branching and conidial germination. The mutant exhibited increased sensitivity to osmotic and oxidative stresses, cell wall-damaging agents, and to rapamycin, while showing increased thermal tolerance. We identified FgMaf1 as one of the FgSch9-interacting proteins that plays an important role in regulating mycotoxin biosynthesis and virulence of F. graminearum. Co-immunoprecipitation and affinity capture-mass spectrometry assays showed that FgSch9 also interacts with FgTor and FgHog1. More importantly, both ΔFgSch9 and FgHog1 null mutant (ΔFgHog1) exhibited increased sensitivity to osmotic and oxidative stresses. This defect was more severe in the FgSch9/FgHog1 double mutant. Taken together, we propose that FgSch9 serves as a mediator of the TOR and high osmolarity glycerol pathways, and regulates vegetative differentiation, multiple stress responses and secondary metabolism in F. graminearum. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  17. Coupling TOR to the Cell Cycle by the Greatwall–Endosulfine–PP2A-B55 Pathway

    Directory of Open Access Journals (Sweden)

    Livia Pérez-Hidalgo

    2017-08-01

    Full Text Available Cell growth and division are two processes tightly coupled in proliferating cells. While Target of Rapamycin (TOR is the master regulator of growth, the cell cycle is dictated by the activity of the cyclin-dependent kinases (CDKs. A long-standing question in cell biology is how these processes may be connected. Recent work has highlighted that regulating the phosphatases that revert CDK phosphorylations is as important as regulating the CDKs for cell cycle progression. At mitosis, maintaining a low level of protein phosphatase 2A (PP2A-B55 activity is essential for CDK substrates to achieve the correct level of phosphorylation. The conserved Greatwall–Endosulfine pathway has been shown to be required for PP2A-B55 inhibition at mitosis in yeasts and multicellular organisms. Interestingly, in yeasts, the Greatwall–Endosulfine pathway is negatively regulated by TOR Complex 1 (TORC1. Moreover, Greatwall–Endosulfine activation upon TORC1 inhibition has been shown to regulate the progression of the cell cycle at different points: the G1 phase in budding yeast, the G2/M transition and the differentiation response in fission yeast, and the entry into quiescence in both budding and fission yeasts. In this review, we discuss the recent findings on how the Greatwall–Endosulfine pathway may provide a connection between cell growth and the cell cycle machinery.

  18. BRAFV600E Negatively Regulates the AKT Pathway in Melanoma Cell Lines

    OpenAIRE

    Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F.; Niu, Huifeng

    2012-01-01

    Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamy...

  19. Genetics Home Reference: androgen insensitivity syndrome

    Science.gov (United States)

    ... Androgen insensitivity syndrome is a condition that affects sexual development before birth and during puberty. People with this ... characteristics or signs of both male and female sexual development. Complete androgen insensitivity syndrome occurs when the body ...

  20. Systemic and Nonrenal Adverse Effects Occurring in Renal Transplant Patients Treated with mTOR Inhibitors

    Directory of Open Access Journals (Sweden)

    Gianluigi Zaza

    2013-01-01

    Full Text Available The mammalian target of rapamycin inhibitors (mTOR-I, sirolimus and everolimus, are immunosuppressive drugs largely used in renal transplantation. The main mechanism of action of these drugs is the inhibition of the mammalian target of rapamycin (mTOR, a regulatory protein kinase involved in lymphocyte proliferation. Additionally, the inhibition of the crosstalk among mTORC1, mTORC2, and PI3K confers the antineoplastic activities of these drugs. Because of their specific pharmacological characteristics and their relative lack of nephrotoxicity, these inhibitors are valid option to calcineurine inhibitors (CNIs for maintenance immunosuppression in renal transplant recipients with chronic allograft nephropathy. However, as other immunosuppressive drugs, mTOR-I may induce the development of several adverse effects that need to be early recognized and treated to avoid severe illness in renal transplant patients. In particular, mTOR-I may induce systemic nonnephrological side effects including pulmonary toxicity, hematological disorders, dysmetabolism, lymphedema, stomatitis, cutaneous adverse effects, and fertility/gonadic toxicity. Although most of the adverse effects are dose related, it is extremely important for clinicians to early recognize them in order to reduce dosage or discontinue mTOR-I treatment avoiding the onset and development of severe clinical complications.

  1. mTORC1 directly phosphorylates and regulates human MAF1.

    OpenAIRE

    Michels Annemieke A; Robitaille Aaron M; Buczynski-Ruchonnet Diane; Hodroj Wassim; Reina Jaime H; Hall Michael N; Hernandez Nouria

    2010-01-01

    mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the p...

  2. Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes.

    Science.gov (United States)

    Gao, Song; Carson, James A

    2016-01-01

    Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes. Copyright © 2016 the American Physiological Society.

  3. Rapamycin Influences the Efficiency of Fertilization and Development in the Mouse: A Role for Autophagic Activation

    Directory of Open Access Journals (Sweden)

    Geun-Kyung Lee

    2016-08-01

    Full Text Available The mammalian target of rapamycin (mTOR regulates cellular processes such as cell growth, metabolism, transcription, translation, and autophagy. Rapamycin is a selective inhibitor of mTOR, and induces autophagy in various systems. Autophagy contributes to clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified-warmed mouse oocytes show acute increases in autophagy during warming, and suggested that it is a natural response to cold stress. In this follow-up study, we examined whether the modulation of autophagy influences survival, fertilization, and developmental rates of vitrified-warmed mouse oocytes. We used rapamycin to enhance autophagy in metaphase II (MII oocytes before and after vitrification. The oocytes were then subjected to in vitro fertilization (IVF. The fertilization and developmental rates of vitrified-warmed oocytes after rapamycin treatment were significantly lower than those for control groups. Modulation of autophagy with rapamycin treatment shows that rapamycin-induced autophagy exerts a negative influence on fertilization and development of vitrified-warmed oocytes.

  4. Switching an O2 sensitive glucose oxidase bioelectrode into an almost insensitive one by cofactor redesign.

    Science.gov (United States)

    Tremey, Emilie; Suraniti, Emmanuel; Courjean, Olivier; Gounel, Sébastien; Stines-Chaumeil, Claire; Louerat, Frédéric; Mano, Nicolas

    2014-06-04

    In the 5-8 mM glucose concentration range, of particular interest for diabetes management, glucose oxidase bioelectrodes are O2 dependent, which decrease their efficiencies. By replacing the natural cofactor of glucose oxidase, we succeeded in turning an O2 sensitive bioelectrode into an almost insensitive one.

  5. Rapamycin Synergizes with Cisplatin in Antiendometrial Cancer Activation by Improving IL-27–Stimulated Cytotoxicity of NK Cells

    Directory of Open Access Journals (Sweden)

    Wen-Jie Zhou

    2018-01-01

    Full Text Available Natural killer (NK cell function is critical for controlling initial tumor growth and determining chemosensitivity of the tumor. A synergistic relationship between rapamycin and cisplatin in uterine endometrial cancer (UEC in vitro has been reported, but the mechanism and the combined therapeutic strategy for endometrial cancer (EC are still unknown. We found a positive correlation between the level of IL-27 and the differentiated stage of UEC. The increase of IL-27 in uterine endometrial cancer cell (UECC lines (Ishikawa, RL95-2 and KLE led to a high cytotoxic activity of NK cells to UECC in the co-culture system. Exposure with rapamycin enhanced the cytotoxicity of NK cells by upregulating the expression of IL-27 in UECC and IL-27 receptors (IL-27Rs: WSX-1 and gp130 on NK cells and further restricted the growth of UEC in Ishikawa-xenografted nude mice. In addition, treatment with rapamycin resulted in an increased autophagy level of UECC, and IL-27 enhanced this ability of rapamycin. Cisplatin-mediated NK cells' cytotoxic activity and anti-UEC activation were independent of IL-27; however, the combination of rapamycin and cisplatin led to a higher cytotoxic activity of NK cells, smaller UEC volume and longer survival rate in vivo. These results suggest that rapamycin and cisplatin synergistically activate the cytotoxicity of NK cells and inhibit the progression of UEC in both an IL-27–dependent and –independent manner. This provides a scientific basis for potential rapamycin-cisplatin combined therapeutic strategies targeted to UEC, especially for the patients with low differentiated stage or abnormally low level of IL-27.

  6. Rapamycin exerts antifungal activity in vitro and in vivo against Mucor circinelloides via FKBP12-dependent inhibition of Tor.

    Science.gov (United States)

    Bastidas, Robert J; Shertz, Cecelia A; Lee, Soo Chan; Heitman, Joseph; Cardenas, Maria E

    2012-03-01

    The zygomycete Mucor circinelloides is an opportunistic fungal pathogen that commonly infects patients with malignancies, diabetes mellitus, and solid organ transplants. Despite the widespread use of antifungal therapy in the management of zygomycosis, the incidence of infections continues to rise among immunocompromised individuals. In this study, we established that the target and mechanism of antifungal action of the immunosuppressant rapamycin in M. circinelloides are mediated via conserved complexes with FKBP12 and a Tor homolog. We found that spontaneous mutations that disrupted conserved residues in FKBP12 conferred rapamycin and FK506 resistance. Disruption of the FKBP12-encoding gene, fkbA, also conferred rapamycin and FK506 resistance. Expression of M. circinelloides FKBP12 (McFKBP12) complemented a Saccharomyces cerevisiae mutant strain lacking FKBP12 to restore rapamycin sensitivity. Expression of the McTor FKBP12-rapamycin binding (FRB) domain conferred rapamycin resistance in S. cerevisiae, and McFKBP12 interacted in a rapamycin-dependent fashion with the McTor FRB domain in a yeast two-hybrid assay, validating McFKBP12 and McTor as conserved targets of rapamycin. We showed that in vitro, rapamycin exhibited potent growth inhibitory activity against M. circinelloides. In a Galleria mellonella model of systemic mucormycosis, rapamycin improved survival by 50%, suggesting that rapamycin and nonimmunosuppressive analogs have the potential to be developed as novel antifungal therapies for treatment of patients with mucormycosis.

  7. Insensitive Munitions Testing

    Data.gov (United States)

    Federal Laboratory Consortium — Insensitive Munitions Testing at RTC is conducted (IAW MILSTD-2105) at Test Area 4. Our engineers and technicians obtain data for hazards classification and safety...

  8. Overexpression of the PP2A regulatory subunit Tap46 leads to enhanced plant growth through stimulation of the TOR signalling pathway

    Science.gov (United States)

    Ahn, Chang Sook; Ahn, Hee-Kyung; Pai, Hyun-Sook

    2015-01-01

    Tap46, a regulatory subunit of protein phosphatase 2A (PP2A), plays an essential role in plant growth and development through a functional link with the Target of Rapamycin (TOR) signalling pathway. Here, we have characterized the molecular mechanisms behind a gain-of-function phenotype of Tap46 and its relationship with TOR to gain further insights into Tap46 function in plants. Constitutive overexpression of Tap46 in Arabidopsis resulted in overall growth stimulation with enlarged organs, such as leaves and siliques. Kinematic analysis of leaf growth revealed that increased cell size was mainly responsible for the leaf enlargement. Tap46 overexpression also enhanced seed size and viability under accelerated ageing conditions. Enhanced plant growth was also observed in dexamethasone (DEX)-inducible Tap46 overexpression Arabidopsis lines, accompanied by increased cellular activities of nitrate-assimilating enzymes. DEX-induced Tap46 overexpression and Tap46 RNAi resulted in increased and decreased phosphorylation of S6 kinase (S6K), respectively, which is a sensitive indicator of endogenous TOR activity, and Tap46 interacted with S6K in planta based on bimolecular fluorescence complementation and co-immunoprecipitation. Furthermore, inactivation of TOR by estradiol-inducible RNAi or rapamycin treatment decreased Tap46 protein levels, but increased PP2A catalytic subunit levels. Real-time quantitative PCR analysis revealed that Tap46 overexpression induced transcriptional modulation of genes involved in nitrogen metabolism, ribosome biogenesis, and lignin biosynthesis. These findings suggest that Tap46 modulates plant growth as a positive effector of the TOR signalling pathway and Tap46/PP2Ac protein abundance is regulated by TOR activity. PMID:25399018

  9. Acute myotube protein synthesis regulation by IL-6-related cytokines.

    Science.gov (United States)

    Gao, Song; Durstine, J Larry; Koh, Ho-Jin; Carver, Wayne E; Frizzell, Norma; Carson, James A

    2017-11-01

    IL-6 and leukemia inhibitory factor (LIF), members of the IL-6 family of cytokines, play recognized paradoxical roles in skeletal muscle mass regulation, being associated with both growth and atrophy. Overload or muscle contractions can induce a transient increase in muscle IL-6 and LIF expression, which has a regulatory role in muscle hypertrophy. However, the cellular mechanisms involved in this regulation have not been completely identified. The induction of mammalian target of rapamycin complex 1 (mTORC1)-dependent myofiber protein synthesis is an established regulator of muscle hypertrophy, but the involvement of the IL-6 family of cytokines in this process is poorly understood. Therefore, we investigated the acute effects of IL-6 and LIF administration on mTORC1 signaling and protein synthesis in C2C12 myotubes. The role of glycoprotein 130 (gp130) receptor and downstream signaling pathways, including phosphoinositide 3-kinase (PI3K)-Akt-mTORC1 and signal transducer and activator of transcription 3 (STAT3)-suppressor of cytokine signaling 3 (SOCS3), was investigated by administration of specific siRNA or pharmaceutical inhibitors. Acute administration of IL-6 and LIF induced protein synthesis, which was accompanied by STAT3 activation, Akt-mTORC1 activation, and increased SOCS3 expression. This induction of protein synthesis was blocked by both gp130 siRNA knockdown and Akt inhibition. Interestingly, STAT3 inhibition or Akt downstream mTORC1 signaling inhibition did not fully block the IL-6 or LIF induction of protein synthesis. SOCS3 siRNA knockdown increased basal protein synthesis and extended the duration of the protein synthesis induction by IL-6 and LIF. These results demonstrate that either IL-6 or LIF can activate gp130-Akt signaling axis, which induces protein synthesis via mTORC1-independent mechanisms in cultured myotubes. However, IL-6- or LIF-induced SOCS3 negatively regulates the activation of myotube protein synthesis. Copyright © 2017 the

  10. Rapamycin Exerts Antifungal Activity In Vitro and In Vivo against Mucor circinelloides via FKBP12-Dependent Inhibition of Tor

    Science.gov (United States)

    Bastidas, Robert J.; Shertz, Cecelia A.; Lee, Soo Chan; Heitman, Joseph

    2012-01-01

    The zygomycete Mucor circinelloides is an opportunistic fungal pathogen that commonly infects patients with malignancies, diabetes mellitus, and solid organ transplants. Despite the widespread use of antifungal therapy in the management of zygomycosis, the incidence of infections continues to rise among immunocompromised individuals. In this study, we established that the target and mechanism of antifungal action of the immunosuppressant rapamycin in M. circinelloides are mediated via conserved complexes with FKBP12 and a Tor homolog. We found that spontaneous mutations that disrupted conserved residues in FKBP12 conferred rapamycin and FK506 resistance. Disruption of the FKBP12-encoding gene, fkbA, also conferred rapamycin and FK506 resistance. Expression of M. circinelloides FKBP12 (McFKBP12) complemented a Saccharomyces cerevisiae mutant strain lacking FKBP12 to restore rapamycin sensitivity. Expression of the McTor FKBP12-rapamycin binding (FRB) domain conferred rapamycin resistance in S. cerevisiae, and McFKBP12 interacted in a rapamycin-dependent fashion with the McTor FRB domain in a yeast two-hybrid assay, validating McFKBP12 and McTor as conserved targets of rapamycin. We showed that in vitro, rapamycin exhibited potent growth inhibitory activity against M. circinelloides. In a Galleria mellonella model of systemic mucormycosis, rapamycin improved survival by 50%, suggesting that rapamycin and nonimmunosuppressive analogs have the potential to be developed as novel antifungal therapies for treatment of patients with mucormycosis. PMID:22210828

  11. Aging Reduces the Activation of the mTORC1 Pathway after Resistance Exercise and Protein Intake in Human Skeletal Muscle: Potential Role of REDD1 and Impaired Anabolic Sensitivity.

    Science.gov (United States)

    Francaux, Marc; Demeulder, Bénédicte; Naslain, Damien; Fortin, Raphael; Lutz, Olivier; Caty, Gilles; Deldicque, Louise

    2016-01-15

    This study was designed to better understand the molecular mechanisms involved in the anabolic resistance observed in elderly people. Nine young (22 ± 0.1 years) and 10 older (69 ± 1.7 years) volunteers performed a one-leg extension exercise consisting of 10 × 10 repetitions at 70% of their 3-RM, immediately after which they ingested 30 g of whey protein. Muscle biopsies were taken from the vastus lateralis at rest in the fasted state and 30 min after protein ingestion in the non-exercised (Pro) and exercised (Pro+ex) legs. Plasma insulin levels were determined at the same time points. No age difference was measured in fasting insulin levels but the older subjects had a 50% higher concentration than the young subjects in the fed state (p young subjects. After Pro+ex, REDD1 expression tended to be higher (p = 0.087) in the older group while AMPK phosphorylation was not modified by any condition. In conclusion, we show that the activation of the mTORC1 pathway is reduced in skeletal muscle of older subjects after resistance exercise and protein ingestion compared with young subjects, which could be partially due to an increased expression of REDD1 and an impaired anabolic sensitivity.

  12. A photocleavable rapamycin conjugate for spatiotemporal control of small GTPase activity.

    Science.gov (United States)

    Umeda, Nobuhiro; Ueno, Tasuku; Pohlmeyer, Christopher; Nagano, Tetsuo; Inoue, Takanari

    2011-01-12

    We developed a novel method to spatiotemporally control the activity of signaling molecules. A newly synthesized photocaged rapamycin derivative induced rapid dimerization of FKBP (FK-506 binding protein) and FRB (FKBP-rapamycin binding protein) upon UV irradiation. With this system and the spatially confined UV irradiation, we achieved subcellularly localized activation of Rac, a member of small GTPases. Our technique offers a powerful approach to studies of dynamic intracellular signaling events.

  13. Autophagy and the nutritional signaling pathway

    Directory of Open Access Journals (Sweden)

    Long HE,Shabnam ESLAMFAM,Xi MA,Defa LI

    2016-09-01

    Full Text Available During their growth and development, animals adapt to tremendous changes in order to survive. These include responses to both environmental and physiological changes and autophagy is one of most important adaptive and regulatory mechanisms. Autophagy is defined as an autolytic process to clear damaged cellular organelles and recycle the nutrients via lysosomic degradation. The process of autophagy responds to special conditions such as nutrient withdrawal. Once autophagy is induced, phagophores form and then elongate and curve to form autophagosomes. Autophagosomes then engulf cargo, fuse with endosomes, and finally fuse with lysosomes for maturation. During the initiation process, the ATG1/ULK1 (unc-51-like kinase 1 and VPS34 (which encodes a class III phosphatidylinositol (PtdIns 3-kinase complexes are critical in recruitment and assembly of other complexes required for autophagy. The process of autophagy is regulated by autophagy related genes (ATGs. Amino acid and energy starvation mediate autophagy by activating mTORC1 (mammalian target of rapamycin and AMP-activated protein kinase (AMPK. AMPK is the energy status sensor, the core nutrient signaling component and the metabolic kinase of cells. This review mainly focuses on the mechanism of autophagy regulated by nutrient signaling especially for the two important complexes, ULK1 and VPS34.

  14. HPV-16 E7 expression up-regulates phospholipase D activity and promotes rapamycin resistance in a pRB-dependent manner.

    Science.gov (United States)

    Rabachini, Tatiana; Boccardo, Enrique; Andrade, Rubiana; Perez, Katia Regina; Nonogaki, Suely; Cuccovia, Iolanda Midea; Villa, Luisa Lina

    2018-04-27

    Human Papillomavirus (HPV) infection is the main risk factor for the development and progression of cervical cancer. HPV-16 E6 and E7 expression is essential for induction and maintenance of the transformed phenotype. These oncoproteins interfere with the function of several intracellular proteins, including those controlling the PI3K/AKT/mTOR pathway in which Phospolipase D (PLD) and Phosphatidic acid (PA) play a critical role. PLD activity was measured in primary human keratinocytes transduced with retroviruses expressing HPV-16 E6, E7 or E7 mutants. The cytostatic effect of rapamycin, a well-known mTOR inhibitor with potential clinical applications, was evaluated in monolayer and organotypic cultures. HPV-16 E7 expression in primary human keratinocytes leads to an increase in PLD expression and activity. Moreover, this activation is dependent on the ability of HPV-16 E7 to induce retinoblastoma protein (pRb) degradation. We also show that cells expressing HPV-16 E7 or silenced for pRb acquire resistance to the antiproliferative effect of rapamycin. This is the first indication that HPV oncoproteins can affect PLD activity. Since PA can interfere with the ability of rapamycin to bind mTOR, the use of combined strategies to target mTOR and PLD activity might be considered to treat HPV-related malignancies.

  15. Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

    Science.gov (United States)

    Stransky, Laura A.; Forgac, Michael

    2015-01-01

    The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly. PMID:26378229

  16. Targeting the phosphatidylinositol 3-kinase/Akt/mechanistic target of rapamycin signaling pathway in B-lineage acute lymphoblastic leukemia: An update.

    Science.gov (United States)

    Simioni, Carolina; Martelli, Alberto M; Zauli, Giorgio; Vitale, Marco; McCubrey, James A; Capitani, Silvano; Neri, Luca M

    2018-04-18

    Despite considerable progress in treatment protocols, B-lineage acute lymphoblastic leukemia (B-ALL) displays a poor prognosis in about 15-20% of pediatric cases and about 60% of adult patients. In addition, life-long irreversible late effects from chemo- and radiation therapy, including secondary malignancies, are a growing problem for leukemia survivors. Targeted therapy holds promising perspectives for cancer treatment as it may be more effective and have fewer side effects than conventional therapies. The phosphatidylinositol 3-phosphate kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway is a key regulatory cascade which controls proliferation, survival and drug-resistance of cancer cells, and it is frequently upregulated in the different subtypes of B-ALL, where it plays important roles in the pathophysiology, maintenance and progression of the disease. Moreover, activation of this signaling cascade portends a poorer prognosis in both pediatric and adult B-ALL patients. Promising preclinical data on PI3K/Akt/mTOR inhibitors have documented their anticancer activity in B-ALL and some of these novel drugs have entered clinical trials as they could lead to a longer event-free survival and reduce therapy-associated toxicity for patients with B-ALL. This review highlights the current status of PI3K/Akt/mTOR inhibitors in B-ALL, with an emphasis on emerging evidence of the superior efficacy of synergistic combinations involving the use of traditional chemotherapeutics or other novel, targeted agents. © 2018 Wiley Periodicals, Inc.

  17. Sustained activation of the mammalian target of rapamycin nutrient sensing pathway is associated with hepatic insulin resistance, but not with steatosis, in mice

    NARCIS (Netherlands)

    Korsheninnikova, E.; van der Zon, G. C. M.; Voshol, P. J.; Janssen, G. M.; Havekes, L. M.; Grefhorst, A.; Kuipers, F.; Reijngoud, D. -J.; Romijn, J. A.; Ouwens, D. M.; Maassen, J. A.

    2006-01-01

    Aims/hypothesis Activation of nutrient sensing through mammalian target of rapamycin (mTOR) has been linked to the pathogenesis of insulin resistance. We examined activation of mTOR-signalling in relation to insulin resistance and hepatic steatosis in mice. Materials and methods Chronic hepatic

  18. Sustained activation of the mammalian target of rapamycin nutrient sensing pathway is associated with hepatic insulin resistance, but not with steatosis, in mice

    NARCIS (Netherlands)

    Korsheninnikova, E.; van der Zon, G. C. M.; Voshol, P. J.; Janssen, G. M.; Havekes, L. M.; Grefhorst, A.; Kuipers, F.; Reijngoud, D.-J.; Romijn, J. A.; Ouwens, D. M.; Maassen, J. A.

    2006-01-01

    Activation of nutrient sensing through mammalian target of rapamycin (mTOR) has been linked to the pathogenesis of insulin resistance. We examined activation of mTOR-signalling in relation to insulin resistance and hepatic steatosis in mice. Chronic hepatic steatosis and hepatic insulin resistance

  19. Expression status and prognostic significance of mammalian target of rapamycin pathway members in urothelial carcinoma of urinary bladder after cystectomy.

    Science.gov (United States)

    Schultz, Luciana; Albadine, Roula; Hicks, Jessica; Jadallah, Sana; DeMarzo, Angelo M; Chen, Ying-Bei; Nielsen, Matthew E; Neilsen, Matthew E; Gonzalgo, Mark L; Sidransky, David; Schoenberg, Mark; Netto, George J

    2010-12-01

    Bladder urothelial carcinoma has high rates of mortality and morbidity. Identifying novel molecular prognostic factors and targets of therapy is crucial. Mammalian target of rapamycin (mTOR) pathway plays a pivotal role in establishing cell shape, migration, and proliferation. Tissue microarrays were constructed from 132 cystectomies (1994-2002). Immunohistochemistry was performed for Pten, c-myc, p27, phosphorylated (phos)Akt, phosS6, and 4E-BP1. Markers were evaluated for pattern, percentage, and intensity of staining. Mean length of follow-up was 62.6 months (range, 1-182 months). Disease progression, overall survival (OS), and disease-specific survival (DSS) rates were 42%, 60%, and 68%, respectively. Pten showed loss of expression in 35% of bladder urothelial carcinoma. All markers showed lower expression in invasive bladder urothelial carcinoma compared with benign urothelium with the exception of 4E-BP1. Pten, p27, phosAkt, phosS6, and 4E-BP1 expression correlated with pathologic stage (pathological stage; P<.03). Pten, 4E-BP1, and phosAkt expression correlated with divergent aggressive histology and invasion. phosS6 expression inversely predicted OS (P=.01), DSS (P=.001), and progression (P=.05). c-myc expression inversely predicted progression (P=.01). In a multivariate analysis model that included TNM stage grouping, divergent aggressive histology, concomitant carcinoma in situ, phosS6, and c-myc expression, phosS6 was an independent predictor of DSS (P=.03; hazard ratio [HR], -0.19), whereas c-myc was an independent predictor of progression (P=.02; HR, -0.38). In a second model substituting organ-confined disease and lymph node status for TNM stage grouping, phosS6 and c-myc remained independent predictors of DSS (P=.03; HR, -0.21) and progression (P=.03; HR, -0.34), respectively. We found an overall down-regulation of mTOR pathway in bladder urothelial carcinoma. phosS6 independently predicted DSS, and c-myc independently predicted progression

  20. Preclinical evaluation of WYE-687, a mTOR kinase inhibitor, as a potential anti-acute myeloid leukemia agent

    International Nuclear Information System (INIS)

    Cheng, Feng; Wang, Lingling; Shen, Yunfeng; Xia, Jun; Chen, Heng; Jiang, Yuanqiang; Lu, Mize

    2016-01-01

    Mammalian target of rapamycin (mTOR) as a potential drug target for treatment of acute myeloid leukemia (AML). Here, we investigated the potential anti-leukemic activity by WYE-687, a potent mTOR kinase inhibitor. We demonstrated that WYE-687 potently inhibited survival and proliferation of established (HL-60, U937, AML-193 and THP-1 lines) and human AML progenitor cells. Yet, same WYE-687 treatment was non-cytotoxic to the primary peripheral blood mononuclear leukocytes (PBMCs) isolated from healthy donors. WYE-687 induced caspase-dependent apoptotic death in above AML cells/progenitor cells. On the other hand, the pan-caspase inhibitor (Z-VAD-FMK), the caspase-3 specific inhibitor (Z-DEVD-FMK) or the caspase-9 specific inhibitor (z-LEHD-fmk) attenuated WYE-687-induced cytotoxicity. At the molecular level, WYE-687 concurrently inhibited activation of mTORC1 (p70S6K1 and S6 phosphorylations) and mTORC2 (AKT Ser-473 and FoxO1/3a phosphorylations), whiling downregulating mTORC1/2-regulated genes (Bcl-xL and hypoxia-inducible factor 1/2α) in both HL-60/U937 cells and human AML progenitor cells. In vivo, oral administration of WYE-687 potently inhibited U937 leukemic xenograft tumor growth in severe combined immunodeficient (SCID) mice, without causing significant toxicities. In summary, our results demonstrate that targeting mTORC1/2 by WYE-687 leads to potent antitumor activity in preclinical models of AML. - Highlights: • WYE-687 inhibits survival and proliferation of human AML cells/progenitor cells. • WYE-687 induces apoptotic death of human AML cells/progenitor cells. • WYE-687 inhibits mTORC1/2 activation in human AML cells/progenitor cells. • WYE-687 inhibits U937 xenograft growth in SCID mice.

  1. Preclinical evaluation of WYE-687, a mTOR kinase inhibitor, as a potential anti-acute myeloid leukemia agent

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Feng; Wang, Lingling; Shen, Yunfeng; Xia, Jun; Chen, Heng; Jiang, Yuanqiang, E-mail: jiangyuanqiangwuxi@163.com; Lu, Mize, E-mail: lumizewuxi9@sina.com

    2016-02-05

    Mammalian target of rapamycin (mTOR) as a potential drug target for treatment of acute myeloid leukemia (AML). Here, we investigated the potential anti-leukemic activity by WYE-687, a potent mTOR kinase inhibitor. We demonstrated that WYE-687 potently inhibited survival and proliferation of established (HL-60, U937, AML-193 and THP-1 lines) and human AML progenitor cells. Yet, same WYE-687 treatment was non-cytotoxic to the primary peripheral blood mononuclear leukocytes (PBMCs) isolated from healthy donors. WYE-687 induced caspase-dependent apoptotic death in above AML cells/progenitor cells. On the other hand, the pan-caspase inhibitor (Z-VAD-FMK), the caspase-3 specific inhibitor (Z-DEVD-FMK) or the caspase-9 specific inhibitor (z-LEHD-fmk) attenuated WYE-687-induced cytotoxicity. At the molecular level, WYE-687 concurrently inhibited activation of mTORC1 (p70S6K1 and S6 phosphorylations) and mTORC2 (AKT Ser-473 and FoxO1/3a phosphorylations), whiling downregulating mTORC1/2-regulated genes (Bcl-xL and hypoxia-inducible factor 1/2α) in both HL-60/U937 cells and human AML progenitor cells. In vivo, oral administration of WYE-687 potently inhibited U937 leukemic xenograft tumor growth in severe combined immunodeficient (SCID) mice, without causing significant toxicities. In summary, our results demonstrate that targeting mTORC1/2 by WYE-687 leads to potent antitumor activity in preclinical models of AML. - Highlights: • WYE-687 inhibits survival and proliferation of human AML cells/progenitor cells. • WYE-687 induces apoptotic death of human AML cells/progenitor cells. • WYE-687 inhibits mTORC1/2 activation in human AML cells/progenitor cells. • WYE-687 inhibits U937 xenograft growth in SCID mice.

  2. Efficacy of Polyphenon E, Red Ginseng, and Rapamycin on Benzo(apyrene-Induced Lung Tumorigenesis in A/J Mice

    Directory of Open Access Journals (Sweden)

    Ying Yan

    2006-01-01

    Full Text Available The objective of this investigation was to determine the efficacy of several novel agents in preventing lung tumorigenesis in mice. We evaluated polyphenon E, red ginseng, and rapamycin in A/J mice treated with the tobacco-specific carcinogen benzo(apyrene for their ability to inhibit pulmonary adenoma formation and growth. We found that treatment with polyphenon E exhibited a significant reduction on both tumor multiplicity and tumor load (tumor multiplicity × tumor volume in a dose-dependent fashion. Polyphenon E (2% wt/wt in the diet reduced tumor multiplicity by 46% and tumor load by 94%. This result provided key evidence in support of a phase II clinical chemoprevention trial of lung cancer. Administration of red ginseng in drinking water decreased tumor multiplicity by 36% and tumor load by 70%. The mammalian target of rapamycin inhibitor rapamycin showed significant efficacy against lung tumor growth in the tumor progression protocol and reduced tumor load by 84%. The results of these investigations demonstrate that polyphenon E, red ginseng, and rapamycin significantly inhibit pulmonary adenoma formation and growth in A/J mice.

  3. Simultaneous inhibition of mTOR-containing complex 1 (mTORC1) and MNK induces apoptosis of cutaneous T-cell lymphoma (CTCL) cells

    DEFF Research Database (Denmark)

    Marzec, Michal Tomasz; Liu, Xiaobin; Wysocka, Maria

    2011-01-01

    mTOR kinase forms the mTORC1 complex by associating with raptor and other proteins and affects a number of key cell functions. mTORC1 activates p70S6kinase 1 (p70S6K1) and inhibits 4E-binding protein 1 (4E-BP1). In turn, p70S6K1 phosphorylates a S6 protein of the 40S ribosomal subunit (S6rp) and 4E...

  4. Male patients with partial androgen insensitivity syndrome

    DEFF Research Database (Denmark)

    Hellmann, Philip; Christiansen, Peter; Johannsen, Trine Holm

    2012-01-01

    To describe the natural history of phenotype, growth and gonadal function in patients with partial androgen insensitivity syndrome.......To describe the natural history of phenotype, growth and gonadal function in patients with partial androgen insensitivity syndrome....

  5. Is reactivation of autophagy a possible therapeutic solution for obesity and metabolic syndrome?

    Science.gov (United States)

    Sciarretta, Sebastiano; Volpe, Massimo; Sadoshima, Junichi

    2012-08-01

    The molecular mechanism regulating the cardiomyocyte response to energy stress has been a hot topic in cardiac research in recent years, since this mechanism could be targeted for treatment of patients with ischemic heart disease. We have shown recently that the activity of RAS homolog enriched in brain (RHEB), a small GTP binding protein, is inhibited in response to glucose deprivation (GD) in cardiomyocytes and ischemia in the mouse heart. This is a physiological adaptation, since it inhibits complex 1 of the mechanistic target of rapamycin (MTORC1) and activates autophagy, thereby promoting cell survival during GD and prolonged ischemia. Importantly, the physiological inhibition of RHEB-MTORC1 signaling during myocardial ischemia is impaired in the presence of obesity and metabolic syndrome caused by high-fat diet (HFD) feeding, leading to a dramatic increase in ischemic injury. Although MTORC1 and autophagy can be regulated through RHEB-independent mechanisms, such as the AMPK-dependent phosphorylation of RPTOR and ULK1, RHEB appears to be critical in the regulation of MTORC1 and autophagy during ischemia in cardiomyocytes, and its dysregulation is relevant to human disease. Here we discuss the biological relevance of the dysregulation of RHEB-MTORC1 signaling and the suppression of autophagy in obesity and metabolic syndrome.

  6. Denervation atrophy is independent from Akt and mTOR activation and is not rescued by myostatin inhibition

    Directory of Open Access Journals (Sweden)

    Elizabeth M. MacDonald

    2014-04-01

    Full Text Available The purpose of our study was to compare two acquired muscle atrophies and the use of myostatin inhibition for their treatment. Myostatin naturally inhibits skeletal muscle growth by binding to ActRIIB, a receptor on the cell surface of myofibers. Because blocking myostatin in an adult wild-type mouse induces profound muscle hypertrophy, we applied a soluble ActRIIB receptor to models of disuse (limb immobilization and denervation (sciatic nerve resection atrophy. We found that treatment of immobilized mice with ActRIIB prevented the loss of muscle mass observed in placebo-treated mice. Our results suggest that this protection from disuse atrophy is regulated by serum and glucocorticoid-induced kinase (SGK rather than by Akt. Denervation atrophy, however, was not protected by ActRIIB treatment, yet resulted in an upregulation of the pro-growth factors Akt, SGK and components of the mTOR pathway. We then treated the denervated mice with the mTOR inhibitor rapamycin and found that, despite a reduction in mTOR activation, there is no alteration of the atrophy phenotype. Additionally, rapamycin prevented the denervation-induced upregulation of the mTORC2 substrates Akt and SGK. Thus, our studies show that denervation atrophy is not only independent from Akt, SGK and mTOR activation but also has a different underlying pathophysiological mechanism than disuse atrophy.

  7. Overexpression of the PP2A regulatory subunit Tap46 leads to enhanced plant growth through stimulation of the TOR signalling pathway.

    Science.gov (United States)

    Ahn, Chang Sook; Ahn, Hee-Kyung; Pai, Hyun-Sook

    2015-02-01

    Tap46, a regulatory subunit of protein phosphatase 2A (PP2A), plays an essential role in plant growth and development through a functional link with the Target of Rapamycin (TOR) signalling pathway. Here, we have characterized the molecular mechanisms behind a gain-of-function phenotype of Tap46 and its relationship with TOR to gain further insights into Tap46 function in plants. Constitutive overexpression of Tap46 in Arabidopsis resulted in overall growth stimulation with enlarged organs, such as leaves and siliques. Kinematic analysis of leaf growth revealed that increased cell size was mainly responsible for the leaf enlargement. Tap46 overexpression also enhanced seed size and viability under accelerated ageing conditions. Enhanced plant growth was also observed in dexamethasone (DEX)-inducible Tap46 overexpression Arabidopsis lines, accompanied by increased cellular activities of nitrate-assimilating enzymes. DEX-induced Tap46 overexpression and Tap46 RNAi resulted in increased and decreased phosphorylation of S6 kinase (S6K), respectively, which is a sensitive indicator of endogenous TOR activity, and Tap46 interacted with S6K in planta based on bimolecular fluorescence complementation and co-immunoprecipitation. Furthermore, inactivation of TOR by estradiol-inducible RNAi or rapamycin treatment decreased Tap46 protein levels, but increased PP2A catalytic subunit levels. Real-time quantitative PCR analysis revealed that Tap46 overexpression induced transcriptional modulation of genes involved in nitrogen metabolism, ribosome biogenesis, and lignin biosynthesis. These findings suggest that Tap46 modulates plant growth as a positive effector of the TOR signalling pathway and Tap46/PP2Ac protein abundance is regulated by TOR activity. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  8. Loss of wobble uridine modification in tRNA anticodons interferes with TOR pathway signaling

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    Viktor Scheidt

    2014-11-01

    Full Text Available Previous work in yeast has suggested that modification of tRNAs, in particular uridine bases in the anticodon wobble position (U34, is linked to TOR (target of rapamycin signaling. Hence, U34 modification mutants were found to be hypersensitive to TOR inhibition by rapamycin. To study whether this involves inappropriate TOR signaling, we examined interaction between mutations in TOR pathway genes (tip41∆, sap190∆, ppm1∆, rrd1∆ and U34 modification defects (elp3∆, kti12∆, urm1∆, ncs2∆ and found the rapamycin hypersensitivity in the latter is epistatic to drug resistance of the former. Epistasis, however, is abolished in tandem with a gln3∆ deletion, which inactivates transcription factor Gln3 required for TOR-sensitive activation of NCR (nitrogen catabolite repression genes. In line with nuclear import of Gln3 being under control of TOR and dephosphorylation by the Sit4 phosphatase, we identify novel TOR-sensitive sit4 mutations that confer rapamycin resistance and importantly, mislocalise Gln3 when TOR is inhibited. This is similar to gln3∆ cells, which abolish the rapamycin hypersensitivity of U34 modification mutants, and suggests TOR deregulation due to tRNA undermodification operates through Gln3. In line with this, loss of U34 modifications (elp3∆, urm1∆ enhances nuclear import of and NCR gene activation (MEP2, GAP1 by Gln3 when TOR activity is low. Strikingly, this stimulatory effect onto Gln3 is suppressed by overexpression of tRNAs that usually carry the U34 modifications. Collectively, our data suggest that proper TOR signaling requires intact tRNA modifications and that loss of U34 modifications impinges on the TOR-sensitive NCR branch via Gln3 misregulation.

  9. Loss of wobble uridine modification in tRNA anticodons interferes with TOR pathway signaling.

    Science.gov (United States)

    Scheidt, Viktor; Jüdes, André; Bär, Christian; Klassen, Roland; Schaffrath, Raffael

    2014-11-29

    Previous work in yeast has suggested that modification of tRNAs, in particular uridine bases in the anticodon wobble position (U34), is linked to TOR (target of rapamycin) signaling. Hence, U34 modification mutants were found to be hypersensitive to TOR inhibition by rapamycin. To study whether this involves inappropriate TOR signaling, we examined interaction between mutations in TOR pathway genes ( tip41 ∆, sap190 ∆, ppm1 ∆, rrd1 ∆) and U34 modification defects ( elp3 ∆, kti 12∆, urm1 ∆, ncs2 ∆) and found the rapamycin hypersensitivity in the latter is epistatic to drug resistance of the former. Epistasis, however, is abolished in tandem with a gln3 ∆ deletion, which inactivates transcription factor Gln3 required for TOR-sensitive activation of NCR (nitrogen catabolite repression) genes. In line with nuclear import of Gln3 being under control of TOR and dephosphorylation by the Sit4 phosphatase, we identify novel TOR-sensitive sit4 mutations that confer rapamycin resistance and importantly, mislocalise Gln3 when TOR is inhibited. This is similar to gln3 ∆ cells, which abolish the rapamycin hypersensitivity of U34 modification mutants, and suggests TOR deregulation due to tRNA undermodification operates through Gln3. In line with this, loss of U34 modifications ( elp3 ∆, urm1 ∆) enhances nuclear import of and NCR gene activation ( MEP2 , GAP1 ) by Gln3 when TOR activity is low. Strikingly, this stimulatory effect onto Gln3 is suppressed by overexpression of tRNAs that usually carry the U34 modifications. Collectively, our data suggest that proper TOR signaling requires intact tRNA modifications and that loss of U34 modifications impinges on the TOR-sensitive NCR branch via Gln3 misregulation.

  10. Rapamycin Eye Drops Suppress Lacrimal Gland Inflammation In a Murine Model of Sjögren's Syndrome

    Science.gov (United States)

    Shah, Mihir; Edman, Maria C.; Reddy Janga, Srikanth; Yarber, Frances; Meng, Zhen; Klinngam, Wannita; Bushman, Jonathan; Ma, Tao; Liu, Siyu; Louie, Stan; Mehta, Arjun; Ding, Chuanqing; MacKay, J. Andrew; Hamm-Alvarez, Sarah F.

    2017-01-01

    Purpose To evaluate the efficacy of topical rapamycin in treating autoimmune dacryoadenitis in a mouse model of Sjögren's syndrome. Methods We developed rapamycin in a poly(ethylene glycol)-distearoyl phosphatidylethanolamine (PEG-DSPE) micelle formulation to maintain solubility. Rapamycin or PEG-DSPE eye drops (vehicle) were administered in a well-established Sjögren's syndrome disease model, the male nonobese diabetic (NOD) mice, twice daily for 12 weeks starting at 8 weeks of age. Mouse tear fluid was collected and tear Cathepsin S, a putative tear biomarker for Sjögren's syndrome, was measured. Lacrimal glands were retrieved for histological evaluation, and quantitative real-time PCR of genes associated with Sjögren's syndrome pathogenesis. Tear secretion was measured using phenol red threads, and corneal fluorescein staining was used to assess corneal integrity. Results Lymphocytic infiltration of lacrimal glands from rapamycin-treated mice was significantly (P = 0.0001) reduced by 3.8-fold relative to vehicle-treated mice after 12 weeks of treatment. Rapamycin, but not vehicle, treatment increased tear secretion and decreased corneal fluorescein staining after 12 weeks. In rapamycin-treated mice, Cathepsin S activity was significantly reduced by 3.75-fold in tears (P eye. PMID:28122086

  11. PARP-1 modulation of mTOR signaling in response to a DNA alkylating agent.

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    Chantal Ethier

    Full Text Available Poly(ADP-ribose polymerase-1 (PARP-1 is widely involved in cell death responses. Depending on the degree of injury and on cell type, PARP activation may lead to autophagy, apoptosis or necrosis. In HEK293 cells exposed to the alkylating agent N-methyl-N'-nitro-N'-nitrosoguanine (MNNG, we show that PARP-1 activation triggers a necrotic cell death response. The massive poly(ADP-ribose (PAR synthesis following PARP-1 activation leads to the modulation of mTORC1 pathway. Shortly after MNNG exposure, NAD⁺ and ATP levels decrease, while AMP levels drastically increase. We characterized at the molecular level the consequences of these altered nucleotide levels. First, AMP-activated protein kinase (AMPK is activated and the mTORC1 pathway is inhibited by the phosphorylation of Raptor, in an attempt to preserve cellular energy. Phosphorylation of the mTORC1 target S6 is decreased as well as the phosphorylation of the mTORC2 component Rictor on Thr1135. Finally, Akt phosphorylation on Ser473 is lost and then, cell death by necrosis occurs. Inhibition of PARP-1 with the potent PARP inhibitor AG14361 prevents all of these events. Moreover, the antioxidant N-acetyl-L-cysteine (NAC can also abrogate all the signaling events caused by MNNG exposure suggesting that reactive oxygen species (ROS production is involved in PARP-1 activation and modulation of mTOR signaling. In this study, we show that PARP-1 activation and PAR synthesis affect the energetic status of cells, inhibit the mTORC1 signaling pathway and possibly modulate the mTORC2 complex affecting cell fate. These results provide new evidence that cell death by necrosis is orchestrated by the balance between several signaling pathways, and that PARP-1 and PAR take part in these events.

  12. Mammalian target of rapamycin activity is required for expansion of CD34(+) hematopoietic progenitor cells

    NARCIS (Netherlands)

    Geest, Christian R.; Zwartkruis, Fried J.; Vellenga, Edo; Coffer, Paul J.; Buitenhuis, Miranda

    Background The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic

  13. Activation of the unfolded protein response in sarcoma cells treated with rapamycin or temsirolimus.

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    Joseph W Briggs

    Full Text Available Activation of the unfolded protein response (UPR in eukaryotic cells represents an evolutionarily conserved response to physiological stress. Here, we report that the mTOR inhibitors rapamycin (sirolimus and structurally related temsirolimus are capable of inducing UPR in sarcoma cells. However, this effect appears to be distinct from the classical role for these drugs as mTOR inhibitors. Instead, we detected these compounds to be associated with ribosomes isolated from treated cells. Specifically, temsirolimus treatment resulted in protection from chemical modification of several rRNA residues previously shown to bind rapamycin in prokaryotic cells. As an application for these findings, we demonstrate maximum tumor cell growth inhibition occurring only at doses which induce UPR and which have been shown to be safely achieved in human patients. These results are significant because they challenge the paradigm for the use of these drugs as anticancer agents and reveal a connection to UPR, a conserved biological response that has been implicated in tumor growth and response to therapy. As a result, eIF2 alpha phosphorylation and Xbp-1 splicing may serve as useful biomarkers of treatment response in future clinical trials using rapamycin and rapalogs.

  14. TOR (target of rapamycin) is a key regulator of triacylglycerol accumulation in microalgae.

    Science.gov (United States)

    Imamura, Sousuke; Kawase, Yasuko; Kobayashi, Ikki; Shimojima, Mie; Ohta, Hiroyuki; Tanaka, Kan

    2016-01-01

    Most microalgae abundantly accumulate lipid droplets (LDs) containing triacylglycerols (TAGs) under several stress conditions, but the underlying molecular mechanism of this accumulation remains unclear. In a recent study, we found that inhibition of TOR (target of rapamycin), a highly conserved protein kinase of eukaryotes, by rapamycin resulted in TAG accumulation in microalgae, indicating that TOR negatively regulates TAG accumulation. Here, we show that formation of intracellular LDs and TAG accumulation were also induced in the unicellular green alga Chlamydomonas reinhardtii after exposure to Torin1 or AZD8055, which are novel TOR inhibitors that inhibit TOR activity in a manner different from rapamycin. These results supported quite well our previous conclusion that TOR is a central regulator of TAG accumulation in microalgae.

  15. Rapamycin Induces Heme Oxygenase-1 in Liver but Inhibits Bile Flow Recovery after Ischemia

    NARCIS (Netherlands)

    Kist, Alwine; Wakkie, Joris; Madu, Max; Versteeg, Ruth; ten Berge, Judith; Nikolic, Andrej; Nieuwenhuijs, Vincent B.; Porte, Robert J.; Padbury, Robert T. A.; Barritt, Greg J.

    Background/Aims. Rapamycin, which is employed in the management of patients undergoing liver surgery, induces the synthesis of heme oxygenase-1 (HO-1) in some non-liver cell types. The aim was to investigate whether rapamycin can induce HO-1 expression in the liver, and to test the effects of

  16. Induction of granzyme B expression in T-cell receptor/CD28-stimulated human regulatory T cells is suppressed by inhibitors of the PI3K-mTOR pathway

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    Kelley Todd W

    2009-11-01

    Full Text Available Abstract Background Regulatory T cells (Tregs can employ a cell contact- and granzyme B-dependent mechanism to mediate suppression of bystander T and B cells. Murine studies indicate that granzyme B is involved in the Treg-mediated suppression of anti-tumor immunity in the tumor microenvironment and in the Treg-mediated maintenance of allograft survival. In spite of its central importance, a detailed study of granzyme B expression patterns in human Tregs has not been performed. Results Our data demonstrated that natural Tregs freshly isolated from the peripheral blood of normal adults lacked granzyme B expression. Tregs subjected to prolonged TCR and CD28 triggering, in the presence of IL-2, expressed high levels of granzyme B but CD3 stimulation alone or IL-2 treatment alone failed to induce granzyme B. Treatment of Tregs with the mammalian target of rapamycin (mTOR inhibitor, rapamycin or the PI3 kinase (PI3K inhibitor LY294002 markedly suppressed granzyme B expression. However, neither rapamycin, as previously reported by others, nor LY294002 inhibited Treg proliferation or induced significant cell death in TCR/CD28/IL-2 stimulated cells. The proliferation rate of Tregs was markedly higher than that of CD4+ conventional T cells in the setting of rapamycin treatment. Tregs expanded by CD3/CD28/IL-2 stimulation without rapamycin demonstrated increased in vitro cytotoxic activity compared to Tregs expanded in the presence of rapamycin in both short term (6 hours and long term (48 hours cytotoxicity assays. Conclusion TCR/CD28 mediated activation of the PI3K-mTOR pathway is important for granyzme B expression but not proliferation in regulatory T cells. These findings may indicate that suppressive mechanisms other than granzyme B are utilized by rapamycin-expanded Tregs.

  17. Construction of a mutant of Actinoplanes sp. N902-109 that produces a new rapamycin analog.

    Science.gov (United States)

    Huang, He; Gao, Ping; Zhao, Qi; Hu, Hai-Feng

    2018-03-01

    In the present study, we introduced point mutations into Ac_rapA which encodes a polyketide synthase responsible for rapamycin biosynthesis in Actinoplanes sp. N902-109, in order to construct a mutant with an inactivated enoylreductase (ER) domain, which was able to synthesize a new rapamycin analog. Based on the homologous recombination induced by double-strand breaks in chromosome mediated by endonuclease I-SceI, the site-directed mutation in the first ER domain of Ac_rapA was introduced using non-replicating plasmid pLYERIA combined with an I-SceI expression plasmid. Three amino acid residues of the active center, Ala-Gly-Gly, were converted to Ala-Ser-Pro. The broth of the mutant strain SIPI-027 was analyzed by HPLC and a new peak with the similar UV spectrum to that of rapamycin was found. The sample of the new peak was prepared by solvent extraction, column chromatography, and crystallization methods. The structure of new compound, named as SIPI-rapxin, was elucidated by determining and analyzing its MS and NMR spectra and its biological activity was assessed using mixed lymphocyte reaction (MLR). An ER domain-deficient mutant of Actinoplanes sp. N902-109, named as SIPI-027, was constructed, which produced a novel rapamycin analog SIPI-rapxin and its structure was elucidated to be 35, 36-didehydro-27-O-demethylrapamycin. The biological activity of SIPI-rapxin was better than that of rapamycin. In conclusion, inactivation of the first ER domain of rapA, one of the modular polyketide synthase responsible for macro-lactone synthesis of rapamycin, gave rise to a mutant capable of producing a novel rapamycin analog, 35, 36-didehydro-27-O-demethylrapamycin, demonstrating that the enoylreductase domain was responsible for the reduction of the double bond between C-35 and C-36 during rapamycin synthesis. Copyright © 2018 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  18. Heat shock protein 70 promotes coxsackievirus B3 translation initiation and elongation via Akt-mTORC1 pathway depending on activation of p70S6K and Cdc2.

    Science.gov (United States)

    Wang, Fengping; Qiu, Ye; Zhang, Huifang M; Hanson, Paul; Ye, Xin; Zhao, Guangze; Xie, Ronald; Tong, Lei; Yang, Decheng

    2017-07-01

    We previously demonstrated that coxsackievirus B3 (CVB3) infection upregulated heat shock protein 70 (Hsp70) and promoted CVB3 multiplication. Here, we report the underlying mechanism by which Hsp70 enhances viral RNA translation. By using an Hsp70-overexpressing cell line infected with CVB3, we found that Hsp70 enhanced CVB3 VP1 translation at two stages. First, Hsp70 induced upregulation of VP1 translation at the initiation stage via upregulation of internal ribosome entry site trans-acting factor lupus autoantigen protein and activation of eIF4E binding protein 1, a cap-dependent translation suppressor. Second, we found that Hsp70 increased CVB3 VP1 translation by enhancing translation elongation. This was mediated by the Akt-mammalian target of rapamycin complex 1 signal cascade, which led to the activation of eukaryotic elongation factor 2 via p70S6K- and cell division cycle protein 2 homolog (Cdc2)-mediated phosphorylation and inactivation of eukaryotic elongation factor 2 kinase. We also determined the position of Cdc2 in this signal pathway, indicating that Cdc2 is regulated by mammalian target of rapamycin complex 1. This signal transduction pathway was validated using a number of specific pharmacological inhibitors, short interfering RNAs (siRNAs) and a dominant negative Akt plasmid. Because Hsp70 is a central component of the cellular network of molecular chaperones enhancing viral replication, these data may provide new strategies to limit this viral infection. © 2017 John Wiley & Sons Ltd.

  19. Vascular Manifestations in Antiphospholipid Syndrome (APS): Is APS a Thrombophilia or a Vasculopathy?

    Science.gov (United States)

    Siddique, Salma; Risse, Jessie; Canaud, Guillaume; Zuily, Stéphane

    2017-09-04

    Antiphospholipid antibody syndrome (APS) is characterized primarily by thrombosis and pregnancy morbidity. Chronic vascular lesions can also occur. While the underlying mechanisms of these vascular lesions are not entirely known, there have been multiple theories describing the potential process of vasculopathy in APS and the various clinical manifestations associated with it. Recently, it has been demonstrated that endothelial proliferation in kidneys can be explained by the activation of the mammalian target of rapamycin complex (mTORC) pathway by antiphospholipid antibodies (aPL). These data support the existence of an APS-related vasculopathy in different locations which can explain-in part-the different manifestations of APS. This review focuses on the various manifestations of APS as a result of APS-related vasculopathy, as well as pathophysiology, current screening, and treatment options for clinicians to be aware of.

  20. Hypoxia induces a phase transition within a kinase signaling network in cancer cells

    Science.gov (United States)

    Wei, Wei; Shi, Qihui; Remacle, Francoise; Qin, Lidong; Shackelford, David B.; Shin, Young Shik; Mischel, Paul S.; Levine, R. D.; Heath, James R.

    2013-01-01

    Hypoxia is a near-universal feature of cancer, promoting glycolysis, cellular proliferation, and angiogenesis. The molecular mechanisms of hypoxic signaling have been intensively studied, but the impact of changes in oxygen partial pressure (pO2) on the state of signaling networks is less clear. In a glioblastoma multiforme (GBM) cancer cell model, we examined the response of signaling networks to targeted pathway inhibition between 21% and 1% pO2. We used a microchip technology that facilitates quantification of a panel of functional proteins from statistical numbers of single cells. We find that near 1.5% pO2, the signaling network associated with mammalian target of rapamycin (mTOR) complex 1 (mTORC1)—a critical component of hypoxic signaling and a compelling cancer drug target—is deregulated in a manner such that it will be unresponsive to mTOR kinase inhibitors near 1.5% pO2, but will respond at higher or lower pO2 values. These predictions were validated through experiments on bulk GBM cell line cultures and on neurosphere cultures of a human-origin GBM xenograft tumor. We attempt to understand this behavior through the use of a quantitative version of Le Chatelier’s principle, as well as through a steady-state kinetic model of protein interactions, both of which indicate that hypoxia can influence mTORC1 signaling as a switch. The Le Chatelier approach also indicates that this switch may be thought of as a type of phase transition. Our analysis indicates that certain biologically complex cell behaviors may be understood using fundamental, thermodynamics-motivated principles. PMID:23530221

  1. Hypoxia induces a phase transition within a kinase signaling network in cancer cells.

    Science.gov (United States)

    Wei, Wei; Shi, Qihui; Remacle, Francoise; Qin, Lidong; Shackelford, David B; Shin, Young Shik; Mischel, Paul S; Levine, R D; Heath, James R

    2013-04-09

    Hypoxia is a near-universal feature of cancer, promoting glycolysis, cellular proliferation, and angiogenesis. The molecular mechanisms of hypoxic signaling have been intensively studied, but the impact of changes in oxygen partial pressure (pO2) on the state of signaling networks is less clear. In a glioblastoma multiforme (GBM) cancer cell model, we examined the response of signaling networks to targeted pathway inhibition between 21% and 1% pO2. We used a microchip technology that facilitates quantification of a panel of functional proteins from statistical numbers of single cells. We find that near 1.5% pO2, the signaling network associated with mammalian target of rapamycin (mTOR) complex 1 (mTORC1)--a critical component of hypoxic signaling and a compelling cancer drug target--is deregulated in a manner such that it will be unresponsive to mTOR kinase inhibitors near 1.5% pO2, but will respond at higher or lower pO2 values. These predictions were validated through experiments on bulk GBM cell line cultures and on neurosphere cultures of a human-origin GBM xenograft tumor. We attempt to understand this behavior through the use of a quantitative version of Le Chatelier's principle, as well as through a steady-state kinetic model of protein interactions, both of which indicate that hypoxia can influence mTORC1 signaling as a switch. The Le Chatelier approach also indicates that this switch may be thought of as a type of phase transition. Our analysis indicates that certain biologically complex cell behaviors may be understood using fundamental, thermodynamics-motivated principles.

  2. Primary intestinal lymphangiectasia treated with rapamycin in a child with tuberous sclerosis complex (TSC).

    Science.gov (United States)

    Pollack, Sarah F; Geffrey, Alexandra L; Thiele, Elizabeth A; Shah, Uzma

    2015-09-01

    Primary intestinal lymphangiectasia (PIL) is a rare protein-losing enteropathy characterized by a congenital malformation of the lymphatic vessels of the small intestine causing insufficient drainage and leakage of lymph fluid. Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder characterized by benign hamartomas in multiple organ systems. While the lymphatic system has been implicated in TSC through lymphangioleiomyomatosis (LAM) and lymphedema, this paper reports the first case of PIL in TSC, a female patient with a TSC2 mutation. She developed persistent and significant abdominal distension with chronic diarrhea during her first year of life. Due to lack of treatment options and the involvement of the mTOR pathway in TSC, a trial of an mTOR inhibitor, rapamycin, was initiated. This treatment was highly effective, with improvement in clinical symptoms of PIL as well as abnormal laboratory values including VEGF-C, which was elevated to over seven times the normal upper limit before treatment. This case suggests that PIL is a rare manifestation of TSC, warranting the use of mTOR inhibitors in future studies. © 2015 Wiley Periodicals, Inc.

  3. Finding a better drug for epilepsy: the mTOR pathway as an antiepileptogenic target.

    NARCIS (Netherlands)

    Galanopoulou, A.S.; Gorter, J.A.; Cepeda, C.

    2012-01-01

    The mammalian target of rapamycin (mTOR) signaling pathway regulates cell growth, differentiation, proliferation, and metabolism. Loss-of-function mutations in upstream regulators of mTOR have been highly associated with dysplasias, epilepsy, and neurodevelopmental disorders. These include tuberous

  4. A polarization-insensitive plasmonic photoconductive terahertz emitter

    KAUST Repository

    Li, Xurong

    2017-11-16

    We present a polarization-insensitive plasmonic photoconductive terahertz emitter that uses a two-dimensional array of nanoscale cross-shaped apertures as the plasmonic contact electrodes. The geometry of the cross-shaped apertures is set to maximize optical pump absorption in close proximity to the contact electrodes. The two-dimensional symmetry of the cross-shaped apertures offers a polarization-insensitive interaction between the plasmonic contact electrodes and optical pump beam. We experimentally demonstrate a polarization-insensitive terahertz radiation from the presented emitter in response to a femtosecond optical pump beam and similar terahertz radiation powers compared to previously demonstrated polarization-sensitive photoconductive emitters with plasmonic contact electrode gratings at the optimum optical pump polarization.

  5. Disruption of mammalian target of rapamycin complex 1 in macrophages decreases chemokine gene expression and atherosclerosis

    NARCIS (Netherlands)

    Ai, Ding; Jiang, Hongfeng; Westerterp, Marit; Murphy, Andrew J.; Wang, Mi; Ganda, Anjali; Abramowicz, Sandra; Welch, Carrie; Almazan, Felicidad; Zhu, Yi; Miller, Yury I.; Tall, Alan R.

    2014-01-01

    The mammalian target of rapamycin complex 1 inhibitor, rapamycin, has been shown to decrease atherosclerosis, even while increasing plasma low-density lipoprotein levels. This suggests an antiatherogenic effect possibly mediated by the modulation of inflammatory responses in atherosclerotic plaques.

  6. Induction of biogenic magnetization and redox control by a component of the target of rapamycin complex 1 signaling pathway.

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    Keiji Nishida

    Full Text Available Most organisms are simply diamagnetic, while magnetotactic bacteria and migratory animals are among organisms that exploit magnetism. Biogenic magnetization not only is of fundamental interest, but also has industrial potential. However, the key factor(s that enable biogenic magnetization in coordination with other cellular functions and metabolism remain unknown. To address the requirements for induction and the application of synthetic bio-magnetism, we explored the creation of magnetism in a simple model organism. Cell magnetization was first observed by attraction towards a magnet when normally diamagnetic yeast Saccharomyces cerevisiae were grown with ferric citrate. The magnetization was further enhanced by genetic modification of iron homeostasis and introduction of ferritin. The acquired magnetizable properties enabled the cells to be attracted to a magnet, and be trapped by a magnetic column. Superconducting quantum interference device (SQUID magnetometry confirmed and quantitatively characterized the acquired paramagnetism. Electron microscopy and energy-dispersive X-ray spectroscopy showed electron-dense iron-containing aggregates within the magnetized cells. Magnetization-based screening of gene knockouts identified Tco89p, a component of TORC1 (Target of rapamycin complex 1, as important for magnetization; loss of TCO89 and treatment with rapamycin reduced magnetization in a TCO89-dependent manner. The TCO89 expression level positively correlated with magnetization, enabling inducible magnetization. Several carbon metabolism genes were also shown to affect magnetization. Redox mediators indicated that TCO89 alters the intracellular redox to an oxidized state in a dose-dependent manner. Taken together, we demonstrated that synthetic induction of magnetization is possible and that the key factors are local redox control through carbon metabolism and iron supply.

  7. Excessive Leucine-mTORC1-Signalling of Cow Milk-Based Infant Formula: The Missing Link to Understand Early Childhood Obesity.

    Science.gov (United States)

    Melnik, Bodo C

    2012-01-01

    Increased protein supply by feeding cow-milk-based infant formula in comparison to lower protein content of human milk is a well-recognized major risk factor of childhood obesity. However, there is yet no conclusive biochemical concept explaining the mechanisms of formula-induced childhood obesity. It is the intention of this article to provide the biochemical link between leucine-mediated signalling of mammalian milk proteins and adipogenesis as well as early adipogenic programming. Leucine has been identified as the predominant signal transducer of mammalian milk, which stimulates the nutrient-sensitive kinase mammalian target of rapamycin complex 1 (mTORC1). Leucine thus functions as a maternal-neonatal relay for mTORC1-dependent neonatal β-cell proliferation and insulin secretion. The mTORC1 target S6K1 plays a pivotal role in stimulation of mesenchymal stem cells to differentiate into adipocytes and to induce insulin resistance. It is of most critical concern that infant formulas provide higher amounts of leucine in comparison to human milk. Exaggerated leucine-mediated mTORC1-S6K1 signalling induced by infant formulas may thus explain increased adipogenesis and generation of lifelong elevated adipocyte numbers. Attenuation of mTORC1 signalling of infant formula by leucine restriction to physiologic lower levels of human milk offers a great chance for the prevention of childhood obesity and obesity-related metabolic diseases.

  8. Rictor positively regulates B cell receptor signaling by modulating actin reorganization via ezrin.

    Directory of Open Access Journals (Sweden)

    Lu Huang

    2017-08-01

    Full Text Available As the central hub of the metabolism machinery, the mammalian target of rapamycin complex 2 (mTORC2 has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. Rictor is involved in B cell development, especially the peripheral development. However, the role of Rictor on B cell receptor (BCR signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell-specfic Rictor knockout (KO mice to investigate how Rictor regulates BCR signaling. We found that the key positive and negative BCR signaling molecules, phosphorylated Brutons tyrosine kinase (pBtk and phosphorylated SH2-containing inositol phosphatase (pSHIP, are reduced and enhanced, respectively, in Rictor KO B cells. This suggests that Rictor positively regulates the early events of BCR signaling. We found that the cellular filamentous actin (F-actin is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration is associated with a decreased humoral immune response in Rictor KO mice. The inhibition of actin polymerization with latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, in which Rictor regulates BCR signaling via actin reorganization.

  9. mTOR inhibition in macrophages of asymptomatic HIV+ persons reverses the decrease in TLR4-mediated TNFα release through prolongation of MAPK pathway activation1

    Science.gov (United States)

    Li, Xin; Han, Xinbing; Llano, Juliana; Bole, Medhavi; Zhou, Xiuqin; Swan, Katharine; Anandaiah, Asha; Nelson, Benjamin; Patel, Naimish R.; Reinach, Peter S.; Koziel, Henry; Tachado, Souvenir D.

    2011-01-01

    Toll-like receptor 4 (TLR4) mediated signaling is significantly impaired in macrophages from HIV+ persons predominantly due to altered MyD88-dependent pathway signaling caused in part by constitutive activation of PI3K. Here we assessed in these macrophages if the blunted increase in TLR4-mediated TNFα release induced by lipid A are associated with PI3K-induced upregulation of mammalian target of rapamycin (mTOR) activity. mTOR inhibition with rapamycin enhanced TLR4-mediated TNFα release, but instead suppressed anti-inflammatory IL-10 release. Targeted gene silencing of mTOR in macrophages resulted in lipid A-induced TNFα and IL-10 release patterns similar to those induced by rapamycin. Rapamycin restored MyD88-IRAK interaction in a dose-dependent manner. Targeted gene silencing of MyD88 (shRNA) and mTOR (RNAi) inhibition resulted in TLR4-mediated p70s6K activation and enhanced TNFα release, whereas IL-10 release was inhibited in both silenced and non-silenced HIV+ macrophages. Furthermore, mTOR inhibition augmented lipid A-induced TNFα release through enhanced and prolonged phosphorylation of ERK1/2 and JNK1/2 MAP kinases, which was associated with time-dependent MKP-1 destabilization. Taken together, impaired TLR4-mediated TNFα release in HIV+ macrophages is attributable in part to mTOR activation by constitutive PI3K expression in a MyD88-dependent signaling pathway. These changes result in MKP-1 stabilization, which shortens and blunts MAP kinase activation. mTOR inhibition may serve as a potential therapeutic target to upregulate macrophage innate immune host defense responsiveness in HIV+ persons. PMID:22025552

  10. Punishment Insensitivity in Early Childhood: A Developmental, Dimensional Approach.

    Science.gov (United States)

    Nichols, Sara R; Briggs-Gowan, Margaret J; Estabrook, Ryne; Burns, James L; Kestler, Jacqueline; Berman, Grace; Henry, David B; Wakschlag, Lauren S

    2015-08-01

    Impairment in learning from punishment ("punishment insensitivity") is an established feature of severe antisocial behavior in adults and youth but it has not been well studied as a developmental phenomenon. In early childhood, differentiating a normal: abnormal spectrum of punishment insensitivity is key for distinguishing normative misbehavior from atypical manifestations. This study employed a novel measure, the Multidimensional Assessment Profile of Disruptive Behavior (MAP-DB), to examine the distribution, dimensionality, and external validity of punishment insensitivity in a large, demographically diverse community sample of preschoolers (3-5 years) recruited from pediatric clinics (N = 1,855). Caregivers completed surveys from which a seven-item Punishment Insensitivity scale was derived. Findings indicated that Punishment Insensitivity behaviors are relatively common in young children, with at least 50 % of preschoolers exhibiting them sometimes. Item response theory analyses revealed a Punishment Insensitivity spectrum. Items varied along a severity continuum: most items needed to occur "Often" in order to be severe and behaviors that were qualitatively atypical or intense were more severe. Although there were item-level differences across sociodemographic groups, these were small. Construct, convergent, and divergent validity were demonstrated via association to low concern for others and noncompliance, motivational regulation, and a disruptive family context. Incremental clinical utility was demonstrated in relation to impairment. Early childhood punishment insensitivity varies along a severity continuum and is atypical when it predominates. Implications for understanding the phenomenology of emergent disruptive behavior are discussed.

  11. Arginine vasopressin antagonizes the effects of prostaglandin E2 on the spontaneous activity of warm-sensitive and temperature-insensitive neurons in the medial preoptic area in rats.

    Science.gov (United States)

    Xu, Jian-Hui; Hou, Xiao-Yu; Tang, Yu; Luo, Rong; Zhang, Jie; Liu, Chang; Yang, Yong-Lu

    2018-01-01

    Arginine vasopressin (AVP) plays an important role in thermoregulation and antipyresis. We have demonstrated that AVP could change the spontaneous activity of thermosensitive and temperature insensitive neurons in the preoptic area. However, whether AVP influences the effects of prostaglandin E 2 (PGE 2 ) on the spontaneous activity of neurons in the medial preoptic area (MPO) remains unclear. Our experiment showed that PGE 2 decreased the spontaneous activity of warm-sensitive neurons, and increased that of low-slope temperature-insensitive neurons in the MPO. AVP attenuated the inhibitory effect of PGE 2 on warm-sensitive neurons, and reversed the excitatory effect of PGE 2 on low-slope temperature-insensitive neurons, demonstrating that AVP antagonized the effects of PGE 2 on the spontaneous activity of these neurons. The effect of AVP was suppressed by an AVP V 1a receptor antagonist, suggesting that V 1a receptor mediated the action of AVP. We also demonstrated that AVP attenuated the PGE 2 -induced decrease in the prepotential's rate of rise in warm-sensitive neurons and the PGE 2 -induced increase in that in low-slope temperature-insensitive neurons through the V 1a receptor. Together, these data indicated that AVP antagonized the PGE 2 -induced change in the spontaneous activity of warm-sensitive and low-slope temperature-insensitive neurons in the MPO partly by reducing the PGE 2 -induced change in the prepotential of these neurons in a V 1a receptor-dependent manner. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Anti-bombing insensitivity life of molybdenum cathode doped with La{sub 2}O{sub 3} and Y{sub 2}O{sub 3}

    Energy Technology Data Exchange (ETDEWEB)

    Wang Jinshu [School of Materials Science and Engineering, Beijing University of Technology, Beijing 100022 (China)]. E-mail: wangjsh@bjut.edu.cn; Wang Yiman [School of Materials Science and Engineering, Beijing University of Technology, Beijing 100022 (China); Zhou Meiling [School of Materials Science and Engineering, Beijing University of Technology, Beijing 100022 (China)

    2006-03-15

    Anti-bombing insensitivity of La{sub 2}O{sub 3}-Y{sub 2}O{sub 3}-Mo secondary emitter has been studied in this paper. The variation of maximum secondary emission coefficient {delta} {sub max} with time was measured. The cathode after life experiment was analyzed by means of HRM, SEM, EDS and XRD. The results showed that {delta} {sub max} of La{sub 2}O{sub 3}-Y{sub 2}O{sub 3}-Mo cathode operating at 1100 deg. C under continuous electron bombardment of 300 W/cm{sup 2} was still about 2.5 after 1000 h operation, indicating that this kind of cathode had good anti-bombing insensitivity. In the internal part of the cathode, RE{sub 2}O{sub 3} (rare earth oxide) and molybdenum grains distributed alternately and there existed a certain relationship between crystallographic orientation of RE{sub 2}O{sub 3} and that of molybdenum. It was found that a RE{sub 2}O{sub 3} layer was formed on the surface after operation. The high {delta} {sub max} of La{sub 2}O{sub 3}-Y{sub 2}O{sub 3}-Mo cathode was related to the RE{sub 2}O{sub 3} layer on the surface and the amount of nanosized La{sub 2}O{sub 3} particles on the Y{sub 2}O{sub 3} layer.

  13. Systemic inhibition of mTOR kinase via rapamycin disrupts consolidation and reconsolidation of auditory fear memory.

    Science.gov (United States)

    Mac Callum, Phillip E; Hebert, Mark; Adamec, Robert E; Blundell, Jacqueline

    2014-07-01

    The mammalian target of rapamycin (mTOR) kinase is a critical regulator of mRNA translation and is known to be involved in various long lasting forms of synaptic and behavioural plasticity. However, information concerning the temporal pattern of mTOR activation and susceptibility to pharmacological intervention during both consolidation and reconsolidation of long-term memory (LTM) remains scant. Male C57BL/6 mice were injected systemically with rapamycin at various time points following conditioning or retrieval in an auditory fear conditioning paradigm, and compared to vehicle (and/or anisomycin) controls for subsequent memory recall. Systemic blockade of mTOR with rapamycin immediately or 12h after training or reactivation impairs both consolidation and reconsolidation of an auditory fear memory. Further behavioural analysis revealed that the enduring effects of rapamycin on reconsolidation are dependent upon reactivation of the memory trace. Rapamycin, however, has no effect on short-term memory or the ability to retrieve an established fear memory. Collectively, our data suggest that biphasic mTOR signalling is essential for both consolidation and reconsolidation-like activities that contribute to the formation, re-stabilization, and persistence of long term auditory-fear memories, while not influencing other aspects of the memory trace. These findings also provide evidence for a cogent treatment model for reducing the emotional strength of established, traumatic memories analogous to those observed in acquired anxiety disorders such as posttraumatic stress disorder (PTSD) and specific phobias, through pharmacologic blockade of mTOR using systemic rapamycin following reactivation. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Depdc5 knockdown causes mTOR-dependent motor hyperactivity in zebrafish.

    Science.gov (United States)

    de Calbiac, Hortense; Dabacan, Adriana; Marsan, Elise; Tostivint, Hervé; Devienne, Gabrielle; Ishida, Saeko; Leguern, Eric; Baulac, Stéphanie; Muresan, Raul C; Kabashi, Edor; Ciura, Sorana

    2018-05-01

    DEPDC5 was identified as a major genetic cause of focal epilepsy with deleterious mutations found in a wide range of inherited forms of focal epilepsy, associated with malformation of cortical development in certain cases. Identification of frameshift, truncation, and deletion mutations implicates haploinsufficiency of DEPDC5 in the etiology of focal epilepsy. DEPDC5 is a component of the GATOR1 complex, acting as a negative regulator of mTOR signaling. Zebrafish represents a vertebrate model suitable for genetic analysis and drug screening in epilepsy-related disorders. In this study, we defined the expression of depdc5 during development and established an epilepsy model with reduced Depdc5 expression. Here we report a zebrafish model of Depdc5 loss-of-function that displays a measurable behavioral phenotype, including hyperkinesia, circular swimming, and increased neuronal activity. These phenotypic features persisted throughout embryonic development and were significantly reduced upon treatment with the mTORC1 inhibitor, rapamycin, as well as overexpression of human WT DEPDC5 transcript. No phenotypic rescue was obtained upon expression of epilepsy-associated DEPDC5 mutations (p.Arg487* and p.Arg485Gln), indicating that these mutations cause a loss of function of the protein. This study demonstrates that Depdc5 knockdown leads to early-onset phenotypic features related to motor and neuronal hyperactivity. Restoration of phenotypic features by WT but not epilepsy-associated Depdc5 mutants, as well as by mTORC1 inhibition confirm the role of Depdc5 in the mTORC1-dependent molecular cascades, defining this pathway as a potential therapeutic target for DEPDC5 -inherited forms of focal epilepsy.

  15. Docosahexaenoic Acid Modulates a HER2-Associated Lipogenic Phenotype, Induces Apoptosis, and Increases Trastuzumab Action in HER2-Overexpressing Breast Carcinoma Cells.

    Science.gov (United States)

    Ravacci, Graziela Rosa; Brentani, Maria Mitzi; Tortelli, Tharcisio Citrângulo; Torrinhas, Raquel Suzana M M; Santos, Jéssica Reis; Logullo, Angela Flávia; Waitzberg, Dan Linetzky

    2015-01-01

    In breast cancer, lipid metabolic alterations have been recognized as potential oncogenic stimuli that may promote malignancy. To investigate whether the oncogenic nature of lipogenesis closely depends on the overexpression of HER2 protooncogene, the normal breast cell line, HB4a, was transfected with HER2 cDNA to obtain HER2-overexpressing HB4aC5.2 cells. Both cell lines were treated with trastuzumab and docosahexaenoic acid. HER2 overexpression was accompanied by an increase in the expression of lipogenic genes involved in uptake (CD36), transport (FABP4), and storage (DGAT) of exogenous fatty acids (FA), as well as increased activation of "de novo" FA synthesis (FASN). We further investigate whether this lipogenesis reprogramming might be regulated by mTOR/PPARγ pathway. Inhibition of the mTORC1 pathway markers, p70S6 K1, SREBP1, and LIPIN1, as well as an increase in DEPTOR expression (the main inhibitor of the mTOR) was detected in HB4aC5.2. Based on these results, a PPARγ selective antagonist, GW9662, was used to treat both cells lines, and the lipogenic genes remained overexpressed in the HB4aC5.2 but not HB4a cells. DHA treatment inhibited all lipogenic genes (except for FABP4) in both cell lines yet only induced death in the HB4aC5.2 cells, mainly when associated with trastuzumab. Neither trastuzumab nor GW9662 alone was able to induce cell death. In conclusion, oncogenic transformation of breast cells by HER2 overexpression may require a reprogramming of lipogenic genetic that is independent of mTORC1 pathway and PPARγ activity. This reprogramming was inhibited by DHA.

  16. Docosahexaenoic Acid Modulates a HER2-Associated Lipogenic Phenotype, Induces Apoptosis, and Increases Trastuzumab Action in HER2-Overexpressing Breast Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Graziela Rosa Ravacci

    2015-01-01

    Full Text Available In breast cancer, lipid metabolic alterations have been recognized as potential oncogenic stimuli that may promote malignancy. To investigate whether the oncogenic nature of lipogenesis closely depends on the overexpression of HER2 protooncogene, the normal breast cell line, HB4a, was transfected with HER2 cDNA to obtain HER2-overexpressing HB4aC5.2 cells. Both cell lines were treated with trastuzumab and docosahexaenoic acid. HER2 overexpression was accompanied by an increase in the expression of lipogenic genes involved in uptake (CD36, transport (FABP4, and storage (DGAT of exogenous fatty acids (FA, as well as increased activation of “de novo” FA synthesis (FASN. We further investigate whether this lipogenesis reprogramming might be regulated by mTOR/PPARγ pathway. Inhibition of the mTORC1 pathway markers, p70S6 K1, SREBP1, and LIPIN1, as well as an increase in DEPTOR expression (the main inhibitor of the mTOR was detected in HB4aC5.2. Based on these results, a PPARγ selective antagonist, GW9662, was used to treat both cells lines, and the lipogenic genes remained overexpressed in the HB4aC5.2 but not HB4a cells. DHA treatment inhibited all lipogenic genes (except for FABP4 in both cell lines yet only induced death in the HB4aC5.2 cells, mainly when associated with trastuzumab. Neither trastuzumab nor GW9662 alone was able to induce cell death. In conclusion, oncogenic transformation of breast cells by HER2 overexpression may require a reprogramming of lipogenic genetic that is independent of mTORC1 pathway and PPARγ activity. This reprogramming was inhibited by DHA.

  17. MEK-ERK inhibition potentiates WAY-600-induced anti-cancer efficiency in preclinical hepatocellular carcinoma (HCC) models

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Kaifeng, E-mail: kaifeng_wangdr@sina.com [Cancer center, the Affiliated Hospital of Hangzhou Normal University, Hangzhou (China); Fan, Yaohua [Oncology Department, No. 1 Hospital of Jiaxing, Zhejiang Province, Jiaxing (China); Chen, Gongying [Oncology Department, The Affiliated Hospital Hangzhou Normal University, Hangzhou (China); Wang, Zhengrong [Taizhou Hospital, Zhejiang Province, Taizhou (China); Kong, Dexin; Zhang, Peng [Oncology Department, Sir Run Run Shaw Hospital, Medical School, Zhejiang University, Hangzhou (China)

    2016-05-27

    The search for novel anti-hepatocellular carcinoma (HCC) agents is important. Mammalian target of rapamycin (mTOR) hyper-activation plays a pivotal role in promoting HCC tumorigenesis and chemoresistance. The current preclinical study evaluated the potential anti-HCC activity by a potent mTOR kinase inhibitor, WAY-600. We showed that WAY-600 inhibited survival and proliferation of HCC cell lines (HepG2 and Huh7) and primary human HCC cells. Caspase-dependent apoptosis was activated by WAY-600 in above HCC cells. Reversely, caspase inhibitors largely attenuated WAY-600's lethality against HCC cells. At the signaling level, WAY-600 blocked mTOR complex 1/2 (mTORC1/2) assemble and activation, yet activated MEK-ERK pathway in HCC cells. MEK-ERK inhibitors, PD-98059 and MEK-162, or MEK1/2 shRNA significantly potentiated WAY-600's cytotoxicity in HCC cells. Further studies showed that WAY-600 intraperitoneal (i.p.) administration in nude mice inhibited p-AKT Ser-473 and displayed significant anti-cancer activity against HepG2 xenografts. Remarkably, co-administration of MEK-162 further potentiated WAY-600's anti-HCC activity in vivo. These preclinical results demonstrate the potent anti-HCC activity by WAY-600, either alone or with MEK-ERK inhibitors. -- Highlights: •WAY-600 inhibits HCC cell survival and proliferation in vitro. •WAY-600 activates caspase-dependent apoptosis in HCC cells. •WAY-600 blocks mTORC1/2 activation, but activates MEK-ERK in HCC cells. •MEK-ERK inhibitors or MEK1/2 shRNA enhances WAY-600's cytotoxicity against HCC cells. •MEK-162 co-administration potentiates WAY-600-induced the anti-HepG2 tumor efficacy.

  18. Differential responses of human regulatory T cells (Treg and effector T cells to rapamycin.

    Directory of Open Access Journals (Sweden)

    Laura Strauss

    Full Text Available BACKGROUND: The immunosuppressive drug rapamycin (RAPA promotes the expansion of CD4(+ CD25(highFoxp3(+ regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-gamma chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA. METHODOLOGY/PRINCIPAL FINDINGS: CD4(+CD25(+ and CD4(+CD25(neg T cells were isolated from PBMC of normal controls (n = 21 using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1-100 nM was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4(+CD25(high and CD4(+CD25(neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4(+CD25(neg or CD8(+CD25(neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4(+CD25(+ T cells in the presence of 1-100 nM RAPA (p<0.001. RAPA-expanded Treg were largely CD4(+CD25(highFoxp3(+ cells and were resistant to apoptosis, while CD4(+CD25(neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4(+CD25(neg cells. Activated Treg+/-RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/mTOR pathway. CONCLUSIONS/SIGNIFICANCE: RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation.

  19. IGF-1 prevents simvastatin-induced myotoxicity in C2C12 myotubes.

    Science.gov (United States)

    Bonifacio, Annalisa; Sanvee, Gerda M; Brecht, Karin; Kratschmar, Denise V; Odermatt, Alex; Bouitbir, Jamal; Krähenbühl, Stephan

    2017-05-01

    Statins are generally well tolerated, but treatment with these drugs may be associated with myopathy. The mechanisms of statin-associated myopathy are not completely understood. Statins inhibit AKT phosphorylation by an unclear mechanism, whereas insulin-like growth factor (IGF-1) activates the IGF-1/AKT signaling pathway and promotes muscle growth. The aims of the study were to investigate mechanisms of impaired AKT phosphorylation by simvastatin and to assess effects of IGF-1 on simvastatin-induced myotoxicity in C2C12 myotubes. C2C12 mouse myotubes were exposed to 10 μM simvastatin and/or 10 ng/mL IGF-1 for 18 h. Simvastatin inhibited the IGF-1/AKT signaling pathway, resulting in increased breakdown of myofibrillar proteins, impaired protein synthesis and increased apoptosis. Simvastatin inhibited AKT S473 phosphorylation, indicating reduced activity of mTORC2. In addition, simvastatin impaired stimulation of AKT T308 phosphorylation by IGF-1, indicating reduced activation of the IGF-1R/PI3K pathway by IGF-1. Nevertheless, simvastatin-induced myotoxicity could be at least partially prevented by IGF-1. The protective effects of IGF-1 were mediated by activation of the IGF-1R/AKT signaling cascade. Treatment with IGF-1 also suppressed muscle atrophy markers, restored protein synthesis and inhibited apoptosis. These results were confirmed by normalization of myotube morphology and protein content of C2C12 cells exposed to simvastatin and treated with IGF-1. In conclusion, impaired activity of AKT can be explained by reduced function of mTORC2 and of the IGF-1R/PI3K pathway. IGF-1 can prevent simvastatin-associated cytotoxicity and metabolic effects on C2C12 cells. The study gives insight into mechanisms of simvastatin-associated myotoxicity and provides potential targets for therapeutic intervention.

  20. Aging and stem cell therapy: AMPK as an applicable pharmacological target for rejuvenation of aged stem cells and achieving higher efficacy in stem cell therapy.

    Science.gov (United States)

    Khorraminejad-Shirazi, Mohammadhossein; Farahmandnia, Mohammad; Kardeh, Bahareh; Estedlal, Alireza; Kardeh, Sina; Monabati, Ahmad

    2017-10-19

    In recent years, tissue regeneration has become a promising field for developing stem cell-based transplantation therapies for human patients. Adult stem cells are affected by the same aging mechanisms that involve somatic cells. One of the mechanisms involved in cellular aging is hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and disruption of 5' adenosine monophosphate-activated protein kinase (AMPK). Aging of stem cells results in their impaired regenerative capacity and depletion of stem cell pools in adult tissue, which results in lower efficacy of stem cell therapy. By utilizing an effective therapeutic intervention for aged stem cells, stem cell therapy can become more promising for future application. mTORC1 inhibition is a practical approach to preserve the stem cell pool. In this article, we review the dynamic interaction between sirtuin (silent mating type information regulation 2 homolog) 1, AMPK, and mTORC1. We propose that using AMPK activators such as 5-aminoimidazole-4-carboxamide ribonucleotide, A769662, metformin, and oxidized nicotinamide adenine dinucleotide (NAD + ) are practical ways to be employed for achieving better optimized results in stem cell-based transplantation therapies. Copyright © 2017 King Faisal Specialist Hospital & Research Centre. Published by Elsevier B.V. All rights reserved.

  1. Transcriptional mechanisms associated with seed dormancy and dormancy loss in the gibberellin-insensitive sly1-2 mutant of Arabidopsis thaliana

    Science.gov (United States)

    While widespread transcriptome changes have been previously observed with seed dormancy loss, this study specifically characterized transcriptional changes associated with the increased seed dormancy and dormancy loss of the gibberellin (GA) hormone-insensitive sleepy1-2 (sly1-2) mutant. The SLY1 g...

  2. The structure of the karrikin-insensitive protein (KAI2) in Arabidopsis thaliana.

    Science.gov (United States)

    Bythell-Douglas, Rohan; Waters, Mark T; Scaffidi, Adrian; Flematti, Gavin R; Smith, Steven M; Bond, Charles S

    2013-01-01

    KARRIKIN INSENSITIVE 2 (KAI2) is an α/β hydrolase involved in seed germination and seedling development. It is essential for plant responses to karrikins, a class of butenolide compounds derived from burnt plant material that are structurally similar to strigolactone plant hormones. The mechanistic basis for the function of KAI2 in plant development remains unclear. We have determined the crystal structure of Arabidopsis thaliana KAI2 in space groups P2(1) 2(1) 2(1) (a =63.57 Å, b =66.26 Å, c =78.25 Å) and P2(1) (a =50.20 Å, b =56.04 Å, c =52.43 Å, β =116.12°) to 1.55 and 2.11 Å respectively. The catalytic residues are positioned within a large hydrophobic pocket similar to that of DAD2, a protein required for strigolactone response in Petunia hybrida. KAI2 possesses a second solvent-accessible pocket, adjacent to the active site cavity, which offers the possibility of allosteric regulation. The structure of KAI2 is consistent with its designation as a serine hydrolase, as well as previous data implicating the protein in karrikin and strigolactone signalling.

  3. The structure of the karrikin-insensitive protein (KAI2 in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Rohan Bythell-Douglas

    Full Text Available KARRIKIN INSENSITIVE 2 (KAI2 is an α/β hydrolase involved in seed germination and seedling development. It is essential for plant responses to karrikins, a class of butenolide compounds derived from burnt plant material that are structurally similar to strigolactone plant hormones. The mechanistic basis for the function of KAI2 in plant development remains unclear. We have determined the crystal structure of Arabidopsis thaliana KAI2 in space groups P2(1 2(1 2(1 (a =63.57 Å, b =66.26 Å, c =78.25 Å and P2(1 (a =50.20 Å, b =56.04 Å, c =52.43 Å, β =116.12° to 1.55 and 2.11 Å respectively. The catalytic residues are positioned within a large hydrophobic pocket similar to that of DAD2, a protein required for strigolactone response in Petunia hybrida. KAI2 possesses a second solvent-accessible pocket, adjacent to the active site cavity, which offers the possibility of allosteric regulation. The structure of KAI2 is consistent with its designation as a serine hydrolase, as well as previous data implicating the protein in karrikin and strigolactone signalling.

  4. Novel mTORC1 and 2 Signaling Pathways in Polycystic Kidney Disease (PKD)

    Science.gov (United States)

    2017-09-01

    30 27 32 2K (g) 0.35 0.53 0.36 * 2K/TBW (%) 1.1 2.0 1.2 * Cyst volume (%) 0 40.1 14.6 * No of cysts/kidney 0 5.3 0.8 * Heart wt (g) 0.3 0.15 0.17...some delay in the project. There were no other delays in the project Changes that had a significant impact on expenditures Delays in hiring PRA... Heart wt (g) 0.3 0.15 0.17 15 BUN (mg/dL) 24 29 25 SCr (mg/dL) 0.22 0.33 0.2* 2K/TBW (%) Two kidney/total body weight, *pɘ.05 vs. Pkd1

  5. The Emerging Role of Branched-Chain Amino Acids in Insulin Resistance and Metabolism

    Directory of Open Access Journals (Sweden)

    Mee-Sup Yoon

    2016-07-01

    Full Text Available Insulin is required for maintenance of glucose homeostasis. Despite the importance of insulin sensitivity to metabolic health, the mechanisms that induce insulin resistance remain unclear. Branched-chain amino acids (BCAAs belong to the essential amino acids, which are both direct and indirect nutrient signals. Even though BCAAs have been reported to improve metabolic health, an increased BCAA plasma level is associated with a high risk of metabolic disorder and future insulin resistance, or type 2 diabetes mellitus (T2DM. The activation of mammalian target of rapamycin complex 1 (mTORC1 by BCAAs has been suggested to cause insulin resistance. In addition, defective BCAA oxidative metabolism might occur in obesity, leading to a further accumulation of BCAAs and toxic intermediates. This review provides the current understanding of the mechanism of BCAA-induced mTORC1 activation, as well as the effect of mTOR activation on metabolic health in terms of insulin sensitivity. Furthermore, the effects of impaired BCAA metabolism will be discussed in detail.

  6. Critical analysis of the potential for therapeutic targeting of mammalian target of rapamycin (mTOR in gastric cancer

    Directory of Open Access Journals (Sweden)

    Inokuchi M

    2014-04-01

    Full Text Available Mikito Inokuchi,1 Keiji Kato,1 Kazuyuki Kojima,2 Kenichi Sugihara1 1Department of Surgical Oncology, 2Department of Minimally Invasive Surgery, Tokyo Medical and Dental University, Tokyo, Japan Abstract: Multidisciplinary treatment including chemotherapy has become the global standard of care for patients with metastatic gastric cancer (mGC; nonetheless, survival remains poor. Although many molecular-targeted therapies have been developed for various cancers, only anti-HER2 treatment has produced promising results in patients with mGC. Mammalian target of rapamycin (mTOR plays a key role in cell proliferation, antiapoptosis, and metastasis in signaling pathways from the tyrosine kinase receptor, and its activation has been demonstrated in gastric cancer (GC cells. This review discusses the clinical relevance of mTOR in GC and examines its potential as a therapeutic target in patients with mGC. Preclinical studies in animal models suggest that suppression of the mTOR pathway inhibits the proliferation of GC cells and delays tumor progression. The mTOR inhibitor everolimus has been evaluated as second- or third-line treatment in clinical trials. Adverse events were well tolerated although the effectiveness of everolimus alone was limited. Everolimus is now being evaluated in combination with chemotherapy in Phase III clinical studies in this subgroup of patients. Two Phase III studies include exploratory biomarker research designed to evaluate the predictive value of the expression or mutation of molecules related to the Akt/mTOR signaling pathway. These biomarker studies may lead to the realization of targeted therapy for selected patients with mGC in the future. Keywords: gastric cancer, mTOR, everolimus

  7. Insensitive detonator apparatus for initiating large failure diameter explosives

    Science.gov (United States)

    Perry, III, William Leroy

    2015-07-28

    A munition according to a preferred embodiment can include a detonator system having a detonator that is selectively coupled to a microwave source that functions to selectively prime, activate, initiate, and/or sensitize an insensitive explosive material for detonation. The preferred detonator can include an explosive cavity having a barrier within which an insensitive explosive material is disposed and a waveguide coupled to the explosive cavity. The preferred system can further include a microwave source coupled to the waveguide such that microwaves enter the explosive cavity and impinge on the insensitive explosive material to sensitize the explosive material for detonation. In use the preferred embodiments permit the deployment and use of munitions that are maintained in an insensitive state until the actual time of use, thereby substantially preventing unauthorized or unintended detonation thereof.

  8. Root Formation in Ethylene-Insensitive Plants1

    Science.gov (United States)

    Clark, David G.; Gubrium, Erika K.; Barrett, James E.; Nell, Terril A.; Klee, Harry J.

    1999-01-01

    Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia × hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more belowground root mass but fewer aboveground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated taproots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli. PMID:10482660

  9. Naringin attenuates granule cell dispersion in the dentate gyrus in a mouse model of temporal lobe epilepsy.

    Science.gov (United States)

    Jang, Hannah; Jeong, Kyoung Hoon; Kim, Sang Ryong

    2016-07-01

    Morphological abnormalities of the dentate gyrus (DG) are an important phenotype in the hippocampus of patients with temporal lobe epilepsy. We recently reported that naringin, a bioflavonoid in grapefruit and citrus fruits, exerts beneficial effects in the kainic acid (KA) mouse model of epilepsy. We found that naringin treatment reduced seizure activities and decreased autophagic stress and neuroinflammation in the hippocampus following in vivo lesion with KA. However, it remains unclear whether naringin may also attenuate seizure-induced morphological changes in the DG, collectively known as granule cell dispersion (GCD). To clarify whether naringin treatment reduces GCD, we evaluated the effects of intraperitoneal injection of naringin on GCD and activation of mammalian target of rapamycin complex 1 (mTORC1), an important regulator of GCD, following intrahippocampal injection of KA. Our results showed that naringin treatment significantly reduced KA-induced GCD and mTORC1 activation, which was confirmed by assessing the phosphorylated form of the mTORC1 substrate, 4E-BP1, in the hippocampus. These results suggest that naringin treatment may help prevent epilepsy-induced hippocampal injury by inhibiting mTORC1 activation and thereby reducing GCD in the hippocampus in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Survival benefit with proapoptotic molecular and pathologic responses from dual targeting of mammalian target of rapamycin and epidermal growth factor receptor in a preclinical model of pancreatic neuroendocrine carcinogenesis.

    Science.gov (United States)

    Chiu, Christopher W; Nozawa, Hiroaki; Hanahan, Douglas

    2010-10-10

    Pancreatic neuroendocrine tumors (PNETs), although rare, often metastasize, such that surgery, the only potentially curative therapy, is not possible. There is no effective systemic therapy for patients with advanced PNETs. Therefore, new strategies are needed. Toward that end, we investigated the potential benefit of dual therapeutic targeting of the epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) kinases, using a preclinical mouse model of PNET. Rapamycin and erlotinib, inhibitors of mTOR and EGFR, respectively, were used to treat RIP-Tag2 transgenic mice bearing advanced multifocal PNET. Tumor growth and survival were monitored, and tumors were surveyed for potential biomarkers of response to the therapeutics. Rapamycin monotherapy was notably efficacious, prolonging survival concomitant with tumor stasis (stable disease). However, the tumors developed resistance, as evidenced by eventual relapse to progressive tumor growth. Erlotinib monotherapy slowed tumor growth and elicited a marginal survival benefit. In combination, there was an unprecedented survival benefit in the face of this aggressive multifocal cancer and, in contrast to either monotherapy, the development of adaptive resistance was not apparent. Additionally, the antiapoptotic protein survivin was implicated as a biomarker of sensitivity and beneficial responses to the dual targeted therapy. Preclinical trials in a mouse model of endogenous PNET suggest that combined targeting of the mTOR and EGFR signaling pathways could have potential clinical benefit in treating PNET. These results have encouraged development of an ongoing phase II clinical trial aimed to evaluate the efficacy of this treatment regimen in human neuroendocrine tumors.

  11. Targeting Glutamatergic Signaling and the PI3 Kinase Pathway to Halt Melanoma Progression

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    Stephen A. Rosenberg

    2015-02-01

    Full Text Available Our group has previously reported that the majority of human melanomas (>60% express the metabotropic glutamate receptor 1 (GRM1 and that the glutamate release inhibitor riluzole, a drug currently used to treat amyotrophic lateral sclerosis, can induce apoptosis in GRM1-expressing melanoma cells. Our group previously reported that in vitro riluzole treatment reduces cell growth in three-dimensional (3D soft agar colony assays by 80% in cells with wildtype phosphoinositide 3-kinase (PI3K pathway activation. However, melanoma cell lines harboring constitutive activating mutations of the PI3K pathway (PTEN and NRAS mutations showed only a 35% to 40% decrease in colony formation in soft agar in the presence of riluzole. In this study, we have continued our preclinical studies of riluzole and its effect on melanoma cells alone and in combination with inhibitors of the PI3 kinase pathway: the AKT inhibitor, API-2, and the mammalian target of rapamycin (mTOR inhibitor, rapamycin. We modeled these combinatorial therapies on various melanoma cell lines in 3D and 2D systems and in vivo. Riluzole combined with mTOR inhibition is more effective at halting melanoma anchorage-independent growth and xenograft tumor progression than either agent alone. PI3K signaling changes associated with this combinatorial treatment shows that 3D (nanoculture modeling of cell signaling more closely resembles in vivo signaling than monolayer models. Riluzole combined with mTOR inhibition is effective at halting tumor cell progression independent of BRAF mutational status. This makes this combinatorial therapy a potentially viable alternative for metastatic melanoma patients who are BRAF WT and are therefore ineligible for vemurafenib therapy.

  12. 10-Hydroxy-2-decenoic Acid, the Major Lipid Component of Royal Jelly, Extends the Lifespan of Caenorhabditis elegans through Dietary Restriction and Target of Rapamycin Signaling

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    Yoko Honda

    2015-01-01

    Full Text Available Royal jelly (RJ produced by honeybees has been reported to possess diverse health-beneficial properties and has been implicated to have a function in longevity across diverse species as well as honeybees. 10-Hydroxy-2-decenoic acid (10-HDA, the major lipid component of RJ produced by honeybees, was previously shown to increase the lifespan of Caenorhabditis elegans. The objective of this study is to elucidate signaling pathways that are involved in the lifespan extension by 10-HDA. 10-HDA further extended the lifespan of the daf-2 mutants, which exhibit long lifespan through reducing insulin-like signaling (ILS, indicating that 10-HDA extended lifespan independently of ILS. On the other hand, 10-HDA did not extend the lifespan of the eat-2 mutants, which show long lifespan through dietary restriction caused by a food-intake defect. This finding indicates that 10-HDA extends lifespan through dietary restriction signaling. We further found that 10-HDA did not extend the lifespan of the long-lived mutants in daf-15, which encodes Raptor, a target of rapamycin (TOR components, indicating that 10-HDA shared some longevity control mechanisms with TOR signaling. Additionally, 10-HDA was found to confer tolerance against thermal and oxidative stress. 10-HDA increases longevity not through ILS but through dietary restriction and TOR signaling in C. elegans.

  13. 10-Hydroxy-2-decenoic Acid, the Major Lipid Component of Royal Jelly, Extends the Lifespan of Caenorhabditis elegans through Dietary Restriction and Target of Rapamycin Signaling.

    Science.gov (United States)

    Honda, Yoko; Araki, Yoko; Hata, Taketoshi; Ichihara, Kenji; Ito, Masafumi; Tanaka, Masashi; Honda, Shuji

    2015-01-01

    Royal jelly (RJ) produced by honeybees has been reported to possess diverse health-beneficial properties and has been implicated to have a function in longevity across diverse species as well as honeybees. 10-Hydroxy-2-decenoic acid (10-HDA), the major lipid component of RJ produced by honeybees, was previously shown to increase the lifespan of Caenorhabditis elegans. The objective of this study is to elucidate signaling pathways that are involved in the lifespan extension by 10-HDA. 10-HDA further extended the lifespan of the daf-2 mutants, which exhibit long lifespan through reducing insulin-like signaling (ILS), indicating that 10-HDA extended lifespan independently of ILS. On the other hand, 10-HDA did not extend the lifespan of the eat-2 mutants, which show long lifespan through dietary restriction caused by a food-intake defect. This finding indicates that 10-HDA extends lifespan through dietary restriction signaling. We further found that 10-HDA did not extend the lifespan of the long-lived mutants in daf-15, which encodes Raptor, a target of rapamycin (TOR) components, indicating that 10-HDA shared some longevity control mechanisms with TOR signaling. Additionally, 10-HDA was found to confer tolerance against thermal and oxidative stress. 10-HDA increases longevity not through ILS but through dietary restriction and TOR signaling in C. elegans.

  14. The impact of photoperiod insensitive Ppd-1a mutations on the photoperiod pathway across the three genomes of hexaploid wheat (Triticum aestivum).

    Science.gov (United States)

    Shaw, Lindsay M; Turner, Adrian S; Laurie, David A

    2012-07-01

    Flowering time is a trait that has been extensively altered during wheat domestication, enabling it to be highly productive in diverse environments and providing a rich source of variation for studying adaptation mechanisms. Hexaploid wheat is ancestrally a long-day plant, but many environments require varieties with photoperiod insensitivity (PI) that can flower in short days. PI results from mutations in the Ppd-1 gene on the A, B or D genomes, with individual mutations conferring different degrees of earliness. The basis of this is poorly understood. Using a common genetic background, the effects of A, B and D genome PI mutations on genes of the circadian clock and photoperiod pathway were studied using genome-specific expression assays. Ppd-1 PI mutations did not affect the clock or immediate clock outputs, but affected TaCO1 and TaFT1, with a reduction in TaCO1 expression as TaFT1 expression increased. Therefore, although Ppd-1 is related to PRR genes of the Arabidopsis circadian clock, Ppd-1 affects flowering by an alternative route, most likely by upregulating TaFT1 with a feedback effect that reduces TaCO1 expression. Individual genes in the circadian clock and photoperiod pathway were predominantly expressed from one genome, and there was no genome specificity in Ppd-1 action. Lines combining PI mutations on two or three genomes had enhanced earliness with higher levels, but not earlier induction, of TaFT1, showing that there is a direct quantitative relationship between Ppd-1 mutations, TaFT1 expression and flowering. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  15. Meta-Analysis of 29 Experiments Evaluating the Effects of Rapamycin on Life Span in the Laboratory Mouse.

    Science.gov (United States)

    Swindell, William R

    2017-08-01

    Rapamycin has favorable effects on aging in mice and may eventually be applied to encourage "healthy aging" in humans. This study analyzed raw data from 29 survival studies of rapamycin- and control-treated mice, with the goals of estimating summary statistics and identifying factors associated with effect size heterogeneity. Meta-analysis demonstrated significant heterogeneity across studies, with hazard ratio (HR) estimates ranging from 0.22 (95% confidence interval [CI]: 0.06-0.82) to 0.92 (95% CI: 0.65-1.28). Sex was the major factor accounting for effect size variation, and mortality was decreased more in females (HR = 0.41; 95% CI: 0.35-0.48) as compared with males (HR = 0.63; 95% CI: 0.55-0.71). Rapamycin effects were also genotype dependent, however, with stronger survivorship increases in hybrid mice (14.4%; 95% CI: 12.5-16.3%) relative to pure inbred strains (8.8%; 95% CI: 6.2-11.6%). Number needed to treat was applied as an effect size metric, which consistently identified early senescence as the age of peak treatment benefit. These results provide synthesis of existing data to support the potential translation of findings from mouse to primate species. Because rapamycin's effect on survival depends on sex and genotype, further work is justified to understand how these factors shape treatment response. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei.

    Science.gov (United States)

    Turrens, J F; Bickar, D; Lehninger, A L

    1986-06-01

    The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase.

  17. Amino acids and autophagy: cross-talk and co-operation to control cellular homeostasis.

    Science.gov (United States)

    Carroll, Bernadette; Korolchuk, Viktor I; Sarkar, Sovan

    2015-10-01

    Maintenance of amino acid homeostasis is important for healthy cellular function, metabolism and growth. Intracellular amino acid concentrations are dynamic; the high demand for protein synthesis must be met with constant dietary intake, followed by cellular influx, utilization and recycling of nutrients. Autophagy is a catabolic process via which superfluous or damaged proteins and organelles are delivered to the lysosome and degraded to release free amino acids into the cytoplasm. Furthermore, autophagy is specifically activated in response to amino acid starvation via two key signaling cascades: the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and the general control nonderepressible 2 (GCN2) pathways. These pathways are key regulators of the integration between anabolic (amino acid depleting) and catabolic (such as autophagy which is amino acid replenishing) processes to ensure intracellular amino acid homeostasis. Here, we discuss the key roles that amino acids, along with energy (ATP, glucose) and oxygen, are playing in cellular growth and proliferation. We further explore how sophisticated methods are employed by cells to sense intracellular amino acid concentrations, how amino acids can act as a switch to dictate the temporal and spatial activation of anabolic and catabolic processes and how autophagy contributes to the replenishment of free amino acids, all to ensure cell survival. Relevance of these molecular processes to cellular and organismal physiology and pathology is also discussed.

  18. Mediobasal hypothalamic overexpression of DEPTOR protects against high-fat diet-induced obesity

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    Alexandre Caron

    2016-02-01

    Full Text Available Background/Objective: The mechanistic target of rapamycin (mTOR is a serine–threonine kinase that functions into distinct protein complexes (mTORC1 and mTORC2 that regulate energy homeostasis. DEP-domain containing mTOR-interacting protein (DEPTOR is part of these complexes and is known to dampen mTORC1 function, consequently reducing mTORC1 negative feedbacks and promoting insulin signaling and Akt/PKB activation in several models. Recently, we observed that DEPTOR is expressed in several structures of the brain including the mediobasal hypothalamus (MBH, a region that regulates energy balance. Whether DEPTOR in the MBH plays a functional role in regulating energy balance and hypothalamic insulin signaling has never been tested. Methods: We have generated a novel conditional transgenic mouse model based on the Cre-LoxP system allowing targeted overexpression of DEPTOR. Mice overexpressing DEPTOR in the MBH were subjected to a metabolic phenotyping and MBH insulin signaling was evaluated. Results: We first report that systemic (brain and periphery overexpression of DEPTOR prevents high-fat diet-induced obesity, improves glucose metabolism and protects against hepatic steatosis. These phenotypes were associated with a reduction in food intake and feed efficiency and an elevation in oxygen consumption. Strikingly, specific overexpression of DEPTOR in the MBH completely recapitulated these phenotypes. DEPTOR overexpression was associated with an increase in hypothalamic insulin signaling, as illustrated by elevated Akt/PKB activation. Conclusion: Altogether, these results support a role for MBH DEPTOR in the regulation of energy balance and metabolism. Keywords: mTOR, DEPTOR, Hypothalamus, Energy balance, Glucose metabolism

  19. The evolution of the TOR pathway and its role in cancer.

    Science.gov (United States)

    Beauchamp, E M; Platanias, L C

    2013-08-22

    The target of rapamycin (TOR) pathway is highly conserved among eukaryotes and has evolved to couple nutrient sensing to cellular growth. TOR is found in two distinct signaling complexes in cells, TOR complex 1 (TORC1) and TOR complex 2 (TORC2). These complexes are differentially regulated and act as effectors for the generation of signals that drive diverse cellular processes such as growth, proliferation, protein synthesis, rearrangement of the cytoskeleton, autophagy, metabolism and survival. Mammalian TOR (mTOR) is very important for development in embryos, while in adult organisms it is linked to aging and lifespan effects. In humans, the mTOR pathway is implicated in the tumorigenesis of multiple cancer types and its deregulation is associated with familial cancer syndromes. Because of its high biological relevance, different therapeutic strategies have been developed to target this signaling cascade, resulting in the emergence of unique pharmacological inhibitors that are either already approved for use in clinical oncology or currently under preclinical or clinical development. Multimodal treatment strategies that simultaneously target multiple nodes of the pathway and/or negative feedback regulatory loops may ultimately provide the best therapeutic advantage in targeting this pathway for the treatment of malignancies.

  20. A randomized controlled trial to establish effects of short-term rapamycin treatment in 24 middle-aged companion dogs.

    Science.gov (United States)

    Urfer, Silvan R; Kaeberlein, Tammi L; Mailheau, Susan; Bergman, Philip J; Creevy, Kate E; Promislow, Daniel E L; Kaeberlein, Matt

    2017-04-01

    Age is the single greatest risk factor for most causes of morbidity and mortality in humans and their companion animals. As opposed to other model organisms used to study aging, dogs share the human environment, are subject to similar risk factors, receive comparable medical care, and develop many of the same age-related diseases humans do. In this study, 24 middle-aged healthy dogs received either placebo or a non-immunosuppressive dose of rapamycin for 10 weeks. All dogs received clinical and hematological exams before, during, and after the trial and echocardiography before and after the trial. Our results showed no clinical side effects in the rapamycin-treated group compared to dogs receiving the placebo. Echocardiography suggested improvement in both diastolic and systolic age-related measures of heart function (E/A ratio, fractional shortening, and ejection fraction) in the rapamycin-treated dogs. Hematological values remained within the normal range for all parameters studied; however, the mean corpuscular volume (MCV) was decreased in rapamycin-treated dogs. Based on these results, we will test rapamycin on a larger dog cohort for a longer period of time in order to validate its effects on cardiac function and to determine whether it can significantly improve healthspan and reduce mortality in companion dogs.

  1. Loss of Folliculin Disrupts Hematopoietic Stem Cell Quiescence and Homeostasis Resulting in Bone Marrow Failure.

    Science.gov (United States)

    Baba, Masaya; Toyama, Hirofumi; Sun, Lei; Takubo, Keiyo; Suh, Hyung-Chan; Hasumi, Hisashi; Nakamura-Ishizu, Ayako; Hasumi, Yukiko; Klarmann, Kimberly D; Nakagata, Naomi; Schmidt, Laura S; Linehan, W Marston; Suda, Toshio; Keller, Jonathan R

    2016-04-01

    Folliculin (FLCN) is an autosomal dominant tumor suppressor gene that modulates diverse signaling pathways required for growth, proliferation, metabolism, survival, motility, and adhesion. FLCN is an essential protein required for murine embryonic development, embryonic stem cell (ESC) commitment, and Drosophila germline stem cell maintenance, suggesting that Flcn may be required for adult stem cell homeostasis. Conditional inactivation of Flcn in adult hematopoietic stem/progenitor cells (HSPCs) drives hematopoietic stem cells (HSC) into proliferative exhaustion resulting in the rapid depletion of HSPC, loss of all hematopoietic cell lineages, acute bone marrow (BM) failure, and mortality after 40 days. HSC that lack Flcn fail to reconstitute the hematopoietic compartment in recipient mice, demonstrating a cell-autonomous requirement for Flcn in HSC maintenance. BM cells showed increased phosphorylation of Akt and mTorc1, and extramedullary hematopoiesis was significantly reduced by treating mice with rapamycin in vivo, suggesting that the mTorc1 pathway was activated by loss of Flcn expression in hematopoietic cells in vivo. Tfe3 was activated and preferentially localized to the nucleus of Flcn knockout (KO) HSPCs. Tfe3 overexpression in HSPCs impaired long-term hematopoietic reconstitution in vivo, recapitulating the Flcn KO phenotype, and supporting the notion that abnormal activation of Tfe3 contributes to the Flcn KO phenotype. Flcn KO mice develop an acute histiocytic hyperplasia in multiple organs, suggesting a novel function for Flcn in macrophage development. Thus, Flcn is intrinsically required to maintain adult HSC quiescence and homeostasis, and Flcn loss leads to BM failure and mortality in mice. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  2. Molecular basis of androgen insensitivity

    NARCIS (Netherlands)

    Brinkmann, A.; Jenster, G.; Ris-Stalpers, C.; van der Korput, H.; Brüggenwirth, H.; Boehmer, A.; Trapman, J.

    1996-01-01

    Male sexual differentiation and development proceed under direct control of androgens. Androgen action is mediated by the intracellular androgen receptor, which belongs to the superfamily of ligand-dependent transcription factors. In the X-linked androgen insensitivity syndrome, defects in the

  3. Inflammatory stress increases hepatic CD36 translational efficiency via activation of the mTOR signalling pathway.

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    Chuan Wang

    Full Text Available Inflammatory stress is an independent risk factor for the development of non-alcoholic fatty liver disease (NAFLD. Although CD36 is known to facilitate long-chain fatty acid uptake and contributes to NAFLD progression, the mechanisms that link inflammatory stress to hepatic CD36 expression and steatosis remain unclear. As the mammalian target of rapamycin (mTOR signalling pathway is involved in CD36 translational activation, this study was undertaken to investigate whether inflammatory stress enhances hepatic CD36 expression via mTOR signalling pathway and the underlying mechanisms. To induce inflammatory stress, we used tumour necrosis factor alpha (TNF-α and interleukin-6 (IL-6 stimulation of the human hepatoblastoma HepG2 cells in vitro and casein injection in C57BL/6J mice in vivo. The data showed that inflammatory stress increased hepatic CD36 protein levels but had no effect on mRNA expression. A protein degradation assay revealed that CD36 protein stability was not different between HepG2 cells treated with or without TNF-α or IL-6. A polysomal analysis indicated that CD36 translational efficiency was significantly increased by inflammatory stress. Additionally, inflammatory stress enhanced the phosphorylation of mTOR and its downstream translational regulators including p70S6K, 4E-BP1 and eIF4E. Rapamycin, an mTOR-specific inhibitor, reduced the phosphorylation of mTOR signalling pathway and decreased the CD36 translational efficiency and protein level even under inflammatory stress resulting in the alleviation of inflammatory stress-induced hepatic lipid accumulation. This study demonstrates that the activation of the mTOR signalling pathway increases hepatic CD36 translational efficiency, resulting in increased CD36 protein expression under inflammatory stress.

  4. Evaluative Conditioning is Insensitive to Blocking

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    Tom Beckers

    2009-01-01

    Full Text Available Evaluative conditioning has been claimed to have a number of functional characteristics that set it apart from other forms of associative learning in humans, such as insensitivity to extinction and contingency, independence of contingency awareness, and insensitivity to modulation. Despite its potential theoretical importance, until now few data are available concerning the susceptibility of evaluative conditioning to cue competition effects such as blocking. In the present study, we assessed the susceptibility of acquired preferences and evaluations to blocking in a candy game. Results suggest that evaluative conditioning is not susceptible to blocking. We discuss this observation in the light of theoretical accounts of evaluative conditioning and associative learning in humans.

  5. Synergistic Effect of Rapamycin and Metformin Against Age-Dependent Oxidative Stress in Rat Erythrocytes.

    Science.gov (United States)

    Singh, Abhishek Kumar; Garg, Geetika; Singh, Sandeep; Rizvi, Syed Ibrahim

    2017-10-01

    Erythrocytes are particularly vulnerable toward age-dependent oxidative stress-mediated damage. Caloric restriction mimetics (CRMs) may provide a novel strategy for the maintenance of redox balance as well as effective treatment of age-associated diseases. Herein, we have investigated the beneficial effect of cotreatment with CRM-candidate drugs, rapamycin (an immunosuppressant drug and inhibitor of mammalian target of rapamycin) and metformin (an antidiabetic biguanide and activator of adenosine monophosphate kinase), against aging-induced oxidative stress in erythrocytes and plasma of aging rats. Male Wistar rats of age 4 (young) and 24 months (old) were coexposed to rapamycin (0.5 mg/kg body weight [b.w.]) and metformin (300 mg/kg b.w.), and data were compared with the response of rats receiving an independent exposure to these chemicals at similar doses. The exposure of individual candidate drugs significantly reversed the age-dependent alterations in the endpoints associated with oxidative stress such as reactive oxygen species, ferric reducing ability of plasma, malondialdehyde, reduced glutathione, plasma membrane redox system, plasma protein carbonyl, and acetyl cholinesterase in erythrocytes and plasma of aging rats. However, the cotreatment with rapamycin and metformin showed a significant augmented effect compared with individual drug interventions on reversal of these age-dependent biomarkers of oxidative stress, suggesting a synergistic response. Thus, the findings open up further possibilities for the design of new combinatorial therapies to prevent oxidative stress- and age-associated health problems.

  6. Repeated functional convergent effects of NaV1.7 on acid insensitivity in hibernating mammals.

    Science.gov (United States)

    Liu, Zhen; Wang, Wei; Zhang, Tong-Zuo; Li, Gong-Hua; He, Kai; Huang, Jing-Fei; Jiang, Xue-Long; Murphy, Robert W; Shi, Peng

    2014-02-07

    Hibernating mammals need to be insensitive to acid in order to cope with conditions of high CO2; however, the molecular basis of acid tolerance remains largely unknown. The African naked mole-rat (Heterocephalus glaber) and hibernating mammals share similar environments and physiological features. In the naked mole-rat, acid insensitivity has been shown to be conferred by the functional motif of the sodium ion channel NaV1.7. There is now an opportunity to evaluate acid insensitivity in other taxa. In this study, we tested for functional convergence of NaV1.7 in 71 species of mammals, including 22 species that hibernate. Our analyses revealed a functional convergence of amino acid sequences, which occurred at least six times independently in mammals that hibernate. Evolutionary analyses determined that the convergence results from both parallel and divergent evolution of residues in the functional motif. Our findings not only identify the functional molecules responsible for acid insensitivity in hibernating mammals, but also open new avenues to elucidate the molecular underpinnings of acid insensitivity in mammals.

  7. Phosphoproteomic Analysis Identifies Signaling Pathways Regulated by Curcumin in Human Colon Cancer Cells.

    Science.gov (United States)

    Sato, Tatsuhiro; Higuchi, Yutaka; Shibagaki, Yoshio; Hattori, Seisuke

    2017-09-01

    Curcumin, a major polyphenol of the spice turmeric, acts as a potent chemopreventive and chemotherapeutic agent in several cancer types, including colon cancer. Although various proteins have been shown to be affected by curcumin, how curcumin exerts its anticancer activity is not fully understood. Phosphoproteomic analyses were performed using SW480 and SW620 human colon cancer cells to identify curcumin-affected signaling pathways. Curcumin inhibited the growth of the two cell lines in a dose-dependent manner. Thirty-nine curcumin-regulated phosphoproteins were identified, five of which are involved in cancer signaling pathways. Detailed analyses revealed that the mTORC1 and p53 signaling pathways are main targets of curcumin. Our results provide insight into the molecular mechanisms of the anticancer activities of curcumin and future molecular targets for its clinical application. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  8. Association of breast cancer risk and the mTOR pathway in women of African ancestry in 'The Root' Consortium.

    Science.gov (United States)

    Wang, Shengfeng; Huo, Dezheng; Ogundiran, Temidayo O; Ojengbede, Oladosu; Zheng, Wei; Nathanson, Katherine L; Nemesure, Barbara; Ambs, Stefan; Olopade, Olufunmilayo I; Zheng, Yonglan

    2017-08-01

    Functional studies have elucidated the role of the mammalian target of rapamycin (mTOR) pathway in breast carcinogenesis, but to date, there is a paucity of data on its contribution to breast cancer risk in women of African ancestry. We examined 47628 SNPs in 61 mTOR pathway genes in the genome wide association study of breast cancer in the African Diaspora study (The Root consortium), which included 3686 participants (1657 cases). Pathway- and gene-level analyses were conducted using the adaptive rank truncated product (ARTP) test for 10994 SNPs that were not highly correlated (r2 studies of breast cancer in the African Diaspora. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  9. Function of SREBP1 in the Milk Fat Synthesis of Dairy Cow Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Nan Li

    2014-09-01

    Full Text Available Sterol regulatory element-binding proteins (SREBPs belong to a family of nuclear transcription factors. The question of which is the most important positive regulator in milk fat synthesis in dairy cow mammary epithelial cells (DCMECs between SREBPs or other nuclear transcription factors, such as peroxisome proliferator-activated receptor γ (PPARγ, remains a controversial one. Recent studies have found that mTORC1 (the mammalian target of rapamycin C1 regulates SREBP1 to promote fat synthesis. Thus far, however, the interaction between the SREBP1 and mTOR (the mammalian target of rapamycin pathways in the regulation of milk fat synthesis remains poorly understood. This study aimed to identify the function of SREBP1 in milk fat synthesis and to characterize the relationship between SREBP1 and mTOR in DCMECs. The effects of SREBP1 overexpression and gene silencing on milk fat synthesis and the effects of stearic acid and serum on SREBP1 expression in the upregulation of milk fat synthesis were investigated in DCMECs using immunostaining, Western blotting, real-time quantitative PCR, lipid droplet staining, and detection kits for triglyceride content. SREBP1 was found to be a positive regulator of milk fat synthesis and was shown to be regulated by stearic acid and serum. These findings indicate that SREBP1 is the key positive regulator in milk fat synthesis.

  10. Aging Reduces the Activation of the mTORC1 Pathway after Resistance Exercise and Protein Intake in Human Skeletal Muscle: Potential Role of REDD1 and Impaired Anabolic Sensitivity

    Directory of Open Access Journals (Sweden)

    Marc Francaux

    2016-01-01

    Full Text Available This study was designed to better understand the molecular mechanisms involved in the anabolic resistance observed in elderly people. Nine young (22 ± 0.1 years and 10 older (69 ± 1.7 years volunteers performed a one-leg extension exercise consisting of 10 × 10 repetitions at 70% of their 3-RM, immediately after which they ingested 30 g of whey protein. Muscle biopsies were taken from the vastus lateralis at rest in the fasted state and 30 min after protein ingestion in the non-exercised (Pro and exercised (Pro+ex legs. Plasma insulin levels were determined at the same time points. No age difference was measured in fasting insulin levels but the older subjects had a 50% higher concentration than the young subjects in the fed state (p < 0.05. While no difference was observed in the fasted state, in response to exercise and protein ingestion, the phosphorylation state of PKB (p < 0.05 in Pro and Pro+ex and S6K1 (p = 0.059 in Pro; p = 0.066 in Pro+ex was lower in the older subjects compared with the young subjects. After Pro+ex, REDD1 expression tended to be higher (p = 0.087 in the older group while AMPK phosphorylation was not modified by any condition. In conclusion, we show that the activation of the mTORC1 pathway is reduced in skeletal muscle of older subjects after resistance exercise and protein ingestion compared with young subjects, which could be partially due to an increased expression of REDD1 and an impaired anabolic sensitivity.

  11. Regulation of cardiac expression of the diabetic marker microRNA miR-29.

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    Nicholas Arnold

    Full Text Available Diabetes mellitus (DM is an independent risk factor for heart disease and its underlying mechanisms are unclear. Increased expression of diabetic marker miR-29 family miRNAs (miR-29a, b and c that suppress the pro-survival protein Myeloid Cell Leukemia 1(MCL-1 is reported in pancreatic β-cells in Type 1 DM. Whether an up-regulation of miR-29 family miRNAs and suppression of MCL-1 (dysregulation of miR-29-MCL-1 axis occurs in diabetic heart is not known. This study tested the hypothesis that insulin regulates cardiac miR-29-MCL-1 axis and its dysregulation correlates with DM progression. In vitro studies with mouse cardiomyocyte HL-1 cells showed that insulin suppressed the expression of miR-29a, b and c and increased MCL-1 mRNA. Conversely, Rapamycin (Rap, a drug implicated in the new onset DM, increased the expression of miR-29a, b and c and suppressed MCL-1 and this effect was reversed by transfection with miR-29 inhibitors. Rap inhibited mammalian target of rapamycin complex 1 (mTORC1 signaling in HL-1 cells. Moreover, inhibition of either mTORC1 substrate S6K1 by PF-4708671, or eIF4E-induced translation by 4E1RCat suppressed MCL-1. We used Zucker diabetic fatty (ZDF rat, a rodent model for DM, to test whether dysregulation of cardiac miR-29-MCL-1 axis correlates with DM progression. 11-week old ZDF rats exhibited significantly increased body weight, plasma glucose, insulin, cholesterol, triglycerides, body fat, heart weight, and decreased lean muscle mass compared to age-matched lean rats. Rap treatment (1.2 mg/kg/day, from 9-weeks to 15-weeks significantly reduced plasma insulin, body weight and heart weight, and severely dysregulated cardiac miR-29-MCL1 axis in ZDF rats. Importantly, dysregulation of cardiac miR-29-MCL-1 axis in ZDF rat heart correlated with cardiac structural damage (disorganization or loss of myofibril bundles. We conclude that insulin and mTORC1 regulate cardiac miR-29-MCL-1 axis and its dysregulation caused by reduced

  12. The effect of G protein-coupled receptor kinase 2 (GRK2) on lactation and on proliferation of mammary epithelial cells from dairy cows.

    Science.gov (United States)

    Hou, Xiaoming; Hu, Hongliu; Lin, Ye; Qu, Bo; Gao, Xuejun; Li, Qingzhang

    2016-07-01

    Milk protein is an important component of milk and a nutritional source for human consumption. To better understand the molecular events underlying synthesis of milk proteins, the global gene expression patterns in mammary glands of dairy cow with high-quality milk (>3% milk protein; >3.5% milk fat) and low-quality milk (milk protein; milk fat) were examined via digital gene expression study. A total of 139 upregulated and 66 downregulated genes were detected in the mammary tissues of lactating cows with high-quality milk compared with the tissues of cows with low-quality milk. A pathway enrichment study of these genes revealed that the top 5 pathways that were differentially affected in the tissues of cows with high- versus low-quality milk involved metabolic pathways, cancer, cytokine-cytokine receptor interactions, regulation of the actin cytoskeleton, and insulin signaling. We also found that the G protein-coupled receptor kinase 2 (GRK2) was one of the most highly upregulated genes in lactating mammary tissue with low-quality milk compared with tissue with high-quality milk. The knockdown of GRK2 in cultured bovine mammary epithelial cells enhanced CSN2 expression and activated signaling molecules related to translation, including protein kinase B, mammalian target of rapamycin, and p70 ribosomal protein S6 kinase 1 (S6K1), whereas overexpression of GRK2 had the opposite effects. However, expression of genes involved in the mitogen-activated protein kinase pathway was positively regulated by GRK2. Therefore, GRK2 seems to act as a negative mediator of milk-protein synthesis via the protein kinase B-mammalian target of rapamycin signaling axis. Furthermore, GRK2 may negatively control milk-protein synthesis by activating the mitogen-activated protein kinase pathway in dairy cow mammary epithelial cells. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  13. Prognostic significance and therapeutic potential of the activation of anaplastic lymphoma kinase/protein kinase B/mammalian target of rapamycin signaling pathway in anaplastic large cell lymphoma

    International Nuclear Information System (INIS)

    Gao, Ju; Yin, Minzhi; Zhu, Yiping; Gu, Ling; Zhang, Yanle; Li, Qiang; Jia, Cangsong; Ma, Zhigui

    2013-01-01

    Activation of the protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway has been demonstrated to be involved in nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-mediated tumorigenesis in anaplastic large cell lymphoma (ALCL) and correlated with unfavorable outcome in certain types of other cancers. However, the prognostic value of AKT/mTOR activation in ALCL remains to be fully elucidated. In the present study, we aim to address this question from a clinical perspective by comparing the expressions of the AKT/mTOR signaling molecules in ALCL patients and exploring the therapeutic significance of targeting the AKT/mTOR pathway in ALCL. A cohort of 103 patients with ALCL was enrolled in the study. Expression of ALK fusion proteins and the AKT/mTOR signaling phosphoproteins was studied by immunohistochemical (IHC) staining. The pathogenic role of ALK fusion proteins and the therapeutic significance of targeting the ATK/mTOR signaling pathway were further investigated in vitro study with an ALK + ALCL cell line and the NPM-ALK transformed BaF3 cells. ALK expression was detected in 60% of ALCLs, of which 79% exhibited the presence of NPM-ALK, whereas the remaining 21% expressed variant-ALK fusions. Phosphorylation of AKT, mTOR, 4E-binding protein-1 (4E-BP1), and 70 kDa ribosomal protein S6 kinase polypeptide 1 (p70S6K1) was detected in 76%, 80%, 91%, and 93% of ALCL patients, respectively. Both phospho-AKT (p-AKT) and p-mTOR were correlated to ALK expression, and p-mTOR was closely correlated to p-AKT. Both p-4E-BP1 and p-p70S6K1 were correlated to p-mTOR, but were not correlated to the expression of ALK and p-AKT. Clinically, ALK + ALCL occurred more commonly in younger patients, and ALK + ALCL patients had a much better prognosis than ALK-ALCL cases. However, expression of p-AKT, p-mTOR, p-4E-BP1, or p-p70S6K1 did not have an impact on the clinical outcome. Overexpression of NPM-ALK in a nonmalignant murine pro-B lymphoid cell line, BaF3, induced the

  14. Characterization of the metabolic phenotype of rapamycin-treated CD8+ T cells with augmented ability to generate long-lasting memory cells.

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    Shan He

    Full Text Available BACKGROUND: Cellular metabolism plays a critical role in regulating T cell responses and the development of memory T cells with long-term protections. However, the metabolic phenotype of antigen-activated T cells that are responsible for the generation of long-lived memory cells has not been characterized. DESIGN AND METHODS: Using lymphocytic choriomeningitis virus (LCMV peptide gp33-specific CD8(+ T cells derived from T cell receptor transgenic mice, we characterized the metabolic phenotype of proliferating T cells that were activated and expanded in vitro in the presence or absence of rapamycin, and determined the capability of these rapamycin-treated T cells to generate long-lived memory cells in vivo. RESULTS: Antigen-activated CD8(+ T cells treated with rapamycin gave rise to 5-fold more long-lived memory T cells in vivo than untreated control T cells. In contrast to that control T cells only increased glycolysis, rapamycin-treated T cells upregulated both glycolysis and oxidative phosphorylation (OXPHOS. These rapamycin-treated T cells had greater ability than control T cells to survive withdrawal of either glucose or growth factors. Inhibition of OXPHOS by oligomycin significantly reduced the ability of rapamycin-treated T cells to survive growth factor withdrawal. This effect of OXPHOS inhibition was accompanied with mitochondrial hyperpolarization and elevation of reactive oxygen species that are known to be toxic to cells. CONCLUSIONS: Our findings indicate that these rapamycin-treated T cells may represent a unique cell model for identifying nutrients and signals critical to regulating metabolism in both effector and memory T cells, and for the development of new methods to improve the efficacy of adoptive T cell cancer therapy.

  15. Searching for the variables that control human rule-governed "insensitivity".

    Science.gov (United States)

    Fox, Adam E; Kyonka, Elizabeth G E

    2017-09-01

    Verbal rules or instructions often exert obvious and meaningful control over human behavior. Sometimes instructions benefit the individual by enabling faster acquisition of a skill or by obviating an aversive consequence. However, research has also suggested a clear disadvantage: "insensitivity" to changing underlying contingencies. The two experiments described here investigated the variables that control initial rule-following behavior and rule-following insensitivity. When the initial rule was inaccurate, behavior was consistent with the rule for approximately half of participants and all participants' behavior was mostly insensitive to changing contingencies. When the initial rule was accurate, behavior of all participants was consistent with it and behavior for nearly all participants was insensitive to changes in underlying contingencies. These findings have implications for how best to establish and maintain rule-following behavior in applied settings when deviant behavior would be more reinforcing to the individual. © 2017 Society for the Experimental Analysis of Behavior.

  16. Mammalian target of rapamycin inhibition in polycystic kidney disease: From bench to bedside

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    Hyun-Jung Kim

    2012-09-01

    Full Text Available Autosomal dominant polycystic kidney disease (ADPKD is the most common life-threatening hereditary disease in the USA resulting in chronic kidney disease and the need for dialysis and transplantation. Approximately 85% of cases of ADPKD are caused by a mutation in the Pkd1 gene that encodes polycystin-1, a large membrane receptor. The Pkd1 gene mutation results in abnormal proliferation in tubular epithelial cells, which plays a crucial role in cyst development and/or growth in PKD. Activation of the proliferative mammalian target of rapamycin (mTOR signaling pathway has been demonstrated in polycystic kidneys from rodents and humans. mTOR inhibition with sirolimus or everolimus decreases cysts in most animal models of PKD including Pkd1 and Pkd2 gene deficient orthologous models of human disease. On the basis of animal studies, human studies were undertaken. Two large randomized clinical trials published in the New England Journal of Medicine of everolimus or sirolimus in ADPKD patients were very unimpressive and associated with a high side-effect profile. Possible reasons for the unimpressive nature of the human studies include their short duration, the high drop-out rate, suboptimal dosing, lack of randomization of “fast” and “slow progressors” and the lack of correlation between kidney size and kidney function in ADPKD. The future of mTOR inhibition in ADPKD is discussed.

  17. Effects of Hd2 in the presence of the photoperiod-insensitive functional allele of Hd1 in rice

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    Zhen-Hua Zhang

    2016-11-01

    Full Text Available The role of photoperiod sensitivity (PS of flowering genes have become well recognized in rice, whereas little attention has been drawn to the non-PS component of these genes, especially to their influence on gene-by-gene interactions. Rice populations in which the photoperiod-sensitive allele at Hd1 has become insensitive to photoperiod but continued to affect heading date (HD were used in this study to fine-map a quantitative trait locus (QTL for HD and analyze its genetic relationship to Hd1. The QTL was delimitated to a 96.3-kb region on the distal end of the long arm of chromosome 7. Sequence comparison revealed that this QTL is identical to Hd2. In the near-isogenic line (NIL populations analyzed, Hd1 and Hd2 were shown to be photoperiod insensitive and have pleiotropic effects for HD, plant height and yield traits. The two genes were found to largely act additively in regulating HD and yield traits. The results indicate that non-PS components of flowering genes involved in photoperiod response play an important role in controlling flowering time and grain yield in rice, which should allow breeders to better manipulate pleiotropic genes for balancing adaptability and high-yielding accumulation.

  18. Inhibition of Cholesterol Synthesis in HepG2 Cells by GINST-Decreasing HMG-CoA Reductase Expression Via AMP-Activated Protein Kinase.

    Science.gov (United States)

    Han, Joon-Seung; Sung, Jong Hwan; Lee, Seung Kwon

    2017-11-01

    GINST, a hydrolyzed ginseng extract, has been reported to have antidiabetic effects and to reduce hyperglycemia and hyperlipidemia. Hypercholesterolemia is caused by diet or genetic factors and can lead to atherosclerosis and coronary heart disease. Thus, the purpose of this study is to determine whether GINST and the ginsenoside metabolite, IH-901 (compound K), reduce cholesterol synthesis in HepG2 cells and the signal transduction pathways involved. Concentrations of cholesterol were measured by using an enzymatic method. Expression levels of sterol regulatory element-binding protein 2 (SREBP2), HMG-CoA reductase (HMGCR), peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer-binding proteins α (C/EBPα), GAPDH, and phosphorylation of AMP-activated protein kinase α (AMPKα), protein kinase B (PKB, also known as Akt), and mechanistic target of rapamycin complex 1 (mTORC1) were measured using western blot. Total cholesterol concentration decreased after GINST treatment for 24 and 48 h. Expression of HMGCR decreased more with GINST than with the inhibitors, U18666A and atorvastatin, after 48 h in a dose-dependent manner. Phosphorylation of AMPKα increased 2.5x by GINST after 360 min of treatment, and phosphorylation of Akt decreased after 120 and 360 min. We separated compound K from GINST extracts flash chromatography. Compound K decreased cholesterol synthesis in HepG2 cells at 24 and 48 h. Therefore, we conclude that GINST inhibits cholesterol synthesis in HepG2 cells by decreasing HMGCR expression via AMPKα activation. GINST, a hydrolyzed ginseng extract, can inhibit cholesterol synthesis in liver cells via activation of AMPKα. IH-901 (compound K), which is the main component with bioactivity in GINST, also has anticholesterol effects. Thus, we suggest that GINST can be used to reduce hypercholesterolemia. © 2017 Institute of Food Technologists®.

  19. Rapamycin blocks the antidepressant effect of ketamine in task-dependent manner

    Czech Academy of Sciences Publication Activity Database

    Holubová, Kristína; Kletečková, Lenka; Škurlová, Martina; Říčný, J.; Stuchlík, Aleš; Valeš, Karel

    2016-01-01

    Roč. 233, č. 11 (2016), s. 2077-2097 ISSN 0033-3158 R&D Projects: GA MZd(CZ) NT13403 Institutional support: RVO:67985823 Keywords : ketamine * rapamycin * antidepressants * anxiety * cognitive deficit * bulbectomy * mTOR * BDNF Subject RIV: FH - Neurology Impact factor: 3.308, year: 2016

  20. MicroRNA-214 Reduces Insulin-like Growth Factor-1 (IGF-1) Receptor Expression and Downstream mTORC1 Signaling in Renal Carcinoma Cells*

    Science.gov (United States)

    Das, Falguni; Dey, Nirmalya; Bera, Amit; Kasinath, Balakuntalam S.; Ghosh-Choudhury, Nandini; Choudhury, Goutam Ghosh

    2016-01-01

    Elevated IGF-1/insulin-like growth factor-1 receptor (IGF-1R) autocrine/paracrine signaling in patients with renal cell carcinoma is associated with poor prognosis of the disease independent of their von Hippel-Lindau (VHL) status. Increased expression of IGF-1R in renal cancer cells correlates with their potency of tumor development and progression. The mechanism by which expression of IGF-1R is increased in renal carcinoma is not known. We report that VHL-deficient and VHL-positive renal cancer cells possess significantly decreased levels of mature, pre-, and pri-miR-214 than normal proximal tubular epithelial cells. We identified an miR-214 recognition element in the 3′UTR of IGF-1R mRNA and confirmed its responsiveness to miR-214. Overexpression of miR-214 decreased the IGF-1R protein levels, resulting in the inhibition of Akt kinase activity in both types of renal cancer cells. IGF-1 provoked phosphorylation and inactivation of PRAS40 in an Akt-dependent manner, leading to the activation of mTORC1 signal transduction to increase phosphorylation of S6 kinase and 4EBP-1. Phosphorylation-deficient mutants of PRAS40 and 4EBP-1 significantly inhibited IGF-1R-driven proliferation of renal cancer cells. Expression of miR-214 suppressed IGF-1R-induced phosphorylation of PRAS40, S6 kinase, and 4EBP-1, indicating inhibition of mTORC1 activity. Finally, miR-214 significantly blocked IGF-1R-forced renal cancer cell proliferation, which was reversed by expression of 3′UTR-less IGF-1R and constitutively active mTORC1. Together, our results identify a reciprocal regulation of IGF-1R levels and miR-214 expression in renal cancer cells independent of VHL status. Our data provide evidence for a novel mechanism for IGF-1R-driven renal cancer cell proliferation involving miR-214 and mTORC1. PMID:27226530

  1. Upregulation of T-type Ca2+ channels in long-term diabetes determines increased excitability of a specific type of capsaicin-insensitive DRG neurons.

    Science.gov (United States)

    Duzhyy, Dmytro E; Viatchenko-Karpinski, Viacheslav Y; Khomula, Eugen V; Voitenko, Nana V; Belan, Pavel V

    2015-05-20

    Previous studies have shown that increased excitability of capsaicin-sensitive DRG neurons and thermal hyperalgesia in rats with short-term (2-4 weeks) streptozotocin-induced diabetes is mediated by upregulation of T-type Ca(2+) current. In longer-term diabetes (after the 8th week) thermal hyperalgesia is changed to hypoalgesia that is accompanied by downregulation of T-type current in capsaicin-sensitive small-sized nociceptors. At the same time pain symptoms of diabetic neuropathy other than thermal persist in STZ-diabetic animals and patients during progression of diabetes into later stages suggesting that other types of DRG neurons may be sensitized and contribute to pain. In this study, we examined functional expression of T-type Ca(2+) channels in capsaicin-insensitive DRG neurons and excitability of these neurons in longer-term diabetic rats and in thermally hypoalgesic diabetic rats. Here we have demonstrated that in STZ-diabetes T-type current was upregulated in capsaicin-insensitive low-pH-sensitive small-sized nociceptive DRG neurons of longer-term diabetic rats and thermally hypoalgesic diabetic rats. This upregulation was not accompanied by significant changes in biophysical properties of T-type channels suggesting that a density of functionally active channels was increased. Sensitivity of T-type current to amiloride (1 mM) and low concentration of Ni(2+) (50 μM) implicates prevalence of Cav3.2 subtype of T-type channels in the capsaicin-insensitive low-pH-sensitive neurons of both naïve and diabetic rats. The upregulation of T-type channels resulted in the increased neuronal excitability of these nociceptive neurons revealed by a lower threshold for action potential initiation, prominent afterdepolarizing potentials and burst firing. Sodium current was not significantly changed in these neurons during long-term diabetes and could not contribute to the diabetes-induced increase of neuronal excitability. Capsaicin-insensitive low-pH-sensitive type

  2. Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress.

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    Scott M Thompson

    Full Text Available Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC, but lesions larger than 2-3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC. Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K/mammalian target of rapamycin (mTOR dependent-protein kinase B (AKT survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3 and prognosis (AKT1. Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin.

  3. Role of the Akt/mTOR signaling pathway in human papillomavirus-associated nasal and sinonasal inverted papilloma.

    Science.gov (United States)

    Liu, Yongliang; Duan, Lihua; Tian, Jie; Song, Daoliang; Zhang, Min; Zhao, Shenlin; Yin, Zhaofu; Xiang, Xinxin; Li, Xuezhong

    2017-12-01

    Nasal and sinonasal inverted papilloma (NSIP) is a benign tumor in which surface epithelial cells grow downward into the underlying supportive tissue with varying degrees of metaplasia. Human papillomavirus (HPV) has been proposed as the causal agent in the pathogenesis of this disease. Many studies have shown that HPV can activate the Akt/mechanistic target of rapamycin (mTOR) signaling pathway, but the role of this pathway in HPV-associated NSIP is largely unknown. In this study, we enrolled 40 control tissue samples and 80 NSIP tissue samples. HPV genotyping showed that 47 of the 80 examined cases of NSIP were HPV-positive (58.8%), and the most common subtype was HPV11 (20/53, 37.7%). The immunohistochemistry showed statistically significant differences in phosphorylated Akt and phosphorylated S6 ribosomal protein staining among control samples, HPV-positive NSIP and HPV-negative NSIP. The HPV11 L1-L2 plasmid increased the proliferation of normal human nasopharyngeal epithelial NP69-SV40T cells and human nasopharyngeal cancer CNE1 cells. Meanwhile, rapamycin, an mTOR inhibitor, reversed the increased cell proliferation induced by the HPV11 L1-L2 plasmid. Western blot analysis showed that Akt/mTOR/S6 were overexpressed in NP69-SV40T cells and CNE1 cells infected with the HPV11 L1-L2 plasmid. These data demonstrate that HPV promotes cell proliferation through the Akt/mTOR signaling pathway in NSIP. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. mTOR pathway inhibition prevents neuroinflammation and neuronal death in a mouse model of cerebral palsy.

    Science.gov (United States)

    Srivastava, Isha N; Shperdheja, Jona; Baybis, Marianna; Ferguson, Tanya; Crino, Peter B

    2016-01-01

    Mammalian target of rapamycin (mTOR) pathway signaling governs cellular responses to hypoxia and inflammation including induction of autophagy and cell survival. Cerebral palsy (CP) is a neurodevelopmental disorder linked to hypoxic and inflammatory brain injury however, a role for mTOR modulation in CP has not been investigated. We hypothesized that mTOR pathway inhibition would diminish inflammation and prevent neuronal death in a mouse model of CP. Mouse pups (P6) were subjected to hypoxia-ischemia and lipopolysaccharide-induced inflammation (HIL), a model of CP causing neuronal injury within the hippocampus, periventricular white matter, and neocortex. mTOR pathway inhibition was achieved with rapamycin (an mTOR inhibitor; 5mg/kg) or PF-4708671 (an inhibitor of the downstream p70S6kinase, S6K, 75 mg/kg) immediately following HIL, and then for 3 subsequent days. Phospho-activation of the mTOR effectors p70S6kinase and ribosomal S6 protein and expression of hypoxia inducible factor 1 (HIF-1α) were assayed. Neuronal cell death was defined with Fluoro-Jade C (FJC) and autophagy was measured using Beclin-1 and LC3II expression. Iba-1 labeled, activated microglia were quantified. Neuronal death, enhanced HIF-1α expression, and numerous Iba-1 labeled, activated microglia were evident at 24 and 48 h following HIL. Basal mTOR signaling, as evidenced by phosphorylated-S6 and -S6K levels, was unchanged by HIL. Rapamycin or PF-4,708,671 treatment significantly reduced mTOR signaling, neuronal death, HIF-1α expression, and microglial activation, coincident with enhanced expression of Beclin-1 and LC3II, markers of autophagy induction. mTOR pathway inhibition prevented neuronal death and diminished neuroinflammation in this model of CP. Persistent mTOR signaling following HIL suggests a failure of autophagy induction, which may contribute to neuronal death in CP. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in

  5. Plasma membrane proteins Slm1 and Slm2 mediate activation of the AGC kinase Ypk1 by TORC2 and sphingolipids in S. cerevisiae.

    Science.gov (United States)

    Niles, Brad J; Powers, Ted

    2012-10-15

    The PH domain-containing proteins Slm1 and Slm2 were originally identified as substrates of the rapamycin-insensitive TOR complex 2 (TORC2) and as mediators of signaling by the lipid second messenger phosphatidyl-inositol-4,5-bisphosphate (PI4,5P2) in budding yeast S. cerevisiae. More recently, these proteins have been identified as critical effectors that facilitate phosphorylation and activation of the AGC kinases Ypk1 and Ypk2 by TORC2. Here, we review the molecular basis for this regulation as well as place it within the context of recent findings that have revealed Slm1/2 and TORC2-dependent phosphorylation of Ypk1 is coupled to the biosynthesis of complex sphingolipids and to their levels within the plasma membrane (PM) as well as other forms of PM stress. Together, these studies reveal the existence of an intricate homeostatic feedback mechanism, whereby the activity of these signaling components is linked to the biosynthesis of PM lipids according to cellular need.

  6. Topical rapamycin as a treatment for fibrofolliculomas in Birt-Hogg-Dubé syndrome: a double-blind placebo-controlled randomized split-face trial.

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    Lieke M C Gijezen

    Full Text Available Birt-Hogg-Dubé syndrome (BHD is a rare autosomal dominant disorder characterised by the occurrence of benign, mostly facial, skin tumours called fibrofolliculomas, multiple lung cysts, spontaneous pneumothorax and an increased renal cancer risk. Current treatments for fibrofolliculomas have high rates of recurrence and carry a risk of complications. It would be desirable to have a treatment that could prevent fibrofolliculomas from growing. Animal models of BHD have previously shown deregulation of mammalian target of rapamycin (mTOR. Topical use of the mTOR inhibitor rapamycin is an effective treatment for the skin tumours (angiofibromas in tuberous sclerosis complex, which is also characterised by mTOR deregulation. In this study we aimed to determine if topical rapamycin is also an effective treatment for fibrofolliculomas in BHD.We performed a double blinded, randomised, facial left-right controlled trial of topical rapamycin 0.1% versus placebo in 19 BHD patients. Trial duration was 6 months. The primary outcome was cosmetic improvement as measured by doctors and patients. Changes in fibrofolliculoma number and size were also measured, as was occurrence of side effects.No change in cosmetic status of fibrofolliculomas was reported in the majority of cases for the rapamycin treated (79% by doctors, 53% by patients as well as the placebo treated facial sides (both 74%. No significant differences between rapamycin and placebo treated facial halves were observed (p = 1.000 for doctors opinion, p = 0.344 for patients opinion. No significant difference in fibrofolliculoma number or change in size of the fibrofolliculomas was seen after 6 months. Side effects occurred more often after rapamycin treatment (68% of patients than after placebo (58% of patients; p = 0.625. A burning sensation, erythema, itching and dryness were most frequently reported.This study provides no evidence that treatment of fibrofolliculomas with topical

  7. AKTivation of the PI3K/AKT/mTOR signaling pathway by KSHV

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    Aadra P Bhatt

    2013-01-01

    Full Text Available As an obligate intracellular parasite, the Kaposi sarcoma-associated herpesvirus (KSHV relies on host cell machinery to meet its needs for survival, viral replication, production, and dissemination of progeny virions. KSHV is a ɣ-herpesvirus that is associated with three different malignancies: Kaposi sarcoma (KS, and two B cell lymphoproliferative disorders, primary effusion lymphoma (PEL and multicentric Castleman disease (MCD. KSHV viral proteins modulate cellular phosphatidylinositol-3-kinase (PI3K/AKT/mammalian target of rapamycin (mTOR signaling pathway, which is a ubiquitous pathway that also controls B lymphocyte proliferation and development. We review the mechanisms by which KSHV manipulates the PI3K/AKT/mTOR pathway, with a specific focus on B cells.

  8. Up-Regulation of the Excitatory Amino Acid Transporters EAAT1 and EAAT2 by Mammalian Target of Rapamycin

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    Abeer Abousaab

    2016-11-01

    Full Text Available Background: The excitatory amino-acid transporters EAAT1 and EAAT2 clear glutamate from the synaptic cleft and thus terminate neuronal excitation. The carriers are subject to regulation by various kinases. The EAAT3 isoform is regulated by mammalian target of rapamycin (mTOR. The present study thus explored whether mTOR influences transport by EAAT1 and/or EAAT2. Methods: cRNA encoding wild type EAAT1 (SLC1A3 or EAAT2 (SLC1A2 was injected into Xenopus oocytes without or with additional injection of cRNA encoding mTOR. Dual electrode voltage clamp was performed in order to determine electrogenic glutamate transport (IEAAT. EAAT2 protein abundance was determined utilizing chemiluminescence. Results: Appreciable IEAAT was observed in EAAT1 or EAAT2 expressing but not in water injected oocytes. IEAAT was significantly increased by coexpression of mTOR. Coexpression of mTOR increased significantly the maximal IEAAT in EAAT1 or EAAT2 expressing oocytes, without significantly modifying affinity of the carriers. Moreover, coexpression of mTOR increased significantly EAAT2 protein abundance in the cell membrane. Conclusions: The kinase mTOR up-regulates the excitatory amino acid transporters EAAT1 and EAAT2.

  9. Cyclin D1 in ASM Cells from Asthmatics Is Insensitive to Corticosteroid Inhibition.

    Science.gov (United States)

    Allen, Jodi C; Seidel, Petra; Schlosser, Tobias; Ramsay, Emma E; Ge, Qi; Ammit, Alaina J

    2012-01-01

    Hyperplasia of airway smooth muscle (ASM) is a feature of the remodelled airway in asthmatics. We examined the antiproliferative effectiveness of the corticosteroid dexamethasone on expression of the key regulator of G(1) cell cycle progression-cyclin D1-in ASM cells from nonasthmatics and asthmatics stimulated with the mitogen platelet-derived growth factor BB. While cyclin D1 mRNA and protein expression were repressed in cells from nonasthmatics in contrast, cyclin D1 expression in asthmatics was resistant to inhibition by dexamethasone. This was independent of a repressive effect on glucocorticoid receptor translocation. Our results corroborate evidence demonstrating that corticosteroids inhibit mitogen-induced proliferation only in ASM cells from subjects without asthma and suggest that there are corticosteroid-insensitive proliferative pathways in asthmatics.

  10. 25-Hydroxycholecalciferol Enhances Male Broiler Breast Meat Yield through the mTOR Pathway.

    Science.gov (United States)

    Vignale, Karen; Greene, Elizabeth S; Caldas, Justina V; England, Judith A; Boonsinchai, Nirun; Sodsee, Phiphob; Pollock, Erik D; Dridi, Sami; Coon, Craig N

    2015-05-01

    In recent years, there has been a growing body of evidence indicating that replacing cholecalciferol (vitamin D₃) with 25-hydroxycholecalciferol [25(OH)D₃] through dietary supplementation enhances breast meat yield in broiler chickens. However, the underlying molecular mechanisms are still unknown. We investigated the effect of 25(OH)D₃ on male broiler growth performance (body weight, feed intake, feed conversion ratio, and breast meat yield), muscle protein synthesis, and the potential underlying molecular mechanisms. Male Cobb 500 broiler chickens were divided into 4 body weight-matched groups and received a control diet with normal cholecalciferol (2760 IU/kg feed) for 42 d, a diet with high concentrations of cholecalciferol (5520 IU/kg feed) for 42 d, or a diet with 25(OH)D₃ (5520 IU/kg feed) for 42 d (HyD-42). A fourth group consumed the HyD-42 for 21 d and then control feed for 21 d (HyD-21) (n = 360 birds, 12 replicates/treatment). Food and clean water were available for ad libitum consumption. At the end of the 42-d experiment, protein turnover was measured by phenylalanine flooding dose. Breast muscle tissues were collected and protein synthesis-related gene and protein expression were measured by real time polymerase chain reaction and Western blot, respectively. Functional studies were performed in vitro with the use of a quail myoblast (QM7) cell line. QM7 cells were treated with 2 doses (1 nM and 10 nM) of cholecalciferol or 25(OH)D₃ alone or in combination with 100 nM rapamycin, and cell proliferation was determined by cell proliferation assay. Protein synthesis-related gene and protein expression were also determined. The HyD-42 increased 25(OH)D₃ circulating concentrations by 126% (P meat yield (P vitro functional studies showed that cells treated with 25(OH)D₃ for 24 h had increased VDR expression, and activated the mechanistic target of rapamycin (mTOR)/S6 kinase (S6K) pathway, enhanced Ki67 protein concentrations, and induced QM7

  11. GDF15 regulates Kv2.1-mediated outward K+ current through the Akt/mTOR signalling pathway in rat cerebellar granule cells.

    Science.gov (United States)

    Wang, Chang-Ying; Huang, An-Qi; Zhou, Meng-Hua; Mei, Yan-Ai

    2014-05-15

    GDF15 (growth/differentiation factor 15), a novel member of the TGFβ (transforming growth factor β) superfamily, plays critical roles in the central and peripheral nervous systems, but the signal transduction pathways and receptor subtypes involved are not well understood. In the present paper, we report that GDF15 specifically increases the IK (delayed-rectifier outward K+ current) in rat CGNs (cerebellar granule neurons) in time- and concentration-dependent manners. The GDF15-induced amplification of the IK is mediated by the increased expression and reduced lysosome-dependent degradation of the Kv2.1 protein, the main α-subunit of the IK channel. Exposure of CGNs to GDF15 markedly induced the phosphorylation of ERK (extracellular-signal-regulated kinase), Akt and mTOR (mammalian target of rapamycin), but the GDF15-induced IK densities and increased expression of Kv2.1 were attenuated only by Akt and mTOR, and not ERK, inhibitors. Pharmacological inhibition of the Src-mediated phosphorylation of TGFβR2 (TGFβ receptor 2), not TGFβR1, abrogated the effect of GDF15 on IK amplification and Kv2.1 induction. Immunoprecipitation assays showed that GDF15 increased the tyrosine phosphorylation of TGFβRII in the CGN lysate. The results of the present study reveal a novel regulation of Kv2.1 by GDF15 mediated through the TGFβRII-activated Akt/mTOR pathway, which is a previously uncharacterized Smad-independent mechanism of GDF15 signalling.

  12. Autophagy in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Piotr Czarny

    2015-01-01

    Full Text Available DNA damage response (DDR involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1. mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADPribose polymerase 1 (PARP-1, Mre11–Rad50–Nbs1 (MRN complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.

  13. Munitions having an insensitive detonator system for initiating large failure diameter explosives

    Science.gov (United States)

    Perry, III, William Leroy

    2015-08-04

    A munition according to a preferred embodiment can include a detonator system having a detonator that is selectively coupled to a microwave source that functions to selectively prime, activate, initiate, and/or sensitize an insensitive explosive material for detonation. The preferred detonator can include an explosive cavity having a barrier within which an insensitive explosive material is disposed and a waveguide coupled to the explosive cavity. The preferred system can further include a microwave source coupled to the waveguide such that microwaves enter the explosive cavity and impinge on the insensitive explosive material to sensitize the explosive material for detonation. In use the preferred embodiments permit the deployment and use of munitions that are maintained in an insensitive state until the actual time of use, thereby substantially preventing unauthorized or unintended detonation thereof.

  14. The TOR Signaling Pathway in Spatial and Temporal Control of Cell Size and Growth

    Directory of Open Access Journals (Sweden)

    Suam Gonzalez

    2017-06-01

    Full Text Available Cell size is amenable by genetic and environmental factors. The highly conserved nutrient-responsive Target of Rapamycin (TOR signaling pathway regulates cellular metabolic status and growth in response to numerous inputs. Timing and duration of TOR pathway activity is pivotal for both cell mass built up as well as cell cycle progression and is controlled and fine-tuned by the abundance and quality of nutrients, hormonal signals, growth factors, stress, and oxygen. TOR kinases function within two functionally and structurally discrete multiprotein complexes, TORC1 and TORC2, that are implicated in temporal and spatial control of cell size and growth respectively; however, recent data indicate that such functional distinctions are much more complex. Here, we briefly review roles of the two complexes in cellular growth and cytoarchitecture in various experimental model systems.

  15. One-step preparation of [2,3-{sup 3}H]1-aminocyclo-propanecarboxylic acid: a useful ligand for strychnine-insensitive glycine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Lewin, A.H.; Lamb, P.B. [Research Triangle Inst., Research Triangle Park, NC (United States); Popik, P.; Skolnick, P. [National Insts. of Health, Bethesda, MD (United States)

    1994-11-01

    Catalytic hydrogenation of 1-aminocyclopropenecarboxylic acid under tritium gas afforded [2,3-{sup 3}H]1-aminocyclopropanecarboxylic acid with specific activity 26 Ci/mmol, determined by a combination of {sup 1}H and {sup 3}H NMR. Pilot radioligand binding assays indicate this compound will be a useful prove for the NMDA receptor-associated strychnine-insensitive glycine receptor. (author).

  16. AVP2, a sequence-divergent, K{sup +}-insensitive H{sup +}-translocating inorganic pyrophosphatase from arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Drozdowicz, Y.M.; Kissinger, J.C.; Rea, P.A.

    2000-05-01

    Plant vacuolar H{sup +}-translocating inorganic pyrophosphatase have been considered to constitute a family of functionally and structurally monotonous intrinsic membrane proteins. Typified by AVPI from Arabidopsis, all characterized plant V-PPases share greater than 84% sequence identity and catalyze K{sup +}-stimulated H{sup +} translocation. Here the authors describe the molecular and biochemical characterization of AVP2, a sequence-divergent K{sup +}-insensitive, Ca{sup 2+}-hypersensitive V-PPase active in both inorganic pyrophosphate hydrolysis and H{sup +} translocation. The differences between AVP2 and AVP1 provide the first indication that plant V-PPase sequences from the same organism fall into two distinct categories. Phylogenetic analyses of these and other V-PPase sequences extend this principle by showing that AVP2, rather than being an isoform of AMP1, is but one representative of a novel category of AVP2-like (type 2) V-PPases that coexist with AVP1-like (type 1) V-PPases not only in plants, but also in apicomplexan protists such as the malarial parasite Plasmodium falciparum.

  17. The PTPN11 loss-of-function mutation Q510E-Shp2 causes hypertrophic cardiomyopathy by dysregulating mTOR signaling.

    Science.gov (United States)

    Schramm, Christine; Fine, Deborah M; Edwards, Michelle A; Reeb, Ashley N; Krenz, Maike

    2012-01-01

    The identification of mutations in PTPN11 (encoding the protein tyrosine phosphatase Shp2) in families with congenital heart disease has facilitated mechanistic studies of various cardiovascular defects. However, the roles of normal and mutant Shp2 in the developing heart are still poorly understood. Furthermore, it remains unclear how Shp2 loss-of-function (LOF) mutations cause LEOPARD Syndrome (also termed Noonan Syndrome with multiple lentigines), which is characterized by congenital heart defects such as pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). In normal hearts, Shp2 controls cardiomyocyte size by regulating signaling through protein kinase B (Akt) and mammalian target of rapamycin (mTOR). We hypothesized that Shp2 LOF mutations dysregulate this pathway, resulting in HCM. For our studies, we chose the Shp2 mutation Q510E, a dominant-negative LOF mutation associated with severe early onset HCM. Newborn mice with cardiomyocyte-specific overexpression of Q510E-Shp2 starting before birth displayed increased cardiomyocyte sizes, heart-to-body weight ratios, interventricular septum thickness, and cardiomyocyte disarray. In 3-mo-old hearts, interstitial fibrosis was detected. Echocardiographically, ventricular walls were thickened and contractile function was depressed. In ventricular tissue samples, signaling through Akt/mTOR was hyperactivated, indicating that the presence of Q510E-Shp2 led to upregulation of this pathway. Importantly, rapamycin treatment started shortly after birth rescued the Q510E-Shp2-induced phenotype in vivo. If rapamycin was started at 6 wk of age, HCM was also ameliorated. We also generated a second mouse model in which cardiomyocyte-specific Q510E-Shp2 overexpression started after birth. In contrast to the first model, these mice did not develop HCM. In summary, our studies establish a role for mTOR signaling in HCM caused by Q510E-Shp2. Q510E-Shp2 overexpression in the cardiomyocyte population alone was sufficient to

  18. Photonic crystal based polarization insensitive flat lens

    International Nuclear Information System (INIS)

    Turduev, M; Bor, E; Kurt, H

    2017-01-01

    The paper proposes a new design of an inhomogeneous artificially created photonic crystal lens structure consisting of annular dielectric rods to efficiently focus both transverse electric and transverse magnetic polarizations of light into the same focal point. The locations of each individual cell that contains the annular dielectric rods are determined according to a nonlinear distribution function. The inner and outer radii of the annular photonic dielectric rods are optimized with respect to the polarization insensitive frequency response of the transmission spectrum of the lens structure. The physical background of the polarization insensitive focusing mechanism is investigated in both spatial and frequency domains. Moreover, polarization independent wavefront transformation/focusing has been explored in detail by investigating the dispersion relation of the structure. Corresponding phase index distribution of the lens is attained for polarization insensitive normalized frequency range of a / λ   =  0.280 and a / λ   =  0.300, where a denotes the lattice constant of the designed structure and λ denotes the wavelength of the incident light. We show the wave transformation performance and focal point movement dynamics for both polarizations of the lens structure by specially adjusting the length of the structure. The 3D finite-difference time domain numerical analysis is also performed to verifiy that the proposed design is able to focus the wave regardless of polarization into approximately the same focal point (difference between focal distances of both polarizations stays below 0.25 λ ) with an operating bandwidth of 4.30% between 1476 nm and 1541 nm at telecom wavelengths. The main superiorities of the proposed lens structure are being all dielectric and compact, and having flat front and back surfaces, rendering the proposed lens design more practical in the photonic integration process in various applications such as optical switch

  19. Three siblings with complete androgen insensitivity syndrome

    African Journals Online (AJOL)

    2013-01-13

    Jan 13, 2013 ... male reference range. A diagnosis of complete androgen insensitivity syndrome was made, based on this clinical ... Laboratory test. Value Normal ..... scenario, incomplete resistance may lead to virilisation during puberty.24, ...

  20. Mechanical Stimulation and IGF-1 Enhance mRNA Translation Rate in Osteoblasts Via Activation of the AKT-mTOR Pathway.

    Science.gov (United States)

    Bakker, Astrid D; Gakes, Tom; Hogervorst, Jolanda M A; de Wit, Gerard M J; Klein-Nulend, Jenneke; Jaspers, Richard T

    2016-06-01

    Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts. © 2015 Wiley Periodicals, Inc.