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Sample records for activating fak signaling

  1. Tumor-secreted LOXL2 Activates Fibroblasts Through FAK Signaling

    OpenAIRE

    Barker, Holly E.; Bird, Demelza; Lang, Georgina; Janine T. Erler

    2013-01-01

    Cancer-associated fibroblasts enhance cancer progression when activated by tumor cells through mechanisms not yet fully understood. Blocking mammary tumor cell-derived lysyl oxidase-like 2 (LOXL2) significantly inhibited mammary tumor cell invasion and metastasis in transgenic and orthotopic mouse models. Here we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown...

  2. FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells.

    OpenAIRE

    2014-01-01

    Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also ...

  3. Activation of the FAK-src molecular scaffolds and p130Cas-JNK signaling cascades by alpha1-integrins during colon cancer cell invasion.

    Science.gov (United States)

    Van Slambrouck, Severine; Grijelmo, Clara; De Wever, Olivier; Bruyneel, Erik; Emami, Shahin; Gespach, Christian; Steelant, Wim F A

    2007-12-01

    Increased src tyrosine kinase expression and activity has been associated with colon cancer cell invasion and survival. Several signaling pathways are involved in the oncogenic activation of src during the adenoma to carcinoma progression and cellular invasion. In the present study, the synthetic ether lipid analog ET-18-OMe was shown to promote invasion of HCT-8/S11 colon cancer cells into collagen type I through the concomitant activation of src by phosphorylation at Tyr416 (5-30 min) in alpha1-integrin immunoprecipitates containing the integrin binding proteins talin and paxillin, as well as the phoshorylated and activated forms of focal adhesion kinase (FAK) at Tyr397 (a FAK kinase activation signal), Tyr576 and Tyr861. This was associated with the lateral redistribution of alpha1-integrins in focal aggregates and persistent activation of the p130Cas/JNK pathways at 5-30 min, with the subsequent induction and activation of the matrix metalloproteinases MMP-2 and MMP-9 (2-12 h). These activated molecular scaffolds and signaling cascades were not observed in immunoprecipitates of alpha2- and beta1-integrins, and tetraspanin CD9, an invasion and metastasis suppressor linked to integrins and FAK signaling. Our data demonstrate that the lateral redistribution and clustering of alpha1-integrins results in the recruitment of the FAK/src motility-promoting signaling complex involved in cancer cell invasion. Disruption of this proinvasive pathway was accomplished by the dominant negative mutant of src (K295R, kinase dead), src pharmacological inhibitor (PP1) and alpha1-integrin function blocking antibodies. These findings support the notion that the alpha1-integrin- and src-dependent signalosome is a relevant therapeutic target against tumor progression in colon cancer patients.

  4. Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK and Mitogen-Activated Protein Kinases (MAP Kinases Signaling Pathway in Keratinocytes

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    Yun-Hee Choi

    2015-11-01

    Full Text Available Mycosporine-like amino acids (MAAs are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS. In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH, Mycosporine-glycine (M-Gly, and Porphyra (P334 were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK, extracellular signal-regulated kinases (ERK, and c-Jun N-terminal kinases (JNK. These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies.

  5. FAK kinase activity is required for the progression of c-Met/β-catenin-driven HCC

    Science.gov (United States)

    Shang, Na; Arteaga, Maribel; Zaidi, Ali; Cotler, Scott J.; Breslin, Peter; Ding, Xianzhong; Kuo, Paul; Nishimura, Michael; Zhang, Jiwang; Qiu, Wei

    2016-01-01

    Background & Aims There is an urgent need to develop new and more effective therapeutic strategies and agents to treat hepatocellular carcinoma (HCC). We have recently found that deletion of Fak in hepatocytes before tumors form inhibits tumor development and prolongs survival of animals in a c-Met (MET)/β-catenin (CAT)-driven HCC mouse model. However, it has yet to be determined whether FAK expression in hepatocytes promotes MET/CAT-induced HCC progression after tumor initiation. In addition, it remains unclear whether FAK promotes HCC development through its kinase activity. Methods We generated hepatocyte-specific inducible Fak-deficient mice (Alb-creERT2; Fakflox/flox) to examine the role of FAK in HCC progression. We re-expressed wild-type and mutant FAK in Fak-deficient mice to determine FAK’s kinase activity in HCC development. We also examined the efficacy of a FAK kinase inhibitor PF-562271 on HCC inhibition. Results We found that deletion of Fak after tumors form significantly repressed MET/CAT-induced tumor progression. Ectopic FAK expression restored HCC formation in hepatocyte-specific Fak-deficient mice. However, overexpression of a FAK kinase-dead mutant led to reduced tumor load compared to mice which express wild-type FAK. Furthermore, PF-562271 significantly suppressed progression of MET/CAT-induced HCC. Conclusion Fak kinase activity is important for MET/CAT-induced HCC progression. Inhibiting FAK kinase activity provides a potential therapeutic strategy to treat HCC. PMID:27142958

  6. αν and β1 Integrins mediate Aβ-induced neurotoxicity in hippocampal neurons via the FAK signaling pathway.

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    Hai-Yan Han

    Full Text Available αν and β1 integrins mediate Aβ-induced neurotoxicity in primary hippocampal neurons. We treated hippocampal neurons with 2.5 µg/mL 17E6 and 5 µg/mL ab58524, which are specific αν and β1 integrin antagonists, respectively, for 42 h prior to 10 µM Aβ treatment. Next, we employed small interfering RNA (siRNA to silence focal adhesion kinase (FAK, a downstream target gene of integrins. The siRNAs were designed with a target sequence, an MOI of 10 and the addition of 5 µg/mL polybrene. Under these conditions, the neurons were transfected and the apoptosis of different cell types was detected. Moreover, we used real-time PCR and Western blotting analyses to detect the expression of FAK and ρFAK genes in different cell types and investigated the underlying mechanism and signal pathway by which αν and β1 integrins mediate Aβ-induced neurotoxicity in hippocampal neurons. An MTT assay showed that both 17E6 and ab58524 significantly increased cell viability compared with the Aβ-treated neurons (P<0.01 and P<0.05, respectively. However, this protective effect was markedly attenuated after transfection with silencing FAK (siFAK. Moreover, TUNEL immunostaining and flow cytometry indicated that both 17E6 and ab58524 significantly protected hippocampal neurons against apoptosis induced by Aβ (P<0.05 compared with the Aβ-treated cells. However, this protective effect was reversed with siFAK treatment. Both the gene and protein expression of FAK increased after Aβ treatment. Interestingly, as the gene and protein levels of FAK decreased, the ρFAK protein expression markedly increased. Furthermore, both the gene and protein expression of FAK and ρFAK were significantly diminished. Thus, we concluded that both αν and β1 integrins interfered with Aβ-induced neurotoxicity in hippocampal neurons and that this mechanism partially contributes to the activation of the Integrin-FAK signaling pathway.

  7. Targeting FAK Radiosensitizes 3-Dimensional Grown Human HNSCC Cells Through Reduced Akt1 and MEK1/2 Signaling

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    Hehlgans, Stephanie [OncoRay-National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden (Germany); Department of Radiotherapy and Oncology, University of Frankfurt, Frankfurt am Main (Germany); Institute of Radiopharmacy, Helmholtz Center Dresden-Rossendorf, Dresden (Germany); Eke, Iris [OncoRay-National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden (Germany); Cordes, Nils, E-mail: Nils.Cordes@OncoRay.de [OncoRay-National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden (Germany); Institute of Radiopharmacy, Helmholtz Center Dresden-Rossendorf, Dresden (Germany); Department of Radiation Oncology, University Hospital and Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden (Germany)

    2012-08-01

    Purpose: Focal adhesion kinase (FAK), a main regulator of integrin signaling and cell migration, is frequently overexpressed and hyperphosphorylated in human head-and-neck squamous cell carcinoma (HNSCC). We have previously shown that pharmacologic FAK inhibition leads to radiosensitization of 3-dimensionally grown HNSCC cell lines. To further evaluate the role of FAK in radioresistance and as a potential cancer target, we examined FAK and FAK downstream signaling in HNSCC cell lines grown in more physiologic extracellular matrix-based 3-dimensional cell cultures. Methods and Materials: Seven HNSCC cell lines were grown in 3-dimensional extracellular matrix and the clonogenic radiation survival, expression, and phosphorylation of FAK, paxillin, Akt1, extracellular signal-regulated kinase (ERK)1/2, and MEK1/2 were analyzed after siRNA-mediated knockdown of FAK, Akt1, MEK1, FAK+Akt1, or FAK+MEK1 compared with controls or stable overexpression of FAK. The role of MEK1/2 for clonogenic survival and signaling was investigated using the MEK inhibitor U0126 with or without irradiation. Results: FAK knockdown moderately or significantly enhanced the cellular radiosensitivity of 3-dimensionally grown HNSCC cells. The FAK downstream targets paxillin, Akt1, and ERK1/2 were substantially dephosphorylated under FAK depletion. FAK overexpression, in contrast, increased radiation survival and paxillin, Akt1, and ERK1/2 phosphorylation. The degree of radiosensitization upon Akt1, ERK1/2, or MEK1 depletion or U0126 was superimposable to FAK knockdown. Combination knockdown conditions (ie, Akt1/FAK, MEK1/FAK, or U0126/FAK) failed to provide additional radiosensitization. Conclusions: Our data provide further evidence for FAK as important determinant of radiation survival, which acts in the same signaling axis as Akt1 and ERK1/2. These data strongly support our hypothesis that FAK is a relevant molecular target for HNSCC radiotherapy.

  8. 1-o-acetylbritannilactone (ABL) inhibits angiogenesis and lung cancer cell growth through regulating VEGF-Src-FAK signaling

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    Zhengfu, He; Hu, Zhang; Huiwen, Miao; Zhijun, Li [Department of Thoracic Surgery, Sir Run Run Shaw Hospital of Zhejiang University School of Medicine, Hangzhou (China); Jiaojie, Zhou [Zhejiang University School of Medicine, Hangzhou (China); Xiaoyi, Yan, E-mail: xiaoyiyan163@163.com [Zhejiang University School of Medicine, Hangzhou (China); Xiujun, Cai, E-mail: xiujuncaomaj@163.com [Sir Run Run Shaw Hospital of Zhejiang University School of Medicine, Hangzhou (China)

    2015-08-21

    The search for safe, effective and affordable therapeutics against non-small cell lung cancer (NSCLC) and other lung cancers is important. Here we explored the potential effect of 1-o-acetylbritannilactone (ABL), a novel extract from Inula britannica-F, on angiogenesis and lung cancer cell growth. We demonstrated that ABL dose-dependently inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration, and capillary structure formation of cultured human umbilical vascular endothelial cells (HUVECs). In vivo, ABL administration suppressed VEGF-induced new vasculature formation in Matrigel plugs. For the mechanism investigations, we found that ABL largely inhibited VEGF-mediated activation of Src kinase and focal adhesion kinase (FAK) in HUVECs. Furthermore, treatment of A549 NSCLC cells with ABL resulted in cell growth inhibition and Src-FAK in-activation. Significantly, administration of a single dose of ABL (12 mg/kg/day) remarkably suppressed growth of A549 xenografts in nude mice. In vivo microvessels formation and Src activation were also significantly inhibited in ABL-treated xenograft tumors. Taken together, our findings suggest that ABL suppresses angiogenesis and lung cancer cell growth possibly via regulating the VEGFR-Src-FAK signaling. - Highlights: • 1-o-acetylbritannilactone (ABL) inhibits VEGF-induced angiogenesis in vivo. • ABL inhibits VEGF-induced HUVEC migration, proliferation, capillary tube formation. • ABL inhibits VEGF-mediated activation of Src and FAK in HUVECs. • ABL inhibits growth and Src-FAK activation in A549 cells. • ABL administration inhibits A549 tumor angiogenesis and growth in nude mice.

  9. PCTK3/CDK18 regulates cell migration and adhesion by negatively modulating FAK activity

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    Matsuda, Shinya; Kawamoto, Kohei; Miyamoto, Kenji; Tsuji, Akihiko; Yuasa, Keizo

    2017-01-01

    PCTAIRE kinase 3 (PCTK3) is a member of the cyclin dependent kinase family, but its physiological function remains unknown. We previously reported that PCTK3-knockdown HEK293T cells showed actin accumulation at the leading edge, suggesting that PCTK3 is involved in the regulation of actin reorganization. In this study, we investigated the physiological function and downstream signal transduction molecules of PCTK3. PCTK3 knockdown in HEK293T cells increased cell motility and RhoA/Rho-associated kinase activity as compared with control cells. We also found that phosphorylation at residue Tyr-397 in focal adhesion kinase (FAK) was increased in PCTK3-knockdown cells. FAK phosphorylation at Tyr-397 was increased in response to fibronectin stimulation, whereas its phosphorylation was suppressed by PCTK3. In addition, excessive expression of PCTK3 led to the formation of filopodia during the early stages of cell adhesion in HeLa cells. These results indicate that PCTK3 controls actin cytoskeleton dynamics by negatively regulating the FAK/Rho signaling pathway. PMID:28361970

  10. Formation of Kv2.1-FAK Complex as a Mechanism of FAK Activation, Cell Polarization and Enhanced Motility

    OpenAIRE

    Wei, Jian-Feng; Wei, Ling; ZHOU, XIN; Lu, Zhong-yang; Francis, Kevin; Hu, Xin-yang; Liu, Yu; Xiong, Wen-Cheng; Zhang, Xiao; Banik, Naren L.; Zheng, Shu-Sen; Yu, Shan Ping

    2008-01-01

    Focal adhesion kinase (FAK) plays key roles in cell adhesion and migration. We now report that the delayed rectifier Kv2.1 potassium channel, through its LD-like motif in N-terminus, may interact with FAK and enhance phosphorylation of FAK397 and FAK576/577. Overlapping distribution of Kv2.1 and FAK was observed on soma and proximal dendrites of cortical neurons. FAK expression promotes a polarized membrane distribution of the Kv2.1 channel. In Kv2.1-transfected CHO cells, formation of the Kv...

  11. Claudin-7 indirectly regulates the integrin/FAK signaling pathway in human colon cancer tissue.

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    Ding, Lei; Wang, Liyong; Sui, Leiming; Zhao, Huanying; Xu, Xiaoxue; Li, Tengyan; Wang, Xiaonan; Li, Wenjing; Zhou, Ping; Kong, Lu

    2016-08-01

    The claudin family of proteins is integral to the structure and function of tight junctions. The role of claudin-7 (Cldn-7, CLDN7) in regulating the integrin/focal adhesion kinase (FAK)/ERK signaling pathway remains poorly understood. Therefore, we investigated differences in gene expression, primarily focusing on CLDN7 and integrin/FAK/ERK signaling pathway genes, between colon cancer and adjacent normal tissues. Quantitative real-time reverse transcription-PCR and immunohistochemistry were utilized to verify the results of mRNA and protein expression, respectively. In silico analysis was used to predict co-regulation between Cldn-7 and integrin/FAK/ERK signaling pathway components, and the STRING database was used to analyze protein-protein interaction pairs among these proteins. Meta-analysis of expression microarrays in The Cancer Genome Atlas (TCGA) database was used to identify significant correlations between Cldn-7 and components of predicted genes in the integrin/FAK/ERK signaling pathway. Our results showed marked cancer stage-specific decreases in the protein expression of Cldn-7, Gelsolin, MAPK1 and MAPK3 in colon cancer samples, and the observed changes for all proteins except Cldn-7 were in agreement with changes in the corresponding mRNA levels. Cldn-7 might indirectly regulate MAPK3 via KRT8 due to KRT8 co-expression with MAPK3 or CLDN7. Our bioinformatics methods supported the hypothesis that Cldn-7 does not directly regulate any genes in the integrin/FAK/ERK signaling pathway. These factors may participate in a common network that regulates cancer progression in which the MAPK pathway serves as the central node.

  12. Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation.

    OpenAIRE

    1996-01-01

    It has been proposed that the focal adhesion kinase (FAK) mediates focal adhesion formation through tyrosine phosphorylation during cell adhesion. We investigated the role of FAK in focal adhesion structure and function. Loading cells with a glutathione-S-transferase fusion protein (GST-Cterm) containing the FAK focal adhesion targeting sequence, but not the kinase domain, decreased the association of endogenous FAK with focal adhesions. This displacement of endogenous FAK in both BALB/c 3T3 ...

  13. Shockwaves increase T-cell proliferation and IL-2 expression through ATP release, P2X7 receptors, and FAK activation.

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    Yu, Tiecheng; Junger, Wolfgang G; Yuan, Changji; Jin, An; Zhao, Yi; Zheng, Xueqing; Zeng, Yanjun; Liu, Jianguo

    2010-03-01

    Shockwaves elicited by transient pressure disturbances are used to treat musculoskeletal disorders. Previous research has shown that shockwave treatment affects T-cell function, enhancing T-cell proliferation and IL-2 expression by activating p38 mitogen-activated protein kinase (MAPK) signaling. Here we investigated the signaling pathway by which shockwaves mediate p38 MAPK phosphorylation. We found that shockwaves at an intensity of 0.18 mJ/mm(2) induce the release of extracellular ATP from human Jurkat T-cells at least in part by affecting cell viability. ATP released into the extracellular space stimulates P2X7-type purinergic receptors that induce the activation of p38 MAPK and of focal adhesion kinase (FAK) by phosphorylation on residues Tyr397 and Tyr576/577. Elimination of released ATP with apyrase or inhibition of P2X7 receptors with the antagonists KN-62 or suramin significantly weakens FAK phosphorylation, p38 MAPK activation, IL-2 expression, and T-cell proliferation. Conversely, addition of exogenous ATP causes phosphorylation of FAK and p38 MAPK. Silencing of FAK expression also reduces these cell responses to shockwave treatment. We conclude that shockwaves enhance p38 MAPK activation, IL-2 expression, and T-cell proliferation via the release of cellular ATP and feedback mechanisms that involve P2X7 receptor activation and FAK phosphorylation.

  14. A FAK-Cas-Rac-lamellipodin signaling module transduces extracellular matrix stiffness into mechanosensitive cell cycling.

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    Bae, Yong Ho; Mui, Keeley L; Hsu, Bernadette Y; Liu, Shu-Lin; Cretu, Alexandra; Razinia, Ziba; Xu, Tina; Puré, Ellen; Assoian, Richard K

    2014-06-17

    Tissue and extracellular matrix (ECM) stiffness is transduced into intracellular stiffness, signaling, and changes in cellular behavior. Integrins and several of their associated focal adhesion proteins have been implicated in sensing ECM stiffness. We investigated how an initial sensing event is translated into intracellular stiffness and a biologically interpretable signal. We found that a pathway consisting of focal adhesion kinase (FAK), the adaptor protein p130Cas (Cas), and the guanosine triphosphatase Rac selectively transduced ECM stiffness into stable intracellular stiffness, increased the abundance of the cell cycle protein cyclin D1, and promoted S-phase entry. Rac-dependent intracellular stiffening involved its binding partner lamellipodin, a protein that transmits Rac signals to the cytoskeleton during cell migration. Our findings establish that mechanotransduction by a FAK-Cas-Rac-lamellipodin signaling module converts the external information encoded by ECM stiffness into stable intracellular stiffness and mechanosensitive cell cycling. Thus, lamellipodin is important not only in controlling cellular migration but also for regulating the cell cycle in response to mechanical signals.

  15. Tivantinib (ARQ-197) exhibits anti-tumor activity with down-regulation of FAK in oral squamous cell carcinoma

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    Xi, Wei-Hong [Department of Oral and Maxillofacial Surgery, First Affiliated Hospital, Nanchang University, Nanchang 330006 (China); Yang, Li-Yun [Department of Blood Transfusion, First Affiliated Hospital, Nanchang University, Nanchang 330006 (China); Cao, Zhong-Yi, E-mail: m18070383032@163.com [Department of Oral and Maxillofacial Surgery, First Affiliated Hospital, Nanchang University, Nanchang 330006 (China); Qian, Yong, E-mail: yfykqkqy@163.com [Department of Oral and Maxillofacial Surgery, First Affiliated Hospital, Nanchang University, Nanchang 330006 (China)

    2015-02-20

    Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide and the 5 years survival rate of the patients is about 60% in the USA, due to acquired chemotherapeutic resistance and metastasis of the disease. In this study, we found that tivantinib, a selective MET inhibitor, suppresses OCSS cell proliferation and colony formation, however, anti-tumor activities induced by tivantinib are independent of the inhibition of MET signaling pathway. In addition, tivantinib cause G2/M cell cycle arrest and caspases-dependent apoptosis in OSCC cell lines. We also found that tivantinib dose-dependently suppressed the activation and expression of FAK. In all, these data suggested that tivantinib may be developed as a chemotherapeutic agent to effectively treat certain cancers including OSCC. - Highlights: • Tivantinib suppresses OSCC cell growth independent of the inhibition of HGF/MET signaling pathway. • Tivantinib blocks cell cycle and induces caspases-mediated apoptosis. • Tivantinib elicits its anti-tumor activity with the inhibition of FAK signaling pathway.

  16. Signaling via ITGB1/FAK and microfilament rearrangement mediates the internalization of Leptospira interrogans in mouse J774A.1 macrophages

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    Zhao Xin

    2015-01-01

    Full Text Available Leptospirosis caused by pathogenic Leptospira species is a worldwide zoonotic 2 infectious disease, but the mechanisms of leptospiral internalization remain poorly understood. Here, we report that mouse J774A.1 macrophages expressed integrin-subfamily proteins (ITGB1, ITGB2 and ITGB3. Antibody blockage and siRNA-based knockdown of ITGB1 decreased the internalization of leptospires into mouse J774A.1 macrophage cells. The internalization required focal adhesion kinase (FAK activation in J774A.1 cells rather than phosphoinositide-3-kinase (PI3K, and microfilament rather than microtubule aggregation during infection. The data indicated that the ITGB1/FAK/microfilament signaling pathway is responsible for leptospiral internalization in mouse macrophages.

  17. Hyperphosphorylated FAK Delocalizes from Focal Adhesions to Membrane Ruffles

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    Abdelkader Hamadi

    2010-01-01

    Full Text Available Cell adhesion and migration are key determinants in tumor metastasis. Adherence of tumor cell to the extracellular matrix is mediated via integrin containing focal adhesions (FAs. Binding of integrins to ECM triggers phosphorylation of two major components of FAs, focal adhesion kinase (FAK and Src, activating downstream signaling pathway which leads to FA disassembly and cell migration. In this paper, we analyze how phosphorylation of FAK regulates its trafficking at FAs in living human astrocytoma cells. Upon pervanadate-induced FAK phosphorylation, phosphorylated FAK appeared highly expressed at newly formed membrane ruffles. This effect was abolished in presence of the specific Src inhibitor PP2. Our findings demonstrate that upon phosphorylation, FAK delocalizes from FAs to membrane ruffles.

  18. PIAS1-FAK Interaction Promotes the Survival and Progression of Non-Small Cell Lung Cancer

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    Jerfiz D. Constanzo

    2016-05-01

    Full Text Available The sequence of genomic alterations acquired by cancer cells during tumor progression and metastasis is poorly understood. Focal adhesion kinase (FAK is a non-receptor tyrosine kinase that integrates cytoskeleton remodeling, mitogenic signaling and cell survival. FAK has previously been reported to undergo nuclear localization during cell migration, cell differentiation and apoptosis. However, the mechanism behind FAK nuclear accumulation and its contribution to tumor progression has remained elusive. We report that amplification of FAK and the SUMO E3 ligase PIAS1 gene loci frequently co-occur in non-small cell lung cancer (NSCLC cells, and that both gene products are enriched in a subset of primary NSCLCs. We demonstrate that endogenous FAK and PIAS1 proteins interact in the cytoplasm and the cell nucleus of NSCLC cells. Ectopic expression of PIAS1 promotes proteolytic cleavage of the FAK C-terminus, focal adhesion maturation and FAK nuclear localization. Silencing of PIAS1 deregulates focal adhesion turnover, increases susceptibility to apoptosis in vitro and impairs tumor xenograft formation in vivo. Nuclear FAK in turn stimulates gene transcription favoring DNA repair, cell metabolism and cytoskeleton regulation. Consistently, ablation of FAK by CRISPR/Cas9 editing, results in basal DNA damage, susceptibility to ionizing radiation and impaired oxidative phosphorylation. Our findings provide insight into a mechanism regulating FAK cytoplasm-nuclear distribution and demonstrate that FAK activity in the nucleus promotes NSCLC survival and progression by increasing cell-ECM interaction and DNA repair regulation.

  19. Somatic mutational analysis of FAK in breast cancer: A novel gain-of-function mutation due to deletion of exon 33

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    Fang, Xu-Qian [Department of Clinical Laboratory, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai (China); Liu, Xiang-Fan [Faculty of Medical Laboratory Science, Shanghai JiaoTong University School of Medicine, Shanghai (China); Yao, Ling [Department of Biochemistry and Molecular Biology, Shanghai JiaoTong University School of Medicine, Shanghai (China); Chen, Chang-Qiang; Gu, Zhi-Dong [Department of Clinical Laboratory, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai (China); Ni, Pei-Hua [Faculty of Medical Laboratory Science, Shanghai JiaoTong University School of Medicine, Shanghai (China); Zheng, Xin-Min [Department of Biochemistry and Molecular Biology, Shanghai JiaoTong University School of Medicine, Shanghai (China); Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY (United States); Fan, Qi-Shi, E-mail: qishifan@126.com [Department of Clinical Laboratory, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai (China)

    2014-01-10

    Highlights: •A novel FAK splicing mutation identified in breast tumor. •FAK-Del33 mutation promotes cell migration and invasion. •FAK-Del33 mutation regulates FAK/Src signal pathway. -- Abstract: Focal adhesion kinase (FAK) regulates cell adhesion, migration, proliferation, and survival. We identified a novel splicing mutant, FAK-Del33 (exon 33 deletion, KF437463), in both breast and thyroid cancers through colony sequencing. Considering the low proportion of mutant transcripts in samples, this mutation was detected by TaqMan-MGB probes based qPCR. In total, three in 21 paired breast tissues were identified with the FAK-Del33 mutation, and no mutations were found in the corresponding normal tissues. When introduced into a breast cell line through lentivirus infection, FAK-Del33 regulated cell motility and migration based on a wound healing assay. We demonstrated that the expression of Tyr397 (main auto-phosphorylation of FAK) was strongly increased in FAK-Del33 overexpressed breast tumor cells compared to wild-type following FAK/Src RTK signaling activation. These results suggest a novel and unique role of the FAK-Del33 mutation in FAK/Src signaling in breast cancer with significant implications for metastatic potential.

  20. Antitumor effects of the flavone chalcone: inhibition of invasion and migration through the FAK/JNK signaling pathway in human gastric adenocarcinoma AGS cells.

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    Lin, Su-Hsuan; Shih, Yuan-Wei

    2014-06-01

    Chalcones (benzylideneacetophenone) are cancer-preventive food components found in a human diet rich in fruits and vegetables. In this study, we first report the chemopreventive effect of chalcone in human gastric adenocarcinoma cell lines: AGS. The results showed that chalcone could inhibit the abilities of the adhesion, invasion, and migration by cell-matrix adhesion assay, Boyden chamber invasion/migration assay, and wound-healing assay. Molecular data showed that the effect of chalcone in AGS cells might be mediated via sustained inactivation of the phosphorylation of focal adhesion kinase (FAK) and c-Jun N-terminal kinase 1 and 2 (JNK1/2) signal involved in the downregulation of the expressions of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Next, chalcone-treated AGS cells showed tremendous decrease in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), the nuclear level of NF-κB, and the binding ability of NF-κB to NF-κB response element. Furthermore, treating FAK small interfering RNA (FAK siRNA) and specific inhibitor for JNK (SP600125) to AGS cells could reduce the phosphorylation of JNK1/2 and the activity of MMP-2 and MMP-9. Our results revealed that chalcone significantly inhibited the metastatic ability of AGS cells by reducing MMP-2 and MMP-9 expressions concomitantly with a marked reduction on cell invasion and migration through suppressing and JNK signaling pathways. We suggest that chalcone may offer the application in clinical medicine.

  1. Epithelial membrane protein-2 promotes endometrial tumor formation through activation of FAK and Src.

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    Maoyong Fu

    Full Text Available Endometrial cancer is the most common gynecologic malignancy diagnosed among women in developed countries. One recent biomarker strongly associated with disease progression and survival is epithelial membrane protein-2 (EMP2, a tetraspan protein known to associate with and modify surface expression of certain integrin isoforms. In this study, we show using a xenograft model system that EMP2 expression is necessary for efficient endometrial tumor formation, and we have started to characterize the mechanism by which EMP2 contributes to this malignant phenotype. In endometrial cancer cells, the focal adhesion kinase (FAK/Src pathway appears to regulate migration as measured through wound healing assays. Manipulation of EMP2 levels in endometrial cancer cells regulates the phosphorylation of FAK and Src, and promotes their distribution into lipid raft domains. Notably, cells with low levels of EMP2 fail to migrate and poorly form tumors in vivo. These findings reveal the pivotal role of EMP2 in endometrial cancer carcinogenesis, and suggest that the association of elevated EMP2 levels with endometrial cancer prognosis may be causally linked to its effect on integrin-mediated signaling.

  2. A synthetic mechano-growth factor E peptide promotes rat tenocyte migration by lessening cell stiffness and increasing F-actin formation via the FAK-ERK1/2 signaling pathway

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    Zhang, Bingyu [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Luo, Qing, E-mail: qing.luo@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Mao, Xinjian [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Xu, Baiyao [Department of Mechanical Science and Engineering, Nagoya University, Nagoya 464-8603 (Japan); Yang, Li [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Ju, Yang [Department of Mechanical Science and Engineering, Nagoya University, Nagoya 464-8603 (Japan); Song, Guanbin, E-mail: song@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China)

    2014-03-10

    Tendon injuries are common in sports and are frequent reasons for orthopedic consultations. The management of damaged tendons is one of the most challenging problems in orthopedics. Mechano-growth factor (MGF), a recently discovered growth repair factor, plays positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury-induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. We used a scratch wound assay in this study to demonstrate that MGF-C25E (a synthetic mechano-growth factor E peptide) promotes the migration of rat tenocytes and that this promotion is accompanied by an elevation in the expression of the following signaling molecules: focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2). Inhibitors of the FAK and ERK1/2 pathways inhibited the MGF-C25E-induced tenocyte migration, indicating that MGF-C25E promotes tenocyte migration through the FAK-ERK1/2 signaling pathway. The analysis of the mechanical properties showed that the Young's modulus of tenocytes was decreased through treatment of MGF-C25E, and an obvious formation of pseudopodia and F-actin was observed in MGF-C25E-treated tenocytes. The inhibition of the FAK or ERK1/2 signals restored the decrease in Young's modulus and inhibited the formation of pseudopodia and F-actin. Overall, our study demonstrated that MGF-C25E promotes rat tenocyte migration by lessening cell stiffness and increasing pseudopodia formation via the FAK-ERK1/2 signaling pathway. - Highlights: • Mechano-growth factor E peptide (MGF-C25E) promotes migration of rat tenocytes. • MGF-C25E activates the FAK-ERK1/2 pathway in rat tenocytes. • MGF-C25E induces the actin remodeling and the formation of pseudopodia, and decreases the stiffness in rat tenocytes. • MGF-C25E promotes tenocyte migration via altering stiffness and forming pseudopodia by the activation of the

  3. Ruthenium Polypyridyl Complex Inhibits Growth and Metastasis of Breast Cancer Cells by Suppressing FAK signaling with Enhancement of TRAIL-induced Apoptosis

    Science.gov (United States)

    Cao, Wenqiang; Zheng, Wenjie; Chen, Tianfeng

    2015-03-01

    Ruthenium-based complexes have emerged as promising antitumor and antimetastatic agents during the past decades. However, the limited understanding of the antimetastatic mechanisms of these agents is a roadblock to their clinical application. Herein, we reported that, RuPOP, a ruthenium polypyridyl complex with potent antitumor activity, was able to effectively inhibit growth and metastasis of MDA-MB-231 cells and synergistically enhance TRAIL-induced apoptosis. The selective intracellular uptake and cytotoxic effect of RuPOP was found associated with transferring receptor (TfR)-mediated endocytosis. Further investigation on intracellular mechanisms reveled that RuPOP notably suppressed FAK-mediated ERK and Akt activation. Pretreatment of cells with ERK inhibitor (U0126) and PI3K inhibitor (LY294002) significantly potentiated the inhibitory effect of RuPOP on cell growth, migration and invasion. Moreover, the alternation in the expression levels of metastatic regulatory proteins, including uPA, MMP-2/-9, and inhibition of VEGF secretion were also observed after RuPOP treatment. These results demonstrate the inhibitory effect of RuPOP on the growth and metastasis of cancer cells and the enhancement of TRAIL-induced apoptosis though suppression of FAK-mediated signaling. Furthermore, RuPOP exhibits the potential to be developed as a metal-based antimetastatic agent and chemosensitizer of TRAIL for the treatment of human metastatic cancers.

  4. Roundabout4 Suppresses Glioma-Induced Endothelial Cell Proliferation, Migration and Tube Formation in Vitro by Inhibiting VEGR2-Mediated PI3K/AKT and FAK Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Heng Cai

    2015-03-01

    Full Text Available Background and Aims: Endothelial cell (EC proliferation, migration, and tube formation are the critical steps for tumor angiogenesis, which is involved in the formation of new tumor blood vessels. Roundabout4 (Robo4, a new member of Robo proteins family, is specifically expressed in endothelial cells. This study aimed to investigate the effects of Robo4 on glioma-induced endothelial cell proliferation, migration and tube formation in vitro. Methods and Results: We found that Robo4 was endogenously expressed in Human Brain Microvascular Endothelial Cells (HBMECs, while Robo4 was significantly down-regulated in endothelial cells cultured in glioma conditioned medium. Robo4 over-expression remarkably suppressed glioma-induced endothelial cell proliferation, migration and tube formation in vitro. In addition, Robo4 influenced the glioma-induced angiogenesis via binding to its ligand Slit2. Further studies demonstrated that the knockdown of Robo4 up-regulated the phosphorylation of VEGFR2, PI3K, AKT and FAK in EC cultured in glioma conditioned medium. VEGFR2 inhibitor SU-1498, AKT inhibitor LY294002 and FAK inhibitor 14 (FAK inhibitor blocked the Robo4 knockdown-mediated alteration in glioma angiogenesis in vitro. Conclusion: Our results proved that Robo4 suppressed glioma-induced endothelial cell proliferation, migration and tube formation in vitro by inhibiting VEGR2-mediated activation of PI3K/AKT and FAK signaling pathways.

  5. Lunasin potentiates the effect of oxaliplatin preventing outgrowth of colon cancer metastasis, binds to α5β1 integrin and suppresses FAK/ERK/NF-κB signaling.

    Science.gov (United States)

    Dia, Vermont P; Gonzalez de Mejia, Elvira

    2011-12-27

    The effect of lunasin on colon cancer metastasis was studied using three human colon cancer cell lines in vitro and a liver metastasis model of colon cancer in vivo. Lunasin bound with α5β1 integrin and internalized into the nucleus of KM12L4 human colon cancer cells. Lunasin (10 μM) inhibited the activation of focal adhesion kinase (FAK) by 28%, 39% and 60% in RKO, HCT-116 and KM12L4 human colon cancer cells, respectively. Lunasin caused an increase in the expression of the inhibitor of kappa B alpha (IκB-α), a decrease in nuclear p50 NF-κB and a reduction in the migration of cancer cells. Lunasin (4 mg/kg bw) inhibited metastasis and potentiated the effect of oxaliplatin by reducing the expression of proliferating cell nuclear antigen. Liver metastatic nodules were reduced from 28 (PBS) to 14 (lunasin, P = 0.047) while combination of lunasin and oxaliplatin to 5 (P = 0.004). The tumor burden was reduced from 0.13 (PBS) to 0.10 (lunasin, P = 0.039) to 0.04 (lunasin + oxaliplatin, P cancer cells by direct binding with α5β1 integrin suppressing FAK/ERK/NF-κB signaling, and potentiated the effect of oxaliplatin in preventing the outgrowth of metastasis.

  6. Ras-induced and extracellular signal-regulated kinase 1 and 2 phosphorylation-dependent isomerization of protein tyrosine phosphatase (PTP)-PEST by PIN1 promotes FAK dephosphorylation by PTP-PEST.

    Science.gov (United States)

    Zheng, Yanhua; Yang, Weiwei; Xia, Yan; Hawke, David; Liu, David X; Lu, Zhimin

    2011-11-01

    Protein tyrosine phosphatase (PTP)-PEST is a critical regulator of cell adhesion and migration. However, the mechanism by which PTP-PEST is regulated in response to oncogenic signaling to dephosphorylate its substrates remains unclear. Here, we demonstrate that activated Ras induces extracellular signal-regulated kinase 1 and 2-dependent phosphorylation of PTP-PEST at S571, which recruits PIN1 to bind to PTP-PEST. Isomerization of the phosphorylated PTP-PEST by PIN1 increases the interaction between PTP-PEST and FAK, which leads to the dephosphorylation of FAK Y397 and the promotion of migration, invasion, and metastasis of v-H-Ras-transformed cells. These findings uncover an important mechanism for the regulation of PTP-PEST in activated Ras-induced tumor progression.

  7. PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

    Science.gov (United States)

    Prolactin-Induced Tyrosine Phosphorylation, Activation and ReceptorAssociation of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells. Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental ProtectionAgency, MD-72, Research Triangle Park, NC 27711, and

  8. Interleukin-2 induces beta2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

    DEFF Research Database (Denmark)

    Brockdorff, J; Kanner, S B; Nielsen, M;

    1998-01-01

    experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in beta2-integrin-positive but not in beta2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fakB......-tyrosine phosphorylation in beta2-integrin-positive T cells. In parallel experiments, IL-2 does not induce or augment tyrosine phosphorylation of p125(FAK). In conclusion, our data indicate that IL-2 induces beta2-integrin-dependent signal transduction events involving the tyrosine kinase substrate fakB....... and a leukocyte adhesion deficiency (LAD) patient. We show that IL-2 induces tyrosine phosphorylation of a 125-kDa protein and homotypic adhesion in beta2 integrin (CD18)-positive but not in beta2-integrin-negative T cells. EDTA, an inhibitor of integrin adhesion, blocks IL-2-induced tyrosine phosphorylation...

  9. Soft matrices downregulate FAK activity to promote growth of tumor-repopulating cells.

    Science.gov (United States)

    Tan, Youhua; Wood, Adam Richard; Jia, Qiong; Zhou, Wenwen; Luo, Junyu; Yang, Fang; Chen, Junwei; Chen, Junjian; Sun, Jian; Seong, Jihye; Tajik, Arash; Singh, Rishi; Wang, Ning

    2017-01-29

    Tumor-repopulating cells (TRCs) are a tumorigenic sub-population of cancer cells that drives tumorigenesis. We have recently reported that soft fibrin matrices maintain TRC growth by promoting histone 3 lysine 9 (H3K9) demethylation and Sox2 expression and that Cdc42 expression influences H3K9 methylation. However, the underlying mechanisms of how soft matrices induce H3K9 demethylation remain elusive. Here we find that TRCs exhibit lower focal adhesion kinase (FAK) and H3K9 methylation levels in soft fibrin matrices than control melanoma cells on 2D rigid substrates. Silencing FAK in control melanoma cells decreases H3K9 methylation, whereas overexpressing FAK in tumor-repopulating cells enhances H3K9 methylation. Overexpressing Cdc42 or RhoA in the presence of FAK knockdown restores H3K9 methylation levels. Importantly, silencing FAK, Cdc42, or RhoA promotes Sox2 expression and proliferation of control melanoma cells in stiff fibrin matrices, whereas overexpressing each gene suppresses Sox2 expression and reduces growth of TRCs in soft but not in stiff fibrin matrices. Our findings suggest that low FAK mediated by soft fibrin matrices downregulates H3K9 methylation through reduction of Cdc42 and RhoA and promotes TRC growth.

  10. Activation of platelet-activating factor receptor and pleiotropic effects on tyrosine phospho-EGFR/Src/FAK/paxillin in ovarian cancer.

    Science.gov (United States)

    Aponte, Margarita; Jiang, Wei; Lakkis, Montaha; Li, Ming-Jiang; Edwards, Dale; Albitar, Lina; Vitonis, Allison; Mok, Samuel C; Cramer, Daniel W; Ye, Bin

    2008-07-15

    Among the proinflammatory mediators, platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is a major primary and secondary messenger involved in intracellular and extracellular communication. Evidence suggests that PAF plays a significant role in oncogenic transformation, tumor growth, angiogenesis, and metastasis. However, PAF, with its receptor (PAFR) and their downstream signaling targets, has not been thoroughly studied in cancer. Here, we characterized the PAFR expression pattern in 4 normal human ovarian surface epithelial (HOSE) cell lines, 13 ovarian cancer cell lines, paraffin blocks (n = 84), and tissue microarrays (n = 230) from patients with ovarian cancer. Overexpression of PAFR was found in most nonmucinous types of ovarian cancer but not in HOSE and mucinous cancer cells. Correspondingly, PAF significantly induced cell proliferation and invasion only in PAFR-positive cells (i.e., OVCA429 and OVCA432), but not in PAFR-negative ovarian cells (HOSE and mucinous RMUG-L). The dependency of cell proliferation and invasion on PAFR was further confirmed using PAFR-specific small interfering RNA gene silencing probes, antibodies against PAFR and PAFR antagonist, ginkgolide B. Using quantitative multiplex phospho-antibody array technology, we found that tyrosine phosphorylation of EGFR/Src/FAK/paxillin was coordinately activated by PAF treatment, which was correlated with the activation of phosphatidylinositol 3-kinase and cyclin D1 as markers for cell proliferation, as well as matrix metalloproteinase 2 and 9 for invasion. Specific tyrosine Src inhibitor (PP2) reversibly blocked PAF-activated cancer cell proliferation and invasion. We suggest that PAFR is an essential upstream target of Src and other signal pathways to control the PAF-mediated cancer progression.

  11. The direct effect of Focal Adhesion Kinase (FAK, dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis

    Directory of Open Access Journals (Sweden)

    Zhang Li

    2009-08-01

    Full Text Available Abstract Background Focal adhesion kinase (FAK is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis. Methods To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD, and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors. Results Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p Conclusion Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal

  12. Fibronectin induces macrophage migration through a SFK-FAK/CSF-1R pathway.

    Science.gov (United States)

    Digiacomo, Graziana; Tusa, Ignazia; Bacci, Marina; Cipolleschi, Maria Grazia; Dello Sbarba, Persio; Rovida, Elisabetta

    2016-09-02

    Integrins, following binding to proteins of the extracellular matrix (ECM) including collagen, laminin and fibronectin (FN), are able to transduce molecular signals inside the cells and to regulate several biological functions such as migration, proliferation and differentiation. Besides activation of adaptor molecules and kinases, integrins transactivate Receptor Tyrosine Kinases (RTK). In particular, adhesion to the ECM may promote RTK activation in the absence of growth factors. The Colony-Stimulating Factor-1 Receptor (CSF-1R) is a RTK that supports the survival, proliferation, and motility of monocytes/macrophages, which are essential components of innate immunity and cancer development. Macrophage interaction with FN is recognized as an important aspect of host defense and wound repair. The aim of the present study was to investigate on a possible cross-talk between FN-elicited signals and CSF-1R in macrophages. FN induced migration in BAC1.2F5 and J774 murine macrophage cell lines and in human primary macrophages. Adhesion to FN determined phosphorylation of the Focal Adhesion Kinase (FAK) and Src Family Kinases (SFK) and activation of the SFK/FAK complex, as witnessed by paxillin phosphorylation. SFK activity was necessary for FAK activation and macrophage migration. Moreover, FN-induced migration was dependent on FAK in either murine macrophage cell lines or human primary macrophages. FN also induced FAK-dependent/ligand-independent CSF-1R phosphorylation, as well as the interaction between CSF-1R and β1. CSF-1R activity was necessary for FN-induced macrophage migration. Indeed, genetic or pharmacological inhibition of CSF-1R prevented FN-induced macrophage migration. Our results identified a new SFK-FAK/CSF-1R signaling pathway that mediates FN-induced migration of macrophages.

  13. Kaempferol-3-O-rutinoside from Afgekia mahidoliae promotes keratinocyte migration through FAK and Rac1 activation.

    Science.gov (United States)

    Petpiroon, Nareerat; Suktap, Chalermlat; Pongsamart, Sunanta; Chanvorachote, Pithi; Sukrong, Suchada

    2015-07-01

    The restoration of the epidermal epithelium through re-epithelialization is a critical process in wound healing. Directed keratinocyte migration to the wound is required, and the retardation of this process may result in a chronic, non-healing wound. The present study contributes to research aiming to identify promising compounds that promote wound healing using a human keratinocyte model. The effects of three kaempferol glycosides from an Afgekia mahidoliae leaf extract, kaempferol-3-O-arabinoside, kaempferol-3-O-glucoside, and kaempferol-3-O-rutinoside, on keratinocyte migration were determined. Interestingly, kaempferol-3-O-rutinoside exhibited a pronounced effect on wound closure in comparison to the parental kaempferol and other glycosides. The mechanism by which kaempferol-3-O-rutinoside enhances cell migration involves the induction of filopodia and lamellipodia formation, increased cellular levels of phosphorylated FAK (Tyr 397) and phosphorylated Akt (Ser 473), and up-regulation of active Rac1-GTP. The data obtained in this study may support the development of this compound for use in wound healing therapies.

  14. Class 3 semaphorin mediates dendrite growth in adult newborn neurons through Cdk5/FAK pathway.

    Directory of Open Access Journals (Sweden)

    Teclise Ng

    Full Text Available Class 3 semaphorins are well-known axonal guidance cues during the embryonic development of mammalian nervous system. However, their activity on postnatally differentiated neurons in neurogenic regions of adult brains has not been characterized. We found that silencing of semaphorin receptors neuropilins (NRP 1 or 2 in neural progenitors at the adult mouse dentate gyrus resulted in newly differentiated neurons with shorter dendrites and simpler branching in vivo. Tyrosine phosphorylation (Tyr 397 and serine phosphorylation (Ser 732 of FAK were essential for these effects. Semaphorin 3A and 3F mediate serine phosphorylation of FAK through the activation of Cdk5. Silencing of either Cdk5 or FAK in newborn neurons phenocopied the defects in dendritic development seen upon silencing of NRP1 or NRP2. Furthermore, in vivo overexpression of Cdk5 or FAK rescued the dendritic phenotypes seen in NRP1 and NRP2 deficient neurons. These results point to a novel role for class 3 semaphorins in promoting dendritic growth and branching during adult hippocampal neurogenesis through the activation of Cdk5-FAK signaling pathway.

  15. CCR9-CCL25 interactions promote cisplatin resistance in breast cancer cell through Akt activation in a PI3K-dependent and FAK-independent fashion

    Directory of Open Access Journals (Sweden)

    Lillard James W

    2011-05-01

    Full Text Available Abstract Background Chemotherapy heavily relies on apoptosis to kill breast cancer (BrCa cells. Many breast tumors respond to chemotherapy, but cells that survive this initial response gain resistance to subsequent treatments. This leads to aggressive cell variants with an enhanced ability to migrate, invade and survive at secondary sites. Metastasis and chemoresistance are responsible for most cancer-related deaths; hence, therapies designed to minimize both are greatly needed. We have recently shown that CCR9-CCL25 interactions promote BrCa cell migration and invasion, while others have shown that this axis play important role in T cell survival. In this study we have shown potential role of CCR9-CCL25 axis in breast cancer cell survival and therapeutic efficacy of cisplatin. Methods Bromodeoxyuridine (BrdU incorporation, Vybrant apoptosis and TUNEL assays were performed to ascertain the role of CCR9-CCL25 axis in cisplatin-induced apoptosis of BrCa cells. Fast Activated Cell-based ELISA (FACE assay was used to quantify In situ activation of PI3Kp85, AktSer473, GSK-3βSer9 and FKHRThr24 in breast cancer cells with or without cisplatin treatment in presence or absence of CCL25. Results CCR9-CCL25 axis provides survival advantage to BrCa cells and inhibits cisplatin-induced apoptosis in a PI3K-dependent and focal adhesion kinase (FAK-independent fashion. Furthermore, CCR9-CCL25 axis activates cell-survival signals through Akt and subsequent glycogen synthase kinase-3 beta (GSK-3β and forkhead in human rhabdomyosarcoma (FKHR inactivation. These results show that CCR9-CCL25 axis play important role in BrCa cell survival and low chemotherapeutic efficacy of cisplatin primarily through PI3K/Akt dependent fashion.

  16. Etk/BMX, a Btk family tyrosine kinase, and Mal contribute to the cross-talk between MyD88 and FAK pathways.

    Science.gov (United States)

    Semaan, Noha; Alsaleh, Ghada; Gottenberg, Jacques-Eric; Wachsmann, Dominique; Sibilia, Jean

    2008-03-01

    MyD88 and focal adhesion kinase (FAK) are key adaptors involved in signaling downstream of TLR2, TLR4, and integrin alpha5beta1, linking pathogen-associated molecule detection to the initiation of proinflammatory response. The MyD88 and integrin pathways are interlinked, but the mechanism of this cross-talk is not yet understood. In this study we addressed the involvement of Etk, which belongs to the Tec family of tyrosine kinases, in the cross-talk between the integrin/FAK and the MyD88 pathways in fibroblast-like synoviocytes (FLS) and in IL-6 synthesis. Using small interfering RNA blockade, we report that Etk plays a major role in LPS- and protein I/II (a model activator of FAK)-dependent IL-6 release by activated FLS. Etk is associated with MyD88, FAK, and Mal as shown by coimmunoprecipitation. Interestingly, knockdown of Mal appreciably inhibited IL-6 synthesis in response to LPS and protein I/II. Our results also indicate that LPS and protein I/II induced phosphorylation of Etk and Mal in rheumatoid arthritis FLS via a FAK-dependent pathway. In conclusion, our data provide support that, in FLS, Etk and Mal are implicated in the cross-talk between FAK and MyD88 and that their being brought into play is clearly dependent on FAK.

  17. Regulation of Stat5 by FAK and PAK1 in Oncogenic FLT3- and KIT-Driven Leukemogenesis

    Directory of Open Access Journals (Sweden)

    Anindya Chatterjee

    2014-11-01

    Full Text Available Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML and myeloproliferative neoplasms (MPNs, and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription, is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis.

  18. FAK dimerization controls its kinase-dependent functions at focal adhesions

    KAUST Repository

    Brami-Cherrier, Karen

    2014-01-30

    Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK\\'s kinase-dependent functions-autophosphorylation of tyrosine-397-requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation. © 2014 The Authors.

  19. FAK deletion accelerates liver regeneration after two-thirds partial hepatectomy

    Science.gov (United States)

    Shang, Na; Arteaga, Maribel; Chitsike, Lennox; Wang, Fang; Viswakarma, Navin; Breslin, Peter; Qiu, Wei

    2016-01-01

    Understanding the molecular mechanisms of liver regeneration is essential to improve the survival rate of patients after surgical resection of large amounts of liver tissue. Focal adhesion kinase (FAK) regulates different cellular functions, including cell survival, proliferation and cell migration. The role of FAK in liver regeneration remains unknown. In this study, we found that Fak is activated and induced during liver regeneration after two-thirds partial hepatectomy (PHx). We used mice with liver-specific deletion of Fak and investigated the role of Fak in liver regeneration in 2/3 PHx model (removal of 2/3 of the liver). We found that specific deletion of Fak accelerates liver regeneration. Fak deletion enhances hepatocyte proliferation prior to day 3 post-PHx but attenuates hepatocyte proliferation 3 days after PHx. Moreover, we demonstrated that the deletion of Fak in liver transiently increases EGFR activation by regulating the TNFα/HB-EGF axis during liver regeneration. Furthermore, we found more apoptosis in Fak-deficient mouse livers compared to WT mouse livers after PHx. Conclusion: Our data suggest that Fak is involved in the process of liver regeneration, and inhibition of FAK may be a promising strategy to accelerate liver regeneration in recipients after liver transplantation. PMID:27677358

  20. Structural Insight into the Mechanisms of Targeting and Signaling of Focal Adhesion Kinase

    OpenAIRE

    2002-01-01

    Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase whose focal adhesion targeting (FAT) domain interacts with other focal adhesion molecules in integrin-mediated signaling. Localization of activated FAK to focal adhesions is indispensable for its function. Here we describe a solution structure of the FAT domain bound to a peptide derived from paxillin, a FAK-binding partner. The FAT domain is composed of four helices that form a “right-turn” elongated bundle; the globular fold is ma...

  1. Endothelial paxillin and focal adhesion kinase (FAK) play a critical role in neutrophil transmigration.

    Science.gov (United States)

    Parsons, Sean A; Sharma, Ritu; Roccamatisi, Dawn L; Zhang, Hong; Petri, Björn; Kubes, Paul; Colarusso, Pina; Patel, Kamala D

    2012-02-01

    During an inflammatory response, endothelial cells undergo morphological changes to allow for the passage of neutrophils from the blood vessel to the site of injury or infection. Although endothelial cell junctions and the cytoskeleton undergo reorganization during inflammation, little is known about another class of cellular structures, the focal adhesions. In this study, we examined several focal adhesion proteins during an inflammatory response. We found that there was selective loss of paxillin and focal adhesion kinase (FAK) from focal adhesions in proximity to transmigrating neutrophils; in contrast the levels of the focal adhesion proteins β1-integrin and vinculin were unaffected. Paxillin was lost from focal adhesions during neutrophil transmigration both under static and flow conditions. Down-regulating endothelial paxillin with siRNA blocked neutrophil transmigration while having no effect on rolling or adhesion. As paxillin dynamics are regulated partly by FAK, the role of FAK in neutrophil transmigration was examined using two complementary methods. siRNA was used to down-regulate total FAK protein while dominant-negative, kinase-deficient FAK was expressed to block FAK signaling. Disruption of the FAK protein or FAK signaling decreased neutrophil transmigration. Collectively, these findings reveal a novel role for endothelial focal adhesion proteins paxillin and FAK in regulating neutrophil transmigration.

  2. Resveratrol suppresses human colon cancer cell proliferation and induces apoptosis via targeting the pentose phosphate and the talin-FAK signaling pathways-A proteomic approach

    Directory of Open Access Journals (Sweden)

    Reddivari Lavanya

    2011-08-01

    Full Text Available Abstract Background We and others have previously reported that resveratrol (RSV suppresses colon cancer cell proliferation and elevates apoptosis in vitro and/or in vivo, however molecular mechanisms are not fully elucidated. Particularly, little information is available on RSV's effects on metabolic pathways and the cell-extra cellular matrix (ECM communication that are critical for cancer cell growth. To identify important targets of RSV, we analyzed whole protein fractions from HT-29 advanced human colon cancer cell line treated with solvent control, IGF-1 (10 nM and RSV (150 μM using LC/MS/MS-Mud PIT (Multidimensional Protein Identification Technology. Results Pentose phosphate pathway (PPP, a vital metabolic pathway for cell cycle progression, was elevated and suppressed by IGF-1 and RSV, respectively in the HT-29 cell line. Enzymatic assays confirmed RSV suppression of glucose-6 phosphate dehydrogenase (rate limiting and transketolase, key enzymes of the PPP. RSV (150 μM suppressed, whereas IGF-1 (10 nM elevated focal adhesion complex (FAC proteins, talin and pFAK, critical for the cell-ECM communication. Western blotting analyses confirmed the suppression or elevation of these proteins in HT-29 cancer cells treated with RSV or IGF-1, respectively. Conclusions Proteomic analysis enabled us to establish PPP and the talin-pFAK as targets of RSV which suppress cancer cell proliferation and induce apoptosis in the colon cancer cell line HT-29. RSV (150 μM suppressed these pathways in the presence and absence of IGF-1, suggesting its role as a chemo-preventive agent even in obese condition.

  3. FAK interacts with MEF2 and drives the stretch-induced activation of an intronic enhancer of phospholamban gene in cardiomyocytes

    OpenAIRE

    2008-01-01

    Resumo: Estudos anteriores demonstraram que em miócitos cardíacos submetidos a estímulos mecânicos ocorre pronta fosforilação e ativação da FAK. Resultados de estudos recentes, realizados em corações de ratos indicaram que em resposta a estímulos mecânicos, a FAK, além de ser ativada, localiza-se no núcleo dos miócitos cardíaco. Estudos conduzidos em corações de ratos Wistar adultos, utilizando a técnica de Imunoprecipitação de cromatina (ChIP) com anticorpo anti- FAK, identificaram uma seqüê...

  4. Prednisone inhibits the focal adhesion kinase/receptor activator of NF-κB ligand/mitogen-activated protein kinase signaling pathway in rats with adriamycin-induced nephropathy.

    Science.gov (United States)

    Ye, Minyuan; Zheng, Jing; Chen, Xiaoying; Chen, Xuelan; Wu, Xinhong; Lin, Xiuqin; Liu, Yafang

    2015-11-01

    The aim of the present study was to investigate the mechanisms underlying the effects of prednisone on adriamycin-induced nephritic rat kidney damage via the focal adhesion kinase (FAK)/receptor activator of nuclear factor-κB ligand (RANKL)/mitogen‑activated protein kinase (MAPK) signaling pathway. An adriamycin‑induced nephritic rat model was established to investigate these mechanisms. A total of 30 healthy male Sprague‑Dawley rats were randomly assigned to the normal, model or prednisone group. Samples of urine were collected over the course of 24 h at days 7, 14, and 28, and renal cortex tissue samples were harvested at days 14, and 28 following nephritic rat model establishment. The total urinary protein content was measured by biuret colorimetry. Pathological changes in the kidney tissue samples were observed using an electron microscope. The mRNA expressions levels of FAK, RANKL, p38, extracellular signal‑regulated kinase (ERK), c‑Jun N‑terminal kinase (JNK), and nephrin were then quantified by reverse transcription‑quantitative polymerase chain reaction. In addition, the protein expressions levels of FAK, RANKL, p38, ERK, JNK, phosphorylated (p)‑FAK, p‑ERK, and p‑JNK were quantified by western blotting. As compared with the normal group, the protein expression levels of FAK, RANKL, p-FAK, p38 and p-ERK in the model group were increased. In the prednisone group, the protein expression levels of p-ERK decreased, as compared with the normal group. In the prednisone group, the urinary protein levels, the protein expression levels of FAK, RANKL, p38, p-FAK, p-p38 and the mRNA expression levels of FAK, p38, RANKL, ERK, JNK decreased, as compared with the model group. In the prednisone group, the mRNA and protein expression levels of nephrin and the serum expression levels of RANKL increased, the serum expression levels of osteoprotegerin (OPG) were decreased, as compared with the model group. No significant changes in the protein expression

  5. FAK通过和EGFR的相互作用调节MAPK和Akt之间的相对平衡%FAK Regulates the Balance between MAPK and Akt Signal Pathways through Interaction with EGFR

    Institute of Scientific and Technical Information of China (English)

    李永林; 祝梅香; 张连峰; 蔡永; 刘先菊; R.M.Lafranie; 沈洁; 王嫣; 张璐; 董伟; 张扬清

    2005-01-01

    目的研究上皮生长因子受体和FAK的相互作用以及对下游信号的影响.方法建立聚集粘连激酶(FAK)缺失突变和绿色荧光蛋白(GFP)融合基因del 1-693FAK-GFP、del 1-100FAK-GFP和FAK-GFP稳定表达细胞系.结果同野生型FAK-GFP相比,N-端1-100氨基酸残基的缺失突变体,缺失1-693氨基酸残基的突变体结合在黏附点的能力被完全抑制.应用等电聚焦和SDS-PAGE双向电泳证明,EGF和纤维连接蛋白诱导FAK磷酸化的位点不同,进一步证实del 1-693FAK-GFP、del 1-100FAK-GFP,抑制MAPK的磷酸化,增强Akt的磷酸化;而FAK-GFP增强MAPK磷酸化,抑制Akt磷酸化.结论FAK通过和EGFR的相互作用调节MAPK和Akt之间的相对平衡.

  6. The roles of FAK and GP Ⅱ b/Ⅲ a in platelet signal transduction%GPⅡb/Ⅲa和FAK在血小板信号转导中的作用

    Institute of Scientific and Technical Information of China (English)

    胡惠静; 刘彦虹; 吴晓岩

    2010-01-01

    GPⅡb/Ⅲa是血小板膜上最重要的糖蛋白,不但参与血小板的黏附、聚集反应,同时介导血小板的信号转导,在维持血小板正常功能中起关键作用.近几年对GPⅡ b/Ⅲa介导的双向信号转导过程的研究有了较大进展,其中信号转导分子--黏着斑激酶在信号转导中所起的重要作用也日益引起人们的重视.%CP Ⅱ b/Ⅲ a is an important glycoprotein that presents on the surface of platelet and is critical for the maintenance of the normal function of platelets. GP Ⅱ b/Ⅲ a plays a key role in the processes of platelet adhesion and aggregation. It also mediates signal transduction. In recent years, great advance has been made in the inside-out and outside-in signal transduction mediated by GP Ⅱ b/Ⅲ a. Furthermore, focal adhesion kinase (FAK), a kind of the signal transduction molecule, is drawing increasing attention in signal transduction mediated by GP II b/Ⅲ a.

  7. EFFECTS OF INTEGRIN ALPHA ⅡbR995A MUTATION ON RECEPTOR AFFINITY AND pp 125 (FAK) PHOSPHORYLATION

    Institute of Scientific and Technical Information of China (English)

    Xue-yuan Tang; Zai-fu Jian; Guo-ping Wang; Hong-hui Yang; Wei Liu

    2004-01-01

    Objective To investigate the role of cytoplasmic domain of integrin alpha Ⅱb in platelet signal transduction.Methods Binding capacity of integrin alpha ⅡbR995Ato antibody platelet activation complex-1 (PAC-1) and pp125focal adhesion kinase (FAK) phosphorylation of cells were detected by flow cytometry, immune precipitation, and Western blotting.Results Without activation, wild-type alpha Ⅱ bbeta3 Chinese hamster ovary (CHO) cells failed to bind to PAC-1, but mutant chimera alpha ⅡbR995Aeta3 CHO cells were able to bind with PAC-1. Furthermore, phosphorylation of pp125 (FAK)in wild-type alpha Ⅱbbeta3 CHO cells occured only when cells were adhered to fibrinogen, but could not be detected in bovine serum albumin suspension. However in the mutant chimera group, it could be detected in both conditions.Conclusion The mutation in integrin alpha ⅡbR995Aalters its affinity state as a receptor, thus also mediating cytoplasmic signal transduction leading to the phosphorylation of pp125 (FAK) without ligand binding.

  8. Hydrogen Sulfide Recruits Macrophage Migration by Integrin β1-Src-FAK/Pyk2-Rac Pathway in Myocardial Infarction

    Science.gov (United States)

    Miao, Lei; Xin, Xiaoming; Xin, Hong; Shen, Xiaoyan; Zhu, Yi-Zhun

    2016-03-01

    Myocardial infarction (MI) triggers an inflammatory reaction, in which macrophages are of key importance for tissue repairing. Infiltration and/or migration of macrophages into the infarct area early after MI is critical for infarct healing, vascularization, and cardiac function. Hydrogen sulfide (H2S) has been demonstrated to possess cardioprotective effects post MI and during the progress of cardiac remodeling. However, the specific molecular and cellular mechanisms involved in macrophage recruitment by H2S remain to be identified. In this study, the NaHS (exogenous sources of H2S) treatment exerted an increased infiltration of macrophages into the infarcted myocardium at early stage of MI cardiac tissues in both wild type (WT) and cystathionine-γ-lyase-knockout (CSE-KO) mice. And NaHS accelerated the migration of macrophage cells in vitro. While, the inhibitors not only significantly diminished the migratory ability in response to NaHS, but also blocked the activation of phospho-Src, -Pyk2, -FAK397, and -FAK925. Furthermore, NaHS induced the internalization of integrin β1 on macrophage surface, but, integrin β1 silencing inhibited macrophage migration and Src signaling activation. These results indicate that H2S may have the potential as an anti-infarct of MI by governing macrophage migration, which was achieved by accelerating internalization of integrin β1 and activating downstream Src-FAK/Pyk2-Rac pathway.

  9. Angiogenic activity of sesamin through the activation of multiple signal pathways

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Byung-Hee [Vascular System Research Center and Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon (Korea, Republic of); Division of Food Biotechnology, School of Biotechnology, Kangwon National University, Chuncheon (Korea, Republic of); Lee, Jung Joon [Center for Molecular Cancer Research, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Kim, Jong-Dai [Division of Food Biotechnology, School of Biotechnology, Kangwon National University, Chuncheon (Korea, Republic of); Jeoung, Dooil; Lee, Hansoo [Division of Life Sciences, Kangwon National University, Chuncheon (Korea, Republic of); Choe, Jongseon; Ha, Kwon-Soo [Vascular System Research Center and Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon (Korea, Republic of); Kwon, Young-Geun [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Kim, Young-Myeong, E-mail: ymkim@kangwon.ac.kr [Vascular System Research Center and Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon (Korea, Republic of)

    2010-01-01

    The natural product sesamin has been known to act as a potent antioxidant and prevent endothelial dysfunction. We here found that sesamin increased in vitro angiogenic processes, such as endothelial cell proliferation, migration, and tube formation, as well as neovascularization in an animal model. This compound elicited the activation of multiple angiogenic signal modulators, such as ERK, Akt, endothelial nitric oxide synthase (eNOS), NO production, FAK, and p38 MAPK, but not Src. The MEK inhibitor PD98059 and the PI3K inhibitor Wortmannin specifically inhibited sesamin-induced activation of the ERK and Akt/eNOS pathways. These inhibitors reduced angiogenic events, with high specificity for MEK/ERK-dependent cell proliferation and migration and PI3K/Akt-mediated tube formation. Moreover, inhibition of p38 MAPK effectively inhibited sesamin-induced cell migration. The angiogenic activity of sesamin was not associated with VEGF expression. Furthermore, this compound did not induce vascular permeability and upregulated ICAM-1 and VCAM-1 expression, which are hallmarks of vascular inflammation. These results suggest that sesamin stimulates angiogenesis in vitro and in vivo through the activation of MEK/ERK-, PI3K/Akt/eNOS-, p125{sup FAK}-, and p38 MAPK-dependent pathways, without increasing vascular inflammation, and may be used for treating ischemic diseases and tissue regeneration.

  10. Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

    Directory of Open Access Journals (Sweden)

    Ohannessian Arthur

    2004-05-01

    Full Text Available Abstract Background Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK. Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK. Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC lines. Methods Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. Results In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. Conclusions We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.

  11. Pharmacological blockade of FAK autophosphorylation decreases human glioblastoma tumor growth and synergizes with temozolomide

    OpenAIRE

    2012-01-01

    Malignant gliomas are characterized by aggressive tumor growth with a mean survival of 15–18 months and frequently developed resistance to temozolomide. Therefore, strategies that sensitize glioma cells to temozolomide have a high translational impact. We have studied focal adhesion kinase (FAK), a tyrosine kinase and emerging therapeutic target that is known to be highly expressed and activated in glioma. In this report we tested the FAK autophosphorylation inhibitor, Y15 in DBTRG and U87 gl...

  12. Sunitinib activates Axl signaling in renal cell cancer.

    Science.gov (United States)

    van der Mijn, Johannes C; Broxterman, Henk J; Knol, Jaco C; Piersma, Sander R; De Haas, Richard R; Dekker, Henk; Pham, Thang V; Van Beusechem, Victor W; Halmos, Balazs; Mier, James W; Jiménez, Connie R; Verheul, Henk M W

    2016-06-15

    Mass spectrometry-based phosphoproteomics provides a unique unbiased approach to evaluate signaling network in cancer cells. The tyrosine kinase inhibitor sunitinib is registered as treatment for patients with renal cell cancer (RCC). We investigated the effect of sunitinib on tyrosine phosphorylation in RCC tumor cells to get more insight in its mechanism of action and thereby to find potential leads for combination treatment strategies. Sunitinib inhibitory concentrations of proliferation (IC50) of 786-O, 769-p and A498 RCC cells were determined by MTT-assays. Global tyrosine phosphorylation was measured by LC-MS/MS after immunoprecipitation with the antiphosphotyrosine antibody p-TYR-100. Phosphoproteomic profiling of 786-O cells yielded 1519 phosphopeptides, corresponding to 675 unique proteins including 57 different phosphorylated protein kinases. Compared to control, incubation with sunitinib at its IC50 of 2 µM resulted in downregulation of 86 phosphopeptides including CDK5, DYRK3, DYRK4, G6PD, PKM and LDH-A, while 94 phosphopeptides including Axl, FAK, EPHA2 and p38α were upregulated. Axl- (y702), FAK- (y576) and p38α (y182) upregulation was confirmed by Western Blot in 786-O and A498 cells. Subsequent proliferation assays revealed that inhibition of Axl with a small molecule inhibitor (R428) sensitized 786-O RCC cells and immortalized endothelial cells to sunitinib up to 3 fold. In conclusion, incubation with sunitinib of RCC cells causes significant upregulation of multiple phosphopeptides including Axl. Simultaneous inhibition of Axl improves the antitumor activity of sunitinib. We envision that evaluation of phosphoproteomic changes by TKI treatment enables identification of new targets for combination treatment strategies.

  13. MICA Expression Is Regulated by Cell Adhesion and Contact in a FAK/Src-Dependent Manner

    Science.gov (United States)

    Moncayo, Gerald; Lin, Da; McCarthy, Michael T.; Watson, Aleksandra A.; O’Callaghan, Christopher A.

    2017-01-01

    MICA is a major ligand for the NKG2D immune receptor, which plays a key role in activating natural killer (NK) cells and cytotoxic T cells. We analyzed NKG2D ligand expression on a range of cell types and could demonstrate that MICA expression levels were closely linked to cellular growth mode. While the expression of other NKG2D ligands was largely independent of cell growth mode, MICA expression was mainly found on cells cultured as adherent cells. In addition, MICA surface expression was reduced through increase in cell–cell contact or loss of cell–matrix adherence. Furthermore, we found that the reduction in MICA expression was modulated by focal adhesion kinase (FAK)/Src signaling and associated with increased susceptibility to NK cell-mediated killing. While the mechanisms of tumor immune evasion are not fully understood, the reduction of MICA expression following loss of attachment poises a potential way by which metastasizing tumor cells avoid immune detection. The role of FAK/Src in this process indicates a potential therapeutic approach to modulate MICA expression and immune recognition of tumor cells during metastasis. PMID:28154561

  14. Ganoderiol A-enriched extract suppresses migration and adhesion of MDA-MB-231 cells by inhibiting FAK-SRC-paxillin cascade pathway.

    Directory of Open Access Journals (Sweden)

    Guo-Sheng Wu

    Full Text Available Cell adhesion, migration and invasion are critical steps for carcinogenesis and cancer metastasis. Ganoderma lucidum, also called Lingzhi in China, is a traditional Chinese medicine, which exhibits anti-proliferation, anti-inflammation and anti-metastasis properties. Herein, GAEE, G. lucidum extract mainly contains ganoderiol A (GA, dihydrogenated GA and GA isomer, was shown to inhibit the abilities of adhesion and migration, while have a slight influence on that of invasion in highly metastatic breast cancer MDA-MB-231 cells at non-toxic doses. Further investigation revealed that GAEE decreased the active forms of focal adhesion kinase (FAK and disrupted the interaction between FAK and SRC, which lead to deactivating of paxillin. Moreover, GAEE treatment downregulated the expressions of RhoA, Rac1, and Cdc42, and decreased the interaction between neural Wiskott-Aldrich Syndrome protein (N-WASP and Cdc42, which impair cell migration and actin assembly. To our knowledge, this is the first report to show that G.lucidum triterpenoids could suppress cell migration and adhesion through FAK-SRC-paxillin signaling pathway. Our study also suggests that GAEE may be a potential agent for treatment of breast cancer.

  15. Hydrostatic Compress Force Enhances the Viability and Decreases the Apoptosis of Condylar Chondrocytes through Integrin-FAK-ERK/PI3K Pathway

    Science.gov (United States)

    Ma, Dandan; Kou, Xiaoxing; Jin, Jing; Xu, Taotao; Wu, Mengjie; Deng, Liquan; Fu, Lusi; Liu, Yi; Wu, Gang; Lu, Haiping

    2016-01-01

    Reduced mechanical stimuli in many pathological cases, such as hemimastication and limited masticatory movements, can significantly affect the metabolic activity of mandibular condylar chondrocytes and the growth of mandibles. However, the molecular mechanisms for these phenomena remain unclear. In this study, we hypothesized that integrin-focal adhesion kinase (FAK)-ERK (extracellular signal–regulated kinase)/PI3K (phosphatidylinositol-3-kinase) signaling pathway mediated the cellular response of condylar chondrocytes to mechanical loading. Primary condylar chondrocytes were exposed to hydrostatic compressive forces (HCFs) of different magnitudes (0, 50, 100, 150, 200, and 250 kPa) for 2 h. We measured the viability, morphology, and apoptosis of the chondrocytes with different treatments as well as the gene, protein expression, and phosphorylation of mechanosensitivity-related molecules, such as integrin α2, integrin α5, integrin β1, FAK, ERK, and PI3K. HCFs could significantly increase the viability and surface area of condylar chondrocytes and decrease their apoptosis in a dose-dependent manner. HCF of 250 kPa resulted in a 1.51 ± 0.02-fold increase of cell viability and reduced the ratio of apoptotic cells from 18.10% ± 0.56% to 7.30% ± 1.43%. HCFs could significantly enhance the mRNA and protein expression of integrin α2, integrin α5, and integrin β1 in a dose-dependent manner, but not ERK1, ERK2, or PI3K. Instead, HCF could significantly increase phosphorylation levels of FAK, ERK1/2, and PI3K in a dose-dependent manner. Cilengitide, the potent integrin inhibitor, could dose-dependently block such effects of HCFs. HCFs enhances the viability and decreases the apoptosis of condylar chondrocytes through the integrin-FAK-ERK/PI3K pathway. PMID:27827993

  16. Heparan sulfate proteoglycans mediate interstitial flow mechanotransduction regulating MMP-13 expression and cell motility via FAK-ERK in 3D collagen.

    Directory of Open Access Journals (Sweden)

    Zhong-Dong Shi

    Full Text Available BACKGROUND: Interstitial flow directly affects cells that reside in tissues and regulates tissue physiology and pathology by modulating important cellular processes including proliferation, differentiation, and migration. However, the structures that cells utilize to sense interstitial flow in a 3-dimensional (3D environment have not yet been elucidated. Previously, we have shown that interstitial flow upregulates matrix metalloproteinase (MMP expression in rat vascular smooth muscle cells (SMCs and fibroblasts/myofibroblasts via activation of an ERK1/2-c-Jun pathway, which in turn promotes cell migration in collagen. Herein, we focused on uncovering the flow-induced mechanotransduction mechanism in 3D. METHODOLOGY/PRINCIPAL FINDINGS: Cleavage of rat vascular SMC surface glycocalyx heparan sulfate (HS chains from proteoglycan (PG core proteins by heparinase or disruption of HS biosynthesis by silencing N-deacetylase/N-sulfotransferase 1 (NDST1 suppressed interstitial flow-induced ERK1/2 activation, interstitial collagenase (MMP-13 expression, and SMC motility in 3D collagen. Inhibition or knockdown of focal adhesion kinase (FAK also attenuated or blocked flow-induced ERK1/2 activation, MMP-13 expression, and cell motility. Interstitial flow induced FAK phosphorylation at Tyr925, and this activation was blocked when heparan sulfate proteoglycans (HSPGs were disrupted. These data suggest that HSPGs mediate interstitial flow-induced mechanotransduction through FAK-ERK. In addition, we show that integrins are crucial for mechanotransduction through HSPGs as they mediate cell spreading and maintain cytoskeletal rigidity. CONCLUSIONS/SIGNIFICANCE: We propose a conceptual mechanotransduction model wherein cell surface glycocalyx HSPGs, in the presence of integrin-mediated cell-matrix adhesions and cytoskeleton organization, sense interstitial flow and activate the FAK-ERK signaling axis, leading to upregulation of MMP expression and cell motility in 3D

  17. Signal focusing through active transport

    Science.gov (United States)

    Godec, Aljaž; Metzler, Ralf

    2015-07-01

    The accuracy of molecular signaling in biological cells and novel diagnostic devices is ultimately limited by the counting noise floor imposed by the thermal diffusion. Motivated by the fact that messenger RNA and vesicle-engulfed signaling molecules transiently bind to molecular motors and are actively transported in biological cells, we show here that the random active delivery of signaling particles to within a typical diffusion distance to the receptor generically reduces the correlation time of the counting noise. Considering a variety of signaling particle sizes from mRNA to vesicles and cell sizes from prokaryotic to eukaryotic cells, we show that the conditions for active focusing—faster and more precise signaling—are indeed compatible with observations in living cells. Our results improve the understanding of molecular cellular signaling and novel diagnostic devices.

  18. Role of focal adhesion tyrosine kinases in GPVI-dependent platelet activation and reactive oxygen species formation.

    Directory of Open Access Journals (Sweden)

    Naadiya Carrim

    Full Text Available We have previously shown the presence of a TRAF4/p47phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI.To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway.Human and mouse washed platelets (from WT or Pyk2 KO mice were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P surface expression and integrin αIIbβ3 activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk, PI3-K and Bruton's tyrosine kinase (Btk and upstream of Rac1, PLCγ2, Ca2+ release, PKC, Hic-5, NOX1 and αIIbβ3 activation.Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway.

  19. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling.

    Science.gov (United States)

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2015-01-15

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3β activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation.

  20. Vinculin modulation of paxillin–FAK interactions regulates ERK to control survival and motility

    Science.gov (United States)

    Subauste, M. Cecilia; Pertz, Olivier; Adamson, Eileen D.; Turner, Christopher E.; Junger, Sachiko; Hahn, Klaus M.

    2004-01-01

    Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin−/−) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal–regulated kinase (ERK). This increase is shown to result from vinculin's modulation of paxillin–FAK interactions. A vinculin fragment (amino acids 811–1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin−/− cells. Both vinY822F and vin−/− cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin−/− and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction. PMID:15138291

  1. Pharmacological blockade of FAK autophosphorylation decreases human glioblastoma tumor growth and synergizes with temozolomide

    Science.gov (United States)

    Golubovskaya, Vita M.; Huang, Grace; Ho, Baotran; Yemma, Michael; Morrison, Carl D.; Lee, Jisook; Eliceiri, Brian P.; Cance, William G.

    2012-01-01

    Malignant gliomas are characterized by aggressive tumor growth with a mean survival of 15–18 months and frequently developed resistance to temozolomide. Therefore, strategies that sensitize glioma cells to temozolomide have a high translational impact. We have studied focal adhesion kinase (FAK), a tyrosine kinase and emerging therapeutic target that is known to be highly expressed and activated in glioma. In this report we tested the FAK autophosphorylation inhibitor, Y15 in DBTRG and U87 glioblastoma cells. Y15 significantly decreased viability and clonogenicity in a dose-dependent manner, increased detachment in a dose and time-dependent manner, caused apoptosis and inhibited cell invasion in both cell lines. In addition, Y15 treatment decreased autophosphorylation of FAK in a dose-dependent manner and changed cell morphology by causing cell rounding in DBTRG and U87 cells. Administration of Y15 significantly decreased subcutaneous DBTRG tumor growth with decreased Y397-FAK autophosphorylation, activated caspase-3 and PARP. Y15 was administered in an orthotopic glioma model, leading to an increase in mouse survival. The combination of Y15 with temozolomide was more effective than either agent alone in decreasing viability and activating caspase-8 in DBTRG and U87 cells in vitro. In addition, the combination of Y15 and temozolomide synergistically blocked U87 brain tumor growth in vivo. Thus, pharmacologic blockade of FAK autophosphorylation with the oral administration of a small molecule inhibitor Y15 has a potential to be an effective therapy approach for glioblastoma either alone or in combination with chemotherapy agents such as temozolomide. PMID:23243059

  2. Pharmacologic blockade of FAK autophosphorylation decreases human glioblastoma tumor growth and synergizes with temozolomide.

    Science.gov (United States)

    Golubovskaya, Vita M; Huang, Grace; Ho, Baotran; Yemma, Michael; Morrison, Carl D; Lee, Jisook; Eliceiri, Brian P; Cance, William G

    2013-02-01

    Malignant gliomas are characterized by aggressive tumor growth with a mean survival of 15 to 18 months and frequently developed resistance to temozolomide. Therefore, strategies that sensitize glioma cells to temozolomide have a high translational impact. We have studied focal adhesion kinase (FAK), a tyrosine kinase and emerging therapeutic target that is known to be highly expressed and activated in glioma. In this report, we tested the FAK autophosphorylation inhibitor, Y15, in DBTRG and U87 glioblastoma cells. Y15 significantly decreased viability and clonogenicity in a dose-dependent manner, increased detachment in a dose- and time-dependent manner, caused apoptosis, and inhibited cell invasion in both cell lines. In addition, Y15 treatment decreased autophosphorylation of FAK in a dose-dependent manner and changed cell morphology by causing cell rounding in DBTRG and U87 cells. Administration of Y15 significantly decreased subcutaneous DBTRG tumor growth with decreased Y397-FAK autophosphorylation, activated caspase-3 and PARP. Y15 was administered in an orthotopic glioma model, leading to an increase in mouse survival. The combination of Y15 with temozolomide was more effective than either agent alone in decreasing viability and activating caspase-8 in DBTRG and U87 cells in vitro. In addition, the combination of Y15 and temozolomide synergistically blocked U87 brain tumor growth in vivo. Thus, pharmacologic blockade of FAK autophosphorylation with the oral administration of a small-molecule inhibitor Y15 has a potential to be an effective therapy approach for glioblastoma either alone or in combination with chemotherapy agents such as temozolomide.

  3. Endothelial FAK as a therapeutic target in disease.

    Science.gov (United States)

    Infusino, Giovanni A; Jacobson, Jeffrey R

    2012-01-01

    Focal adhesions (FA) are important mediators of endothelial cytoskeletal interactions with the extracellular matrix (ECM) via transmembrane receptors, integrins and integrin-associated intracellular proteins. This communication is essential for a variety of cell processes including EC barrier regulation and is mediated by the non-receptor protein tyrosine kinase, focal adhesion kinase (FAK). As FA mediate the basic response of EC to a variety of stimuli and FAK is essential to these responses, the idea of targeting EC FAK as a therapeutic strategy for an assortment of diseases is highly promising. In particular, inhibition of FAK could prove beneficial in a variety of cancers via effects on EC proliferation and angiogenesis, in acute lung injury (ALI) via the attenuation of lung vascular permeability, and in rheumatoid arthritis via reductions in synovial angiogenesis. In addition, there are potential therapeutic benefits of FAK inhibition in cardiovascular disease and diabetic nephropathy as well. Several drugs that target EC FAK are now in existence and include agents currently under investigation in preclinical models as well as drugs that are readily available such as the sphingolipid analog FTY720 and statins. As the role of EC FAK in the pathogenesis of a variety of diseases continues to be explored and new insights are revealed, drug targeting of FAK will continue to be an important area of investigation and may ultimately lead to highly novel and effective strategies to treat these diseases.

  4. The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

    LENUS (Irish Health Repository)

    Krause-Gruszczynska, Malgorzata

    2011-12-28

    Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-\\/-, integrin-beta1-\\/-, focal adhesion kinase (FAK)-\\/- and Src\\/Yes\\/Fyn-\\/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1\\/2-\\/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker

  5. RNase L Suppresses Androgen Receptor Signaling, Cell Migration and Matrix Metalloproteinase Activity in Prostate Cancer Cells.

    Science.gov (United States)

    Dayal, Shubham; Zhou, Jun; Manivannan, Praveen; Siddiqui, Mohammad Adnan; Ahmad, Omaima Farid; Clark, Matthew; Awadia, Sahezeel; Garcia-Mata, Rafael; Shemshedini, Lirim; Malathi, Krishnamurthy

    2017-03-01

    The interferon antiviral pathways and prostate cancer genetics converge on a regulated endoribonuclease, RNase L. Positional cloning and linkage studies mapped Hereditary Prostate Cancer 1 (HPC1) to RNASEL. To date, there is no correlation of viral infections with prostate cancer, suggesting that RNase L may play additional roles in tumor suppression. Here, we demonstrate a role of RNase L as a suppressor of androgen receptor (AR) signaling, cell migration and matrix metalloproteinase activity. Using RNase L mutants, we show that its nucleolytic activity is dispensable for both AR signaling and migration. The most prevalent HPC1-associated mutations in RNase L, R462Q and E265X, enhance AR signaling and cell migration. RNase L negatively regulates cell migration and attachment on various extracellular matrices. We demonstrate that RNase L knockdown cells promote increased cell surface expression of integrin β1 which activates Focal Adhesion Kinase-Sarcoma (FAK-Src) pathway and Ras-related C3 botulinum toxin substrate 1-guanosine triphosphatase (Rac1-GTPase) activity to increase cell migration. Activity of matrix metalloproteinase (MMP)-2 and -9 is significantly increased in cells where RNase L levels are ablated. We show that mutations in RNase L found in HPC patients may promote prostate cancer by increasing expression of AR-responsive genes and cell motility and identify novel roles of RNase L as a prostate cancer susceptibility gene.

  6. 大麻素受体1、FAK mRNA在小鼠肝纤维化形成过程中肝组织中的表达及相互关系%Hepatic expression of CB1 in rats with fibrosis and the relationship with FAK

    Institute of Scientific and Technical Information of China (English)

    杨莉; 赵召霞; 侯军良; 刘玉珍; 姜惠卿; 戴二黑

    2011-01-01

    Objective To evaluate hepatic expression of CBl mRNA in rats during liver fibrogenesis and study the relationship between CB1 and FAK. Methods Liver fibrosis model was prepared by intraperitoneal injection of carbon tetrachloride ( 10% ). Liver tissues and serum samples were collected at 2, 4, 6 and 8 week. The scores of fibrosis stage (S) were performed. The mRNA levels of CB1 mRNA and FAK were determined by quantitative RT - PCR. The levels of serum TGFβ1 were detected by ELISA. Results Compared with normal control group, CBI mRNA and FAK mRNA levels in every model group were significantly increased ( P < O.05 ). With the modeling time prolonged, CB1 and FAK mRNA levels gradually increased (P <0.05 ). CB1 mRNA level in liver tissue not only had correlation with the degree of liver fibrosis ( r = O. 747, P < 0.01 ), but also with FAK mRNA level in liver tissue ( r = 0.907, P < O. 01 ). With the modeling time prolonged, serum TGFβ1 level gradually increased (P < O. 05 ). CBI mRNA level in liver tissue had positive correlation with serum TGFβ1 level ( r = O. 542, P < 0. O1 ). Conclusion CB1 may promote the activation of FAK in rats with fibrosis, and induce HSC proliferation and secrete plentiful TGFβ1 by PI3K signal transduction. CB1 may promote progression of liver fibrosis.%目的 研究大麻素受体1(CB1)mRNA在肝纤维化形成过程中表达的变化,从基因转录水平探讨其与黏着斑激酶(FAK)的关系.方法 采用10%四氯化碳腹腔注射制备肝纤维化模型,分别于造模第2、4、6、8周留取小鼠的肝组织及血清.通过肝组织病理对肝纤维化程度进行评分,荧光定量PCR检测CB1 mRNA和FAK mRNA水平,ELISA方法检测血清中转化生长因子(TGF)β1的含量.结果 与正常对照组相比,各模型组小鼠肝组织中CBI mRNA和FAK mRNA含量显著升高(P<0.05),而且随造模时间的延长,CB1 mRNA和FAK mRNA含量亦逐渐增高,各组之间差异有统计学意义(P<0.05).CBI mRNA的含

  7. The relative roles of collagen adhesive receptor DDR2 activation and matrix stiffness on the downregulation of focal adhesion kinase in vascular smooth muscle cells.

    Science.gov (United States)

    Bhadriraju, Kiran; Chung, Koo-Hyun; Spurlin, Tighe A; Haynes, Ross J; Elliott, John T; Plant, Anne L

    2009-12-01

    Cells within tissues derive mechanical anchorage and specific molecular signals from the insoluble extracellular matrix (ECM) that surrounds them. Understanding the role of different cues that extracellular matrices provide cells is critical for controlling and predicting cell response to scaffolding materials. Using an engineered extracellular matrix of Type I collagen we examined how the stiffness, supramolecular structure, and glycosylation of collagen matrices influence the protein levels of cellular FAK and the activation of myosin II. Our results show that (1) cellular FAK is downregulated on collagen fibrils, but not on a non-fibrillar monolayer of collagen, (2) the downregulation of FAK is independent of the stiffness of the collagen fibrils, and (3) FAK levels are correlated with levels of tyrosine phosphorylation of the collagen adhesion receptor DDR2. Further, siRNA depletion of DDR2 blocks FAK downregulation. Our results suggest that the collagen receptor DDR2 is involved in the regulation of FAK levels in vSMC adhered to Type I collagen matrices, and that regulation of FAK levels in these cells appears to be independent of matrix stiffness.

  8. The cysteine-cluster motif of c-Yes, Lyn and FAK as a suppressive module for the kinases.

    Science.gov (United States)

    Rahman, Mohammad Aminur; Senga, Takeshi; Oo, Myat Lin; Hasegawa, Hitoki; Biswas, Md Helal Uddin; Mon, Naing Naing; Huang, Pengyu; Ito, Satoko; Yamamoto, Tadashi; Hamaguchi, Michinari

    2008-04-01

    The Src family of non-receptor protein tyrosine kinases plays a critical role in the progression of human cancers so that the development of its specific inhibitors is important as a therapeutic tool. We previously reported that cysteine residues in the cysteine-cluster (CC) motif of v-Src were critical for the kinase inactivation by the SH-alkylating agents such as N-(9-acridinyl) maleimide (NAM), whereas other cysteine residues were dispensable. We found similar CC-motifs in other Src-family kinases and a non-Src-family kinase, FAK. In this study, we explored the function of the CC-motif in Yes, Lyn and FAK. While Src has four cysteines in the CC-motif, c-Yes and Lyn have three and two of the four cysteines, respectively. Two conserved cysteines of the Src family kinases, corresponding to Cys487 and Cys498 of Src, were essential for the resistance to the inactivation of the kinase activity by NAM, whereas the first cysteine of c-Yes, which is absent in Lyn, was less important. FAK has similar CC-motifs with two cysteines and both cysteines were again essential for the resistance to the inactivation of the kinase activity by NAM. Taken together, modification of cysteine residues of the CC-motif causes a repressor effect on the catalytic activity of the Src family kinases and FAK.

  9. Gene Expression Profiling Identifies Important Genes Affected by R2 Compound Disrupting FAK and P53 Complex

    Directory of Open Access Journals (Sweden)

    Vita M. Golubovskaya

    2014-01-01

    Full Text Available Focal Adhesion Kinase (FAK is a non-receptor kinase that plays an important role in many cellular processes: adhesion, proliferation, invasion, angiogenesis, metastasis and survival. Recently, we have shown that Roslin 2 or R2 (1-benzyl-15,3,5,7-tetraazatricyclo[3.3.1.1~3,7~]decane compound disrupts FAK and p53 proteins, activates p53 transcriptional activity, and blocks tumor growth. In this report we performed a microarray gene expression analysis of R2-treated HCT116 p53+/+ and p53−/− cells and detected 1484 genes that were significantly up- or down-regulated (p < 0.05 in HCT116 p53+/+ cells but not in p53−/− cells. Among up-regulated genes in HCT p53+/+ cells we detected critical p53 targets: Mdm-2, Noxa-1, and RIP1. Among down-regulated genes, Met, PLK2, KIF14, BIRC2 and other genes were identified. In addition, a combination of R2 compound with M13 compound that disrupts FAK and Mmd-2 complex or R2 and Nutlin-1 that disrupts Mdm-2 and p53 decreased clonogenicity of HCT116 p53+/+ colon cancer cells more significantly than each agent alone in a p53-dependent manner. Thus, the report detects gene expression profile in response to R2 treatment and demonstrates that the combination of drugs targeting FAK, Mdm-2, and p53 can be a novel therapy approach.

  10. The Expression of Integrinβ1 and FAK in Pituitary Adenomas and Their Correlation with Invasiveness

    Institute of Scientific and Technical Information of China (English)

    Feng WAN; Kai SHU; Ting LEI; Delin XUE

    2008-01-01

    Summary: The expression and possible role of integrin-focal adhesion kinase signal pathway in invasive pituitary adenomas were explored. Forty-nine human pituitary adenomas were detected for the expression of integrinβ1 (INTβ1) and focal adhesion kinase (FAK) by immunohistochemistry, and their correlation with the invasiveness of pituitary adenomas as well as between themselves was analyzed. The results showed that INTβ1 was expressed in 46 cases (93.9%) and FAK in 36 cases (73.5%), respectively, and their expression levels were highly correlated with tumor invasiveness, but not with the tumor types. It was suggested that the integrin-focal adhesion kinase signal pathway plays a role in the invasiveness of pituitary adenomas.

  11. Brain-derived neurotrophic factor induces migration of endothelial cells through a TrkB-ERK-integrin αVβ3-FAK cascade.

    Science.gov (United States)

    Matsuda, Shinji; Fujita, Tsuyoshi; Kajiya, Mikihito; Takeda, Katsuhiro; Shiba, Hideki; Kawaguchi, Hiroyuki; Kurihara, Hidemi

    2012-05-01

    Brain-derived neurotrophic factor (BDNF) promotes the regeneration of periodontal tissue. Since angiogenesis is important for tissue regeneration, investigating effect of BDNF on endothelial cell function may help to reveal its mechanism, whereby, BDNF promotes periodontal tissue regeneration. In this study, we examined the influence of BDNF on migration in human microvascular endothelial cells (HMVECs), focusing on the effects on extracellular signal-regulated kinase (ERK), integrin α(V)β(3), and focal adhesion kinase (FAK). The migration of endothelial cells was assessed with a modified Boyden chamber and a wound healing assay. The expression of integrin α(V)β(3) and the phosphorylation of ERK and FAK were analyzed by immunoblotting and immunofluorescence microscopy. BDNF (25 ng/ml) induced cell migration. PD98059, an ERK inhibitor, K252a, a specific inhibitor for TrkB, a high affinity receptor of BDNF, and an anti-integrin α(V)β(3) antibody suppressed the BDNF-induced migration. BDNF increased the levels of integrin α(V)β(3) and phosphorylated ERK1/2 and FAK. The ERK inhibitor and TrkB inhibitor also reduced levels of integrin α(V)β(3) and phosphorylated FAK. We propose that BDNF stimulates endothelial cell migration by a process involving TrkB/ERK/integrin α(V)β(3)/FAK, and this may help to enhance the regeneration of periodontal tissue.

  12. CDPK Activation in PRR Signaling.

    Science.gov (United States)

    Seybold, Heike; Boudsocq, Marie; Romeis, Tina

    2017-01-01

    Calcium-dependent protein kinases undergo a rapid biochemical activation in response to an intracellular Ca increase induced by the PRR-dependent perception of a pathogen-related stimulus. Based on SDS gel resolution, the in-gel kinase assay allows the analysis of multiple in vivo protein samples in parallel, combining the advantage of protein separation according to molecular mass with the activity read-out of a protein kinase assay. It thus enables to follow the transient CDPK activation and inactivation in response to in vivo elicitation with a time-wise resolution. In addition, changes of CDPK phosphorylation activity often correlate with slight shifts in the enzyme's apparent molecular mass, indicating posttranslational modifications and a conformational change of the active enzyme compared to its inactive resting form. These band shifts can be detected by a simple immunoblotting to monitor CDPK activation.

  13. Research progress on focal adhesion kinase in malignant tumors%FAK 在恶性肿瘤中的研究进展

    Institute of Scientific and Technical Information of China (English)

    赵美娜(综述); 陈公琰(审校)

    2016-01-01

    Focal adhesion kinase( FAK) ,a cytoplasmic non-receptor protein tyrosine kinase,serves as both a molecular scaffold and a mediator participating in multiple signal transduction pathways.FAK is involved in tumor cell survival,proliferation,migration and metastasis.Recent studies have shown that FAK is expressed in many tumor cells.Currently,FAK has been regarded as a potential target for cancer therapy.This review is to summarize the relationship between FAK and tumor progression.%黏着斑激酶( Focal adhesion kinase,FAK)是细胞内重要的骨架蛋白,属于一种非受体型酪氨酸蛋白激酶,也是多种信号通路的关键性分子。在肿瘤发生、发展、迁移以及侵袭的各个阶段FAK都具有重要作用。近年来,人们对FAK的研究越来越多,综合国内外研究资料表明,FAK在许多肿瘤组织中表达增高,提示FAK可能与肿瘤的发生发展密切相关,可能是肿瘤治疗的潜在靶点。该综述将对FAK分子结构及功能特点,与肿瘤的关系进行系统阐述。

  14. Overexpression of integrin αv facilitates proliferation and invasion of oral squamous cell carcinoma cells via MEK/ERK signaling pathway that is activated by interaction of integrin αvβ8 with type Ⅰ collagen.

    Science.gov (United States)

    Hayashido, Yasutaka; Kitano, Hisataka; Sakaue, Taishi; Fujii, Takahiko; Suematsu, Mirei; Sakurai, Shigeru; Okamoto, Tetsuji

    2014-11-01

    To examine the role of integrin αv subunit in the progression of squamous cell carcinoma (SCC), oral SCC cells were stably transfected with integrin αv cDNA. Integrin αv transfectants exhibited the enhancement of proliferation on type Ⅰ collagen, and seemed to have a high ability to invade type Ⅰ collagen gel. Overexpression of integrin αv led to rapid phosphorylation of focal adhesion kinase (FAK), mitogen‑activated protein kinase kinase 1/2 (MEK1/2) and extracellular signal‑regulated kinase 1/2 (ERK1/2) in SCC cells on type Ⅰ collagen. The downregulation of integrin β8 in integrin αv transfectants by its specific antisense oligonucleotide led to a decrease in type Ⅰ collagen‑stimulated activation of FAK and the MEK/ERK signaling pathway, and also suppressed the proliferation on type Ⅰ collagen and the invasiveness into type Ⅰ collagen gel. Moreover, the expression of integrin β8 was induced following transfection with integrin αv cDNA. These results indicated that the overexpression of integrin αv induces integrin αvβ8 heterodimer formation and the binding of integrin αvβ8 to type Ⅰ collagen might enhance the proliferation and invasion of SCC cells via the activation of the MEK/ERK signaling pathway.

  15. The Src kinase Yes is activated in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters, but not pancreatic growth factors, which stimulate its association with numerous other signaling molecules.

    Science.gov (United States)

    Sancho, Veronica; Nuche-Berenguer, Bernardo; Jensen, R T

    2012-08-01

    For growth factors, cytokines, G-protein-coupled receptors and numerous other stimuli, the Src Family of kinases (SFK) play a central signaling role. SFKs also play an important role in pancreatic acinar cell function including metabolism, secretion, endocytosis, growth and cytoskeletal integrity, although the specific SFKs involved are not fully known. In the present study we used specific antibodies for the SFK, Yes, to determine its presence, activation by pancreatic secretagogues or growth factors, and interaction with cellular signaling cascades mediated by CCK in which Yes participates in to cause acinar cell responses. Yes was identified in acini and secretagogues known to activate phospholipase C (PLC) [CCK, carbachol, bombesin] as well as post-receptor stimulants activating PKC [TPA] or mobilizing cellular calcium [thapsigargin/calcium ionophore (A23187)] each activated Yes. Secretin, which activates adenylate cyclase did not stimulate Yes, nor did pancreatic growth factors. CCK activation of Yes required both high- and low-affinity CCK(1)-receptor states. TPA-/CCK-stimulated Yes activation was completely inhibited by thapsigargin and the PKC inhibitor, GF109203X. CCK/TPA stimulated the association of Yes with focal adhesion kinases (Pyk2, FAK) and its autophosphorylated forms (pY397FAK, pY402Pyk2). Moreover, CCK/TPA stimulated Yes interacted with a number of other signaling proteins, including Shc, PKD, p130(Cas), PI3K and PTEN. This study demonstrates that in rat pancreatic acini, the SFK member Yes is expressed and activated by CCK and other gastrointestinal hormones/neurotransmitters. Because its activation results in the direct activation of many cellular signaling cascades that have been shown to mediate CCK's effect in acinar cell function our results suggest that it is one of the important pancreatic SFKs mediating these effects.

  16. Gene Expression Profiling Identifies Important Genes Affected by R2 Compound Disrupting FAK and P53 Complex

    Energy Technology Data Exchange (ETDEWEB)

    Golubovskaya, Vita M., E-mail: Vita.Golubovskaya@roswellpark.org; Ho, Baotran [Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States); Conroy, Jeffrey [Genomics Shared Resource, Center for Personalized Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States); Liu, Song; Wang, Dan [Bioinformatics Core Facility, Biostatistics, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States); Cance, William G. [Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States)

    2014-01-21

    Focal Adhesion Kinase (FAK) is a non-receptor kinase that plays an important role in many cellular processes: adhesion, proliferation, invasion, angiogenesis, metastasis and survival. Recently, we have shown that Roslin 2 or R2 (1-benzyl-15,3,5,7-tetraazatricyclo[3.3.1.1~3,7~]decane) compound disrupts FAK and p53 proteins, activates p53 transcriptional activity, and blocks tumor growth. In this report we performed a microarray gene expression analysis of R2-treated HCT116 p53{sup +/+} and p53{sup −/−} cells and detected 1484 genes that were significantly up- or down-regulated (p < 0.05) in HCT116 p53{sup +/+} cells but not in p53{sup −/−} cells. Among up-regulated genes in HCT p53{sup +/+} cells we detected critical p53 targets: Mdm-2, Noxa-1, and RIP1. Among down-regulated genes, Met, PLK2, KIF14, BIRC2 and other genes were identified. In addition, a combination of R2 compound with M13 compound that disrupts FAK and Mmd-2 complex or R2 and Nutlin-1 that disrupts Mdm-2 and p53 decreased clonogenicity of HCT116 p53{sup +/+} colon cancer cells more significantly than each agent alone in a p53-dependent manner. Thus, the report detects gene expression profile in response to R2 treatment and demonstrates that the combination of drugs targeting FAK, Mdm-2, and p53 can be a novel therapy approach.

  17. FAK Inhibition Decreases Hepatoblastoma Survival Both In Vitro and In Vivo12

    Science.gov (United States)

    Gillory, Lauren A; Stewart, Jerry E; Megison, Michael L; Nabers, Hugh C; Mroczek-Musulman, Elizabeth; Beierle, Elizabeth A

    2013-01-01

    Hepatoblastoma is the most frequently diagnosed liver tumor of childhood, and children with advanced, metastatic or relapsed disease have a disease-free survival rate under 50%. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. FAK has been found in other pediatric solid tumors and in adult hepatocellular carcinoma, leading us to hypothesize that FAK would be present in hepatoblastoma and would impact its cellular survival. In the current study, we showed that FAK was present and phosphorylated in human hepatoblastoma tumor specimens. We also examined the effects of FAK inhibition upon hepatoblastoma cells using a number of parallel approaches to block FAK including RNAi and small molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion, and migration and increased apoptosis. Further, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse xenograft model of hepatoblastoma. The findings from this study will help to further our understanding of the regulation of hepatoblastoma tumorigenesis and may provide desperately needed novel therapeutic strategies and targets for aggressive, recurrent, or metastatic hepatoblastomas. PMID:23544173

  18. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol

    Science.gov (United States)

    Donejko, Magdalena; Rysiak, Edyta; Galicka, Elżbieta; Terlikowski, Robert; Głażewska, Edyta Katarzyna; Przylipiak, Andrzej

    2017-01-01

    Aim The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA) on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK), and the influence of HA on those processes. Materials and methods Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Results Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. Conclusion This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol. PMID:28293103

  19. Integrin activation and focal complex formation in cardiac hypertrophy

    Science.gov (United States)

    Laser, M.; Willey, C. D.; Jiang, W.; Cooper, G. 4th; Menick, D. R.; Zile, M. R.; Kuppuswamy, D.

    2000-01-01

    Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.

  20. Anti-tumor effect in human breast cancer by TAE226, a dual inhibitor for FAK and IGF-IR in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Kurio, Naito [Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700-8525 (Japan); Shimo, Tsuyoshi, E-mail: shimotsu@md.okayama-u.ac.jp [Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700-8525 (Japan); Fukazawa, Takuya; Takaoka, Munenori [Department of General Surgery, Kawasaki Medical School, Okayama, 700-0821 (Japan); Okui, Tatsuo; Hassan, Nur Mohammad Monsur; Honami, Tatsuki [Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700-8525 (Japan); Hatakeyama, Shinji [Novartis Institutes for BioMedical Research, Basel (Switzerland); Ikeda, Masahiko [Department of Surgery, Fukuyama City Hospital, Fukuyama, 720-8511 (Japan); Naomoto, Yoshio [Department of General Surgery, Kawasaki Medical School, Okayama, 700-0821 (Japan); Sasaki, Akira [Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700-8525 (Japan)

    2011-05-01

    Focal adhesion kinase (FAK) is a 125-kDa non-receptor type tyrosine kinase that localizes to focal adhesions. FAK overexpression is frequently found in invasive and metastatic cancers of the breast, colon, thyroid, and prostate, but its role in osteolytic metastasis is not well understood. In this study, we have analyzed anti-tumor effects of the novel FAK Tyr{sup 397} inhibitor TAE226 against bone metastasis in breast cancer by using TAE226. Oral administration of TAE226 in mice significantly decreased bone metastasis and osteoclasts involved which were induced by MDA-MB-231 breast cancer cells and increased the survival rate of the mouse models of bone metastasis. TAE226 also suppressed the growth of subcutaneous tumors in vivo and the proliferation and migration of MDA-MB-231 cells in vitro. Significantly, TAE226 inhibited the osteoclast formation in murine pre-osteoclastic RAW264.7 cells, and actin ring and pit formation in mature osteoclasts. Moreover, TAE226 inhibited the receptor activator for nuclear factor {kappa} B Ligand (RANKL) gene expression induced by parathyroid hormone-related protein (PTHrP) in bone stromal ST2 cells and blood free calcium concentration induced by PTHrP administration in vivo. These findings suggest that FAK was critically involved in osteolytic metastasis and activated in tumors, pre-osteoclasts, mature osteoclasts, and bone stromal cells and TAE226 can be effectively used for the treatment of cancer induced bone metastasis and other bone diseases.

  1. Lung carcinoma signaling pathways activated by smoking

    Institute of Scientific and Technical Information of China (English)

    Jing Wen; Jian-Hua Fu; Wei Zhang; Ming Guo

    2011-01-01

    Lung cancer is the leading cause of cancer death in men and women worldwide, with over a million deaths annually. Tobacco smoke is the major etiologic risk factor for lung cancer in current or previous smokers and has been strongly related to certain types of lung cancer, such as small cell lung carcinoma and squamous cell lung carcinoma. In recent years, there has been an increased incidence of lung adenocarcinoma. This change is strongly associated with changes in smoking behavior and cigarette design. Carcinogens present in tobacco products and their intermediate metabolites can activate multiple signaling pathways that contribute to lung cancer carcinogenesis. In this review, we summarize the smoking-activated signaling pathways involved in lung cancer.

  2. αvβ5 Integrin/FAK/PGC-1α Pathway Confers Protective Effects on Retinal Pigment Epithelium.

    Directory of Open Access Journals (Sweden)

    Murilo F Roggia

    Full Text Available To elucidate the mechanism of the induction of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α by photoreceptor outer segments (POS and its effects on retinal pigment epithelium (RPE.PGC-1α upregulation by POS was confirmed in ARPE-19 cells and in RPE ex vivo. To elucidate the mechanism, siRNAs against β5 integrin, CD36, Mer tyrosine kinase (MerTK, and Atg5, blocking antibodies against CD36 and MerTK, and a specific inhibitor for focal adhesion kinase (FAK were used. We examined the effect of POS-induced PGC-1α upregulation on the levels of reactive oxygen species (ROS, mitochondrial biogenesis, senescence-associated β-galactosidase (SA-β-gal after H2O2 treatment, and lysosomal activity. Lysosomal activity was evaluated through transcriptional factor EB and its target genes, and the activity of cathepsin D. Lipid metabolism after POS treatment was assessed using Oil Red O and BODIPY C11. RPE phenotypes of PGC-1α-deficient mice were examined.POS-induced PGC-1α upregulation was suppressed by siRNA against β5 integrin and a FAK inhibitor. siRNAs and blocking antibodies against CD36 and MerTK enhanced the effect of POS on PGC-1α. The upregulation of PGC-1α increased the levels of mRNA for antioxidant enzymes and stimulated mitochondrial biogenesis, decreased ROS levels, and reduced SA-β-gal staining in H2O2-treated ARPE-19 cells. PGC-1α was critical for lysosomal activity and lipid metabolism after POS treatment. PGC-1α-deficient mice demonstrated an accumulation of type 2 lysosomes in RPE, thickening of Bruch's membrane, and poor choriocapillaris vasculature.The binding, but not the internalization of POS confers protective effects on RPE cells through the αvβ5 integrin/FAK/PGC-1α pathway.

  3. Cell Volume Regulation and Signaling in 3T3-L1 Pre-adipocytes and Adipocytes

    DEFF Research Database (Denmark)

    Eduardsen, Kathrine; Larsen, Susanne; Novak, Ivana;

    2011-01-01

    for either RVD or RVI in pre-adipocytes. The insulin receptor (InsR) localizes to caveolae and its expression dramatically increases upon adipocyte differentiation. In pre-adipocytes, InsR and its effectors focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK1/2) localized to focal...... adhesions and were activated by a 5 min exposure to insulin (100 nM). Osmotic shrinkage transiently inhibited InsR Y(146)-phosphorylation, followed by an increase at t=15 min; a similar pattern was seen for ERK1/2 and FAK, in a manner unaffected by cholesterol depletion. In contrast, cell swelling had...... is not required for volume regulation. Given the relationship between hyperosmotic stress and insulin signaling, the finding that cell volume regulation is dramatically altered upon adipocyte differentiation may be relevant for the understanding of insulin resistance and metabolic syndrome....

  4. Disentangling stellar activity and planetary signals

    CERN Document Server

    Boisse, I; Hebrard, G; Bonfils, X; Santos, N C; Vauclair, S

    2010-01-01

    Photospheric stellar activity might be an important source of noise and confusion in the radial-velocity measurements. RV planet search surveys as well as follow-up of photometric transit surveys require a deeper understanding and characterization of the effects of stellar activities to disentangle it from planetary signals. We simulate dark spots on a rotating stellar photosphere. The variations of the photometry, RV and spectral line shapes are characterized and analyzed according to the stellar inclination, the latitude and the number of spots. The Lomb-Scargle periodograms of the RV variations induced by activity present power at the rotational period Prot of the star and its two-first harmonics Prot/2 and Prot/3. Three adjusted sinusoids fixed at Prot and its two-first harmonics allow to remove about 90% of the RV jitter amplitude. We apply and validate our approach on four known active planet-host stars: HD189733, GJ674, CoRoT-7 and iHor. We succeed in fitting simultaneously activity and planetary signa...

  5. [Study on FAK regulation of migration of vascular endothelial cells depending upon focal adhesion proteins].

    Science.gov (United States)

    Gao, Min; Liu, Xiaoheng; Sun, Heng; Ren, Hongyi; Wang, Lijuan; Shen, Yang

    2013-06-01

    Tumor angiogenesis induced by vascular endothelial cells (VECs) migration is a necessary condition for tumor growth and metastasis. The purpose of this study is to investigate the effect of focal adhesion kinase (FAK) inhibitor (50nmol/mL) on the adhesion and migration of endothelial cells(ECs) and the expression of focal adhesion proteins vinculin, talin and paxillin. Scratch wound migration assay was performed to examine the effect of FAK inhibitor with 50nmol/mL on ECs migration at 0, 5, 10, 30, 60 and 120min, respectively. And immunofluorescence analysis was performed to detect the expression of F-actin in ECs treated with FAK inhibitor within 2h. Western blot was carried out to determine the effect of FAK inhibitor on expression of vinculin, talin and paxillin proteins. The results showed that the migration distance and the expression of F-actin in ECs treated with FAK inhibitor decreased significantly compared with that of the controls, and the level of vinculin showed no significant difference with increasing of treated time of FAK inhibitor. However, the talin and paxillin showed an identical decreasing tendency in 5-10min, but slowly going up in 30min and then after subsequently decreasing. The results of this study proved that blocking phosphorylation of FAK could inhibit VECs adhesion and migration by downregulating focal adhesion proteins so that it may inhibit tumor angiogenesis. This may provide a new approach for tumor therapy.

  6. Disentangling stellar activity and planetary signals

    Directory of Open Access Journals (Sweden)

    Santos N.C.

    2011-02-01

    Full Text Available Photospheric stellar activity (i.e. dark spots or bright plages might be an important source of noise and confusion in the radial-velocity (RV measurements. Radial-velocimetry planet search surveys as well as follow-up of photometric transit surveys require a deep understanding and precise characterization of the effects of stellar activity, in order to disentangle it from planetary signals. We simulate dark spots on a rotating stellar photosphere. The variations of the RV are characterized and analyzed according to the stellar inclination, the latitude and the number of spots. The Lomb-Scargle periodograms of the RV variations induced by activity present power at the rotational period Prot of the star and its two-first harmonics Prot/2 and Prot/3. Three adjusted sinusoids fixed at the fundamental period and its two-first harmonics allow to remove about 90% of the RV jitter amplitude. We apply and validate our approach on four known active planet-host stars: HD 189733, GJ 674, CoRoT-7 and ι Hor.

  7. Alterations in ovarian cancer cell adhesion drive taxol resistance by increasing microtubule dynamics in a FAK-dependent manner.

    Science.gov (United States)

    McGrail, Daniel J; Khambhati, Niti N; Qi, Mark X; Patel, Krishan S; Ravikumar, Nithin; Brandenburg, Chandler P; Dawson, Michelle R

    2015-04-17

    Chemorefractory ovarian cancer patients show extremely poor prognosis. Microtubule-stabilizing Taxol (paclitaxel) is a first-line treatment against ovarian cancer. Despite the close interplay between microtubules and cell adhesion, it remains unknown if chemoresistance alters the way cells adhere to their extracellular environment, a process critical for cancer metastasis. To investigate this, we isolated Taxol-resistant populations of OVCAR3 and SKOV3 ovarian cancer cell lines. Though Taxol-resistant cells neither effluxed more drug nor gained resistance to other chemotherapeutics, they did display increased microtubule dynamics. These changes in microtubule dynamics coincided with faster attachment rates and decreased adhesion strength, which correlated with increased surface β1-integrin expression and decreased focal adhesion formation, respectively. Adhesion strength correlated best with Taxol-sensitivity, and was found to be independent of microtubule polymerization but dependent on focal adhesion kinase (FAK), which was up-regulated in Taxol-resistant cells. FAK inhibition also decreased microtubule dynamics to equal levels in both populations, indicating alterations in adhesive signaling are up-stream of microtubule dynamics. Taken together, this work demonstrates that Taxol-resistance dramatically alters how ovarian cancer cells adhere to their extracellular environment causing down-stream increases in microtubule dynamics, providing a therapeutic target that may improve prognosis by not only recovering drug sensitivity, but also decreasing metastasis.

  8. The Microarray Gene Profiling Analysis of Glioblastoma Cancer Cells Reveals Genes Affected by FAK Inhibitor Y15 and Combination of Y15 and Temozolomide

    OpenAIRE

    2014-01-01

    Focal adhesion is known to be highly expressed and activated in glioma cells. Recently, we demonstrated that FAK autophosphorylation inhibitor, Y15 significantly decreased tumor growth of DBTRG and U87 cells, especially in combination with temozolomide. In the present report, we performed gene expression analysis in these cells to reveal genes affected by Y15, temozolomide and combination of Y15 and temozolomide. We tested the effect of Y15 on gene expression by Illumina Human HT12v4 microarr...

  9. CCR9 interactions support ovarian cancer cell survival and resistance to cisplatin-induced apoptosis in a PI3K-dependent and FAK-independent fashion

    Directory of Open Access Journals (Sweden)

    Johnson Erica L

    2010-06-01

    Full Text Available Abstract Background Cisplatin is more often used to treat ovarian cancer (OvCa, which provides modest survival advantage primarily due to chemo-resistance and up regulated anti-apoptotic machineries in OvCa cells. Therefore, targeting the mechanisms responsible for cisplatin resistance in OvCa cell may improve therapeutic outcomes. We have shown that ovarian cancer cells express CC chemokine receptor-9 (CCR9. Others have also shown that CCL25, the only natural ligand for CCR9, up regulates anti-apoptotic proteins in immature T lymphocytes. Hence, it is plausible that CCR9-mediated cell signals might be involved in OvCa cell survival and inhibition of cisplatin-induced apoptosis. In this study, we investigated the potential role and molecular mechanisms of CCR9-mediated inhibition of cisplatin-induced apoptosis in OvCa cells. Methods Cell proliferation, vibrant apoptosis, and TUNEL assays were performed with or without cisplatin treatment in presence or absence of CCL25 to determine the role of the CCR9-CCL25 axis in cisplatin resistance. In situ Fast Activated cell-based ELISA (FACE assays were performed to determine anti-apoptotic signaling molecules responsible for CCL25-CCR9 mediated survival. Results Our results show interactions between CCR9 and CCL25 increased anti-apoptotic signaling cascades in OvCa cells, which rescued cells from cisplatin-induced cell death. Specifically, CCL25-CCR9 interactions mediated Akt, activation as well as GSK-3β and FKHR phosphorylation in a PI3K-dependent and FAK-independent fashion. Conclusions Our results suggest the CCR9-CCL25 axis plays an important role in reducing cisplatin-induced apoptosis of OvCa cells.

  10. Targeting adhesion signaling in KRAS, LKB1 mutant lung adenocarcinoma

    Science.gov (United States)

    Konen, Jessica; Koo, Junghui; Robinson, Brian S.; Wiles, Walter Guy; Huang, Chunzi; Martin, W. David; Behera, Madhusmita; Smith, Geoffrey H.; Hill, Charles E.; Rossi, Michael R.; Sica, Gabriel L.; Rupji, Manali; Chen, Zhengjia; Kowalski, Jeanne; Kasinski, Andrea L.; Ramalingam, Suresh S.; Khuri, Fadlo R.; Marcus, Adam I.

    2017-01-01

    Loss of LKB1 activity is prevalent in KRAS mutant lung adenocarcinoma and promotes aggressive and treatment-resistant tumors. Previous studies have shown that LKB1 is a negative regulator of the focal adhesion kinase (FAK), but in vivo studies testing the efficacy of FAK inhibition in LKB1 mutant cancers are lacking. Here, we took a pharmacologic approach to show that FAK inhibition is an effective early-treatment strategy for this high-risk molecular subtype. We established a lenti-Cre–induced Kras and Lkb1 mutant genetically engineered mouse model (KLLenti) that develops 100% lung adenocarcinoma and showed that high spatiotemporal FAK activation occurs in collective invasive cells that are surrounded by high levels of collagen. Modeling invasion in 3D, loss of Lkb1, but not p53, was sufficient to drive collective invasion and collagen alignment that was highly sensitive to FAK inhibition. Treatment of early, stage-matched KLLenti tumors with FAK inhibitor monotherapy resulted in a striking effect on tumor progression, invasion, and tumor-associated collagen. Chronic treatment extended survival and impeded local lymph node spread. Lastly, we identified focally upregulated FAK and collagen-associated collective invasion in KRAS and LKB1 comutated human lung adenocarcinoma patients. Our results suggest that patients with LKB1 mutant tumors should be stratified for early treatment with FAK inhibitors.

  11. Focal adhesion kinase (FAK) mediates the induction of pro-oncogenic and fibrogenic phenotypes in hepatitis C virus (HCV)-infected cells.

    Science.gov (United States)

    Alisi, Anna; Arciello, Mario; Petrini, Stefania; Conti, Beatrice; Missale, Gabriele; Balsano, Clara

    2012-01-01

    Hepatitis C Virus (HCV) infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC). The pivotal role of hepatic stellate cells (HCSs) and extracellular matrix (ECM) in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP). These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in HSCs.

  12. Inhibition of Focal Adhesion Kinase (FAK) Leads to Abrogation of the Malignant Phenotype in Aggressive Pediatric Renal Malignancies

    Science.gov (United States)

    Megison, Michael L.; Gillory, Lauren A.; Stewart, Jerry E.; Nabers, Hugh C.; Mrozcek-Musulman, Elizabeth; Beierle, Elizabeth A.

    2014-01-01

    Despite the tremendous advances in the treatment of childhood kidney tumors, there remain subsets of pediatric renal tumors that continue to pose a therapeutic challenge, mainly malignant rhabdoid kidney tumors and non-osseous renal Ewing sarcoma. Children with advanced, metastatic or relapsed disease have a disease-free survival rate under 30%. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. FAK has been found in other pediatric solid tumors and in adult renal cellular carcinoma, leading us to hypothesize that FAK would be present in pediatric kidney tumors and would impact their cellular survival. In the current study, we showed that FAK was present and phosphorylated in pediatric kidney tumor specimens. We also examined the effects of FAK inhibition upon G401 and SK-NEP-1 cell lines utilizing a number of parallel approaches to block FAK including RNAi and small molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion and migration, and increased apoptosis. Further, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse SK-NEP-1 xenograft model. The findings from this study will help to further our understanding of the regulation of tumorigenesis in rare pediatric renal tumors, and may provide desperately needed novel therapeutic strategies and targets for these rare, but difficult to treat, malignancies. PMID:24464916

  13. PKCθ activation in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors is needed for stimulation of numerous important cellular signaling cascades

    Science.gov (United States)

    Sancho, Veronica; Berna, Marc J.; Thill, Michelle; Jensen, R. T.

    2011-01-01

    The novel PKCθ isoform is highly expressed in T-cells, brain and skeletal muscle and originally thought to have a restricted distribution. It has been extensively studied in T-cells and shown to be important for apoptosis, T-cell activation and proliferation. Recent studies showed its presence in other tissues and importance in insulin signaling, lung surfactant secretion, intestinal barrier permeability, platelet and mast-cell functions. However, little information is available for PKCθ activation by gastrointestinal(GI) hormones/neurotransmitters and growth factors. In the present study we used rat pancreatic acinar cells to explore their ability to activate PKCθ and the possible interactions with important cellular mediators of their actions. Particular attention was paid to cholecystokinin(CCK), a physiological regulator of pancreatic function and important in pathological processes affecting acinar function, like pancreatitis. PKCθ-protein/mRNA were present in the pancreatic acini, and T538-PKCθ phosphorylation/activation was stimulated only by hormones/neurotransmitters activating phospholipase C. PKCθ was activated in time- and dose-related manner by CCK, mediated 30% by high-affinity CCKA-receptor activation. CCK stimulated PKCθ translocation from cytosol to membrane. PKCθ inhibition (by pseudostrate-inhibitor or dominant negative) inhibited CCK- and TPA-stimulation of PKD, Src, RafC, PYK2, p125FAK and IKKα/β, but not basal/stimulated enzyme secretion. Also CCK- and TPA-induced PKCθ activation produced an increment in PKCθ’s direct association with AKT, RafA, RafC and Lyn. These results show for the first time PKCθ presence in pancreatic acinar cells, its activation by some GI hormones/neurotransmitters and involvement in important cell signaling pathways mediating physiological responses (enzyme secretion, proliferation, apoptosis, cytokine expression, and pathological responses like pancreatitis and cancer growth). PMID:21810446

  14. Human ECG signal parameters estimation during controlled physical activity

    Science.gov (United States)

    Maciejewski, Marcin; Surtel, Wojciech; Dzida, Grzegorz

    2015-09-01

    ECG signal parameters are commonly used indicators of human health condition. In most cases the patient should remain stationary during the examination to decrease the influence of muscle artifacts. During physical activity, the noise level increases significantly. The ECG signals were acquired during controlled physical activity on a stationary bicycle and during rest. Afterwards, the signals were processed using a method based on Pan-Tompkins algorithms to estimate their parameters and to test the method.

  15. Hypergravity Stimulates the Extracellular Matrix/Integrin-Signaling Axis and Proliferation in Primary Osteoblasts

    Science.gov (United States)

    Parra, M.; Vercoutere, W.; Roden, C.; Banerjee, I.; Krauser, W.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

    2003-01-01

    We set out to determine the molecular mechanisms involved in the proliferative response of primary rat osteoblasts to mechanical stimulation using cell culture centrifugation as a model for hypergravity. We hypothesized that this proliferative response is mediated by specific integrin/Extracellular Matrix (ECM) interactions. To investigate this question we developed a cell culture centrifuge and an automated system that performs cell fixation during hypergravity loading. We generated expression vectors for various focal adhesion and cytoskeletal proteins fused to GFP or dsRed and visualized these structures in transfected (or infected) osteoblasts. The actin cytoskeleton was also visualized using rhodamine-phalloidin staining and Focal Adhesion Kinase (FAK) levels were assessed biochemically. We observed that a 24 hour exposure to 50-g stimulated proliferation compared to the 1-g control when cells were plated on fibronectin, collagen Type I , and collagen Type IV, but not on uncoated tissue culture plastic surfaces. This proliferative response was greatest for osteoblasts grown on fibronectin (2-fold increase over 1-g control) and collagen Type I (1.4 fold increase over 1-g control), suggesting that specific matrices and integrins are involved in the signaling pathways required for proliferation. Exposing osteoblasts grown on different matrices to 10-g or 25-g showed that effects on proliferation depended on both matrix type and loading level. We found that osteoblasts exposed to a short pulse of hypergravity during adhesion spread further and had more GFP-FAK containing focal adhesions compared to their 1-g controls. While overall levels of FAK did not change, more FAK was in the active (phosphorylated) form under hypergravity than in the 1-g controls. Cytoskeletal F-actin organization into filaments was also more prominent after brief exposures to hypergravity during the first five minutes of adhesion. These results suggest that specific integrins sense

  16. Activation of DNA damage response signaling by condensed chromatin.

    Science.gov (United States)

    Burgess, Rebecca C; Burman, Bharat; Kruhlak, Michael J; Misteli, Tom

    2014-12-11

    The DNA damage response (DDR) occurs in the context of chromatin, and architectural features of chromatin have been implicated in DNA damage signaling and repair. Whereas a role of chromatin decondensation in the DDR is well established, we show here that chromatin condensation is integral to DDR signaling. We find that, in response to DNA damage chromatin regions transiently expand before undergoing extensive compaction. Using a protein-chromatin-tethering system to create defined chromatin domains, we show that interference with chromatin condensation results in failure to fully activate DDR. Conversely, forced induction of local chromatin condensation promotes ataxia telangiectasia mutated (ATM)- and ATR-dependent activation of upstream DDR signaling in a break-independent manner. Whereas persistent chromatin compaction enhanced upstream DDR signaling from irradiation-induced breaks, it reduced recovery and survival after damage. Our results demonstrate that chromatin condensation is sufficient for activation of DDR signaling and is an integral part of physiological DDR signaling.

  17. Paracrine SDF-1α signaling mediates the effects of PSCs on GEM chemoresistance through an IL-6 autocrine loop in pancreatic cancer cells.

    Science.gov (United States)

    Zhang, Hui; Wu, Huanwen; Guan, Jian; Wang, Li; Ren, Xinyu; Shi, Xiaohua; Liang, Zhiyong; Liu, Tonghua

    2015-02-20

    Pancreatic cancer exhibits the poorest prognosis among all tumors and is characterized by high resistance to the currently available chemotherapeutic agents. Our previous studies have suggested that stromal components could promote the chemoresistance of pancreatic cancer cells (PCCs). Here, we explored the roles of pancreatic stellate cells (PSCs) and the SDF-1α/CXCR4 axis in pancreatic cancer chemoresitance. Our results showed that primary PSCs typically expressed SDF-1α, whereas its receptor CXCR4 was highly expressed in PCCs. PSC-conditioned medium (PSC-CM) inhibited Gemcitabine (GEM)-induced cytotoxicity and apoptosis in the human PCC line Panc-1, which was antagonized by an SDF-1α neutralizing Ab. Recombinant human SDF-1α (rhSDF-1α) increased IL-6 expression and secretion in Panc-1 cells in a time and dose-dependent manner, and this effect was suppressed by the CXCR4 antagonist AMD3100. rhSDF-1α protected Panc-1 cells from GEM-induced apoptosis, and the protective effect was significantly reduced by blocking IL-6 using a neutralizing antibody. Moreover, rhSDF-1α increased FAK, ERK1/2, AKT and P38 phosphorylation in Panc-1 cells, and either FAK or ERK1/2 inhibition suppressed SDF-1α-upregulated IL-6 expression. SDF-1α-induced AKT activation was almost completely blocked by FAK inhibition. In conclusion, we demonstrate for the first time that PSCs promote the chemoresistance of PCCs to GEM, and this effect is mediated by paracrine SDF-1α/CXCR4 signaling-induced activation of the intracellular FAK-AKT and ERK1/2 signaling pathways and a subsequent IL-6 autocrine loop in PCCs. Our findings indicate that blocking the PSC-PCC interaction by inhibiting SDF-1α/CXCR4 signaling may be a promising therapeutic strategy for overcoming chemoresistance in pancreatic cancer.

  18. Mitogen-activated protein kinase signaling in plants

    DEFF Research Database (Denmark)

    Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

    2010-01-01

    Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses. MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation of subs...... the Arabidopsis thaliana MAPKs MPK3, 4, and 6 and MAP2Ks MKK1, 2, 4, and 5. Future work needs to focus on identifying substrates of MAPKs, and on understanding how specificity is achieved among MAPK signaling pathways....

  19. Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Pengxiang; Nedelcu, Daniel; Watanabe, Miyako; Jao, Cindy; Kim, Youngchang; Liu, Jing; Salic, Adrian

    2016-08-01

    In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol.

  20. Activation of endothelial β-catenin signaling induces heart failure

    Science.gov (United States)

    Nakagawa, Akito; Naito, Atsuhiko T.; Sumida, Tomokazu; Nomura, Seitaro; Shibamoto, Masato; Higo, Tomoaki; Okada, Katsuki; Sakai, Taku; Hashimoto, Akihito; Kuramoto, Yuki; Oka, Toru; Lee, Jong-Kook; Harada, Mutsuo; Ueda, Kazutaka; Shiojima, Ichiro; Limbourg, Florian P.; Adams, Ralf H.; Noda, Tetsuo; Sakata, Yasushi; Akazawa, Hiroshi; Komuro, Issei

    2016-01-01

    Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure. PMID:27146149

  1. Focal adhesion kinase regulates pathogen-killing capability and life span of neutrophils via mediating both adhesion-dependent and -independent cellular signals.

    Science.gov (United States)

    Kasorn, Anongnard; Alcaide, Pilar; Jia, Yonghui; Subramanian, Kulandayan K; Sarraj, Bara; Li, Yitang; Loison, Fabien; Hattori, Hidenori; Silberstein, Leslie E; Luscinskas, William F; Luo, Hongbo R

    2009-07-15

    Various neutrophil functions such as phagocytosis, superoxide production, and survival are regulated by integrin signaling. Despite the essential role of focal adhesion kinase (FAK) in mediating this signaling pathway, its exact function in neutrophils is ill defined. In this study, we investigated the role of FAK in neutrophils using a myeloid-specific conditional FAK knockout mouse. As reported in many other cell types, FAK is required for regulation of focal adhesion dynamics when neutrophils adhere to fibronectin or ICAM-1. Adhesion on VCAM-1-coated surfaces and chemotaxis after adhesion were not altered in FAK null neutrophils. In addition, we observed significant reduction in NADPH oxidase-mediated superoxide production and complement-mediated phagocytosis in FAK null neutrophils. As a result, these neutrophils displayed decreased pathogen killing capability both in vitro and in vivo in a mouse peritonitis model. In adherent cells, the defects associated with FAK deficiency are likely due to suppression of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) signaling and chemoattractant-elicited calcium signaling. Disruption of FAK also reduced chemoattractant-elicited superoxide production in suspended neutrophils in the absence of cell adhesion. This may be solely caused by suppression of PtdIns(3,4,5)P3 signaling in these cells, because the fMLP-elicited calcium signal was not altered. Consistent with decreased PtdIns(3,4,5)P3/Akt signaling in FAK null neutrophils, we also observed accelerated spontaneous death in these cells. Taken together, our results revealed previously unrecognized roles of FAK in neutrophil function and provided a potential therapeutic target for treatment of a variety of infectious and inflammatory diseases.

  2. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol

    Directory of Open Access Journals (Sweden)

    Donejko M

    2017-03-01

    Full Text Available Magdalena Donejko,1 Edyta Rysiak,2 Elżbieta Galicka,1 Robert Terlikowski,3 Edyta Katarzyna Głażewska,1 Andrzej Przylipiak1 1Department of Esthetic Medicine, 2Department of Medicinal Chemistry, Faculty of Pharmacy, 3Department of Health Restoration, Medical University of Białystok, Białystok, Poland Aim: The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK, and the influence of HA on those processes. Materials and methods: Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Results: Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. Conclusion: This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol. Keywords: apoptosis, skin fibroblast, focal adhesion kinase, hyaluronic acid, ethanol

  3. Hypotonic shock mediation by p38 MAPK, JNK, PKC, FAK, OSR1 and SPAK in osmosensing chloride secreting cells of killifish opercular epithelium

    DEFF Research Database (Denmark)

    Marshall, W. S.; Ossum, Carlo Gunnar; Hoffmann, Else Kay

    2005-01-01

    at the basolateral membrane. The protein tyrosine kinase inhibitor genistein (100 µmol l-1) inhibited Cl- secretion that was high, increased Cl- secretion that was low and reduced immunocytochemical staining for phosphorylated FAK. We present a model for rapid control of CFTR and NKCC in chloride cells that includes......: (1) activation of NKCC and CFTR via cAMP/PKA, (2) activation of NKCC by PKC, myosin light chain kinase (MLCK), p38, OSR1 and SPAK, (3) deactivation of NKCC by hypotonic cell swelling, Ca2+ and an as yet unidentified protein phosphatase and (4) involvement of protein tyrosine kinase (PTK) acting...

  4. Tetrandrine inhibits migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes through down-regulating the expressions of Rac1, Cdc42, and RhoA GTPases and activation of the PI3K/Akt and JNK signaling pathways.

    Science.gov (United States)

    Lv, Qi; Zhu, Xian-Yang; Xia, Yu-Feng; Dai, Yue; Wei, Zhi-Feng

    2015-11-01

    Tetrandrine (Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet (0.3, 1 μmol·L(-1)) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC (against PI3K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK.

  5. Activation of the ERK1/2 Signaling Pathway during the Osteogenic Differentiation of Mesenchymal Stem Cells Cultured on Substrates Modified with Various Chemical Groups

    Directory of Open Access Journals (Sweden)

    Bing Bai

    2013-01-01

    Full Text Available The current study examined the influence of culture substrates modified with the functional groups –OH, –COOH, –NH2, and –CH3 using SAMs technology, in conjunction with TAAB control, on the osteogenic differentiation of rabbit BMSCs. The CCK-8 assay revealed that BMSCs exhibited substrate-dependent cell viability. The cells plated on –NH2- and –OH-modified substrates were well spread and homogeneous, but those on the –COOH- and –CH3-modified substrates showed more rounded phenotype. The mRNA expression of BMSCs revealed that –NH2-modified substrate promoted the mRNA expression and osteogenic differentiation of the BMSCs. The contribution of ERK1/2 signaling pathway to the osteogenic differentiation of BMSCs cultured on the –NH2-modified substrate was investigated in vitro. The –NH2-modified substrate promoted the expression of integrins; the activation of FAK and ERK1/2. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked ERK1/2 activation in a dose-dependent manner, as revealed for expression of Cbfα-1 and ALP. Blockade of ERK1/2 phosphorylation in BMSCs by PD98059 suppressed osteogenic differentiation on chemical surfaces. These findings indicate a potential role for ERK in the osteogenic differentiation of BMSCs on surfaces modified by specific chemical functional groups, indicating that the microenvironment affects the differentiation of BMSCs. This observation has important implications for bone tissue engineering.

  6. Cholinergic signals in mouse barrel cortex during active whisker sensing.

    Science.gov (United States)

    Eggermann, Emmanuel; Kremer, Yves; Crochet, Sylvain; Petersen, Carl C H

    2014-12-11

    Internal brain states affect sensory perception, cognition, and learning. Many neocortical areas exhibit changes in the pattern and synchrony of neuronal activity during quiet versus active behaviors. Active behaviors are typically associated with desynchronized cortical dynamics. Increased thalamic firing contributes importantly to desynchronize mouse barrel cortex during active whisker sensing. However, a whisking-related cortical state change persists after thalamic inactivation, which is mediated at least in part by acetylcholine, as we show here by using whole-cell recordings, local pharmacology, axonal calcium imaging, and optogenetic stimulation. During whisking, we find prominent cholinergic signals in the barrel cortex, which suppress spontaneous cortical activity. The desynchronized state of barrel cortex during whisking is therefore driven by at least two distinct signals with opposing functions: increased thalamic activity driving glutamatergic excitation of the cortex and increased cholinergic input suppressing spontaneous cortical activity.

  7. Cholinergic Signals in Mouse Barrel Cortex during Active Whisker Sensing

    Directory of Open Access Journals (Sweden)

    Emmanuel Eggermann

    2014-12-01

    Full Text Available Internal brain states affect sensory perception, cognition, and learning. Many neocortical areas exhibit changes in the pattern and synchrony of neuronal activity during quiet versus active behaviors. Active behaviors are typically associated with desynchronized cortical dynamics. Increased thalamic firing contributes importantly to desynchronize mouse barrel cortex during active whisker sensing. However, a whisking-related cortical state change persists after thalamic inactivation, which is mediated at least in part by acetylcholine, as we show here by using whole-cell recordings, local pharmacology, axonal calcium imaging, and optogenetic stimulation. During whisking, we find prominent cholinergic signals in the barrel cortex, which suppress spontaneous cortical activity. The desynchronized state of barrel cortex during whisking is therefore driven by at least two distinct signals with opposing functions: increased thalamic activity driving glutamatergic excitation of the cortex and increased cholinergic input suppressing spontaneous cortical activity.

  8. Inhibition of tumor vasculogenic mimicry and prolongation of host survival in highly aggressive gallbladder cancers by norcantharidin via blocking the ephrin type a receptor 2/focal adhesion kinase/paxillin signaling pathway.

    Directory of Open Access Journals (Sweden)

    Hui Wang

    Full Text Available Vasculogenic mimicry (VM is a newly-defined tumor microcirculation pattern in highly aggressive malignant tumors. We recently reported tumor growth and VM formation of gallbladder cancers through the contribution of the ephrin type a receptor 2 (EphA2/focal adhesion kinase (FAK/Paxillin signaling pathways. In this study, we further investigated the anti-VM activity of norcantharidin (NCTD as a VM inhibitor for gallbladder cancers and the underlying mechanisms. In vivo and in vitro experiments to determine the effects of NCTD on tumor growth, host survival, VM formation of GBC-SD nude mouse xenografts, and vasculogenic-like networks, malignant phenotypes i.e., proliferation, apoptosis, invasion and migration of GBC-SD cells. Expression of VM signaling-related markers EphA2, FAK and Paxillin in vivo and in vitro were examined by immunofluorescence, western blotting and real-time polymerase chain reaction (RT-PCR, respectively. The results showed that after treatment with NCTD, GBC-SD cells were unable to form VM structures when injecting into nude mouse, growth of the xenograft was inhibited and these observations were confirmed by facts that VM formation by three-dimensional (3-D matrix, proliferation, apoptosis, invasion, migration of GBC-SD cells were affected; and survival time of the xenograft mice was prolonged. Furthermore, expression of EphA2, FAK and Paxillin proteins/mRNAs of the xenografts was downregulated. Thus, we concluded that NCTD has potential anti-VM activity against human gallbladder cancers; one of the underlying mechanisms may be via blocking the EphA2/FAK/Paxillin signaling pathway.

  9. Correlation between Gli2, FAK expression in colonic adenocarcinoma tissue with different clinical pathological characteristics and cancer cell proliferation, invasion

    Institute of Scientific and Technical Information of China (English)

    Zhe Su

    2017-01-01

    Objective:To study the correlation between glioma-associated oncogene homologue 2 (Gli2), focal adhesion kinase (FAK) expression in colonic adenocarcinoma tissue with different clinical pathological characteristics and cancer cell proliferation, invasion.Methods: 56 patients with colonic adenocarcinoma who received surgical resection in our hospital between May 2012 and December 2015 were selected, cancer tissue and para-carcinoma tissue were collected respectively, immunohistochemical staining was used to detect the Gli2 and FAK protein-positive rate, and fluorescence quantitative PCR was used to determine the mRNA expression of Gli2 and FAK as well as the proliferation and invasionn genes.Results:Gli2 and FAK mRNA expression and protein-positive rate in colonic adenocarcinoma tissues were significantly higher than those in para-carcinoma tissues (P<0.05); Gli2 and FAK mRNA expression and protein-positive rate in colonic adenocarcinoma tissues with low differentiation, no differentiation, extraserosal infiltration and Dukes stage D were significantly higher than those in colonic adenocarcinoma tissues with high differentiation, medium differentiation, intraserosal infiltration, Dukes stage B-C (P<0.05); CyclinD1, CDK4, c-myc, N-cadherin and vimentin mRNA expression in Gli2- and FAK-positive colonic adenocarcinoma tissues were significantly higher than those in Gli2- and FAK-negative colonic adenocarcinoma tissues (P<0.05).Conclusions:Gli2 and FAK expression are high in colonic adenocarcinoma tissues and associated with the clinical pathological staging of tumor, and highly expressed Gli2 and FAK can promote cell proliferation and invasion.

  10. Coco is a dual activity modulator of TGFβ signaling

    Science.gov (United States)

    Deglincerti, Alessia; Haremaki, Tomomi; Warmflash, Aryeh; Sorre, Benoit; Brivanlou, Ali H.

    2015-01-01

    The TGFβ signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFβ ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFβ1 signaling. Coco synergizes with TGFβ1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFβ1 and enhances TGFβ1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFβ signaling. PMID:26116664

  11. Modeled Microgravity Disrupts Collagen I/Integrin Signaling During Osteoblastic Differentiation of Human Mesenchymal Stem Cells

    Science.gov (United States)

    Meyers, Valerie E.; Zayzafoon, Majd; Gonda, Steven R.; Gathings, William E.; McDonald, Jay M.

    2004-01-01

    Spaceflight leads to reduced bone mineral density in weight bearing bones that is primarily attributed to a reduction in bone formation. We have previously demonstrated severely reduced osteoblastogenesis of human mesenchymal stem cells (hMSC) following seven days culture in modeled microgravity. One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of modeled microgravity on integrin expression and function in hMSC. We demonstrate that seven days of culture in modeled microgravity leads to reduced expression of the extracellular matrix protein, type I collagen (Col I). Conversely, modeled microgravity consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin sub-unit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-MAPK pathway is evidenced by a reduction in Ras and ERK activation. Taken together, our findings indicate that modeled microgravity decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.

  12. Modulation of β-catenin signaling by glucagon receptor activation.

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    Jiyuan Ke

    Full Text Available The glucagon receptor (GCGR is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R and glucagon-like peptide 1 (GLP-1R receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5 is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1 or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

  13. Digital signaling decouples activation probability and population heterogeneity

    DEFF Research Database (Denmark)

    Kellogg, Ryan A; Tian, Chengzhe; Lipniacki, Tomasz;

    2015-01-01

    Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-κB system, we studied transcription factor dynamic...... and uniform dynamics. These results show that digital NF-κB signaling enables multidimensional control of cellular phenotype via input profile, allowing parallel and independent control of single-cell activation probability and population heterogeneity....

  14. Cellular phosphatases facilitate combinatorial processing of receptor-activated signals

    Directory of Open Access Journals (Sweden)

    Siddiqui Zaved

    2008-09-01

    Full Text Available Abstract Background Although reciprocal regulation of protein phosphorylation represents a key aspect of signal transduction, a larger perspective on how these various interactions integrate to contribute towards signal processing is presently unclear. For example, a key unanswered question is that of how phosphatase-mediated regulation of phosphorylation at the individual nodes of the signaling network translates into modulation of the net signal output and, thereby, the cellular phenotypic response. Results To address the above question we, in the present study, examined the dynamics of signaling from the B cell antigen receptor (BCR under conditions where individual cellular phosphatases were selectively depleted by siRNA. Results from such experiments revealed a highly enmeshed structure for the signaling network where each signaling node was linked to multiple phosphatases on the one hand, and each phosphatase to several nodes on the other. This resulted in a configuration where individual signaling intermediates could be influenced by a spectrum of regulatory phosphatases, but with the composition of the spectrum differing from one intermediate to another. Consequently, each node differentially experienced perturbations in phosphatase activity, yielding a unique fingerprint of nodal signals characteristic to that perturbation. This heterogeneity in nodal experiences, to a given perturbation, led to combinatorial manipulation of the corresponding signaling axes for the downstream transcription factors. Conclusion Our cumulative results reveal that it is the tight integration of phosphatases into the signaling network that provides the plasticity by which perturbation-specific information can be transmitted in the form of a multivariate output to the downstream transcription factor network. This output in turn specifies a context-defined response, when translated into the resulting gene expression profile.

  15. Adipocyte activation of cancer stem cell signaling in breast cancer

    Institute of Scientific and Technical Information of China (English)

    Benjamin; Wolfson; Gabriel; Eades; Qun; Zhou

    2015-01-01

    Signaling within the tumor microenvironment has a critical role in cancer initiation and progression. Adipocytes, one of the major components of the breast microenvironment,have been shown to provide pro-tumorigenic signals that promote cancer cell proliferation and invasiveness in vitro and tumorigenicity in vivo. Adipocyte secreted factors such as leptin and interleukin-6(IL-6) have a paracrine effect on breast cancer cells. In adipocyte-adjacent breast cancer cells, the leptin and IL-6 signaling pathways activate janus kinase 2/signal transducer and activatorof transcription 5, promoting the epithelial-mesenchymal transition, and upregulating stemness regulators such as Notch, Wnt and the Sex determining region Y-box 2/octamer binding transcription factor 4/Nanog signaling axis. In this review we will summarize the major signaling pathways that regulate cancer stem cells in breast cancer and describe the effects that adipocyte secreted IL-6 and leptin have on breast cancer stem cell signaling. Finally we will introduce a new potential treatment paradigm of inhibiting the adipocyte-breast cancer cell signaling via targeting the IL-6 or leptin pathways.

  16. Determination of strongly overlapping signaling activity from microarray data

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    Bidaut Ghislain

    2006-02-01

    Full Text Available Abstract Background As numerous diseases involve errors in signal transduction, modern therapeutics often target proteins involved in cellular signaling. Interpretation of the activity of signaling pathways during disease development or therapeutic intervention would assist in drug development, design of therapy, and target identification. Microarrays provide a global measure of cellular response, however linking these responses to signaling pathways requires an analytic approach tuned to the underlying biology. An ongoing issue in pattern recognition in microarrays has been how to determine the number of patterns (or clusters to use for data interpretation, and this is a critical issue as measures of statistical significance in gene ontology or pathways rely on proper separation of genes into groups. Results Here we introduce a method relying on gene annotation coupled to decompositional analysis of global gene expression data that allows us to estimate specific activity on strongly coupled signaling pathways and, in some cases, activity of specific signaling proteins. We demonstrate the technique using the Rosetta yeast deletion mutant data set, decompositional analysis by Bayesian Decomposition, and annotation analysis using ClutrFree. We determined from measurements of gene persistence in patterns across multiple potential dimensionalities that 15 basis vectors provides the correct dimensionality for interpreting the data. Using gene ontology and data on gene regulation in the Saccharomyces Genome Database, we identified the transcriptional signatures of several cellular processes in yeast, including cell wall creation, ribosomal disruption, chemical blocking of protein synthesis, and, criticially, individual signatures of the strongly coupled mating and filamentation pathways. Conclusion This works demonstrates that microarray data can provide downstream indicators of pathway activity either through use of gene ontology or transcription

  17. A small physiological electric field mediated responses of extravillous trophoblasts derived from HTR8/SVneo cells: involvement of activation of focal adhesion kinase signaling.

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    Juan Zhang

    Full Text Available Moderate invasion of trophoblast cells into endometrium is essential for the placental development and normal pregnancy. Electric field (EF-induced effects on cellular behaviors have been observed in many cell types. This study was to investigate the effect of physiological direct current EF (dc EF on cellular responses such as elongation, orientation and motility of trophoblast cells. Immortalized first trimester extravillous trophoblast cells (HTR-8/SVneo were exposed to the dc EF at physiological magnitude. Cell images were recorded and analyzed by image analyzer. Cell lysates were used to detect protein expression by Western blot. Cultured in the dc EFs the cells showed elongation, orientation and enhanced migration rate compared with non-EF stimulated cells at field strengths of 100 mV/mm to 200 mV/mm. EF exposure increased focal adhesion kinase (FAK phosphorylation in a time-dependent manner and increased expression levels of MMP-2. Pharmacological inhibition of FAK impaired the EF-induced responses including motility and abrogated the elevation of MMP-2 expression. However, the expression levels of integrins like integrin α1, α5, αV and β1 were not affected by EF stimulation. Our results demonstrate the importance of FAK activation in migration/motility of trophobalst cells driven by EFs. In addition, it raises the feasibility of using applied EFs to promote placentation through effects on trophoblast cells.

  18. Focal adhesion kinase (FAK mediates the induction of pro-oncogenic and fibrogenic phenotypes in hepatitis C virus (HCV-infected cells.

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    Anna Alisi

    Full Text Available Hepatitis C Virus (HCV infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC. The pivotal role of hepatic stellate cells (HCSs and extracellular matrix (ECM in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP. These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in

  19. Active Finger Recognition from Surface EMG Signal Using Bayesian Filter

    Science.gov (United States)

    Araki, Nozomu; Hoashi, Yuki; Konishi, Yasuo; Mabuchi, Kunihiko; Ishigaki, Hiroyuki

    This paper proposed an active finger recognition method using Bayesian filter in order to control a myoelectric hand. We have previously proposed a finger joint angle estimation method based on measured surface electromyography (EMG) signals and a linear model. However, when we estimate 2 or more finger angles by this estimation method, the estimation angle of the inactive finger is not accurate. This is caused by interference of surface EMG signal. To solve this interference problem, we proposed active finger recognition method from the amplitude spectrum of surface EMG signal using Bayesian filter. To confirm the effectiveness of this recognition method, we developed a myoelectric hand simulator that implements proposed recognition algorithm and carried out real-time recognition experiment.

  20. CDH1 and IL1-beta expression dictates FAK and MAPKK-dependent cross-talk between cancer cells and human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Al-toub, Mashael; Vishnubalaji, Radhakrishnan; Hamam, Rimi;

    2015-01-01

    INTRODUCTION: Tumor microenvironment conferred by stromal (mesenchymal) stem cells (MSCs) plays a key role in tumor development, progression, and response to therapy. Defining the role of MSCs in tumorigenesis is crucial for their safe utilization in regenerative medicine. Herein, we conducted...... was dependent on direct cell-cell contact. Our data also revealed transfer of cellular components between cancer cells and hMSCs as one possible mechanism for intercellular communication. Global gene expression analysis of sorted hMSCs following co-culturing with MCF7 and BT-20 cells revealed enrichment...... in signaling pathways related to bone formation, FAK and MAPKK signaling. Co-culturing hMSCs with MCF7 cells increased their growth evidenced by increase in Ki67 and PCNA staining in tumor cells in direct contact with hMSCs niche. On the other hand, co-culturing hMSCs with FaDu, HT-29 or MDA-MB-231 cells led...

  1. Biased signaling by peptide agonists of protease activated receptor 2.

    Science.gov (United States)

    Jiang, Yuhong; Yau, Mei-Kwan; Kok, W Mei; Lim, Junxian; Wu, Kai-Chen; Liu, Ligong; Hill, Timothy A; Suen, Jacky Y; Fairlie, David P

    2017-02-07

    Protease activated receptor 2 (PAR2) is associated with metabolism, obesity, inflammatory, respiratory and gastrointestinal disorders, pain, cancer and other diseases. The extracellular N-terminus of PAR2 is a common target for multiple proteases, which cleave it at different sites to generate different N-termini that activate different PAR2-mediated intracellular signaling pathways. There are no synthetic PAR2 ligands that reproduce the same signaling profiles and potencies as proteases. Structure-activity relationships here for 26 compounds spanned a signaling bias over 3 log units, culminating in three small ligands as biased agonist tools for interrogating PAR2 functions. DF253 (2f-LAAAAI-NH2) triggered PAR2-mediated calcium release (EC50 2 μM) but not ERK1/2 phosphorylation (EC50 > 100 μM) in CHO cells transfected with hPAR2. AY77 (Isox-Cha-Chg-NH2) was a more potent calcium-biased agonist (EC50 40 nM, Ca2+; EC50 2 μM, ERK1/2), while its analogue AY254 (Isox-Cha-Chg-A-R-NH2) was an ERK-biased agonist (EC50 2 nM, ERK1/2; EC50 80 nM, Ca2+). Signaling bias led to different functional responses in human colorectal carcinoma cells (HT29). AY254, but not AY77 or DF253, attenuated cytokine-induced caspase 3/8 activation, promoted scratch-wound healing and induced IL-8 secretion, all via PAR2-ERK1/2 signaling. Different ligand components were responsible for different PAR2 signaling and functions, clues that can potentially lead to drugs that modulate different pathway-selective cellular and physiological responses.

  2. Mislocalized activation of oncogenic RTKs switches downstream signaling outcomes

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Olsen, Jesper V; Brandts, Christian

    2009-01-01

    Inappropriate activation of oncogenic kinases at intracellular locations is frequently observed in human cancers, but its effects on global signaling are incompletely understood. Here, we show that the oncogenic mutant of Flt3 (Flt3-ITD), when localized at the endoplasmic reticulum (ER), aberrant...

  3. Mitogen-activated protein kinase and abscisic acid signal transduction

    NARCIS (Netherlands)

    Heimovaara-Dijkstra, S.; Testerink, C.; Wang, M.

    1998-01-01

    The phytohormone abscisic acid (ABA) is a classical plant hormone, responsible for regulation of abscission, diverse aspects of plant and seed development, stress responses and germination. It was found that ABA signal transduction in plants can involve the activity of type 2C-phosphatases (PP2C), c

  4. Enhanced Multistatic Active Sonar via Innovative Signal Processing

    Science.gov (United States)

    2015-09-30

    Department of Electrical and Computer Engineering , P.O. Box 116130 University of Florida, Gainesville, FL 32611 phone: (352) 392-2642 fax: (352...Range-Doppler Imaging and Target Parameter Estimation in Multistatic Active Sonar Systems," IEEE Journal of Oceanic Engineering , Vol. 39, No. 2, pp...3. DATES COVERED (From - To) Oct. 01, 2014-Sept. 30, 2015 4. TITLE AND SUBTITLE Enhanced Multistatic Active Sonar via Innovative Signal

  5. Loss of signal transducer and activator of transcription 3 (STAT3) signaling during elevated activity causes vulnerability in hippocampal neurons.

    Science.gov (United States)

    Murase, Sachiko; Kim, Eunyoung; Lin, Lin; Hoffman, Dax A; McKay, Ronald D

    2012-10-31

    Chronically altered levels of network activity lead to changes in the morphology and functions of neurons. However, little is known of how changes in neuronal activity alter the intracellular signaling pathways mediating neuronal survival. Here, we use primary cultures of rat hippocampal neurons to show that elevated neuronal activity impairs phosphorylation of the serine/threonine kinase, Erk1/2, and the activation of signal transducer and activator of transcription 3 (STAT3) by phosphorylation of serine 727. Chronically stimulated neurons go through apoptosis when they fail to activate another serine/threonine kinase, Akt. Gain- and loss-of-function experiments show that STAT3 plays the key role directly downstream from Erk1/2 as the alternative survival pathway. Elevated neuronal activity resulted in increased expression of a tumor suppressor, p53, and its target gene, Bax. These changes are observed in Kv4.2 knock-out mouse hippocampal neurons, which are also sensitive to the blockade of TrkB signaling, confirming that the alteration occurs in vivo. Thus, this study provides new insight into a mechanism by which chronic elevation of activity may cause neurodegeneration.

  6. Vascular growth responses to chronic arterial occlusion are unaffected by myeloid specific focal adhesion kinase (FAK) deletion

    Science.gov (United States)

    Heuslein, Joshua L.; Murrell, Kelsey P.; Leiphart, Ryan J.; Llewellyn, Ryan A.; Meisner, Joshua K.; Price, Richard J.

    2016-05-01

    Arteriogenesis, or the lumenal expansion of pre-existing arterioles in the presence of an upstream occlusion, is a fundamental vascular growth response. Though alterations in shear stress stimulate arteriogenesis, the migration of monocytes into the perivascular space surrounding collateral arteries and their differentiation into macrophages is critical for this vascular growth response to occur. Focal adhesion kinase’s (FAK) role in regulating cell migration has recently been expanded to primary macrophages. We therefore investigated the effect of the myeloid-specific conditional deletion of FAK on vascular remodeling in the mouse femoral arterial ligation (FAL) model. Using laser Doppler perfusion imaging, whole mount imaging of vascular casted gracilis muscles, and immunostaining for CD31 in gastrocnemius muscles cross-sections, we found that there were no statistical differences in perfusion recovery, arteriogenesis, or angiogenesis 28 days after FAL. We therefore sought to determine FAK expression in different myeloid cell populations. We found that FAK is expressed at equally low levels in Ly6Chi and Ly6Clo blood monocytes, however expression is increased over 2-fold in bone marrow derived macrophages. Ultimately, these results suggest that FAK is not required for monocyte migration to the perivascular space and that vascular remodeling following arterial occlusion occurs independently of myeloid specific FAK.

  7. Survey of activated FLT3 signaling in leukemia.

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    Ting-lei Gu

    Full Text Available Activating mutations of FMS-like tyrosine kinase-3 (FLT3 are found in approximately 30% of patients with acute myeloid leukemia (AML. FLT3 is therefore an attractive drug target. However, the molecular mechanisms by which FLT3 mutations lead to cell transformation in AML remain unclear. To develop a better understanding of FLT3 signaling as well as its downstream effectors, we performed detailed phosphoproteomic analysis of FLT3 signaling in human leukemia cells. We identified over 1000 tyrosine phosphorylation sites from about 750 proteins in both AML (wild type and mutant FLT3 and B cell acute lymphoblastic leukemia (normal and amplification of FLT3 cell lines. Furthermore, using stable isotope labeling by amino acids in cell culture (SILAC, we were able to quantified over 400 phosphorylation sites (pTyr, pSer, and pThr that were responsive to FLT3 inhibition in FLT3 driven human leukemia cell lines. We also extended this phosphoproteomic analysis on bone marrow from primary AML patient samples, and identify over 200 tyrosine and 800 serine/threonine phosphorylation sites in vivo. This study showed that oncogenic FLT3 regulates proteins involving diverse cellular processes and affects multiple signaling pathways in human leukemia that we previously appreciated, such as Fc epsilon RI-mediated signaling, BCR, and CD40 signaling pathways. It provides a valuable resource for investigation of oncogenic FLT3 signaling in human leukemia.

  8. Platycodin D inhibits tumor growth by antiangiogenic activity via blocking VEGFR2-mediated signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Luan, Xin; Gao, Yun-Ge; Guan, Ying-Yun; Xu, Jian-Rong; Lu, Qin [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Zhao, Mei [Department of Pharmacy, Shanghai Institute of Health Sciences and Health School Attached to SJTU-SM, 279 Zhouzhu Road, Shanghai 201318 (China); Liu, Ya-Rong; Liu, Hai-Jun [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Fang, Chao, E-mail: fangchao100@hotmail.com [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Chen, Hong-Zhuan, E-mail: hongzhuan_chen@hotmail.com [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China)

    2014-11-15

    Platycodin D (PD) is an active component mainly isolated from the root of Platycodon grandiflorum. Recent studies proved that PD exhibited inhibitory effect on proliferation, migration, invasion and xenograft growth of diverse cancer cell lines. However, whether PD is suppressive for angiogenesis, an important hallmark in cancer development, remains unknown. Here, we found that PD could dose-dependently inhibit human umbilical vein endothelial cell (HUVEC) proliferation, motility, migration and tube formation. PD also significantly inhibited angiogenesis in the chick embryo chorioallantoic membrane (CAM). Moreover, the antiangiogenic activity of PD contributed to its in vivo anticancer potency shown in the decreased microvessel density and delayed growth of HCT-15 xenograft in mice with no overt toxicity. Western blot analysis indicated that PD inhibited the phosphorylation of VEGFR2 and its downstream protein kinase including PLCγ1, JAK2, FAK, Src, and Akt in endothelial cells. Molecular docking simulation showed that PD formed hydrogen bonds and hydrophobic interactions within the ATP binding pocket of VEGFR2 kinase domain. The present study firstly revealed the high antiangiogenic activity and the underlying molecular basis of PD, suggesting that PD may be a potential antiangiogenic agent for angiogenesis-related diseases. - Highlights: • Platycodin D inhibits HUVEC proliferation, motility, migration and tube formation. • Platycodin D inhibits the angiogenesis in chick embryo chorioallantoic membrane. • Platycodin D suppresses the angiogenesis and growth of HCT-15 xenograft in mice. • Platycodin D inhibits the phosphorylation of VEGFR2 and downstream kinases in HUVEC.

  9. Physical parameters activating electrical signal distortions in polluted soils

    Directory of Open Access Journals (Sweden)

    R. Angelini

    2002-06-01

    Full Text Available Laboratory investigations and field measurements show that the electrical behaviour of polluted soils is strongly non-linear at low frequencies. This phenomenon can be related to the class and the amount of pollutants. To measure this non-linearity, we used only monochromatic voltage waveform as input signal and analysed the current signals at first by means of the classical spectral analysis. In particular, the Total Harmonic Distortion % (THD% and the Harmonic Distortion %(? measure the non-linearity level and identify the frequency interval where the non-linear electrical behaviour is activated. This frequency interval can be related to the pollutant molecular size. Open interpretative problems were the following: 1 phase localization of the signal deformation; 2 «local» amplitude of the applied signal activating the distortion, and 3 numerical fit of the distortion. We employed the wavelet analysis to study the phenomenon. The wavelet technique breaks up a signal into shifted and scaled versions of the original wavelet, which is a waveform of limited duration. These features of the wavelets allow us to obtain current components that can be interpreted on the bases of a real physical meaning. By using the wavelet analysis, we obtained the phase localization of the ‘oscillations’ of the details and consequently the phase and amplitude of the applied signal. The sum of nine details provides a good numerical fit of the distorted signal. Starting from the wavelet analysis, we determined the physical conditions activating each distortion, testing some parameters on experimental data. The parameters that resulted most significant are the phase ? of the distortion activation and the product Vin?t (Vs (where ?t is the time interval corresponding to the said ? and Vin is the integral tension applied to the sample on ?t. The latter parameter is in a very good agreement with field data of Advanced Monochromatic Spectral Induced Polarization (AMSIP

  10. Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

    Science.gov (United States)

    Fang, Jung-Da; Lee, Sheau-Ling

    2014-07-01

    Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

  11. BMP2 Transfer to Neighboring Cells and Activation of Signaling.

    Science.gov (United States)

    Alborzinia, Hamed; Shaikhkarami, Marjan; Hortschansky, Peter; Wölfl, Stefan

    2016-09-01

    Morphogen gradients and concentration are critical features during early embryonic development and cellular differentiation. Previously we reported the preparation of biologically active, fluorescently labeled BMP2 and quantitatively analyzed their binding to the cell surface and followed BMP2 endocytosis over time on the level of single endosomes. Here we show that this internalized BMP2 can be transferred to neighboring cells and, moreover, also activates downstream BMP signaling in adjacent cells, indicated by Smad1/5/8 phosphorylation and activation of the downstream target gene id1. Using a 3D matrix to modulate cell-cell contacts in culture we could show that direct cell-cell contact significantly increased BMP2 transfer. Using inhibitors of vesicular transport, transfer was strongly inhibited. Interestingly, cotreatment with the physiological BMP inhibitor Noggin increased BMP2 uptake and transfer, albeit activation of Smad signaling in neighboring cells was completely suppressed. Our findings present a novel and interesting mechanism by which morphogens such as BMP2 can be transferred between cells and how this is modulated by BMP antagonists such as Noggin, and how this influences activation of Smad signaling by BMP2 in neighboring cells.

  12. ATP release and purinergic signaling in NLRP3 inflammasome activation

    Directory of Open Access Journals (Sweden)

    Isabelle eCOUILLIN

    2013-01-01

    Full Text Available The NLRP3 inflammasome is a protein complex involved in IL-1β and IL-18 processing that senses pathogen- and danger-associated molecular patterns. One step- or two step- models have been proposed to explain the tight regulation of IL-1β production during inflammation. Moreover, cellular stimulation triggers ATP release and subsequent activation of purinergic receptors at the cell surface. Importantly some studies have reported roles for extracellular ATP (eATP, in NLRP3 inflammasome activation in response to PAMPs and DAMPs. In this mini review, we will discuss the link between active ATP release, purinergic signaling and NLRP3 inflammasome activation. We will focus on the role of autocrine or paracrine ATP export in particle-induced NLRP3 inflammasome activation and discuss how particle activators are competent to induce maturation and secretion of IL-1β through a process that involves, as a first event, extracellular release of endogenous ATP through hemichannel opening, and as a second event, signaling through purinergic receptors that trigger NLRP3 inflammasome activation. Finally, we will review the evidence for ATP as a key proinflammatory mediator released by dying cells. In particular we will discuss how cancer cells dying via autophagy trigger ATP-dependent NLRP3 inflammasome activation in the macrophages engulfing them, eliciting an immunogenic response against tumors.

  13. Integration of the beta-catenin-dependent Wnt pathway with integrin signaling through the adaptor molecule Grb2.

    Directory of Open Access Journals (Sweden)

    Steve P Crampton

    Full Text Available BACKGROUND: THE COMPLEXITY OF WNT SIGNALING LIKELY STEMS FROM TWO SOURCES: multiple pathways emanating from frizzled receptors in response to wnt binding, and modulation of those pathways and target gene responsiveness by context-dependent signals downstream of growth factor and matrix receptors. Both rac1 and c-jun have recently been implicated in wnt signaling, however their upstream activators have not been identified. METHODOLOGY/PRINCIPAL FINDINGS: Here we identify the adapter protein Grb2, which is itself an integrator of multiple signaling pathways, as a modifier of beta-catenin-dependent wnt signaling. Grb2 synergizes with wnt3A, constitutively active (CA LRP6, Dvl2 or CA-beta-catenin to drive a LEF/TCF-responsive reporter, and dominant negative (DN Grb2 or siRNA to Grb2 block wnt3A-mediated reporter activity. MMP9 is a target of beta-catenin-dependent wnt signaling, and an MMP9 promoter reporter is also responsive to signals downstream of Grb2. Both a jnk inhibitor and DN-c-jun block transcriptional activation downstream of Dvl2 and Grb2, as does DN-rac1. Integrin ligation by collagen also synergizes with wnt signaling as does overexpression of Focal Adhesion Kinase (FAK, and this is blocked by DN-Grb2. CONCLUSIONS/SIGNIFICANCE: These data suggest that integrin ligation and FAK activation synergize with wnt signaling through a Grb2-rac-jnk-c-jun pathway, providing a context-dependent mechanism for modulation of wnt signaling.

  14. Energetics of neuronal signaling and fMRI activity.

    Science.gov (United States)

    Maandag, Natasja J G; Coman, Daniel; Sanganahalli, Basavaraju G; Herman, Peter; Smith, Arien J; Blumenfeld, Hal; Shulman, Robert G; Hyder, Fahmeed

    2007-12-18

    Energetics of resting and evoked fMRI signals were related to localized ensemble firing rates (nu) measured by electrophysiology in rats. Two different unstimulated, or baseline, states were established by anesthesia. Halothane and alpha-chloralose established baseline states of high and low energy, respectively, in which forepaw stimulation excited the contralateral primary somatosensory cortex (S1). With alpha-chloralose, forepaw stimulation induced strong and reproducible fMRI activations in the contralateral S1, where the ensemble firing was dominated by slow signaling neurons (SSN; nu range of 1-13 Hz). Under halothane, weaker and less reproducible fMRI activations were observed in the contralateral S1 and elsewhere in the cortex, but ensemble activity in S1 was dominated by rapid signaling neurons (RSN; nu range of 13-40 Hz). For both baseline states, the RSN activity (i.e., higher frequencies, including the gamma band) did not vary upon stimulation, whereas the SSN activity (i.e., alpha band and lower frequencies) did change. In the high energy baseline state, a large majority of total oxidative energy [cerebral metabolic rate of oxygen consumption (CMR(O2))] was devoted to RSN activity, whereas in the low energy baseline state, it was roughly divided between SSN and RSN activities. We hypothesize that in the high energy baseline state, the evoked changes in fMRI activation in areas beyond S1 are supported by rich intracortical interactions represented by RSN. We discuss implications for interpreting fMRI data where stimulus-specific DeltaCMR(O2) is generally small compared with baseline CMR(O2).

  15. Different activation signals induce distinct mast cell degranulation strategies

    Science.gov (United States)

    Sibilano, Riccardo; Marichal, Thomas; Reber, Laurent L.; Cenac, Nicolas; McNeil, Benjamin D.; Dong, Xinzhong; Hernandez, Joseph D.; Sagi-Eisenberg, Ronit; Hammel, Ilan; Roers, Axel; Valitutti, Salvatore; Tsai, Mindy

    2016-01-01

    Mast cells (MCs) influence intercellular communication during inflammation by secreting cytoplasmic granules that contain diverse mediators. Here, we have demonstrated that MCs decode different activation stimuli into spatially and temporally distinct patterns of granule secretion. Certain signals, including substance P, the complement anaphylatoxins C3a and C5a, and endothelin 1, induced human MCs rapidly to secrete small and relatively spherical granule structures, a pattern consistent with the secretion of individual granules. Conversely, activating MCs with anti-IgE increased the time partition between signaling and secretion, which was associated with a period of sustained elevation of intracellular calcium and formation of larger and more heterogeneously shaped granule structures that underwent prolonged exteriorization. Pharmacological inhibition of IKK-β during IgE-dependent stimulation strongly reduced the time partition between signaling and secretion, inhibited SNAP23/STX4 complex formation, and switched the degranulation pattern into one that resembled degranulation induced by substance P. IgE-dependent and substance P–dependent activation in vivo also induced different patterns of mouse MC degranulation that were associated with distinct local and systemic pathophysiological responses. These findings show that cytoplasmic granule secretion from MCs that occurs in response to different activating stimuli can exhibit distinct dynamics and features that are associated with distinct patterns of MC-dependent inflammation. PMID:27643442

  16. Activation and signaling of the p38 MAP kinase pathway

    Institute of Scientific and Technical Information of China (English)

    Tyler ZARUBIN; Jiahuai HAN

    2005-01-01

    The family members of the mitogen-activated protein (MAP) kinases mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the four main sub-groups, the p38 group of MAP kinases, serve as a nexus for signal transduction and play a vital role in numerous biological processes. In this review, we highlight the known characteristics and components of the p38 pathway along with the mechanism and consequences of p38 activation. We focus on the role of p38 as a signal transduction mediator and examine the evidence linking p38 to inflammation, cell cycle, cell death, development, cell differentiation, senescence and tumorigenesis in specific cell types. Upstream and downstream components of p38 are described and questions remaining to be answered are posed. Finally, we propose several directions for future research on p38.

  17. Artifact suppression and analysis of brain activities with electroencephalography signals

    Institute of Scientific and Technical Information of China (English)

    Md. Rashed-Al-Mahfuz; Md. Rabiul Islam; Keikichi Hirose; Md. Khademul Islam Molla

    2013-01-01

    Brain-computer interface is a communication system that connects the brain with computer (or other devices) but is not dependent on the normal output of the brain (i.e., peripheral nerve and muscle). Electro-oculogram is a dominant artifact which has a significant negative influence on further analysis of real electroencephalography data. This paper presented a data adaptive technique for artifact suppression and brain wave extraction from electroencephalography signals to detect regional brain activities. Empirical mode decomposition based adaptive thresholding approach was employed here to suppress the electro-oculogram artifact. Fractional Gaussian noise was used to determine the threshold level derived from the analysis data without any training. The purified electroencephalography signal was composed of the brain waves also called rhythmic components which represent the brain activities. The rhythmic components were extracted from each electroencephalography channel using adaptive wiener filter with the original scale. The regional brain activities were mapped on the basis of the spatial distribution of rhythmic components, and the results showed that different regions of the brain are activated in response to different stimuli. This research analyzed the activities of a single rhythmic component, alpha with respect to different motor imaginations. The experimental results showed that the proposed method is very efficient in artifact suppression and identifying individual motor imagery based on the activities of alpha component.

  18. The microarray gene profiling analysis of glioblastoma cancer cells reveals genes affected by FAK inhibitor Y15 and combination of Y15 and temozolomide.

    Science.gov (United States)

    Huang, Grace; Ho, Baotran; Conroy, Jeffrey; Liu, Song; Qiang, Hu; Golubovskaya, Vita

    2014-01-01

    Focal adhesion is known to be highly expressed and activated in glioma cells. Recently, we demonstrated that FAK autophosphorylation inhibitor, Y15 significantly decreased tumor growth of DBTRG and U87 cells, especially in combination with temozolomide. In the present report, we performed gene expression analysis in these cells to reveal genes affected by Y15, temozolomide and combination of Y15 and temozolomide. We tested the effect of Y15 on gene expression by Illumina Human HT12v4 microarray assay and detected 8087 and 6555 genes, which were significantly either up- or down-regulated by Y15-treatment in DBTRG and U87 cells, respectively (ptemozolomide and by combination of Y15 and temozolomide treatment in U87 cells. Among genes up-regulated by Y15 and temozolomide more significantly than by each agent alone were: COX7B; interferon, gamma-inducible transcript: IFI16; DDIT4; GADD45G and down-regulated: KIF3A, AKT1; ABL; JAK1, GLI3 and ALDH1A3. Thus, microarray gene expression analysis can be effective in establishing genes affected in response to FAK inhibitor alone and in response to combination of Y15 with temozolomide that is important for glioblastoma therapy.

  19. MicroRNA-151 and its hosting gene FAK (focal adhesion kinase) regulate tumor cell migration and spreading of hepatocellular carcinoma.

    Science.gov (United States)

    Luedde, Tom

    2010-09-01

    Recurrent chromosomal aberrations are often observed in hepatocellular carcinoma (HCC), but little is known about the functional non-coding sequences, particularly microRNAs (miRNAs), at the chromosomal breakpoints in HCC. Here we show that 22 miRNAs are often amplified or deleted in HCC. MicroRNA-151 (miR-151), a frequently amplified miRNA on 8q24.3, is correlated with intrahepatic metastasis of HCC. We further show that miR-151, which is often expressed together with its host gene FAK, encoding focal adhesion kinase, significantly increases HCC cell migration and invasion in vitro and in vivo, mainly through miR-151-5p, but not through miR-151-3p. Moreover, miR-151 exerts this function by directly targeting RhoGDIA, a putative metastasis suppressor in HCC, thus leading to the activation of Rac1, Cdc42 and Rho GTPases. In addition, miR-151 can function synergistically with FAK to enhance HCC cell motility and spreading. Thus, our findings indicate that chromosome gain of miR-151 is a crucial stimulus for tumour invasion and metastasis of HCC.

  20. TLR signaling augments macrophage bactericidal activity through mitochondrial ROS

    OpenAIRE

    West, A. Phillip; Brodsky, Igor E.; Rahner, Christoph; Woo, Dong Kyun; Erdjument-Bromage, Hediye; Tempst, Paul; Walsh, Matthew C; Choi, Yongwon; Shadel, Gerald S.; Ghosh, Sankar

    2011-01-01

    Reactive oxygen species (ROS) are essential components of the innate immune response against intracellular bacteria, and it is thought that professional phagocytes generate ROS primarily via the phagosomal NADPH oxidase (Phox) machinery 1 . However, recent studies have suggested that mitochondrial ROS (mROS) also contribute to macrophage bactericidal activity, although the mechanisms linking innate immune signaling to mitochondria for mROS generation remain unclear 2-4 . Here we demonstrate t...

  1. Ephrin-B reverse signaling induces expression of wound healing associated genes in IEC-6 intestinal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Christian Hafner; Stefanie Meyer; Ilja Hagen; Bernd Becker; Alexander Roesch; Michael Landthaler; Thomas Vogt

    2005-01-01

    AIM: Eph receptors and ephrin ligands play a pivotal role in development and tissue maintenance. Since previous data have indicated an involvement of ephrin-B2 in epithelial healing, we investigated the gene expression and downstream signaling pathways induced by ephrin-B mediated cell-cell signaling in intestinal epithelial cells.METHODS: Upon stimulation of ephrin-B pathways in IFC-6 cells with recombinant rat EphB1-Fc, gene expression was analyzed by Affymetrix(R) rat genome 230 high density arrays at different time points. Differentially expressed genes were confirmed by real-time RT-PCR. In addition, MAP kinase pathways and focal adhesion kinase (FAK) activation downstream of ephrin-B were investigated by immunoblotting and fluorescence microscopy.RESULTS: Stimulation of the ephrin-B reverse signaling pathway in IEC-6 cells induces predominant expression of genes known to be involved into wound healing/cell migration, antiapoptotic pathways, host defense and inflammation. Cox-2, c-Fos, Egr-1, Egr-2, and MCP-1 were found among the most significantly regulated genes.Furthermore, we show that the expression of repairrelated genes is also accompanied by activation of the ERK1/2 MAP kinase pathway and FAK, two key regulators of epithelial restitution.CONCLUSION: Stimulation of the ephrin-B reverse signaling pathway induces a phenotype characterized by upregulation of repair-related genes, which may partially be mediated by ERK1/2 pathways.

  2. Wnt signaling activates Shh signaling in early postnatal intervertebral discs, and re-activates Shh signaling in old discs in the mouse.

    Science.gov (United States)

    Winkler, Tamara; Mahoney, Eric J; Sinner, Debora; Wylie, Christopher C; Dahia, Chitra Lekha

    2014-01-01

    Intervertebral discs (IVDs) are strong fibrocartilaginous joints that connect adjacent vertebrae of the spine. As discs age they become prone to failure, with neurological consequences that are often severe. Surgical repair of discs treats the result of the disease, which affects as many as one in seven people, rather than its cause. An ideal solution would be to repair degenerating discs using the mechanisms of their normal differentiation. However, these mechanisms are poorly understood. Using the mouse as a model, we previously showed that Shh signaling produced by nucleus pulposus cells activates the expression of differentiation markers, and cell proliferation, in the postnatal IVD. In the present study, we show that canonical Wnt signaling is required for the expression of Shh signaling targets in the IVD. We also show that Shh and canonical Wnt signaling pathways are down-regulated in adult IVDs. Furthermore, this down-regulation is reversible, since re-activation of the Wnt or Shh pathways in older discs can re-activate molecular markers of the IVD that are lost with age. These data suggest that biological treatments targeting Wnt and Shh signaling pathways may be feasible as a therapeutic for degenerative disc disease.

  3. Disentangling stellar activity from exoplanetary signals with interferometry

    Directory of Open Access Journals (Sweden)

    Ligi Roxanne

    2015-01-01

    Full Text Available Stellar activity can express as many forms at stellar surfaces: dark spots, convective cells, bright plages. Particularly, dark spots and bright plages add noise on photometric data or radial velocity measurements used to detect exoplanets, and thus lead to false detection or disrupt their derived parameters. Since interferometry provides a very high angular resolution, it may constitute an interesting solution to distinguish the signal of a transiting exoplanet and that of stellar activity. It has also been shown that granulation adds bias in visibility and closure phase measurements, affecting in turn the derived stellar parameters. We analyze the noises generated by dark spots on interferometric observables and compare them to exoplanet signals. We investigate the current interferometric instruments able to measure and disentangle these signals, and show that there is a lack in spatial resolution. We thus give a prospective of the improvements to be brought on future interferometers, which would also significantly extend the number of available targets.

  4. Signal integration by Ca2+ regulates intestinal stem cell activity

    Science.gov (United States)

    Deng, Hansong; Gerencser, Akos A.; Jasper, Heinrich

    2015-01-01

    Summary Somatic stem cells (SCs) maintain tissue homeostasis by dynamically adjusting proliferation and differentiation in response to stress and metabolic cues. Here, we identify Ca2+ signaling as a central regulator of intestinal SC (ISC) activity in Drosophila. We find that dietary L-glutamate stimulates ISC division and gut growth. The metabotropic glutamate receptor (mGluR) is required in ISCs for this response and for an associated modulation of cytosolic Ca2+ oscillations that results in sustained high cytosolic Ca2+ concentrations. High cytosolic Ca2+ induces ISC proliferation by regulating Calcineurin and CREB - regulated transcriptional co-activator (CRTC). In response to a wide range of dietary and stress stimuli, ISCs reversibly transition between Ca2+ oscillation states that represent poised or activated modes of proliferation, respectively. We propose that the dynamic regulation of intracellular Ca2+ levels allows effective integration of diverse mitogenic signals in ISCs to tailor their proliferative activity to the needs of the tissue. PMID:26633624

  5. SNIP1: a new activator of HSE signaling pathway.

    Science.gov (United States)

    Li, Qiang; An, Jian; Liu, Xianghua; Zhang, Mingjun; Ling, Yichen; Wang, Chenji; Zhao, Jing; Yu, Long

    2012-03-01

    In the last 10 years, more and more attention has been focused on SNIP1 (Smad nuclear interacting protein 1), which functions as a transcriptional coactivator. We report here that through quantitative real-time PCR analysis in 18 different human tissues, SNIP1 was found to be expressed ubiquitously. When overexpressed in HeLa cells, SNIP1-EGFP fused protein exhibited a nuclear localization with a characteristic subnuclear distribution in speckles or formed larger discrete nuclear bodies in some cells. Reporter gene assay showed that overexpression of SNIP1 in HEK 293 cells or H1299 cells strongly activated the HSE signaling pathway. Moreover, SNIP1 could selectively regulate the transcription of HSP70A1A and HSP27. Taken together, our findings suggest that SNIP1 might also be a positive regulator of HSE signaling pathway.

  6. Blocking FAK Signaling in the Tumor or Stroma and Effects on Metastasis

    Science.gov (United States)

    2010-04-01

    PEG400, polyethylene glycol 400; PI, propidium iodide; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis...directional cell movement.2,3 To assess if PND-1186 would inhibit 4T1 cell migration, time lapse wound healing assays were performed in the presence...using vernier calipers and determined by length x width2/2) were grouped (n = 8 per group) and PND-1186 solubilized in polyeth- ylene glycol 400 (PEG400

  7. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    Directory of Open Access Journals (Sweden)

    Koul Sweaty

    2004-06-01

    Full Text Available Abstract The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy.

  8. Thrombin-Mediated Direct Activation of Proteinase-Activated Receptor-2: Another Target for Thrombin Signaling.

    Science.gov (United States)

    Mihara, Koichiro; Ramachandran, Rithwik; Saifeddine, Mahmoud; Hansen, Kristina K; Renaux, Bernard; Polley, Danny; Gibson, Stacy; Vanderboor, Christina; Hollenberg, Morley D

    2016-05-01

    Thrombin is known to signal to cells by cleaving/activating a G-protein-coupled family of proteinase-activated receptors (PARs). The signaling mechanism involves the proteolytic unmasking of an N-terminal receptor sequence that acts as a tethered receptor-activating ligand. To date, the recognized targets of thrombin cleavage and activation for signaling are PAR1 and PAR4, in which thrombin cleaves at a conserved target arginine to reveal a tethered ligand. PAR2, which like PAR1 is also cleaved at an N-terminal arginine to unmask its tethered ligand, is generally regarded as a target for trypsin but not for thrombin signaling. We now show that thrombin, at concentrations that can be achieved at sites of acute injury or in a tumor microenvironment, can directly activate PAR2 vasorelaxation and signaling, stimulating calcium and mitogen-activated protein kinase responses along with triggeringβ-arrestin recruitment. Thus, PAR2 can be added alongside PAR1 and PAR4 to the targets, whereby thrombin can affect tissue function.

  9. Monocyte Signal Transduction Receptors in Active and Latent Tuberculosis

    Directory of Open Access Journals (Sweden)

    Magdalena Druszczynska

    2013-01-01

    Full Text Available The mechanisms that promote either resistance or susceptibility to TB disease remain insufficiently understood. Our aim was to compare the expression of cell signaling transduction receptors, CD14, TLR2, CD206, and β2 integrin LFA-1 on monocytes from patients with active TB or nonmycobacterial lung disease and healthy individuals with M.tb latency and uninfected controls to explain the background of the differences between clinical and subclinical forms of M.tb infection. A simultaneous increase in the expression of the membrane bound mCD14 receptor and LFA-1 integrin in patients with active TB may be considered a prodrome of breaking immune control by M.tb bacilli in subjects with the latent TB and absence of clinical symptoms.

  10. Rapid actions of plasma membrane estrogen receptors regulate motility of mouse embryonic stem cells through a profilin-1/cofilin-1-directed kinase signaling pathway.

    Science.gov (United States)

    Yun, Seung Pil; Ryu, Jung Min; Kim, Mi Ok; Park, Jae Hong; Han, Ho Jae

    2012-08-01

    Long-term estrogen actions are vital for driving cell growth, but more recent evidence suggests that estrogen mediates more rapid cellular effects. However, the function of estradiol-17β (E(2))-BSA in mouse embryonic stem cells has not been reported. Therefore, we examined the role of E(2)-BSA in mouse embryonic stem cell motility and its related signal pathways. E(2)-BSA (10(-8) m) significantly increased motility after 24 h incubation and increased filamentous (F)-actin expression; these effects were inhibited by the estrogen receptor antagonist ICI 182,780, indicating that E(2)-BSA bound membrane estrogen receptors and initiated a signal. E(2)-BSA increased c-Src and focal adhesion kinase (FAK) phosphorylation, which was attenuated by ICI 182,780. The E(2)-BSA-induced increase in epidermal growth factor receptor (EGFR) phosphorylation was inhibited by Src inhibitor PP2. As a downstream signal molecule, E(2)-BSA activated cdc42 and increased formation of a complex with the neural Wiskott-Aldrich syndrome protein (N-WASP)/cdc42/transducer of cdc42-dependent actin assembly-1 (TOCA-1), which was inhibited by FAK small interfering RNA (siRNA) and EGFR inhibitor AG 1478. In addition, E(2)-BSA increased profilin-1 expression and cofilin-1 phosphorylation, which was blocked by cdc42 siRNA. Subsequently, E(2)-BSA induced an increase in F-actin expression, and cell motility was inhibited by each signal pathway-related siRNA molecule or inhibitors but not by cofilin-1 siRNA. A combined treatment of cofilin-1 siRNA and E(2)-BSA increased F-actin expression and cell motility more than that of E(2)-BSA alone. These data demonstrate that E(2)-BSA stimulated motility by interacting with profilin-1/cofilin-1 and F-actin through FAK- and c-Src/EGFR transactivation-dependent N-WASP/cdc42/TOCA-1 complex.

  11. Active Sonar Detection in Reverberation via Signal Subspace Extraction Algorithm

    Directory of Open Access Journals (Sweden)

    Ma Xiaochuan

    2010-01-01

    Full Text Available This paper presents a new algorithm called Signal Subspace Extraction (SSE for detecting and estimating target echoes in reverberation. The new algorithm can be taken as an extension of the Principal Component Inverse (PCI and maintains the benefit of PCI algorithm and moreover shows better performance due to a more reasonable reverberation model. In the SSE approach, a best low-rank estimate of a target echo is extracted by decomposing the returns into short duration subintervals and by invoking the Eckart-Young theorem twice. It was assumed that CW is less efficiency in lower Doppler than broadband waveforms in spectrum methods; however, the subspace methods show good performance in detection whatever the respective Doppler is. Hence, the signal emitted by active sonar is CW in the new algorithm which performs well in detection and estimation even when low Doppler is low. Further, a block forward matrix is proposed to extend the algorithm to the sensor array problem. The comparison among the block forward matrix, the conventional matrix, and the three-mode array is discussed. Echo separation is also provided by the new algorithm. Examples are presented using both real, active-sonar data and simulated data.

  12. You've found a safety signal--now what?: regulatory implications of industry signal detection activities.

    Science.gov (United States)

    Kahn, Sidney N

    2007-01-01

    Signals detected by measuring disproportionality of drug-event combinations are only statistical indicators of possible real safety issues, and are not per se necessarily medically important. Nevertheless, once a signal is observed, sponsors are obligated by regulations and ethical considerations to determine whether it represents a new product-associated risk by additional analysis, validation and evaluation of its clinical relevance. Signal strength does not necessarily correlate with medical significance. Strong signals most often represent known, expected and/or medically trivial adverse reactions or confounding by treatment indication, common co-morbidities or other common concomitant treatments. Conversely, any product with reasonably extensive clinical use and reporting of suspected adverse reactions is likely to manifest many weak but clinically unimportant signals, creating significant background 'noise'. Since relatively rare, medically important adverse drug reactions are often likely to manifest as weak signals, sponsors face a potentially onerous burden of evaluating multiple signals in order to distinguish true, clinically important events of concern from spurious signals. This paper discusses the regulatory, clinical and potential legal liability issues that confront industry as a consequence of signal identification activities, including: current and anticipated regulatory requirements for detection, assessment and reporting; the reliability of the data used for signal generation; assessment of clinical relevance; organisational approaches and responses to observed signals; targeted clinical and scientific responses to observed signals; and potential regulatory, legal and commercial impact.

  13. Transfer function between EEG and BOLD signals of epileptic activity

    Directory of Open Access Journals (Sweden)

    Marco eLeite

    2013-01-01

    Full Text Available Simultaneous EEG-fMRI recordings have seen growing application in the evaluation of epilepsy, namely in the characterization of brain networks related to epileptic activity. In EEG-correlated fMRI studies, epileptic events are usually described as boxcar signals based on the timing information retrieved from the EEG, and subsequently convolved with a heamodynamic response function to model the associated BOLD changes. Although more flexible approaches may allow a higher degree of complexity for the haemodynamics, the issue of how to model these dynamics based on the EEG remains an open question. In this work, a new methodology for the integration of simultaneous EEG-fMRI data in epilepsy is proposed, which incorporates a transfer function from the EEG to the BOLD signal. Independent component analysis (ICA of the EEG is performed, and a number of metrics expressing different models of the EEG-BOLD transfer function are extracted from the resulting time courses. These metrics are then used to predict the fMRI data and to identify brain areas associated with the EEG epileptic activity. The methodology was tested on both ictal and interictal EEG-fMRI recordings from one patient with a hypothalamic hamartoma. When compared to the conventional analysis approach, plausible, consistent and more significant activations were obtained. Importantly, frequency-weighted EEG metrics yielded superior results than those weighted solely on the EEG power, which comes in agreement with previous literature. Reproducibility, specificity and sensitivity should be addressed in an extended group of patients in order to further validate the proposed methodology and generalize the presented proof of concept.

  14. Bisphenol A (BPA) stimulates the interferon signaling and activates the inflammasome activity in myeloid cells.

    Science.gov (United States)

    Panchanathan, Ravichandran; Liu, Hongzhu; Leung, Yuet-Kin; Ho, Shuk-mei; Choubey, Divaker

    2015-11-05

    Environmental factors contribute to the development of autoimmune diseases, including systemic lupus erythematosus (SLE), which exhibits a strong female bias (female-to-male ratio 9:1). However, the molecular mechanisms remain largely unknown. Because a feedforward loop between the female sex hormone estrogen (E2) and type I interferon (IFN-α/β)-signaling induces the expression of certain p200-family proteins (such as murine p202 and human IFI16) that regulate innate immune responses and modify lupus susceptibility, we investigated whether treatment of myeloid cells with bisphenol A (BPA), an environmental estrogen, could regulate the p200-family proteins and activate innate immune responses. We found that treatment of murine bone marrow-derived cells (BMCs) and human peripheral blood mononuclear cells with BPA induced the expression of ERα and IFN-β, activated the IFN-signaling, and stimulated the expression of the p202 and IFI16 proteins. Further, the treatment increased levels of the NLRP3 inflammasome and stimulated its activity. Accordingly, BPA-treatment of BMCs from non lupus-prone C57BL/6 and the lupus-prone (NZB×NZW)F1 mice activated the type I IFN-signaling, induced the expression of p202, and activated an inflammasome activity. Our study demonstrates that BPA-induced signaling in the murine and human myeloid cells stimulates the type I IFN-signaling that results in an induction of the p202 and IFI16 innate immune sensors for the cytosolic DNA and activates an inflammasome activity. These observations provide novel molecular insights into the role of environmental BPA exposures in potentiating the development of certain autoimmune diseases such as SLE.

  15. DMPD: Macrophage activation by endogenous danger signals. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18161744 Macrophage activation by endogenous danger signals. Zhang X, Mosser DM. J ...Pathol. 2008 Jan;214(2):161-78. (.png) (.svg) (.html) (.csml) Show Macrophage activation by endogenous dange...r signals. PubmedID 18161744 Title Macrophage activation by endogenous danger signals. Authors Zhang X, Moss

  16. Lunasin Sensitivity in Non-Small Cell Lung Cancer Cells Is Linked to Suppression of Integrin Signaling and Changes in Histone Acetylation

    Directory of Open Access Journals (Sweden)

    Junichi Inaba

    2014-12-01

    Full Text Available Lunasin is a plant derived bioactive peptide with both cancer chemopreventive and therapeutic activity. We recently showed lunasin inhibits non-small cell lung cancer (NSCLC cell proliferation in a cell-line-specific manner. We now compared the effects of lunasin treatment of lunasin-sensitive (H661 and lunasin-insensitive (H1299 NSCLC cells with respect to lunasin uptake, histone acetylation and integrin signaling. Both cell lines exhibited changes in histone acetylation, with H661 cells showing a unique increase in H4K16 acetylation. Proximity ligation assays demonstrated lunasin interacted with integrins containing αv, α5, β1 and β3 subunits to a larger extent in the H661 compared to H1299 cells. Moreover, lunasin specifically disrupted the interaction of β1 and β3 subunits with the downstream signaling components phosphorylated Focal Adhesion Kinase (pFAK, Kindlin and Intergrin Linked Kinase in H661 cells. Immunoblot analyses demonstrated lunasin treatment of H661 resulted in reduced levels of pFAK, phosphorylated Akt and phosphorylated ERK1/2 whereas no changes were observed in H1299 cells. Silencing of αv expression in H661 cells confirmed signaling through integrins containing αv is essential for proliferation. Moreover, lunasin was unable to further inhibit proliferation in αv-silenced H661 cells. This indicates antagonism of integrin signaling via αv-containing integrins is an important component of lunasin’s mechanism of action.

  17. The satiety signaling neuropeptide perisulfakinin inhibits the activity of central neurons promoting general activity

    Directory of Open Access Journals (Sweden)

    Dieter Wicher

    2007-12-01

    Full Text Available The metabolic state is one of the determinants of the general activity level. Satiety is related to resting or sleep whereas hunger correlates to wakefulness and activity. The counterpart to the mammalian satiety signal cholecystokinin (CCK in insects are the sulfakinins. The aim of this study was to resolve the mechanism by which the antifeedant activity of perisulfakinin (PSK in Periplaneta americana is mediated. We identified the sources of PSK which is used both as hormone and as paracrine messenger. PSK is found in the neurohemal organ of the brain and in nerve endings throughout the central nervous system. To correlate the distributions of PSK and its receptor (PSKR, we cloned the gene coding for PSKR and provide evidence for its expression within the nervous system. It occurs only in a few neurons, among them are the dorsal unpaired median (DUM neurons which release octopamine thereby regulating the general level of activity. Application of PSK to DUM neurons attenuated the spiking frequency (EC50=11pM due to reduction of a pacemaker Ca2+ current through cAMP-inhibited pTRPγ channels. PSK increased the intracellular cAMP level while decreasing the intracellular Ca2+ concentration in DUM neurons. Thus, the satiety signal conferred by PSK acts antagonistically to the hunger signal, provided by the adipokinetic hormone (AKH: PSK depresses the electrical activity of DUM neurons by inhibiting the pTRPγ channel that is activated by AKH under conditions of food shortage.

  18. Signaling lymphocyte activating molecule (SLAM) expression in subacute sclerosing panencephalitis.

    Science.gov (United States)

    Piskin, A Kevser; Akpinar, Pinar; Muftuoglu, Sevda; Anlar, Banu

    2007-08-01

    Signaling lymphocyte activating molecule (SLAM) is a receptor for measles virus which also has immunomodulatory activity. We analyzed SLAM expression in mononuclear cells (MNC) of patients with SSPE (n=7) and control subjects (n=7) from the same population. Native 10% PAGE analysis in cell and brain tissue extracts followed by Western blotting using monoclonal anti-human SLAM showed four types of bands. Differences in the type and amount of SLAM expression were observed between SSPE and control cases. Lymphocytes of SSPE patients showed two types of SLAM bands in comparison to only one in control lymphocytes. Stimulation of cells with lipopolysaccharide (80 u/ml) and concanavalin A (1 microg/ml) in vitro led to the appearance of a second isoform in both groups. Brain homogenates of SSPE patients (n=2) displayed all four types of SLAM isoforms at significantly higher levels than those of control brains (n=2). Our results show native PAGE enables the detection of all SLAM isotypes. The expression of SLAM is increased in lymphocytes, monocytes, and brain tissues of SSPE patients.

  19. Simvastatin impairs growth hormone-activated signal transducer and activator of transcription (STAT signaling pathway in UMR-106 osteosarcoma cells.

    Directory of Open Access Journals (Sweden)

    María Claudia Sandoval-Usme

    Full Text Available Recent studies have demonstrated that statins reduce cell viability and induce apoptosis in various types of cancer cells. The molecular mechanisms underlying these effects are poorly understood. The JAK/STAT pathway plays an important role in the regulation of proliferation and apoptosis in many tissues, and its deregulation is believed to be involved in tumorigenesis and cancer. The physiological activation of STAT proteins by GH is rapid but transient in nature and its inactivation is regulated mainly by the expression of SOCS proteins. UMR-106 osteosarcoma cells express a GH-responsive JAK2/STAT5 signaling pathway, providing an experimental model to study the influence of statins on this system. In this study we investigated the actions of simvastatin on cell proliferation, migration, and invasion on UMR-106 cells and examined whether alterations in GH-stimulated JAK/STAT/SOCS signaling may be observed. Results showed that treatment of osteosarcoma cells with simvastatin at 3 to 10 µM doses decreases cell proliferation, migration, and invasion in a time- and dose-dependent manner. At the molecular level, although the mechanisms used by simvastatin are not entirely clear, the effect of the statin on the reduction of JAK2 and STAT5 phosphorylation levels may partially explain the decrease in the GH-stimulated STAT5 transcriptional activity. This effect correlated with a time- and dose-dependent increase of SOCS-3 expression levels in cells treated with simvastatin, a regulatory role that has not been previously described. Furthermore, the finding that simvastatin is capable of inducing SOCS-3 and CIS genes expression shows the potential of the JAK/STAT pathway as a therapeutic target, reinforcing the efficacy of simvastatin as chemotherapeutic drug for the treatment of osteosarcoma.

  20. Eight paths of ERK1/2 signalling pathway regulating hepatocyte proliferation in rat liver regeneration

    Indian Academy of Sciences (India)

    J. W. Li; G. P. Wang; J. Y. Fan; C. F. Chang; C. S. Xu

    2011-12-01

    Although it is known that hormones, growth factors and integrin promote hepatocyte proliferation in liver regeneration (LR) through ERK1/2 signalling pathway, reports about regulating processes of its intracellular paths in hepatocytes of LR are limited. This study aims at exploring which paths of ERK1/2 signalling pathway participate in the regulation of rat LR, especially in hepatocyte proliferation, and how they do so. In all, 14 paths and 165 genes are known to be involved in ERK1/2 signalling pathway. Of them, 161 genes are included in Rat Genome 230 2.0 Array. This array was used to detect expression changes of genes related to ERK1/2 signalling pathway in isolated hepatocytes of rat LR, showing that 60 genes were related to hepatocytes of LR. In addition, bioinformatics and systems biology methods were used to analyse the roles of 14 above paths in regenerating hepatocytes. We found that three paths, RTK → SHC → GRB2/SOS → RAS → RAF, Integrin → FAK → RAC → PAK → RAF and G → PI3K → RAC → PAK → RAF, promoted the G1 phase progression of hepatocytes by activating ERK1/2. A further four paths, Gq → PLC → PKC → SRC/PYK2 → GRB2/SOS → RAS → RAF, RTK → PLC → PKC → SRC/PYK2 → GRB2/SOS → RAS → RAF, Integrin → FAK/SRC → GRB2/SOS → RAS → RAF and Integrin → FAK → RAC → PAK → RAF, advanced the cell progression of S phase and G2/M checkpoint by activating ERK1/2, and so did PP1/2 → Mek1/2 by decreasing the negative influence on ERK1/2. At the late phase of LR, Gs → AC → EPAC → Rap1 → Raf blocked hepatocyte proliferation by decreasing the activity of ERK1/2 and so did PP1/2 → Mek1/2. In summary, 60 genes and 8 paths of ERK1/2 signalling pathway regulated hepatocyte proliferation in rat LR.

  1. Signal transduction pathways in liver and the influence of hepatitis C virus infection on their activities

    Institute of Scientific and Technical Information of China (English)

    Magdalena M Dabrowska; Anatol Panasiuk; Robert Flisiak

    2009-01-01

    In liver, the most intensively studied transmembrane and intracellular signal transduction pathways are the Janus kinase signal transduction pathway, the mitogen-activated protein kinases signal transduction pathway, the transforming growth factor b signal transduction pathway, the tumor necrosis factor a signal transduction pathway and the recently discovered sphingolipid signal transduction pathway. All of them are activated by many different cytokines and growth factors. They regulate specific cell mechanisms such as hepatocytes proliferation, growth, differentiation, adhesion, apoptosis, and synthesis and degradation of the extracellular matrix. The replication cycle of hepatitis C virus (HCV) is intracellular and requires signal transduction to the nucleus to regulate transcription of its genes. Moreover, HCV itself, by its structural and nonstructural proteins, could influence the activity of the second signal messengers. Thus, the inhibition of the transmembrane and intracellular signal transduction pathways could be a new therapeutic target in chronic hepatitis C treatment.

  2. Sinulariolide Suppresses Human Hepatocellular Carcinoma Cell Migration and Invasion by Inhibiting Matrix Metalloproteinase-2/-9 through MAPKs and PI3K/Akt Signaling Pathways.

    Science.gov (United States)

    Wu, Yu-Jen; Neoh, Choo-Aun; Tsao, Chia-Yu; Su, Jui-Hsin; Li, Hsing-Hui

    2015-07-20

    Sinulariolide is an active compound isolated from the cultured soft coral Sinularia flexibilis. In this study, we investigate the migration and invasion effects of sinulariolide in hepatocellular carcinoma cell HA22T. Sinulariolide inhibited the migration and invasion effects of hepatocellular carcinoma cells in a concentration-dependent manner. The results of zymography assay showed that sinulariolide suppressed the activities of matrix metalloproteinase (MMP)-2 and MMP-9. Moreover, protein levels of MMP-2, MMP-9, and urokinase-type plasminogen activator (uPA) were reduced by sinulariolide in a concentration-dependent manner. Sinulariolide also exerted an inhibitory effect on phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK), phosphatidylinositol 3-kinase (PI3K), Akt, Focal adhesion kinase (FAK), growth factor receptor-bound protein 2 (GRB2). Taken together, these results demonstrated that sinulariolide could inhibit hepatocellular carcinoma cell migration and invasion and alter HA22T cell metastasis by reduction of MMP-2, MMP-9, and uPA expression through the suppression of MAPKs, PI3K/Akt, and the FAK/GRB2 signaling pathway. These findings suggest that sinulariolide merits further evaluation as a chemotherapeutic agent for human hepatocellular carcinoma.

  3. Agonist-biased signaling via proteinase activated receptor-2: differential activation of calcium and mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Ramachandran, Rithwik; Mihara, Koichiro; Mathur, Maneesh; Rochdi, Moulay Driss; Bouvier, Michel; Defea, Kathryn; Hollenberg, Morley D

    2009-10-01

    We evaluated the ability of different trypsin-revealed tethered ligand (TL) sequences of rat proteinase-activated receptor 2 (rPAR(2)) and the corresponding soluble TL-derived agonist peptides to trigger agonist-biased signaling. To do so, we mutated the proteolytically revealed TL sequence of rPAR(2) and examined the impact on stimulating intracellular calcium transients and mitogen-activated protein (MAP) kinase. The TL receptor mutants, rPAR(2)-Leu(37)Ser(38), rPAR(2)-Ala(37-38), and rPAR(2)-Ala(39-42) were compared with the trypsin-revealed wild-type rPAR(2) TL sequence, S(37)LIGRL(42)-. Upon trypsin activation, all constructs stimulated MAP kinase signaling, but only the wt-rPAR(2) and rPAR(2)-Ala(39-42) triggered calcium signaling. Furthermore, the TL-derived synthetic peptide SLAAAA-NH2 failed to cause PAR(2)-mediated calcium signaling but did activate MAP kinase, whereas SLIGRL-NH2 triggered both calcium and MAP kinase signaling by all receptors. The peptides AAIGRL-NH2 and LSIGRL-NH2 triggered neither calcium nor MAP kinase signals. Neither rPAR(2)-Ala(37-38) nor rPAR(2)-Leu(37)Ser(38) constructs recruited beta-arrestins-1 or -2 in response to trypsin stimulation, whereas both beta-arrestins were recruited to these mutants by SLIGRL-NH2. The lack of trypsin-triggered beta-arrestin interactions correlated with impaired trypsin-activated TL-mutant receptor internalization. Trypsin-stimulated MAP kinase activation by the TL-mutated receptors was not blocked by inhibitors of Galpha(i) (pertussis toxin), Galpha(q) [N-cyclohexyl-1-(2,4-dichlorophenyl)-1,4-dihydro-6-methylindeno[1,2-c]pyrazole-3-carboxamide (GP2A)], Src kinase [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], or the epidermal growth factor (EGF) receptor [4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478)], but was inhibited by the Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl (Y27362). The data indicate that the

  4. NMDA receptor activation regulates sociability by its effect on mTOR signaling activity.

    Science.gov (United States)

    Burket, Jessica A; Benson, Andrew D; Tang, Amy H; Deutsch, Stephen I

    2015-07-01

    Tuberous Sclerosis Complex is one example of a syndromic form of autism spectrum disorder associated with disinhibited activity of mTORC1 in neurons (e.g., cerebellar Purkinje cells). mTORC1 is a complex protein possessing serine/threonine kinase activity and a key downstream molecule in a signaling cascade beginning at the cell surface with the transduction of neurotransmitters (e.g., glutamate and acetylcholine) and nerve growth factors (e.g., Brain-Derived Neurotrophic Factor). Interestingly, the severity of the intellectual disability in Tuberous Sclerosis Complex may relate more to this metabolic disturbance (i.e., overactivity of mTOR signaling) than the density of cortical tubers. Several recent reports showed that rapamycin, an inhibitor of mTORC1, improved sociability and other symptoms in mouse models of Tuberous Sclerosis Complex and autism spectrum disorder, consistent with mTORC1 overactivity playing an important pathogenic role. NMDA receptor activation may also dampen mTORC1 activity by at least two possible mechanisms: regulating intraneuronal accumulation of arginine and the phosphorylation status of a specific extracellular signal regulating kinase (i.e., ERK1/2), both of which are "drivers" of mTORC1 activity. Conceivably, the prosocial effects of targeting the NMDA receptor with agonists in mouse models of autism spectrum disorders result from their ability to dampen mTORC1 activity in neurons. Strategies for dampening mTORC1 overactivity by NMDA receptor activation may be preferred to its direct inhibition in chronic neurodevelopmental disorders, such as autism spectrum disorders.

  5. A Active Micromachined Scalp Electrode Array for Eeg Signal Recording.

    Science.gov (United States)

    Alizadeh-Taheri, Babak

    This thesis describes the design, microfabrication, and testing of an active scalp EEG (electroencephalograph) electrode that has several distinct advantages over existing technologies. These advantages are: (1) no electrolyte used, (2) no skin preparation, (3) significantly reduced sensor size, and (4) compatibility with EEG monitoring systems. The active electrode array is an integrated system made of an array of capacitive sensors with local integrated circuitry housed in a package with batteries to power the circuitry. This level of integration was required to achieve the functional performance obtained by the electrode. The electrode consists of a silicon sensor substrate fabricated at UCD and a custom circuit substrate fabricated at Orbit Semiconductors, using a 2 μm analog CMOS technology. The circuitry was designed for low 1/f noise. One side of the sensor substrate holds four capacitive sensors with rm Si_3N _4 as the dielectric material. The opposite side holds aluminum pads for bonding to the circuit substrate. A via hole technology was developed to make electrical contact to both sides of the sensor substrate. The via holes are 200 μm square openings etched through the silicon by a reactive ion etching (RIE) process using an rm SF_6/O_2 gas mixture, oxidized, and then filled with sputtered aluminum for contacts through the substrate. The via holes have an aspect ratio of 2:1 (length of opening to depth of hole). Silicon RIE etch rates of up to 18 mu/hr were obtained under optimum conditions, using a 0.8 μm aluminum mask. The circuit and sensor substrates were bonded with silver adhesive, and wire bonding was used to make electrical contacts between the substrates. The two substrates were then integrated in a custom package for testing. The electrode was tested on an electrical test bench and on human subjects in four modalities of EEG activity, namely: (1) spontaneous EEG, (2) sensory event-related potentials, (3) brain stem potentials, and (4

  6. Akt1 signaling coordinates BMP signaling and β-catenin activity to regulate second heart field progenitor development.

    Science.gov (United States)

    Luo, Wen; Zhao, Xia; Jin, Hengwei; Tao, Lichan; Zhu, Jingai; Wang, Huijuan; Hemmings, Brian A; Yang, Zhongzhou

    2015-02-15

    Second heart field (SHF) progenitors exhibit continued proliferation and delayed differentiation, which are modulated by FGF4/8/10, BMP and canonical Wnt/β-catenin signaling. PTEN-Akt signaling regulates the stem cell/progenitor cell homeostasis in several systems, such as hematopoietic stem cells, intestinal stem cells and neural progenitor cells. To address whether PTEN-Akt signaling is involved in regulating cardiac progenitors, we deleted Pten in SHF progenitors. Deletion of Pten caused SHF expansion and increased the size of the SHF derivatives, the right ventricle and the outflow tract. Cell proliferation of cardiac progenitors was enhanced, whereas cardiac differentiation was unaffected by Pten deletion. Removal of Akt1 rescued the phenotype and early lethality of Pten deletion mice, suggesting that Akt1 was the key downstream target that was negatively regulated by PTEN in cardiac progenitors. Furthermore, we found that inhibition of FOXO by Akt1 suppressed the expression of the gene encoding the BMP ligand (BMP7), leading to dampened BMP signaling in the hearts of Pten deletion mice. Cardiac activation of Akt also increased the Ser552 phosphorylation of β-catenin, thus enhancing its activity. Reducing β-catenin levels could partially rescue heart defects of Pten deletion mice. We conclude that Akt signaling regulates the cell proliferation of SHF progenitors through coordination of BMP signaling and β-catenin activity.

  7. ECM Signaling Regulates Collective Cellular Dynamics to Control Pancreas Branching Morphogenesis.

    Science.gov (United States)

    Shih, Hung Ping; Panlasigui, Devin; Cirulli, Vincenzo; Sander, Maike

    2016-01-12

    During pancreas development, epithelial buds undergo branching morphogenesis to form an exocrine and endocrine gland. Proper morphogenesis is necessary for correct lineage allocation of pancreatic progenitors; however, the cellular events underlying pancreas morphogenesis are unknown. Here, we employed time-lapse microscopy and fluorescent labeling of cells to analyze cell behaviors associated with pancreas morphogenesis. We observed that outer bud cells adjacent to the basement membrane are pleomorphic and rearrange frequently; additionally, they largely remain in the outer cell compartment even after mitosis. These cell behaviors and pancreas branching depend on cell contacts with the basement membrane, which induce actomyosin cytoskeleton remodeling via integrin-mediated activation of FAK/Src signaling. We show that integrin signaling reduces E-cadherin-mediated cell-cell adhesion in outer cells and provide genetic evidence that this regulation is necessary for initiation of branching. Our study suggests that regulation of cell motility and adhesion by local niche cues initiates pancreas branching morphogenesis.

  8. MUC16 contributes to the metastasis of pancreatic ductal adenocarcinoma through focal adhesion mediated signaling mechanism

    Science.gov (United States)

    Chugh, Seema; Rachagani, Satyanarayana; Lakshmanan, Imayavaramban; Gupta, Suprit; Seshacharyulu, Parthasarathy; Smith, Lynette M.; Ponnusamy, Moorthy P.; Batra, Surinder K.

    2016-01-01

    MUC16, a heavily glycosylated type-I transmembrane mucin is overexpressed in several cancers including pancreatic ductal adenocarcinoma (PDAC). Previously, we have shown that MUC16 is significantly overexpressed in human PDAC tissues. However, the functional consequences and its role in PDAC is poorly understood. Here, we show that MUC16 knockdown decreases PDAC cell proliferation, colony formation and migration in vitro. Also, MUC16 knockdown decreases the tumor formation and metastasis in orthotopic xenograft mouse model. Mechanistically, immunoprecipitation and immunofluorescence analyses confirms MUC16 interaction with galectin-3 and mesothelin in PDAC cells. Adhesion assay displayed decreased cell attachment of MUC16 knockdown cells with recombinant galectin-1 and galectin-3 protein. Further, CRISPR/Cas9-mediated MUC16 knockout cells show decreased tumor-associated carbohydrate antigens (T and Tn) in PDAC cells. Importantly, carbohydrate antigens were decreased in the region that corresponds to MUC16 and suggests for the decreased MUC16-galectin interactions. Co-immunoprecipitation also revealed a novel interaction between MUC16 and FAK in PDAC cells. Interestingly, we observed decreased expression of mesenchymal and increased expression of epithelial markers in MUC16-silenced cells. Additionally, MUC16 loss showed a decreased FAK-mediated Akt and ERK/MAPK activation. Altogether, these findings suggest that MUC16-focal adhesion signaling may play a critical role in facilitating PDAC growth and metastasis. PMID:27382435

  9. A third-order active-R filter with feedforward input signal

    Indian Academy of Sciences (India)

    G N Shinde; P B Patil; P R Mirkute

    2003-12-01

    A realization of voltage-mode transfer functions with feedforward input signal for third-order active-R filter using an oprational amplifier has been presented. This filter is useful for high frequency operation, monolithic IC implementation and is easy to design. The single circuit gives three filter functions, low pass, high pass and band pass. This filter circuit can be used for different and f0 with high passband gain. This gives better stop band attenuation and sharper cut-off at the edge of the passband.

  10. Twist promotes reprogramming of glucose metabolism in breast cancer cells through PI3K/AKT and p53 signaling pathways.

    Science.gov (United States)

    Yang, Li; Hou, Yixuan; Yuan, Jie; Tang, Shifu; Zhang, Hailong; Zhu, Qing; Du, Yan-e; Zhou, Mingli; Wen, Siyang; Xu, Liyun; Tang, Xi; Cui, Xiaojiang; Liu, Manran

    2015-09-22

    Twist, a key regulator of epithelial-mesenchymal transition (EMT), plays an important role in the development of a tumorigenic phenotype. Energy metabolism reprogramming (EMR), a newly discovered hallmark of cancer cells, potentiates cancer cell proliferation, survival, and invasion. Currently little is known about the effects of Twist on tumor EMR. In this study, we found that glucose consumption and lactate production were increased and mitochondrial mass was decreased in Twist-overexpressing MCF10A mammary epithelial cells compared with vector-expressing MCF10A cells. Moreover, these Twist-induced phenotypic changes were augmented by hypoxia. The expression of some glucose metabolism-related genes such as PKM2, LDHA, and G6PD was also found to be upregulated. Mechanistically, activated β1-integrin/FAK/PI3K/AKT/mTOR and suppressed P53 signaling were responsible for the observed EMR. Knockdown of Twist reversed the effects of Twist on EMR in Twist-overexpressing MCF10A cells and Twist-positive breast cancer cells. Furthermore, blockage of the β1-integrin/FAK/PI3K/AKT/mTOR pathway by siRNA or specific chemical inhibitors, or rescue of p53 activation can partially reverse the switch of glucose metabolism and inhibit the migration of Twist-overexpressing MCF10A cells and Twist-positive breast cancer cells. Thus, our data suggest that Twist promotes reprogramming of glucose metabolism in MCF10A-Twist cells and Twist-positive breast cancer cells via activation of the β1-integrin/FAK/PI3K/AKT/mTOR pathway and inhibition of the p53 pathway. Our study provides new insight into EMR.

  11. 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

  12. The Search for signals of technological activities in the galaxy

    CERN Document Server

    Lemarchand, Guillermo A

    2010-01-01

    In this article an analysis of the fundamentals used to search for extraterrestrial artificial signals in the galaxy, which have been developing for more than five decades, is presented. It is shown that the key factor for the success of these research projects is given by the technological civilizations lifetimes. Assuming the Principle of Mediocrity, estimations are made to determine the minimum number of civilizations that may co-exist in the galaxy and the probability of detecting a signal from them.

  13. Design of excitation signals for active system monitoring in a performance assessment setup

    DEFF Research Database (Denmark)

    Green, Torben; Izadi-Zamanabadi, Roozbeh; Niemann, Hans Henrik

    2011-01-01

    This paper investigates how the excitation signal should be chosen for a active performance setup. The signal is used in a setup where the main purpose is to detect whether a parameter change of the controller has changed the global performance significantly. The signal has to be able to excite...

  14. Loss of STAT3 signaling during elevated activity causes vulnerability in hippocampal neurons

    OpenAIRE

    2012-01-01

    Chronically altered levels of network activity lead to changes in the morphology and functions of neurons. However, little is known of how changes in neuronal activity alter the intracellular signaling pathways mediating neuronal survival. Here we use primary cultures of rat hippocampal neurons to show that elevated neuronal activity impairs phosphorylation of the serine/threonine kinase, Erk1/2 and the activation of signal transducer and activator of transcription 3 (STAT3) by phosphorylatio...

  15. Artemisinin inhibits neuroblastoma proliferation through activation of AHP-activated protein kinase (AMPK) signaling.

    Science.gov (United States)

    Tan, Wei-Qiang; Chen, Gang; Jia, Bing; Ye, Ming

    2014-06-01

    Recent population studies suggest that the use of artemisinin is associated with reduced incidence and improved prognosis of certain cancers. In the current study, we assessed the effect of artemisinin on neuroblastoma cells using SHSY5Y cells. We found that artemisinin inhibited growth and modulated expression of cell-cycle regulators in these cells. Treatment with artemisinin was also associated with activation of AMP kinase and inhibition of mTOR/p70S6K/pS6 signaling in SHSY5Y cells. In addition, inhibition of AMPK signaling reversed impact on the anti-proliferative roles of artemisinin. Taken together, these results provide evidence for a mechanism that may contribute to the antineoplastic effects of artemisinin suggested by recent population studies and justify further work to explore its potential roles in neuroblastoma prevention and treatment.

  16. Focal adhesion kinase modulates activation of NF-κB by flow in endothelial cells

    OpenAIRE

    Petzold, Tobias; Orr, A. Wayne; Hahn, Cornelia; Jhaveri, Krishna A.; Parsons, J Thomas; Schwartz, Martin Alexander

    2009-01-01

    Atherogenesis involves activation of NF-κB in endothelial cells by fluid shear stress. Because this pathway involves integrins, we investigated the involvement of focal adhesion kinase (FAK). We found that FAK was not required for flow-stimulated translocation of the p65 NF-κB subunit to the nucleus but was essential for phosphorylation of p65 on serine 536 and induction of ICAM-1, an NF-κB-dependent gene. NF-κB activation by TNF-α or hydrogen peroxide was FAK independent. Events upstream of ...

  17. uPA and PAI-1-Related Signaling Pathways Differ between Primary Breast Cancers and Lymph Node Metastases12

    Science.gov (United States)

    Malinowsky, Katharina; Wolff, Claudia; Berg, Daniela; Schuster, Tibor; Walch, Axel; Bronger, Holger; Mannsperger, Heiko; Schmidt, Christian; Korf, Ulrike; Höfler, Heinz; Becker, Karl-Friedrich

    2012-01-01

    The supporting role of urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor 1 (PAI-1) in migration and invasion is well known. In addition, both factors are key components in cancer cell-related signaling. However, little information is available for uPA and PAI-1-associated signaling pathways in primary cancers and corresponding lymph node metastases. The aim of this study was to compare the expression of uPA and PAI-1-associated signaling proteins in 52 primary breast cancers and corresponding metastases. Proteins were extracted from formalin-fixed paraffin-embedded tissue samples of the primary tumors and metastases. Protein lysates were subsequently analyzed by reverse phase protein array for the expression of members of the PI3K/AKT (FAK, GSK3-β, ILK, pGSK3-β, PI3K, and ROCK) and the MAPK pathways (pp38, pSTAT3, and p38). A solid correlation of uPA expression existed between primary tumors and metastases, whereas PAI-1 expression did not significantly correlate between them. The correlations of uPA and PAI-1 with signaling pathways found in primary tumors did not persist in metastases. Analysis of single molecules revealed that some correlated well between tumors and metastases (FAK, pGSK3-β, ILK, Met, PI3K, ROCK, uPA, p38, and pp38), whereas others did not (PAI-1 and GSK3-β). Whether the expression of a protein correlated between tumor and metastasis or not was independent of the pathway the protein is related to. These findings hint at a complete deregulation of uPA and PAI-1-related signaling in metastases, which might be the reason why uPA and PAI-1 reached clinical relevance only for lymph node-negative breast cancer tissues. PMID:22496926

  18. uPA and PAI-1-Related Signaling Pathways Differ between Primary Breast Cancers and Lymph Node Metastases.

    Science.gov (United States)

    Malinowsky, Katharina; Wolff, Claudia; Berg, Daniela; Schuster, Tibor; Walch, Axel; Bronger, Holger; Mannsperger, Heiko; Schmidt, Christian; Korf, Ulrike; Höfler, Heinz; Becker, Karl-Friedrich

    2012-04-01

    The supporting role of urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor 1 (PAI-1) in migration and invasion is well known. In addition, both factors are key components in cancer cell-related signaling. However, little information is available for uPA and PAI-1-associated signaling pathways in primary cancers and corresponding lymph node metastases. The aim of this study was to compare the expression of uPA and PAI-1-associated signaling proteins in 52 primary breast cancers and corresponding metastases. Proteins were extracted from formalin-fixed paraffin-embedded tissue samples of the primary tumors and metastases. Protein lysates were subsequently analyzed by reverse phase protein array for the expression of members of the PI3K/AKT (FAK, GSK3-β, ILK, pGSK3-β, PI3K, and ROCK) and the MAPK pathways (pp38, pSTAT3, and p38). A solid correlation of uPA expression existed between primary tumors and metastases, whereas PAI-1 expression did not significantly correlate between them. The correlations of uPA and PAI-1 with signaling pathways found in primary tumors did not persist in metastases. Analysis of single molecules revealed that some correlated well between tumors and metastases (FAK, pGSK3-β, ILK, Met, PI3K, ROCK, uPA, p38, and pp38), whereas others did not (PAI-1 and GSK3-β). Whether the expression of a protein correlated between tumor and metastasis or not was independent of the pathway the protein is related to. These findings hint at a complete deregulation of uPA and PAI-1-related signaling in metastases, which might be the reason why uPA and PAI-1 reached clinical relevance only for lymph node-negative breast cancer tissues.

  19. Focal Adhesion Kinase Inhibitors in Combination with Erlotinib Demonstrate Enhanced Anti-Tumor Activity in Non-Small Cell Lung Cancer.

    Directory of Open Access Journals (Sweden)

    Grant A Howe

    Full Text Available Blockade of epidermal growth factor receptor (EGFR activity has been a primary therapeutic target for non-small cell lung cancers (NSCLC. As patients with wild-type EGFR have demonstrated only modest benefit from EGFR tyrosine kinase inhibitors (TKIs, there is a need for additional therapeutic approaches in patients with wild-type EGFR. As a key component of downstream integrin signalling and known receptor cross-talk with EGFR, we hypothesized that targeting focal adhesion kinase (FAK activity, which has also been shown to correlate with aggressive stage in NSCLC, would lead to enhanced activity of EGFR TKIs. As such, EGFR TKI-resistant NSCLC cells (A549, H1299, H1975 were treated with the EGFR TKI erlotinib and FAK inhibitors (PF-573,228 or PF-562,271 both as single agents and in combination. We determined cell viability, apoptosis and 3-dimensional growth in vitro and assessed tumor growth in vivo. Treatment of EGFR TKI-resistant NSCLC cells with FAK inhibitor alone effectively inhibited cell viability in all cell lines tested; however, its use in combination with the EGFR TKI erlotinib was more effective at reducing cell viability than either treatment alone when tested in both 2- and 3-dimensional assays in vitro, with enhanced benefit seen in A549 cells. This increased efficacy may be due in part to the observed inhibition of Akt phosphorylation when the drugs were used in combination, where again A549 cells demonstrated the most inhibition following treatment with the drug combination. Combining erlotinib with FAK inhibitor was also potent in vivo as evidenced by reduced tumor growth in the A549 mouse xenograft model. We further ascertained that the enhanced sensitivity was irrespective of the LKB1 mutational status. In summary, we demonstrate the effectiveness of combining erlotinib and FAK inhibitors for use in known EGFR wild-type, EGFR TKI resistant cells, with the potential that a subset of cell types, which includes A549, could be

  20. Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans.

    Science.gov (United States)

    Liu, Zhiyu; Wang, Bin; He, Ruijun; Zhao, Yanmei; Miao, Long

    2014-02-01

    In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.

  1. Kurarinol induces hepatocellular carcinoma cell apoptosis through suppressing cellular signal transducer and activator of transcription 3 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Shu, Guangwen; Yang, Jing; Zhao, Wenhao; Xu, Chan; Hong, Zongguo; Mei, Zhinan; Yang, Xinzhou, E-mail: xinzhou_yang@hotmail.com

    2014-12-01

    Kurarinol is a flavonoid isolated from roots of the medical plant Sophora flavescens. However, its cytotoxic activity against hepatocellular carcinoma (HCC) cells and toxic effects on mammalians remain largely unexplored. Here, the pro-apoptotic activities of kurarinol on HCC cells and its toxic impacts on tumor-bearing mice were evaluated. The molecular mechanisms underlying kurarinol-induced HCC cell apoptosis were also investigated. We found that kurarinol dose-dependently provoked HepG2, Huh-7 and H22 HCC cell apoptosis. In addition, kurarinol gave rise to a considerable decrease in the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) in HCC cells. Suppression of STAT3 signaling is involved in kurarinol-induced HCC cell apoptosis. In vivo studies showed that kurarinol injection substantially induced transplanted H22 cell apoptosis with low toxic impacts on tumor-bearing mice. Similarly, the transcriptional activity of STAT3 in transplanted tumor tissues was significantly suppressed after kurarinol treatment. Collectively, our current research demonstrated that kurarinol has the capacity of inducing HCC cell apoptosis both in vitro and in vivo with undetectable toxic impacts on the host. Suppressing STAT3 signaling is implicated in kurarinol-mediated HCC cell apoptosis. - Highlights: • Kurarinol induces hepatocellular carcinoma (HCC) cell apoptosis. • Kurarinol induces HCC cell apoptosis via inhibiting STAT3. • Kurarinol exhibits low toxic effects on tumor-bearing animals.

  2. Caffeine Induces Cell Death via Activation of Apoptotic Signal and Inactivation of Survival Signal in Human Osteoblasts

    Directory of Open Access Journals (Sweden)

    Wen-Hsiung Chan

    2008-05-01

    Full Text Available Caffeine consumption is a risk factor for osteoporosis, but the precise regulatory mechanisms are currently unknown. Here, we show that cell viability decreases in osteoblasts treated with caffeine in a dose-dependent manner. This cell death is attributed primarily to apoptosis and to a smaller extent, necrosis. Moreover, caffeine directly stimulates intracellular oxidative stress. Our data support caffeine-induced apoptosis in osteoblasts via a mitochondria-dependent pathway. The apoptotic biochemical changes were effectively prevented upon pretreatment with ROS scavengers, indicating that ROS plays a critical role as an upstream controller in the caffeine-induced apoptotic cascade. Additionally, p21-activated protein kinase 2 (PAK2 and c-Jun N-terminal kinase (JNK were activated in caffeine-treated osteoblasts. Experiments further found that PAK2 activity is required for caffeine-induced JNK activation and apoptosis. Importantly, our data also show that caffeine triggers cell death via inactivation of the survival signal, including the ERK- and Akt-mediated anti-apoptotic pathways. Finally, exposure of rats to dietary water containing 10~20 μM caffeine led to bone mineral density loss. These results demonstrate for the first time that caffeine triggers apoptosis in osteoblasts via activation of mitochondria-dependent cell death signaling and inactivation of the survival signal, and causes bone mineral density loss in vivo.

  3. Activation of the Notch signaling pathway promotes neurovascular repair after traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    Qi-shan Ran; Yun-hu Yu; Xiao-hong Fu; Yuan-chao Wen

    2015-01-01

    The Notch signaling pathway plays a key role in angiogenesis and endothelial cell formation, but it remains unclear whether it is involved in vascular repair by endothelial progenitor cells after traumatic brain injury. Therefore, in the present study, we controlled the Notch signaling path-way using overexpression and knockdown constructs. Activation of the Notch signaling pathway by Notch1 or Jagged1 overexpression enhanced the migration, invasiveness and angiogenic ability of endothelial progenitor cells. Suppression of the Notch signaling pathway with Notch1 or Jagged1 siRNAs reduced the migratory capacity, invasiveness and angiogenic ability of endo-thelial progenitor cells. Activation of the Notch signaling pathwayin vivo in a rat model of mild traumatic brain injury promoted neurovascular repair. These ifndings suggest that the activation of the Notch signaling pathway promotes blood vessel formation and tissue repair after brain trauma.

  4. Signal transducer and activator of transcription 3 regulation by novel binding partners

    Institute of Scientific and Technical Information of China (English)

    Tadashi; Matsuda; Ryuta; Muromoto; Yuichi; Sekine; Sumihito; Togi; Yuichi; Kitai; Shigeyuki; Kon; Kenji; Oritani

    2015-01-01

    Signal transducers and activators of transcription(STATs) mediate essential signals for various biological processes,including immune responses,hematopoiesis,and neurogenesis. STAT3,for example,is involved in the pathogenesis of various human diseases,including cancers,autoimmune and inflammatory disorders. STAT3 activation is therefore tightly regulated at multiple levels to prevent these pathological conditions. A number of proteins have been reported to associate with STAT3 and regulate its activity. These STAT3-interacting proteins function to modulate STAT3-mediated signaling at various steps and mediate the crosstalk of STAT3 with other cellular signaling pathways. This article reviews the roles of novel STAT3 binding partners such as DAXX,zipperinteracting protein kinase,Krüppel-associated box-associated protein 1,Y14,PDZ and LIM domain 2 and signal transducing adaptor protein-2,in the regulation of STAT3-mediated signaling.

  5. Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling

    Directory of Open Access Journals (Sweden)

    Kumar N Alagramam

    2014-01-01

    Full Text Available Noise-induced hearing loss (NIHL is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway.

  6. The Search for signals of technological activities in the galaxy

    OpenAIRE

    Lemarchand, Guillermo A.

    2010-01-01

    In this article an analysis of the fundamentals used to search for extraterrestrial artificial signals in the galaxy, which have been developing for more than five decades, is presented. It is shown that the key factor for the success of these research projects is given by the technological civilizations lifetimes. Assuming the Principle of Mediocrity, estimations are made to determine the minimum number of civilizations that may co-exist in the galaxy and the probability of detecting a signa...

  7. D-pinitol Inhibits Prostate Cancer Metastasis through Inhibition of αVβ3 Integrin by Modulating FAK, c-Src and NF-κB Pathways

    Directory of Open Access Journals (Sweden)

    Chih-Hsin Tang

    2013-05-01

    Full Text Available Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to the bone. D-pinitol, a 3-methoxy analogue of d-chiro-inositol, was identified as an active principle in soy foods and legumes, and it has been proven to induce tumor apoptosis and metastasis of cancer cells. In this study, we investigated the anti-metastasis effects of D-pinitol in human prostate cancer cells. We found that D-pinitol reduced the migration and the invasion of prostate cancer cells (PC3 and DU145 at noncytotoxic concentrations. Integrins are the major adhesive molecules in mammalian cells and have been associated with the metastasis of cancer cells. Treatment of prostate cancer cells with D-pinitol reduced mRNA and cell surface expression of αvβ3 integrin. In addition, D-pinitol exerted its inhibitory effects by reducing focal adhesion kinase (FAK phosphorylation, c-Src kinase activity and NF-kB activation. Thus, D-pinitol may be a novel anti-metastasis agent for the treatment of prostate cancer metastasis.

  8. D-pinitol inhibits prostate cancer metastasis through inhibition of αVβ3 integrin by modulating FAK, c-Src and NF-κB pathways.

    Science.gov (United States)

    Lin, Tien-Huang; Tan, Tzu-Wei; Tsai, Tsung-Hsun; Chen, Chi-Cheng; Hsieh, Teng-Fu; Lee, Shang-Sen; Liu, Hsin-Ho; Chen, Wen-Chi; Tang, Chih-Hsin

    2013-05-08

    Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to the bone. D-pinitol, a 3-methoxy analogue of d-chiro-inositol, was identified as an active principle in soy foods and legumes, and it has been proven to induce tumor apoptosis and metastasis of cancer cells. In this study, we investigated the anti-metastasis effects of D-pinitol in human prostate cancer cells. We found that D-pinitol reduced the migration and the invasion of prostate cancer cells (PC3 and DU145) at noncytotoxic concentrations. Integrins are the major adhesive molecules in mammalian cells and have been associated with the metastasis of cancer cells. Treatment of prostate cancer cells with D-pinitol reduced mRNA and cell surface expression of αvβ3 integrin. In addition, D-pinitol exerted its inhibitory effects by reducing focal adhesion kinase (FAK) phosphorylation, c-Src kinase activity and NF-kB activation. Thus, D-pinitol may be a novel anti-metastasis agent for the treatment of prostate cancer metastasis.

  9. Integration of light and metabolic signals for stem cell activation at the shoot apical meristem

    Science.gov (United States)

    Pfeiffer, Anne; Janocha, Denis; Dong, Yihan; Medzihradszky, Anna; Schöne, Stefanie; Daum, Gabor; Suzaki, Takuya; Forner, Joachim; Langenecker, Tobias; Rempel, Eugen; Schmid, Markus; Wirtz, Markus; Hell, Rüdiger; Lohmann, Jan U

    2016-01-01

    A major feature of embryogenesis is the specification of stem cell systems, but in contrast to the situation in most animals, plant stem cells remain quiescent until the postembryonic phase of development. Here, we dissect how light and metabolic signals are integrated to overcome stem cell dormancy at the shoot apical meristem. We show on the one hand that light is able to activate expression of the stem cell inducer WUSCHEL independently of photosynthesis and that this likely involves inter-regional cytokinin signaling. Metabolic signals, on the other hand, are transduced to the meristem through activation of the TARGET OF RAPAMYCIN (TOR) kinase. Surprisingly, TOR is also required for light signal dependent stem cell activation. Thus, the TOR kinase acts as a central integrator of light and metabolic signals and a key regulator of stem cell activation at the shoot apex. DOI: http://dx.doi.org/10.7554/eLife.17023.001 PMID:27400267

  10. Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm-egg interactions and anaphase resumption.

    Science.gov (United States)

    McGinnis, Lynda K; Luo, Jinping; Kinsey, William H

    2013-04-01

    Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.

  11. CREB and the CRTC co-activators: sensors for hormonal and metabolic signals.

    Science.gov (United States)

    Altarejos, Judith Y; Montminy, Marc

    2011-03-01

    The cyclic AMP-responsive element-binding protein (CREB) is phosphorylated in response to a wide variety of signals, yet target gene transcription is only increased in a subset of cases. Recent studies indicate that CREB functions in concert with a family of latent cytoplasmic co-activators called cAMP-regulated transcriptional co-activators (CRTCs), which are activated through dephosphorylation. A dual requirement for CREB phosphorylation and CRTC dephosphorylation is likely to explain how these activator-co-activator cognates discriminate between different stimuli. Following their activation, CREB and CRTCs mediate the effects of fasting and feeding signals on the expression of metabolic programmes in insulin-sensitive tissues.

  12. Alternative splicing of MALT1 controls signalling and activation of CD4(+) T cells.

    Science.gov (United States)

    Meininger, Isabel; Griesbach, Richard A; Hu, Desheng; Gehring, Torben; Seeholzer, Thomas; Bertossi, Arianna; Kranich, Jan; Oeckinghaus, Andrea; Eitelhuber, Andrea C; Greczmiel, Ute; Gewies, Andreas; Schmidt-Supprian, Marc; Ruland, Jürgen; Brocker, Thomas; Heissmeyer, Vigo; Heyd, Florian; Krappmann, Daniel

    2016-04-12

    MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4(+) T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation.

  13. Signal Modulation of Super Read Only Memory with Thermally Activated Aperture Model

    Science.gov (United States)

    Kim, June Seo; Kwak, Keumcheol; You, Chun-Yeol

    2008-07-01

    We describe the signal modulation of super read only memory (ROM) with thermally activated aperture model using a three-dimensional finite-difference time-domain method. The thermally activated aperture is modeled using a spatially varied refractive indices of the GeSbTe layer. No meaningful signal modulation is observed without thermally activated aperture below the resolution limit of 120 nm. When we open the thermally activated aperture by considering the temperature dependence of the refractive indices in the GeSbTe layer, the 2.8 and 1.7% signal modulations are observed for 120 and 80 nm pits, respectively. The experimentally observed signal modulation under the resolution limit can be explained using the thermally activated aperture model.

  14. Molecular mechanism of cellular reception of ionizing radiation and of activation of signal transduction pathway

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Keiji [Nagasaki Univ. (Japan). Faculty of Pharmaceutical Sciences

    1997-03-01

    The author reviewed what in cells receives ionizing radiation as a stress and which signal transduction pathway is activated to induce the stress reaction in the following order: Activation of protein kinase C (PKC) pathway by radiation, activation of MAP kinase superfamily by radiation, induction of p53 function by radiation, and radiation exposure and stress reaction pathway. Conclusion was as follows: Cellular receptors to radiation can be cell membrane and DNA. Membrane reception of radiation induces activation of tyrosine kinase and sphingomyelinase, which resulting in activation of PKC- and MAP kinase-mediated signal transduction. The signal generated in the nucleus participates in regulation of cell cycle and in DNA repair. Therefore, it seems that irradiation of ionizing radiation gives energy to various cellular receptor sites as well as DNA, which generate various independent signals to be transduced and accumulated in the nucleus, and leading to cellular response. (K.H.). 63 refs.

  15. Activation of the Notch signaling pathway promotes neurovascular repair after traumatic brain injury

    OpenAIRE

    2015-01-01

    The Notch signaling pathway plays a key role in angiogenesis and endothelial cell formation, but it remains unclear whether it is involved in vascular repair by endothelial progenitor cells after traumatic brain injury. Therefore, in the present study, we controlled the Notch signaling pathway using overexpression and knockdown constructs. Activation of the Notch signaling pathway by Notch1 or Jagged1 overexpression enhanced the migration, invasiveness and angiogenic ability of endothelial pr...

  16. Regulation of promyogenic signal transduction by cell-cell contact and adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Robert S., E-mail: Robert.Krauss@mssm.edu [Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY 10029 (United States)

    2010-11-01

    Skeletal myoblast differentiation involves acquisition of the muscle-specific transcriptional program and morphological changes, including fusion into multinucleated myofibers. Differentiation is regulated by extracellular signaling cues, including cell-cell contact and adhesion. Cadherin and Ig adhesion receptors have been implicated in distinct but overlapping stages of myogenesis. N-cadherin signals through the Ig receptor Cdo to activate p38 MAP kinase, while the Ig receptor neogenin signals to activate FAK; both processes promote muscle-specific gene expression and myoblast fusion. M-cadherin activates Rac1 to enhance fusion. Specific Ig receptors (Kirre and Sns) are essential for myoblast fusion in Drosophila, also signaling through Rac, and vertebrate orthologs of Kirre and Sns have partially conserved function. Mice lacking specific cytoplasmic signaling factors activated by multiple receptors (e.g., Rac1) have strong muscle phenotypes in vivo. In contrast, mice lacking individual adhesion receptors that lie upstream of these factors have modest phenotypes. Redundancy among receptors may account for this. Many of the mammalian Ig receptors and cadherins associate with each other, and multivalent interactions within these complexes may require removal of multiple components to reveal dramatic defects in vivo. Nevertheless, it is possible that the murine adhesion receptors rate-limiting in vivo have not yet been identified or fully assessed.

  17. Pleiotropic patterning response to activation of Shh signaling in the limb apical ectodermal ridge.

    Science.gov (United States)

    Wang, Chi-Kuang Leo; Tsugane, Mizuyo H; Scranton, Victoria; Kosher, Robert A; Pierro, Louis J; Upholt, William B; Dealy, Caroline N

    2011-05-01

    Sonic hedgehog (Shh) signaling in the limb plays a central role in coordination of limb patterning and outgrowth. Shh expression in the limb is limited to the cells of the zone of polarizing activity (ZPA), located in posterior limb bud mesoderm. Shh is not expressed by limb ectoderm or apical ectodermal ridge (AER), but recent studies suggest a role for AER-Shh signaling in limb patterning. Here, we have examined the effects of activation of Shh signaling in the AER. We find that targeted expression of Shh in the AER activates constitutive Shh signaling throughout the AER and subjacent limb mesoderm, and causes a range of limb patterning defects with progressive severity from mild polydactyly, to polysyndactyly with proximal defects, to severe oligodactyly with phocomelia and partial limb ventralization. Our studies emphasize the importance of control of the timing, level and location of Shh pathway signaling for limb anterior-posterior, proximal-distal, and dorsal-ventral patterning.

  18. An Apodized Kepler Periodogram for Separating Planetary and Stellar Activity Signals

    CERN Document Server

    Gregory, Philip C

    2016-01-01

    A new apodized Keplerian (AK) model is proposed for the analysis of precision radial velocity (RV) data to model both planetary and stellar activity (SA) induced RV signals. A symmetrical Gaussian apodization function with unknown width and center can distinguish planetary signals from SA signals on the basis of the span of the apodization window. The general model for $m$ apodized Keplerian signals includes a linear regression term between RV and the stellar activity diagnostic $\\log(R'hk)$, as well as an extra Gaussian noise term with unknown standard deviation. The model parameters are explored using a Bayesian fusion MCMC code. A differential version of the Generalized Lomb-Scargle periodogram that employs a control diagnostic provides an additional way of distinguishing SA signals and helps guide the choice of new periods. Results are reported for a recent international RV blind challenge which included multiple state of the art simulated data sets supported by a variety of stellar activity diagnostics. ...

  19. Discovery of novel small molecule activators of β-catenin signaling.

    Directory of Open Access Journals (Sweden)

    Folkert Verkaar

    Full Text Available Wnt/β-catenin signaling plays a major role in embryonic development and adult stem cell maintenance. Reduced activation of the Wnt/β-catenin pathway underlies neurodegenerative disorders and aberrations in bone formation. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed bona fide activators of β-catenin signaling. The compounds stabilized cytoplasmic β-catenin and activated β-catenin-dependent reporter gene activity. Although the mechanism through which the compounds activate β-catenin signaling has yet to be determined, several key regulators of Wnt/β-catenin signaling, including glycogen synthase kinase 3 and Frizzled receptors, were excluded as the molecular target. The compounds displayed remarkable selectivity, as they only induced β-catenin signaling in a human osteosarcoma U2OS cell line and not in a variety of other cell lines examined. Our data indicate that differences in cellular Wnt/β-catenin signaling machinery can be exploited to identify cell type-specific activators of Wnt/β-catenin signaling.

  20. Remote activation of the Wnt/β-catenin signalling pathway using functionalised magnetic particles.

    Science.gov (United States)

    Rotherham, Michael; El Haj, Alicia J

    2015-01-01

    Wnt signalling pathways play crucial roles in developmental biology, stem cell fate and tissue patterning and have become an attractive therapeutic target in the fields of tissue engineering and regenerative medicine. Wnt signalling has also been shown to play a role in human Mesenchymal Stem Cell (hMSC) fate, which have shown potential as a cell therapy in bone and cartilage tissue engineering. Previous work has shown that biocompatible magnetic nanoparticles (MNP) can be used to stimulate specific mechanosensitive membrane receptors and ion channels in vitro and in vivo. Using this strategy, we determined the effects of mechano-stimulation of the Wnt Frizzled receptor on Wnt pathway activation in hMSC. Frizzled receptors were tagged using anti-Frizzled functionalised MNP (Fz-MNP). A commercially available oscillating magnetic bioreactor (MICA Biosystems) was used to mechanically stimulate Frizzled receptors remotely. Our results demonstrate that Fz-MNP can activate Wnt/β-catenin signalling at key checkpoints in the signalling pathway. Immunocytochemistry indicated nuclear localisation of the Wnt intracellular messenger β-catenin after treatment with Fz-MNP. A Wnt signalling TCF/LEF responsive luciferase reporter transfected into hMSC was used to assess terminal signal activation at the nucleus. We observed an increase in reporter activity after treatment with Fz-MNP and this effect was enhanced after mechano-stimulation using the magnetic array. Western blot analysis was used to probe the mechanism of signalling activation and indicated that Fz-MNP signal through an LRP independent mechanism. Finally, the gene expression profiles of stress response genes were found to be similar when cells were treated with recombinant Wnt-3A or Fz-MNP. This study provides proof of principle that Wnt signalling and Frizzled receptors are mechanosensitive and can be remotely activated in vitro. Using magnetic nanoparticle technology it may be possible to modulate Wnt signalling

  1. Remote activation of the Wnt/β-catenin signalling pathway using functionalised magnetic particles.

    Directory of Open Access Journals (Sweden)

    Michael Rotherham

    Full Text Available Wnt signalling pathways play crucial roles in developmental biology, stem cell fate and tissue patterning and have become an attractive therapeutic target in the fields of tissue engineering and regenerative medicine. Wnt signalling has also been shown to play a role in human Mesenchymal Stem Cell (hMSC fate, which have shown potential as a cell therapy in bone and cartilage tissue engineering. Previous work has shown that biocompatible magnetic nanoparticles (MNP can be used to stimulate specific mechanosensitive membrane receptors and ion channels in vitro and in vivo. Using this strategy, we determined the effects of mechano-stimulation of the Wnt Frizzled receptor on Wnt pathway activation in hMSC. Frizzled receptors were tagged using anti-Frizzled functionalised MNP (Fz-MNP. A commercially available oscillating magnetic bioreactor (MICA Biosystems was used to mechanically stimulate Frizzled receptors remotely. Our results demonstrate that Fz-MNP can activate Wnt/β-catenin signalling at key checkpoints in the signalling pathway. Immunocytochemistry indicated nuclear localisation of the Wnt intracellular messenger β-catenin after treatment with Fz-MNP. A Wnt signalling TCF/LEF responsive luciferase reporter transfected into hMSC was used to assess terminal signal activation at the nucleus. We observed an increase in reporter activity after treatment with Fz-MNP and this effect was enhanced after mechano-stimulation using the magnetic array. Western blot analysis was used to probe the mechanism of signalling activation and indicated that Fz-MNP signal through an LRP independent mechanism. Finally, the gene expression profiles of stress response genes were found to be similar when cells were treated with recombinant Wnt-3A or Fz-MNP. This study provides proof of principle that Wnt signalling and Frizzled receptors are mechanosensitive and can be remotely activated in vitro. Using magnetic nanoparticle technology it may be possible to modulate

  2. Accelerometer signal-based human activity recognition using augmented autoregressive model coefficients and artificial neural nets.

    Science.gov (United States)

    Khan, A M; Lee, Y K; Kim, T S

    2008-01-01

    Automatic recognition of human activities is one of the important and challenging research areas in proactive and ubiquitous computing. In this work, we present some preliminary results of recognizing human activities using augmented features extracted from the activity signals measured using a single triaxial accelerometer sensor and artificial neural nets. The features include autoregressive (AR) modeling coefficients of activity signals, signal magnitude areas (SMA), and title angles (TA). We have recognized four human activities using AR coefficients (ARC) only, ARC with SMA, and ARC with SMA and TA. With the last augmented features, we have achieved the recognition rate above 99% for all four activities including lying, standing, walking, and running. With our proposed technique, real time recognition of some human activities is possible.

  3. Activator of G-protein signaling 8 is involved in VEGF-mediated signal processing during angiogenesis.

    Science.gov (United States)

    Hayashi, Hisaki; Al Mamun, Abdullah; Sakima, Miho; Sato, Motohiko

    2016-03-15

    Activator of G-protein signaling 8 (AGS8, also known as FNDC1) is a receptor-independent accessory protein for the Gβγ subunit, which was isolated from rat heart subjected to repetitive transient ischemia with the substantial development of collaterals. Here, we report the role of AGS8 in vessel formation by endothelial cells. Knockdown of AGS8 by small interfering RNA (siRNA) inhibited vascular endothelial growth factor (VEGF)-induced tube formation, as well as VEGF-stimulated cell growth and migration. VEGF stimulated the phosphorylation of the VEGF receptor-2 (VEGFR-2, also known as KDR), ERK1/2 and p38 MAPK; however, knockdown of AGS8 inhibited these signaling events. Signal alterations by AGS8 siRNA were associated with a decrease of cell surface VEGFR-2 and an increase of VEGFR-2 in the cytosol. Endocytosis blockers did not influence the decrease of VEGFR-2 by AGS8 siRNA, suggesting the involvement of AGS8 in VEGFR-2 trafficking to the plasma membrane. VEGFR-2 formed a complex with AGS8 in cells, and a peptide designed to disrupt AGS8-Gβγ interaction inhibited VEGF-induced tube formation. These data suggest a potential role for AGS8-Gβγ in VEGF signal processing. AGS8 might play a key role in tissue adaptation by regulating angiogenic events.

  4. SDF-1/CXCR4 axis induces human dental pulp stem cell migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways.

    Science.gov (United States)

    Li, Mingwei; Sun, Xuefei; Ma, Liang; Jin, Lu; Zhang, Wenfei; Xiao, Min; Yu, Qing

    2017-01-09

    SDF-1 (stromal cell derived factor-1) has been found to be widely expressed during dental pulp inflammation, while hDPSCs (human dental pulp stem cells) contribute to the repair of dental pulp. We showed that the migration of hDPSCs was induced by SDF-1 in a concentration-dependent manner and could be inhibited with siCXCR4 (C-X-C chemokine receptor type 4) and siCDC42 (cell division control protein 42), as well as drug inhibitors such as AMD3100 (antagonist of CXCR4), LY294002 (inhibitor of PI3K) and PF573228 (inhibitor of FAK). It was also confirmed that SDF-1 regulated the phosphorylation of FAK (focal adhesion kinases) on cell membranes and the translocation of β-catenin into the cell nucleus. Subsequent experiments confirmed that the expression of CXCR4 and β-catenin and the phosphorylation of FAK, PI3K (phosphoinositide 3-kinase), Akt and GSK3β (glycogen synthase kinase-3β) were altered significantly with SDF-1 stimulation. FAK and PI3K worked in coordination during this process. Our findings provide direct evidence that SDF-1/CXCR4 axis induces hDPSCs migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways, implicating a novel mechanism of dental pulp repair and a possible application of SDF-1 for the treatment of pulpitis.

  5. The Polycomb group protein RING1B is overexpressed in ductal breast carcinoma and is required to sustain FAK steady state levels in breast cancer epithelial cells

    Science.gov (United States)

    Bosch, Almudena; Panoutsopoulou, Konstantina; Corominas, Josep Maria; Gimeno, Ramón; Moreno-Bueno, Gema; Martín-Caballero, Juan; Morales, Saleta; Lobato, Tania; Martínez-Romero, Carles; Farias, Eduardo F.; Mayol, Xavier; Cano, Amparo; Hernández-Muáoz, Inmaculada

    2014-01-01

    In early stages of metastasis malignant cells must acquire phenotypic changes to enhance their migratory behavior and their ability to breach the matrix surrounding tumors and blood vessel walls. Epigenetic regulation of gene expression allows the acquisition of these features that, once tumoral cells have escape from the primary tumor, can be reverted. Here we report that the expression of the Polycomb epigenetic repressor Ring1B is enhanced in tumoral cells that invade the stroma in human ductal breast carcinoma and its expression is coincident with that of Fak in these tumors. Ring1B knockdown in breast cancer cell lines revealed that Ring1B is required to sustain Fak expression in basal conditions as well as in Tgfβ-treated cells. Functionally, endogenous Ring1B is required for cell migration and invasion in vitro and for in vivo invasion of the mammary fat pad by tumoral cells. Finally we identify p63 as a target of Ring1B to regulate Fak expression: Ring1B depletion results in enhanced p63 expression, which in turns represses Fak expression. Importantly, Fak downregulation upon Ring1B depletion is dependent on p63 expression. Our findings provide new insights in the biology of the breast carcinoma and open new avenues for breast cancer prognosis and therapy. PMID:24742605

  6. EGF stimulates the activation of EGF receptors and the selective activation of major signaling pathways during mitosis.

    Science.gov (United States)

    Wee, Ping; Shi, Huaiping; Jiang, Jennifer; Wang, Yuluan; Wang, Zhixiang

    2015-03-01

    Mitosis and epidermal growth factor (EGF) receptor (EGFR) are both targets for cancer therapy. The role of EGFR signaling in mitosis has been rarely studied and poorly understood. The limited studies indicate that the activation of EGFR and downstream signaling pathways is mostly inhibited during mitosis. However, we recently showed that EGFR is phosphorylated in response to EGF stimulation in mitosis. Here we studied EGF-induced EGFR activation and the activation of major signaling pathways downstream of EGFR during mitosis. We showed that EGFR was strongly activated by EGF during mitosis as all the five major tyrosine residues including Y992, Y1045, Y1068, Y1086, and Y1173 were phosphorylated to a level similar to that in the interphase. We further showed that the activated EGFR is able to selectively activate some downstream signaling pathways while avoiding others. Activated EGFR is able to activate PI3K and AKT2, but not AKT1, which may be responsible for the observed effects of EGF against nocodazole-induced cell death. Activated EGFR is also able to activate c-Src, c-Cbl and PLC-γ1 during mitosis. However, activated EGFR is unable to activate ERK1/2 and their downstream substrates RSK and Elk-1. While it activated Ras, EGFR failed to fully activate Raf-1 in mitosis due to the lack of phosphorylation at Y341 and the lack of dephosphorylation at pS259. We conclude that contrary to the dogma, EGFR is activated by EGF during mitosis. Moreover, EGFR-mediated cell signaling is regulated differently from the interphase to specifically serve the needs of the cell in mitosis.

  7. Notch2 signaling promotes osteoclast resorption via activation of PYK2.

    Science.gov (United States)

    Jin, Won Jong; Kim, Bongjun; Kim, Jung-Wook; Kim, Hong-Hee; Ha, Hyunil; Lee, Zang Hee

    2016-05-01

    Notch signaling plays a central role in various cell fate decisions, including skeletal development. Recently, Notch signaling was implicated in osteoclast differentiation and maturation, including the resorption activity of osteoclasts. However, the specific involvement of notch signaling in resorption activity was not fully investigated. Here, we investigated the roles of Notch signaling in the resorption activity of osteoclasts by use of the gamma-secretase inhibitor dibenzazepine (DBZ). Attenuating Notch signaling by DBZ suppressed the expression of NFATc1, a master transcription factor for osteoclast differentiation. However, overexpression of a constitutively active form of NFATc1 did not fully rescue the effects of DBZ. DBZ suppressed the autophosphorylation of PYK2, which is essential for the formation of the podosome belt and sealing zone, with reduced c-Src/PYK2 interaction. We found that RANKL increases PYK2 activation accompanied by increased NICD2 production in osteoclasts. Overexpression of NICD2 in osteoclasts rescued DBZ-mediated suppression of resorption activity with promotion of PYK2 autophosphorylation and microtubule acetylation. Consistent with the in vitro results, DBZ strongly suppressed bone destruction in an interleukin-1-induced bone loss model. Collectively, these results demonstrate that Notch2 in osteoclasts plays a role in the control of resorption activity via the PYK2-c-Src-microtubule signaling pathway.

  8. Lymphocyte signaling and activation by the CARMA1-BCL10-MALT1 signalosome.

    Science.gov (United States)

    Meininger, Isabel; Krappmann, Daniel

    2016-12-01

    The CARMA1-BCL10-MALT1 (CBM) signalosome triggers canonical NF-κB signaling and lymphocyte activation upon antigen-receptor stimulation. Genetic studies in mice and the analysis of human immune pathologies unveiled a critical role of the CBM complex in adaptive immune responses. Great progress has been made in elucidating the fundamental mechanisms that dictate CBM assembly and disassembly. By bridging proximal antigen-receptor signaling to downstream signaling pathways, the CBM complex exerts a crucial scaffolding function. Moreover, the MALT1 subunit confers a unique proteolytic activity that is key for lymphocyte activation. Deregulated 'chronic' CBM signaling drives constitutive NF-κB signaling and MALT1 activation, which contribute to the development of autoimmune and inflammatory diseases as well as lymphomagenesis. Thus, the processes that govern CBM activation and function are promising targets for the treatment of immune disorders. Here, we summarize the current knowledge on the functions and mechanisms of CBM signaling in lymphocytes and how CBM deregulations contribute to aberrant signaling in malignant lymphomas.

  9. Nandrolone reduces activation of Notch signaling in denervated muscle associated with increased Numb expression

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xin-Hua [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States); Yao, Shen; Qiao, Rui-Fang; Levine, Alice C. [Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States); Kirschenbaum, Alexander [Department of Urology, Mount Sinai School of Medicine, New York, NY 10029 (United States); Pan, Jiangping; Wu, Yong [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Qin, Weiping [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States); Bauman, William A. [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States); Rehabilitation Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States); Cardozo, Christopher P., E-mail: chris.cardozo@mssm.edu [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States); Rehabilitation Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States)

    2011-10-14

    Highlights: {yields} Nerve transection increased Notch signaling in paralyzed muscle. {yields} Nandrolone prevented denervation-induced Notch signaling. {yields} Nandrolone induced the expression of an inhibitor of the Notch signaling, Numb. {yields} Reduction of denervation-induced Notch signaling by nandrolone is likely through upregulation of Numb. -- Abstract: Nandrolone, an anabolic steroid, slows denervation-atrophy in rat muscle. The molecular mechanisms responsible for this effect are not well understood. Androgens and anabolic steroids activate Notch signaling in animal models of aging and thereby mitigate sarcopenia. To explore the molecular mechanisms by which nandrolone prevents denervation-atrophy, we investigated the effects of nandrolone on Notch signaling in denervated rat gastrocnemius muscle. Denervation significantly increased Notch activity reflected by elevated levels of nuclear Notch intracellular domain (NICD) and expression of Hey1 (a Notch target gene). Activation was greatest at 7 and 35 days after denervation but remained present at 56 days after denervation. Activation of Notch in denervated muscle was prevented by nandrolone associated with upregulated expression of Numb mRNA and protein. These data demonstrate that denervation activates Notch signaling, and that nandrolone abrogates this response associated with increased expression of Numb, suggesting a potential mechanism by which nandrolone reduces denervation-atrophy.

  10. Danger signals activating innate immunity in graft-versus-host disease.

    Science.gov (United States)

    Zeiser, Robert; Penack, Olaf; Holler, Ernst; Idzko, Marco

    2011-09-01

    Extensive cell death with consecutive release of danger signals can cause immune-mediated tissue destruction. The abundance of cell death is likely to determine the relevance of the danger signals as physiological mechanisms that counteract immune activation may be overruled. Such constellation is conceivable in chemo-/radiotherapy-induced tissue damage, reperfusion injury, trauma, and severe infection. Studies on graft-versus-host disease (GvHD) development have to consider the effects of chemo-/radiotherapy-related tissue damage leading to the release of exogenous and endogenous danger signals. Our previous work has demonstrated a role for adenosine-5'-triphosphate (ATP) as an endogenous danger signal in GvHD. Besides ATP, uric acid or soluble extracellular matrix components are functional danger signals that activate the NLRP3 inflammasome when released from dying cells or from extracellular matrix. In contrast to sterile inflammation, GvHD is more complex since bacterial components that leak through damaged intestinal barriers and the skin can activate pattern recognition receptors and directly contribute to GvHD pathogenesis. These exogenous danger signals transmit immune activation via toll-like receptors and NOD-like receptors of the innate immune system. This review covers both the impact of endogenous and exogenous danger signals activating innate immunity in GvHD.

  11. Towards brain-activity-controlled information retrieval: Decoding image relevance from MEG signals.

    Science.gov (United States)

    Kauppi, Jukka-Pekka; Kandemir, Melih; Saarinen, Veli-Matti; Hirvenkari, Lotta; Parkkonen, Lauri; Klami, Arto; Hari, Riitta; Kaski, Samuel

    2015-05-15

    We hypothesize that brain activity can be used to control future information retrieval systems. To this end, we conducted a feasibility study on predicting the relevance of visual objects from brain activity. We analyze both magnetoencephalographic (MEG) and gaze signals from nine subjects who were viewing image collages, a subset of which was relevant to a predetermined task. We report three findings: i) the relevance of an image a subject looks at can be decoded from MEG signals with performance significantly better than chance, ii) fusion of gaze-based and MEG-based classifiers significantly improves the prediction performance compared to using either signal alone, and iii) non-linear classification of the MEG signals using Gaussian process classifiers outperforms linear classification. These findings break new ground for building brain-activity-based interactive image retrieval systems, as well as for systems utilizing feedback both from brain activity and eye movements.

  12. Bi-Static Active Microwave Remote Sensing of Reflected Signals-of-Opportunity Project

    Data.gov (United States)

    National Aeronautics and Space Administration — We propose to demonstrate the use of these so-called signals-of-opportunity (SOP) to perform bi-static active microwave remote sensing of land surfaces. Specially,...

  13. Saturated fatty acids activate TLR-mediated proinflammatory signaling pathways.

    Science.gov (United States)

    Huang, Shurong; Rutkowsky, Jennifer M; Snodgrass, Ryan G; Ono-Moore, Kikumi D; Schneider, Dina A; Newman, John W; Adams, Sean H; Hwang, Daniel H

    2012-09-01

    Toll-like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report suggested that SFA-induced TLR activation in cell culture systems is due to contaminants in BSA used for solubilizing fatty acids. This report raised doubt about proinflammatory effects of SFAs. Our studies herein demonstrate that sodium palmitate (C16:0) or laurate (C12:0) without BSA solubilization induced phosphorylation of inhibitor of nuclear factor-κB α, c-Jun N-terminal kinase (JNK), p44/42 mitogen-activated-kinase (ERK), and nuclear factor-κB subunit p65, and TLR target gene expression in THP1 monocytes or RAW264.7 macrophages, respectively, when cultured in low FBS (0.25%) medium. C12:0 induced NFκB activation through TLR2 dimerized with TLR1 or TLR6, and through TLR4. Because BSA was not used in these experiments, contaminants in BSA have no relevance. Unlike in suspension cells (THP-1), BSA-solubilized C16:0 instead of sodium C16:0 is required to induce TLR target gene expression in adherent cells (RAW264.7). C16:0-BSA transactivated TLR2 dimerized with TLR1 or TLR6 and through TLR4 as seen with C12:0. These results and additional studies with the LPS sequester polymixin B and in MyD88(-/-) macrophages indicated that SFA-induced activation of TLR2 or TLR4 is a fatty acid-specific effect, but not due to contaminants in BSA or fatty acid preparations.

  14. Transient epileptiform signaling during neuronal network development: regulation by external stimulation and bimodal GABAergic activity.

    Science.gov (United States)

    Zemianek, Jill M; Shultz, Abraham M; Lee, Sangmook; Guaraldi, Mary; Yanco, Holly A; Shea, Thomas B

    2013-04-01

    A predominance of excitatory activity, with protracted appearance of inhibitory activity, accompanies cortical neuronal development. It is unclear whether or not inhibitory neuronal activity is solicited exclusively by excitatory neurons or whether the transient excitatory activity displayed by developing GABAergic neurons contributes to an excitatory threshold that fosters their conversion to inhibitory activity. We addressed this possibility by culturing murine embryonic neurons on multi-electrode arrays. A wave of individual 0.2-0.4 mV signals ("spikes") appeared between approx. 20-30 days in culture, then declined. A transient wave of high amplitude (>0.5 mV) epileptiform activity coincided with the developmental decline in spikes. Bursts (clusters of ≥3 low-amplitude spikes within 0.7s prior to returning to baseline) persisted following this decline. Addition of the GABAergic antagonist bicuculline initially had no effect on signaling, consistent with delayed development of GABAergic synapses. This was followed by a period in which bicuculline inhibited overall signaling, confirming that GABAergic neurons initially display excitatory activity in ex vivo networks. Following the transient developmental wave of epileptiform signaling, bicuculline induced a resurgence of epileptiform signaling, indicating that GABAergic neurons at this point displayed inhibitory activity. The appearance of transition after the developmental and decline of epileptiform activity, rather than immediately after the developmental decline in lower-amplitude spikes, suggests that the initial excitatory activity of GABAergic neurons contributes to their transition into inhibitory neurons, and that inhibitory GABAergic activity is essential for network development. Prior studies indicate that a minority (25%) of neurons in these cultures were GABAergic, suggesting that inhibitory neurons regulate multiple excitatory neurons. A similar robust increase in signaling following cessation of

  15. Adaptive and innate immune reactions regulating mast cell activation: from receptor-mediated signaling to responses

    DEFF Research Database (Denmark)

    Tkaczyk, Christine; Jensen, Bettina M; Iwaki, Shoko

    2006-01-01

    differentially activate multiple signaling pathways within the mast cells required for the generation and/or release of inflammatory mediators. Thus, the composition of the suite of mediators released and the physiologic ramifications of these responses are dependent on the stimuli and the microenvironment...... activation. The exact interconnections between the signaling pathways initiated by the surface receptors described in this article remain to be completely worked out; thus, this remains a topic for future investigation....

  16. Activation of MyD88 Signaling upon Staphylococcal Enterotoxin Binding to MHC Class II Molecules

    Science.gov (United States)

    2011-01-20

    Activation of MyD88 Signaling upon Staphylococcal Enterotoxin Binding to MHC Class II Molecules Teri L. Kissner, Gordon Ruthel, Shahabuddin Alam...mediated signaling, which activates pro-inflammatory cytokine responses. Recently we reported that staphylococcal enterotoxins (SEA or SEB), which...upon Staphylococcal Enterotoxin Binding to MHC Class II Molecules. PLoS ONE 6(1): e15985. doi:10.1371/journal.pone.0015985 Editor: Jacques Zimmer

  17. Enhanced Multistatic Active Sonar via Innovative Signal Processing

    Science.gov (United States)

    2014-09-30

    detection line. This is due to the ridge induced range-Doppler ambiguity associated with the Doppler- tolerant LFM waveforms, as the same target is...PAS) and continuous active sonar (CAS) in the presence of strong direct blast are studied for the Doppler- tolerant linear frequency modulation...and limitations of PAS and CAS, as well as Doppler- tolerant and Doppler- sensitive waveforms in the presence of a strong delay and Doppler-spread

  18. Mechanistic pathways and biological roles for receptor-independent activators of G-protein signaling.

    Science.gov (United States)

    Blumer, Joe B; Smrcka, Alan V; Lanier, Stephen M

    2007-03-01

    Signal processing via heterotrimeric G-proteins in response to cell surface receptors is a central and much investigated aspect of how cells integrate cellular stimuli to produce coordinated biological responses. The system is a target of numerous therapeutic agents and plays an important role in adaptive processes of organs; aberrant processing of signals through these transducing systems is a component of various disease states. In addition to G-protein coupled receptor (GPCR)-mediated activation of G-protein signaling, nature has evolved creative ways to manipulate and utilize the Galphabetagamma heterotrimer or Galpha and Gbetagamma subunits independent of the cell surface receptor stimuli. In such situations, the G-protein subunits (Galpha and Gbetagamma) may actually be complexed with alternative binding partners independent of the typical heterotrimeric Galphabetagamma. Such regulatory accessory proteins include the family of regulator of G-protein signaling (RGS) proteins that accelerate the GTPase activity of Galpha and various entities that influence nucleotide binding properties and/or subunit interaction. The latter group of proteins includes receptor-independent activators of G-protein signaling (AGS) proteins that play surprising roles in signal processing. This review provides an overview of our current knowledge regarding AGS proteins. AGS proteins are indicative of a growing number of accessory proteins that influence signal propagation, facilitate cross talk between various types of signaling pathways, and provide a platform for diverse functions of both the heterotrimeric Galphabetagamma and the individual Galpha and Gbetagamma subunits.

  19. Active transport improves the precision of linear long distance molecular signalling

    Science.gov (United States)

    Godec, Aljaž; Metzler, Ralf

    2016-09-01

    Molecular signalling in living cells occurs at low copy numbers and is thereby inherently limited by the noise imposed by thermal diffusion. The precision at which biochemical receptors can count signalling molecules is intimately related to the noise correlation time. In addition to passive thermal diffusion, messenger RNA and vesicle-engulfed signalling molecules can transiently bind to molecular motors and are actively transported across biological cells. Active transport is most beneficial when trafficking occurs over large distances, for instance up to the order of 1 metre in neurons. Here we explain how intermittent active transport allows for faster equilibration upon a change in concentration triggered by biochemical stimuli. Moreover, we show how intermittent active excursions induce qualitative changes in the noise in effectively one-dimensional systems such as dendrites. Thereby they allow for significantly improved signalling precision in the sense of a smaller relative deviation in the concentration read-out by the receptor. On the basis of linear response theory we derive the exact mean field precision limit for counting actively transported molecules. We explain how intermittent active excursions disrupt the recurrence in the molecular motion, thereby facilitating improved signalling accuracy. Our results provide a deeper understanding of how recurrence affects molecular signalling precision in biological cells and novel medical-diagnostic devices.

  20. Activation of the yeast Hippo pathway by phosphorylation-dependent assembly of signaling complexes.

    Science.gov (United States)

    Rock, Jeremy M; Lim, Daniel; Stach, Lasse; Ogrodowicz, Roksana W; Keck, Jamie M; Jones, Michele H; Wong, Catherine C L; Yates, John R; Winey, Mark; Smerdon, Stephen J; Yaffe, Michael B; Amon, Angelika

    2013-05-17

    Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.

  1. Role of cell adhesion signal molecules in hepatocellular carcinoma cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Jian-Min Su; Li-Ying Wang; Yu-Long Liang; Xi-Liang Zha

    2005-01-01

    AIM: Cell adhesion molecules and their signal molecules play a very important role in carcinogenesis. The aim of this study is to elucidate the role of these molecules and the signal molecules of integrins and E-cadherins, such as (focal adhesion kinase) FAK, (integrin linked kinase)ILK, and β-catenin in hepatocellular carcinoma cell apoptosis.METHODS: We first synthesized the small molecular compound, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and identified it, by element analysis and 1H NMR. To establish the apoptosis model of the SMMC-7721 hepatocellular carcinoma cell, we treated cells with DCVC in EBSS for different concentrations or for various length times in the presence of 20 μmol/L N,N-diphenyl-p-phenylenediamine,which blocks necrotic cell death and identified this model by flow cytometry and DNA ladder. Then we studied the changes of FAK, ILK, β-catenin, and PKB in this apoptotic model by Western blot.RESULTS: We found that the loss or decrease of cell adhesion signal molecules is an important reason in apoptosis of SMMC-7721 hepatocellular carcinoma cell and the apoptosis of SMMC-7721 cell was preceded by the loss or decrease of FAK, ILK, PKB, and β-catenin or the damage of cell-matrix and cell-cell adhesion.CONCLUSION: Our results suggested that the decrease of adhesion signal molecules, FAK, ILK, PKB, and β-catenin,could induce hepatocellular carcinoma cell apoptosis.

  2. Microglial Immunoreceptor Tyrosine-Based Activation and Inhibition Motif Signaling in Neuroinflammation

    Directory of Open Access Journals (Sweden)

    Bettina Linnartz

    2010-01-01

    Full Text Available Elimination of extracellular aggregates and apoptotic neural membranes without inflammation is crucial for brain tissue homeostasis. In the mammalian central nervous system, essential molecules in this process are the Fc receptors and the DAP12-associated receptors which both trigger the microglial immunoreceptor tyrosine-based activation motif- (ITAM- Syk-signaling cascade. Microglial triggering receptor expressed on myeloid cells-2 (TREM2, signal regulatory protein-1, and complement receptor-3 (CD11b/CD18 signal via the adaptor protein DAP12 and activate phagocytic activity of microglia. Microglial ITAM-signaling receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif- (ITIM- signaling molecules such as sialic acid-binding immunoglobulin superfamily lectins (Siglecs. Siglecs can suppress the proinflammatory and phagocytic activity of microglia via ITIM signaling. Moreover, microglial neurotoxicity is alleviated via interaction of Siglec-11 with sialic acids on the neuronal glycocalyx. Thus, ITAM- and ITIM-signaling receptors modulate microglial phagocytosis and cytokine expression during neuroinflammatory processes. Their dysfunction could lead to impaired phagocytic clearance and neurodegeneration triggered by chronic inflammation.

  3. Active control of shocks and sonic boom ground signal

    Science.gov (United States)

    Yagiz, Bedri

    The manipulation of a flow field to obtain a desired change is a much heightened subject. Active flow control has been the subject of the major research areas in fluid mechanics for the past two decades. It offers new solutions for mitigation of shock strength, sonic boom alleviation, drag minimization, reducing blade-vortex interaction noise in helicopters, stall control and the performance maximization of existing designs to meet the increasing requirements of the aircraft industries. Despite the wide variety of the potential applications of active flow control, the majority of studies have been performed at subsonic speeds. The active flow control cases were investigated in transonic speed in this study. Although the active flow control provides significant improvements, the sensibility of aerodynamic performance to design parameters makes it a nontrivial and expensive problem, so the designer has to optimize a number of different parameters. For the purpose of gaining understanding of the active flow control concepts, an automated optimization cycle process was generated. Also, the optimization cycle reduces cost and turnaround time. The mass flow coefficient, location, width and angle were chosen as design parameters to maximize the aerodynamic performance of an aircraft. As the main contribution of this study, a detailed parametric study and optimization process were presented. The second step is to appraise the practicability of weakening the shock wave and thereby reducing the wave drag in transonic flight regime using flow control devices such as two dimensional contour bump, individual jet actuator, and also the hybrid control which includes both control devices together, thereby gaining the desired improvements in aerodynamic performance of the air-vehicle. After this study, to improve the aerodynamic performance, the flow control and shape parameters are optimized separately, combined, and in a serial combination. The remarkable part of all these

  4. DMPD: Multiple signaling pathways leading to the activation of interferon regulatoryfactor 3. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12213596 Multiple signaling pathways leading to the activation of interferon regula...(.html) (.csml) Show Multiple signaling pathways leading to the activation of interferon regulatoryfactor 3.... PubmedID 12213596 Title Multiple signaling pathways leading to the activation of

  5. RKIP regulates MAP kinase signaling in cells with defective B-Raf activity.

    Science.gov (United States)

    Zeng, Lingchun; Ehrenreiter, Karin; Menon, Jyotsana; Menard, Ray; Kern, Florian; Nakazawa, Yoko; Bevilacqua, Elena; Imamoto, Akira; Baccarini, Manuela; Rosner, Marsha Rich

    2013-05-01

    MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.

  6. An Efficient Method to Identify Conditionally Activated Transcription Factors and their Corresponding Signal Transduction Pathway Segments

    Directory of Open Access Journals (Sweden)

    Haiyan Hu

    2009-11-01

    Full Text Available A signal transduction pathway (STP is a cascade composed of a series of signal transferring steps, which often activate one or more transcription factors (TFs to control the transcription of target genes. Understanding signaling pathways is important to our understanding of the molecular mechanisms of disease. Many condition-annotated pathways have been deposited in public databases. However, condition-annotated pathways are far from complete, considering the large number of possible conditions. Computational methods to assist in the identification of conditionally activated pathways are greatly needed. In this paper, we propose an efficient method to identify conditionally activated pathway segments starting from the identification of conditionally activated TFs, by incorporating protein-DNA binding data, gene expression data and protein interaction data. Applying our methods on several microarray datasets, we have discovered many significantly activated TFs and their corresponding pathway segments, which are supported by evidence in the literature.

  7. Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirose, Yoshikazu [Laboratory of Cell Growth and Differentiation, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Itoh, Tohru, E-mail: itohru@iam.u-tokyo.ac.jp [Laboratory of Cell Growth and Differentiation, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Miyajima, Atsushi [Laboratory of Cell Growth and Differentiation, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan)

    2009-09-10

    Hedgehog (Hh) signaling plays crucial roles in development and homeostasis of various organs. In the adult liver, it regulates proliferation and/or viability of several types of cells, particularly under injured conditions, and is also implicated in stem/progenitor cell maintenance. However, the role of this signaling pathway during the normal developmental process of the liver remains elusive. Although Sonic hedgehog (Shh) is expressed in the ventral foregut endoderm from which the liver derives, the expression disappears at the onset of the liver bud formation, and its possible recurrence at the later stages has not been investigated. Here we analyzed the activation and functional relevance of Hh signaling during the mouse fetal liver development. At E11.5, Shh and an activation marker gene for Hh signaling, Gli1, were expressed in Dlk{sup +} hepatoblasts, the fetal liver progenitor cells, and the expression was rapidly decreased thereafter as the development proceeded. In the culture of Dlk{sup +} hepatoblasts isolated from the E11.5 liver, activation of Hh signaling stimulated their proliferation and this effect was cancelled by a chemical Hh signaling inhibitor, cyclopamine. In contrast, hepatocyte differentiation of Dlk{sup +} hepatoblasts in vitro as manifested by the marker gene expression and acquisition of ammonia clearance activity was significantly inhibited by forced activation of Hh signaling. Taken together, these results demonstrate the temporally restricted manner of Hh signal activation and its role in promoting the hepatoblast proliferation, and further suggest that the pathway needs to be shut off for the subsequent hepatic differentiation of hepatoblasts to proceed normally.

  8. Identification of Domains for Efficient Notch Signaling Activity in Immobilized Notch Ligand Proteins.

    Science.gov (United States)

    Liu, Ledi; Wada, Hiroe; Matsubara, Natsuki; Hozumi, Katsuto; Itoh, Motoyuki

    2017-04-01

    Notch is a critical signaling pathway that controls cell fate and tissue homeostasis, but the functional characterization of Notch ligand domains that activate Notch receptors remains incomplete. Here, we established a method for immobilizing Notch ligand proteins onto beads to measure time-dependent Notch activity after the addition of Notch ligand-coated beads. A comparison between activities by the Notch ligand found on the cell surface to that of the ligand immobilized on beads showed that immobilized Notch ligand protein produces comparable signal activity during the first 10 h. Follow-up truncation studies showed that the N-terminal epidermal growth factor (EGF) repeat three region of delta like canonical Notch ligand 4 (DLL4) or jagged 1 (JAG1) is the minimum region for activating Notch signaling, and the DLL4 EGF repeat three domain may have a role in activation through a mechanism other than by increasing binding affinity. In addition, we found that reconstruction of the DLL4 delta and OSM-11 (DOS) motif (N257P) resulted in an increase in both binding affinity and signaling activity, which suggests that the role of the DOS motif is conserved among Notch ligands. Furthermore, active DLL4 protein on beads promoted T cell differentiation or inhibited B cell differentiation in vitro, whereas JAG1 proteins on beads did not have any effect. Taken together, our findings provide unambiguous evidence for the role of different Notch ligands and their domains in Notch signal activation, and may be potential tools for controlling Notch signaling activation. J. Cell. Biochem. 118: 785-796, 2017. © 2016 Wiley Periodicals, Inc.

  9. Hedgehog target genes: mechanisms of carcinogenesis induced by aberrant hedgehog signaling activation.

    Science.gov (United States)

    Katoh, Y; Katoh, M

    2009-09-01

    Hedgehog signaling is aberrantly activated in glioma, medulloblastoma, basal cell carcinoma, lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, and other tumors. Hedgehog signals activate GLI family members via Smoothened. RTK signaling potentiates GLI activity through PI3K-AKT-mediated GSK3 inactivation or RAS-STIL1-mediated SUFU inactivation, while GPCR signaling to Gs represses GLI activity through adenylate cyclase-mediated PKA activation. GLI activators bind to GACCACCCA motif to regulate transcription of GLI1, PTCH1, PTCH2, HHIP1, MYCN, CCND1, CCND2, BCL2, CFLAR, FOXF1, FOXL1, PRDM1 (BLIMP1), JAG2, GREM1, and Follistatin. Hedgehog signals are fine-tuned based on positive feedback loop via GLI1 and negative feedback loop via PTCH1, PTCH2, and HHIP1. Excessive positive feedback or collapsed negative feedback of Hedgehog signaling due to epigenetic or genetic alterations leads to carcinogenesis. Hedgehog signals induce cellular proliferation through upregulation of N-Myc, Cyclin D/E, and FOXM1. Hedgehog signals directly upregulate JAG2, indirectly upregulate mesenchymal BMP4 via FOXF1 or FOXL1, and also upregulate WNT2B and WNT5A. Hedgehog signals induce stem cell markers BMI1, LGR5, CD44 and CD133 based on cross-talk with WNT and/or other signals. Hedgehog signals upregulate BCL2 and CFLAR to promote cellular survival, SNAI1 (Snail), SNAI2 (Slug), ZEB1, ZEB2 (SIP1), TWIST2, and FOXC2 to promote epithelial-to-mesenchymal transition, and PTHLH (PTHrP) to promote osteolytic bone metastasis. KAAD-cyclopamine, Mu-SSKYQ-cyclopamine, IPI-269609, SANT1, SANT2, CUR61414 and HhAntag are small-molecule inhibitors targeted to Smoothened, GANT58, GANT61 to GLI1 and GLI2, and Robot-nikinin to SHH. Hedgehog signaling inhibitors should be used in combination with RTK inhibitors, GPCR modulators, and/or irradiation for cancer therapy.

  10. Signal transducer and activator of transcription 3 activation is associated with bladder cancer cell growth and survival

    Directory of Open Access Journals (Sweden)

    Hsieh Fu-Chuan

    2008-10-01

    Full Text Available Abstract Background Constitutive activation of signal transducer and activator of transcription 3 (Stat3 signaling pathway plays an important role in several human cancers. Activation of Stat3 is dependent on the phosphorylation at the tyrosine residue 705 by upstream kinases and subsequent nuclear translocation after dimerization. It remains unclear whether oncogenic Stat3 signaling pathway is involved in the oncogenesis of bladder cancer. Results We found that elevated Stat3 phosphorylation in 19 of 100 (19% bladder cancer tissues as well as bladder cancer cell lines, WH, UMUC-3 and 253J. To explore whether Stat3 activation is associated with cell growth and survival of bladder cancer, we targeted the Stat3 signaling pathway in bladder cancer cells using an adenovirus-mediated dominant-negative Stat3 (Y705F and a small molecule compound, STA-21. Both prohibited cell growth and induction of apoptosis in these bladder cancer cell lines but not in normal bladder smooth muscle cell (BdSMC. The survival inhibition might be mediated through apoptotic caspase 3, 8 and 9 pathways. Moreover, down-regulation of anti-apoptotic genes (Bcl-2, Bcl-xL and survivin and a cell cycle regulating gene (cyclin D1 was associated with the cell growth inhibition and apoptosis. Conclusion These results indicated that activation of Stat3 is crucial for bladder cancer cell growth and survival. Therefore, interference of Stat3 signaling pathway emerges as a potential therapeutic approach for bladder cancer.

  11. Carbon nanotube-assisted optical activation of TGF-β signalling by near-infrared light

    Science.gov (United States)

    Lin, Liang; Liu, Ling; Zhao, Bing; Xie, Ran; Lin, Wei; Li, He; Li, Yaya; Shi, Minlong; Chen, Ye-Guang; Springer, Timothy A.; Chen, Xing

    2015-05-01

    Receptor-mediated signal transduction modulates complex cellular behaviours such as cell growth, migration and differentiation. Although photoactivatable proteins have emerged as a powerful tool for controlling molecular interactions and signalling cascades at precise times and spaces using light, many of these light-sensitive proteins are activated by ultraviolent or visible light, which has limited tissue penetration. Here, we report a single-walled carbon nanotube (SWCNT)-assisted approach that enables near-infrared light-triggered activation of transforming growth factor β (TGF-β) signal transduction, an important signalling pathway in embryonic development and cancer progression. The protein complex of TGF-β and its latency-associated peptide is conjugated onto SWCNTs, where TGF-β is inactive. Upon near-infrared irradiation, TGF-β is released through the photothermal effect of SWCNTs and becomes active. The released TGF-β activates downstream signal transduction in live cells and modulates cellular behaviours. Furthermore, preliminary studies show that the method can be used to mediate TGF-β signalling in living mice.

  12. Fibronectin-integrin mediated signaling in human cervical cancer cells (SiHa).

    Science.gov (United States)

    Maity, Gargi; Fahreen, Shabana; Banerji, Aniruddha; Roy Choudhury, Paromita; Sen, Triparna; Dutta, Anindita; Chatterjee, Amitava

    2010-03-01

    Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation, and migration, including tumor development and invasion of tumor cells. Matrix metalloproteinases (MMPs) are a family of metalloproteinases capable of digesting ECM components and are important molecules for cell migration. Binding of ECM to integrins initiates cascades of cell signaling events modulating expression and activity of different MMPs. The aim of this study is to investigate fibronectin-integrin-mediated signaling and modulation of MMPs. Our findings indicated that culture of human cervical cancer cell (SiHa) on fibronectin-coated surface perhaps sends signals via fibronectin-integrin-mediated signaling pathways recruiting focal adhesion kinase (FAK) extracellular signal regulated kinase (ERK), phosphatidyl inositol 3 kinase (PI-3K), integrin-linked kinase (ILK), nuclear factor-kappa B (NF-kappaB), and modulates expression and activation of mainly pro-MMP-9, and moderately pro-MMP-2 in serum-free culture medium.

  13. Photolysis of caged compounds: studying Ca(2+) signaling and activation of Ca(2+)-dependent ion channels.

    Science.gov (United States)

    Almassy, Janos; Yule, David I

    2013-01-01

    A wide variety of signaling molecules have been chemically modified by conjugation to a photolabile chromophore to render the substance temporarily biologically inert. Subsequent exposure to ultraviolet (UV) light can release the active moiety from the "caged" precursor in an experimentally controlled manner. This allows the concentration of active molecule to be precisely manipulated in both time and space. These techniques are particularly useful in experimental protocols designed to investigate the mechanisms underlying Ca(2+) signaling and the activation of Ca(2+)-dependent effectors.

  14. Concurrent inhibition of kit- and FcepsilonRI-mediated signaling: coordinated suppression of mast cell activation

    DEFF Research Database (Denmark)

    Jensen, Bettina M; Beaven, Michael A; Iwaki, Shoko;

    2008-01-01

    be explained by its inhibition of Kit kinase activity, whereas the inhibitory effects on FcepsilonRI-dependent signaling were at the level of Btk activation. Because hypothemycin also significantly reduced the mouse passive cutaneous anaphylaxis response in vivo, these data provide proof of principle...

  15. Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function

    Science.gov (United States)

    Muhammad, Karzan; Reddy-Alla, Suneel; Driller, Jan H; Schreiner, Dietmar; Rey, Ulises; Böhme, Mathias A.; Hollmann, Christina; Ramesh, Niraja; Depner, Harald; Lützkendorf, Janine; Matkovic, Tanja; Götz, Torsten; Bergeron, Dominique D.; Schmoranzer, Jan; Goettfert, Fabian; Holt, Mathew; Wahl, Markus C.; Hell, Stefan W.; Scheiffele, Peter; Walter, Alexander M.; Loll, Bernhard; Sigrist, Stephan J.

    2015-01-01

    Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic neurexin/neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to neurexin. Here we report that the scaffold protein spinophilin binds to the C-terminal portion of neurexin and is needed to limit neurexin/neuroligin signalling by acting antagonistic to Syd-1. Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. We conclude that presynaptic spinophilin fine-tunes neurexin/neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis. PMID:26471740

  16. Crosstalk and signalling switches in mitogen-activated protein kinase cascades

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    Dirk eFey

    2012-09-01

    Full Text Available Mitogen-activated protein kinase (MAPK cascades control cell fate decisions, such as proliferation, differentiation and apoptosis by integrating and processing intra- and extracellular cues. However, similar MAPK kinetic profiles can be associated with opposing cellular decisions depending on cell type, signal strength and dynamics. This implies that signalling by each individual MAPK cascade has to be considered in the context of the entire MAPK network. Here, we develop a dynamic model of feedback and crosstalk for the three major MAPK cascades; extracellular signal-regulated kinase (ERK, p38 mitogen-activated protein kinase (p38, c-Jun N-terminal kinase (JNK, and also include input from protein kinase B (AKT. Focusing on the bistable activation characteristics of the JNK pathway, this model explains how pathway crosstalk harmonises different MAPK responses resulting in pivotal cell fate decisions. We show that JNK can switch from a transient to sustained activity due to multiple positive feedback loops. Once activated, positive feedback locks JNK in a highly active state and promotes cell death. The switch is modulated by the ERK, p38 and AKT pathways. ERK activation enhances the dual specificity phosphatase (DUSP mediated dephosphorylation of JNK and shifts the threshold of the apoptotic switch to higher inputs. Activation of p38 restores the threshold by inhibiting ERK activity via the PP1 or PP2A phosphatases. Finally, AKT activation inhibits the JNK positive feedback, thus abrogating the apoptotic switch and allowing only proliferative signalling. Our model facilitates understanding of how cancerous deregulations disturb MAPK signal processing and provides explanations for certain drug resistances. We highlight a critical role of DUSP1 and DUSP2 expression patterns in facilitating the switching of JNK activity and show how oncogene induced ERK hyperactivity prevents the normal apoptotic switch explaining the failure ocertain drugs to

  17. Analysis of EMG Signals in Aggressive and Normal Activities by Using Higher-Order Spectra

    Directory of Open Access Journals (Sweden)

    Necmettin Sezgin

    2012-01-01

    Full Text Available The analysis and classification of electromyography (EMG signals are very important in order to detect some symptoms of diseases, prosthetic arm/leg control, and so on. In this study, an EMG signal was analyzed using bispectrum, which belongs to a family of higher-order spectra. An EMG signal is the electrical potential difference of muscle cells. The EMG signals used in the present study are aggressive or normal actions. The EMG dataset was obtained from the machine learning repository. First, the aggressive and normal EMG activities were analyzed using bispectrum and the quadratic phase coupling of each EMG episode was determined. Next, the features of the analyzed EMG signals were fed into learning machines to separate the aggressive and normal actions. The best classification result was 99.75%, which is sufficient to significantly classify the aggressive and normal actions.

  18. Danger signal-dependent activation of human dendritic cells by plasma-derived factor VIII products.

    Science.gov (United States)

    Miller, L; Weissmüller, S; Ringler, E; Crauwels, P; van Zandbergen, G; Seitz, R; Waibler, Z

    2015-08-01

    Treatment of haemophilia A by infusions of the clotting factor VIII (FVIII) results in the development of inhibitors/anti-drug antibodies in up to 25 % of patients. Mechanisms leading to immunogenicity of FVIII products are not yet fully understood. Amongst other factors, danger signals as elicited upon infection or surgery have been proposed to play a role. In the present study, we focused on effects of danger signals on maturation and activation of dendritic cells (DC) in the context of FVIII application. Human monocyte-derived DC were treated with FVIII alone, with a danger signal alone or a combination of both. By testing more than 60 different healthy donors, we show that FVIII and the bacterial danger signal lipopolysaccharide synergise in increasing DC activation, as characterised by increased expression of co-stimulatory molecules and secretion of pro-inflammatory cytokines. The degree and frequency of this synergistic activation correlate with CD86 expression levels on immature DC prior to stimulation. In our assay system, plasma-derived but not recombinant FVIII products activate human DC in a danger signal-dependent manner. Further tested danger signals, such as R848 also induced DC activation in combination with FVIII, albeit not in every tested donor. In our hands, human DC but not human B cells or macrophages could be activated by FVIII in a danger signal-dependent manner. Our results suggest that immunogenicity of FVIII is a result of multiple factors including the presence of danger, predisposition of the patient, and the choice of a FVIII product for treatment.

  19. Vitamin D controls T cell antigen receptor signaling and activation of human T cells

    DEFF Research Database (Denmark)

    von Essen, Marina Rode; Kongsbak-Wismann, Martin; Schjerling, Peter

    2010-01-01

    Phospholipase C (PLC) isozymes are key signaling proteins downstream of many extracellular stimuli. Here we show that naive human T cells had very low expression of PLC-gamma1 and that this correlated with low T cell antigen receptor (TCR) responsiveness in naive T cells. However, TCR triggering...... led to an upregulation of approximately 75-fold in PLC-gamma1 expression, which correlated with greater TCR responsiveness. Induction of PLC-gamma1 was dependent on vitamin D and expression of the vitamin D receptor (VDR). Naive T cells did not express VDR, but VDR expression was induced by TCR...... signaling via the alternative mitogen-activated protein kinase p38 pathway. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-gamma1, which are required for subsequent classical TCR signaling and T cell activation....

  20. Biophysical assay for tethered signaling reactions reveals tether-controlled activity for the phosphatase SHP-1

    Science.gov (United States)

    Goyette, Jesse; Salas, Citlali Solis; Coker-Gordon, Nicola; Bridge, Marcus; Isaacson, Samuel A.; Allard, Jun; Dushek, Omer

    2017-01-01

    Tethered enzymatic reactions are ubiquitous in signaling networks but are poorly understood. A previously unreported mathematical analysis is established for tethered signaling reactions in surface plasmon resonance (SPR). Applying the method to the phosphatase SHP-1 interacting with a phosphorylated tether corresponding to an immune receptor cytoplasmic tail provides five biophysical/biochemical constants from a single SPR experiment: two binding rates, two catalytic rates, and a reach parameter. Tether binding increases the activity of SHP-1 by 900-fold through a binding-induced allosteric activation (20-fold) and a more significant increase in local substrate concentration (45-fold). The reach parameter indicates that this local substrate concentration is exquisitely sensitive to receptor clustering. We further show that truncation of the tether leads not only to a lower reach but also to lower binding and catalysis. This work establishes a new framework for studying tethered signaling processes and highlights the tether as a control parameter in clustered receptor signaling.

  1. Dynamic regulation of integrin activation by intracellular and extracellular signals controls oligodendrocyte morphology

    Directory of Open Access Journals (Sweden)

    Olsen Inger

    2005-11-01

    Full Text Available Abstract Background Myelination requires precise control of oligodendrocyte morphology and myelin generation at each of the axons contacted by an individual cell. This control must involve the integration of extracellular cues, such as those on the axon surface, with intrinsic developmental programmes. We asked whether integrins represent one class of oligodendrocyte cell-surface receptors able to provide this integration. Results Integrins signal via a process of activation, a conformational change that can be induced either by "outside-in" signals comprising physiological extracellular matrix ligands (mimicked by the pharmacological use of the divalent cation manganese or "inside-out" signalling molecules such as R-Ras. Increasing levels of outside-in signalling via the laminin receptor α6β1 integrin were found to promote oligodendrocyte processing and myelin sheet formation in culture. Similar results were obtained when inside-out signalling was increased by the expression of a constitutively-active R-Ras. Inhibiting inside-out signalling by using dominant-negative R-Ras reduces processes and myelin sheets; importantly, this can be partially rescued by the co-stimulation of outside-in signalling using manganese. Conclusion The balance of the equilibrium between active and inactive integrins regulates oligodendrocyte morphology, which is itself regulated by extrinsic and intrinsic cues so providing a mechanism of signal integration. As laminins capable of providing outside-in signals are present on axons at the time of myelination, a mechanism exists by which morphology and myelin generation might be regulated independently in each oligodendrocyte process.

  2. Integrin-dependent activation of the JNK signaling pathway by mechanical stress.

    Directory of Open Access Journals (Sweden)

    Andrea Maria Pereira

    Full Text Available Mechanical force is known to modulate the activity of the Jun N-terminal kinase (JNK signaling cascade. However, the effect of mechanical stresses on JNK signaling activation has previously only been analyzed by in vitro detection methods. It still remains unknown how living cells activate the JNK signaling cascade in response to mechanical stress and what its functions are in stretched cells.We assessed in real-time the activity of the JNK pathway in Drosophila cells by Fluorescence Lifetime Imaging Microscopy (FLIM, using an intramolecular phosphorylation-dependent dJun-FRET (Fluorescence Resonance Energy Transfer biosensor. We found that quantitative FRET-FLIM analysis and confocal microscopy revealed sustained dJun-FRET biosensor activation and stable morphology changes in response to mechanical stretch for Drosophila S2R+ cells. Further, these cells plated on different substrates showed distinct levels of JNK activity that associate with differences in cell morphology, integrin expression and focal adhesion organization.These data imply that alterations in the cytoskeleton and matrix attachments may act as regulators of JNK signaling, and that JNK activity might feed back to modulate the cytoskeleton and cell adhesion. We found that this dynamic system is highly plastic; at rest, integrins at focal adhesions and talin are key factors suppressing JNK activity, while multidirectional static stretch leads to integrin-dependent, and probably talin-independent, Jun sensor activation. Further, our data suggest that JNK activity has to coordinate with other signaling elements for the regulation of the cytoskeleton and cell shape remodeling associated with stretch.

  3. Microglial Immunoreceptor Tyrosine-Based Activation and Inhibition Motif Signaling in Neuroinflammation

    OpenAIRE

    Bettina Linnartz; Yiner Wang; Harald Neumann

    2010-01-01

    Elimination of extracellular aggregates and apoptotic neural membranes without inflammation is crucial for brain tissue homeostasis. In the mammalian central nervous system, essential molecules in this process are the Fc receptors and the DAP12-associated receptors which both trigger the microglial immunoreceptor tyrosine-based activation motif- (ITAM-) Syk-signaling cascade. Microglial triggering receptor expressed on myeloid cells-2 (TREM2), signal regulatory protein- 1, and complement re...

  4. Quercitrin attenuates osteoporosis in ovariectomized rats by regulating mitogen-activated protein kinase (MAPK) signaling pathways.

    Science.gov (United States)

    Xing, Li-Zhi; Ni, Huai-Jun; Wang, Yu-Ling

    2017-03-13

    MAPK signaling pathways are crucial in regulating osteogenesis, a genetic disorder affecting the bones. Quercitrin, a type of flavonoid, is widely distributed in nature and involved in many pharmacological activities. But its osteoprotective functions and mechanism in osteoporosis are far from being understood clearly. In this paper, the MAPK upregulation was observed in the ovariectomy-induced bone loss. Quercitrin was found to downregulate MAPK signaling pathways and prevent the ovariectomy-induced deterioration of bone mineral density (BMD), trabecular microstructure, and bone mechanical characteristics. In this study, quercitrin was seen to prevent the progression of the postmenopausal osteoporosis among the rats, which may be mediated by the downregulated MAPK signaling pathways.

  5. Silica nanoparticles induce endoplasmic reticulum stress response and activate mitogen activated kinase (MAPK signalling

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    Verena Christen

    2016-01-01

    Full Text Available Humans may be exposed to engineered silica nanoparticles (SiO2-NPs but potential adverse effects are poorly understood, in particular in relation to cellular effects and modes of action. Here we studied effects of SiO2-NPs on cellular function in human hepatoma cells (Huh7. Exposure for 24 h to 10 and 50 μg/ml SiO2-NPs led to induction of endoplasmic reticulum (ER stress as demonstrated by transcriptional induction of DNAJB9, GADD34, CHOP, as well as CHOP target genes BIM, CHAC-1, NOXA and PUMA. In addition, CHOP protein was induced. In addition, SiO2-NPs induced an inflammatory response as demonstrated by induction of TNF-α and IL-8. Activation of MAPK signalling was investigated employing a PCR array upon exposure of Huh7 cells to SiO2-NPs. Five of 84 analysed genes, including P21, P19, CFOS, CJUN and KSR1 exhibited significant transcriptional up-regulation, and 18 genes a significant down-regulation. Strongest down-regulation occurred for the proto-oncogene BRAF, MAPK11, one of the four p38 MAPK genes, and for NFATC4. Strong induction of CFOS, CJUN, FRA1 and CMYC was found after exposure to 50 μg/ml SiO2-NPs for 24 h. To analyse for effects derived from up-regulation of TNF-α, Huh7 cells were exposed to SiO2-NPs in the presence of the TNF-α inhibitor sauchinone, which reduced the induction of the TNF-α transcript by about 50%. These data demonstrate that SiO2-NPs induce ER stress, MAPK pathway and lead to inflammatory reaction in human hepatoma cells. Health implications of SiO2-NPs exposure should further be investigated for a risk assessment of these frequently used nanoparticles.

  6. Constitutive activation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor κB signaling in glioblastoma cancer stem cells regulates the Notch pathway.

    Science.gov (United States)

    Garner, Jo Meagan; Fan, Meiyun; Yang, Chuan He; Du, Ziyun; Sims, Michelle; Davidoff, Andrew M; Pfeffer, Lawrence M

    2013-09-06

    Malignant gliomas are locally aggressive, highly vascular tumors that have a dismal prognosis, and present therapies provide little improvement in the disease course and outcome. Many types of malignancies, including glioblastoma, originate from a population of cancer stem cells (CSCs) that are able to initiate and maintain tumors. Although CSCs only represent a small fraction of cells within a tumor, their high tumor-initiating capacity and therapeutic resistance drives tumorigenesis. Therefore, it is imperative to identify pathways associated with CSCs to devise strategies to selectively target them. In this study, we describe a novel relationship between glioblastoma CSCs and the Notch pathway, which involves the constitutive activation of STAT3 and NF-κB signaling. Glioma CSCs were isolated and maintained in vitro using an adherent culture system, and the biological properties were compared with the traditional cultures of CSCs grown as multicellular spheres under nonadherent culture conditions. Interestingly, both adherent and spheroid glioma CSCs show constitutive activation of the STAT3/NF-κB signaling pathway and up-regulation of STAT3- and NF-κB-dependent genes. Gene expression profiling also identified components of the Notch pathway as being deregulated in glioma CSCs, and the deregulated expression of these genes was sensitive to treatment with STAT3 and NF-κB inhibitors. This finding is particularly important because Notch signaling appears to play a key role in CSCs in a variety of cancers and controls cell fate determination, survival, proliferation, and the maintenance of stem cells. The constitutive activation of STAT3 and NF-κB signaling pathways that leads to the regulation of Notch pathway genes in glioma CSCs identifies novel therapeutic targets for the treatment of glioma.

  7. Activation of Melatonin Signaling Promotes β-Cell Survival and Function.

    Science.gov (United States)

    Costes, Safia; Boss, Marti; Thomas, Anthony P; Matveyenko, Aleksey V

    2015-05-01

    Type 2 diabetes mellitus (T2DM) is characterized by pancreatic islet failure due to loss of β-cell secretory function and mass. Studies have identified a link between a variance in the gene encoding melatonin (MT) receptor 2, T2DM, and impaired insulin secretion. This genetic linkage raises the question whether MT signaling plays a role in regulation of β-cell function and survival in T2DM. To address this postulate, we used INS 832/13 cells to test whether activation of MT signaling attenuates proteotoxicity-induced β-cell apoptosis and through which molecular mechanism. We also used nondiabetic and T2DM human islets to test the potential of MT signaling to attenuate deleterious effects of glucotoxicity and T2DM on β-cell function. MT signaling in β-cells (with duration designed to mimic typical nightly exposure) significantly enhanced activation of the cAMP-dependent signal transduction pathway and attenuated proteotoxicity-induced β-cell apoptosis evidenced by reduced caspase-3 cleavage (∼40%), decreased activation of stress-activated protein kinase/Jun-amino-terminal kinase (∼50%) and diminished oxidative stress response. Activation of MT signaling in human islets was shown to restore glucose-stimulated insulin secretion in islets exposed to chronic hyperglycemia as well as in T2DM islets. Our data suggest that β-cell MT signaling is important for the regulation of β-cell survival and function and implies a preventative and therapeutic potential for preservation of β-cell mass and function in T2DM.

  8. Task control signals in pediatric Tourette syndrome show evidence of immature and anomalous functional activity

    Directory of Open Access Journals (Sweden)

    Jessica A Church

    2009-11-01

    Full Text Available Tourette Syndrome (TS is a pediatric movement disorder that may affect control signaling in the brain. Previous work has proposed a dual-networks architecture of control processing involving a task-maintenance network and an adaptive control network (Dosenbach et al., 2008. A prior resting-state functional connectivity MRI (rs-fcMRI analysis in TS has revealed functional immaturity in both putative control networks, with “anomalous” correlations (i.e. correlations outside the typical developmental range limited to the adaptive control network (Church et al., 2009. The present study used functional MRI (fMRI to study brain activity related to adaptive control (by studying start-cues signals, and to task-maintenance (by studying signals sustained across a task set. Two hypotheses from the previous rs-fcMRI results were tested. First, adaptive control (i.e., start-cue activity will be altered in TS, including activity inconsistent with typical development (“anomalous”. Second, group differences found in task maintenance (i.e., sustained activity will be consistent with functional immaturity in TS. We examined regions found through a direct comparison of adolescents with and without TS, as well as regions derived from a previous investigation that showed differences between unaffected children and adults. The TS group showed decreased start-cue signal magnitude in regions where start-cue activity is unchanged over typical development, consistent with anomalous adaptive control. The TS group also had higher magnitude sustained signals in frontal cortex regions that overlapped with regions showing differences over typical development, consistent with immature task maintenance in TS. The results demonstrate task-related fMRI signal differences anticipated by the atypical functional connectivity found previously in adolescents with TS, strengthening the evidence for functional immaturity and anomalous signaling in control networks in adolescents

  9. Differences in integrin expression and signaling within human breast cancer cells

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    Liu Yongqing

    2011-07-01

    more stress fibers and focal adhesions and only exhibited adhesion-induced activation of pMEK and pFAK. All cells expressed the urokinase receptor, but MCF7 cells had markedly higher VEGFR expression. Adhesion induced differential expression of pFAK, pMEK and pERK. Conclusions This study demonstrates that breast cancers vary in their expression of integrins, their capacity to form focal adhesion and to signal through integrins. These differences likely contribute to phenotypic variations between cancer lines and account for some of the heterogeneity of breast cancer.

  10. Cyr61 induces the expression of monocyte chemoattractant protein-1 via the integrin ανβ3, FAK, PI3K/Akt, and NF-κB pathways in retinal vascular endothelial cells.

    Science.gov (United States)

    You, Jian-Jang; Yang, Chang-Hao; Yang, Chung-May; Chen, Muh-Shy

    2014-01-01

    Diabetes causes a number of metabolic and physiological abnormalities in the retina. Many of the molecular and physiological abnormalities that develop during diabetic retinopathy are due to inflammation. Monocyte chemoattractant protein-1 (MCP-1) is an important factor involved in diabetic retinopathy. In a previous study, we found that cysteine-rich 61 (Cyr61), an important angiogenic factor, also plays an important role in diabetic retinopathy. In addition to the direct effects of Cyr61, we observed that Cyr61 can induce the expression of MCP-1. However, the mechanism through which this occurs is not completely understood in chorioretinal vascular endothelial cells. We therefore investigated the effects of Cyr61 on MCP-1 expression in this cell type. Cyr61 stimulated the expression of MCP-1 at the mRNA, protein, and secreted protein levels in a dose-dependent and time-dependent manner. Both total MCP-1 levels and secreted MCP-1 levels were attenuated during the response to Cyr61 stimulation by pretreatment with integrin ανβ3-blocking antibodies, a FAK inhibitor (PF573228), a PI3K inhibitor (LY294002), and an Akt inhibitor (A6730). Electrophoretic mobility shift assays revealed that the above inhibitors suppressed the activation of NF-κB. Additionally, deletion of the NF-κB-binding element in the MCP-1 gene promoter led to a decrease in expression in luciferase reporter assays. These results show that the induction of MCP-1 by Cyr61 is mediated through the activation of the integrin ανβ3, FAK, PI3K/Akt, and IKK/NF-κB pathways in chorioretinal vascular endothelial cells.

  11. Methylglyoxal activates the target of rapamycin complex 2-protein kinase C signaling pathway in Saccharomyces cerevisiae.

    Science.gov (United States)

    Nomura, Wataru; Inoue, Yoshiharu

    2015-04-01

    Methylglyoxal is a typical 2-oxoaldehyde derived from glycolysis. We show here that methylglyoxal activates the Pkc1-Mpk1 mitogen-activated protein (MAP) kinase cascade in a target of rapamycin complex 2 (TORC2)-dependent manner in the budding yeast Saccharomyces cerevisiae. We demonstrate that TORC2 phosphorylates Pkc1 at Thr(1125) and Ser(1143). Methylglyoxal enhanced the phosphorylation of Pkc1 at Ser(1143), which transmitted the signal to the downstream Mpk1 MAP kinase cascade. We found that the phosphorylation status of Pkc1(T1125) affected the phosphorylation of Pkc1 at Ser(1143), in addition to its protein levels. Methylglyoxal activated mammalian TORC2 signaling, which, in turn, phosphorylated Akt at Ser(473). Our results suggest that methylglyoxal is a conserved initiator of TORC2 signaling among eukaryotes.

  12. A role for Raptor phosphorylation in the mechanical activation of mTOR signaling.

    Science.gov (United States)

    Frey, John W; Jacobs, Brittany L; Goodman, Craig A; Hornberger, Troy A

    2014-02-01

    The activation of mTOR signaling is necessary for mechanically-induced changes in skeletal muscle mass, but the mechanisms that regulate the mechanical activation of mTOR signaling remain poorly defined. In this study, we set out to determine if changes in the phosphorylation of Raptor contribute to the mechanical activation of mTOR. To accomplish this goal, mouse skeletal muscles were subjected to mechanical stimulation via a bout of eccentric contractions (EC). Using mass spectrometry and Western blot analysis, we found that ECs induced an increase in Raptor S696, T706, and S863 phosphorylation, and this effect was not inhibited by rapamycin. This observation suggested that changes in Raptor phosphorylation might be an upstream event in the pathway through which mechanical stimuli activate mTOR. To test this, we employed a phospho-defective mutant of Raptor (S696A/T706A/S863A) and found that the EC-induced activation of mTOR signaling was significantly blunted in muscles expressing this mutant. Furthermore, mutation of the three phosphorylation sites altered the interactions of Raptor with PRAS40 and p70(S6k), and it also prevented the EC-induced dissociation of Raptor from p70(S6k). Combined, these results suggest that changes in the phosphorylation of Raptor play an important role in the pathway through which mechanical stimuli activate mTOR signaling.

  13. PARP1 orchestrates variant histone exchange in signal-mediated transcriptional activation.

    Science.gov (United States)

    O'Donnell, Amanda; Yang, Shen-Hsi; Sharrocks, Andrew D

    2013-12-01

    Transcriptional activation is accompanied by multiple molecular events that remodel the local chromatin environment in promoter regions. These molecular events are often orchestrated in response to the activation of signalling pathways, as exemplified by the response of immediate early genes such as FOS to ERK MAP kinase signalling. Here, we demonstrate that inducible NFI recruitment permits PARP1 binding to the FOS promoter by a mutually reinforcing loop. PARP1 and its poly(ADP-ribosyl)ation activity are required for maintaining FOS activation kinetics. We also show that the histone variant H2A.Z associates with the FOS promoter and acts in a transcription-suppressive manner. However, in response to ERK pathway signalling, H2A.Z is replaced by H2A; PARP1 activity is required to promote this exchange. Thus, our work has revealed an additional facet of PARP1 function in promoting dynamic remodelling of promoter-associated nucleosomes to allow transcriptional activation in response to cellular signalling.

  14. Eviprostat Activates cAMP Signaling Pathway and Suppresses Bladder Smooth Muscle Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Masayuki Takeda

    2013-06-01

    Full Text Available Eviprostat is a popular phytotherapeutic agent for the treatment of lower urinary tract symptoms (LUTS. At present, the signaling mechanisms underlying its therapeutic effects are still poorly understood. Given that cAMP has been reported to suppress cell hyperplasia and hypertrophy in various pathological situations, we asked whether the effect of Eviprostat could be ascribed to the activation of the cAMP signaling pathway. In the study, exposure of cAMP response element (CRE-secreted alkaline phosphatase (SEAP (CRE-SEAP-reporter cells to Eviprostat elevated SEAP secretion, which was associated with an increased phosphorylation of vasodilator-stimulated phosphoprotein (VASP and cAMP-response element-binding protein (CREB, as well as enhanced expression of CRE-regulated protein connexin43, indicating an activation of the cAMP signaling pathway. Consistent with these observations, Eviprostat-induced expression of Cx43 was abolished in the presence of adenylyl cyclase inhibitor SQ22536 or PKA inhibitor H89, whereas it was mimicked by adenylyl cyclase activator, forskolin. Further analysis demonstrated that Eviprostat significantly potentiated the effect of phosphodiesterase 3 (PDE3 inhibitor, but not that of PDE4 inhibitor, on CRE activation. Moreover, Eviprostat suppressed PDGF-induced activation of ERK and Akt and inhibited cell proliferation and hillock formation in both mesangial cells and bladder smooth muscle cells. Collectively, activation of the cAMP signaling pathway could be an important mechanism by which Eviprostat exerts its therapeutic effects for LUTS.

  15. The lack of autophagy triggers precocious activation of Notch signaling during Drosophila oogenesis

    Directory of Open Access Journals (Sweden)

    Barth Julia MI

    2012-12-01

    Full Text Available Abstract Background The proper balance of autophagy, a lysosome-mediated degradation process, is indispensable for oogenesis in Drosophila. We recently demonstrated that egg development depends on autophagy in the somatic follicle cells (FC, but not in the germline cells (GCs. However, the lack of autophagy only affects oogenesis when FCs are autophagy-deficient but GCs are wild type, indicating that a dysfunctional signaling between soma and germline may be responsible for the oogenesis defects. Thus, autophagy could play an essential role in modulating signal transduction pathways during egg development. Results Here, we provide further evidence for the necessity of autophagy during oogenesis and demonstrate that autophagy is especially required in subsets of FCs. Generation of autophagy-deficient FCs leads to a wide range of phenotypes that are similar to mutants with defects in the classical cell-cell signaling pathways in the ovary. Interestingly, we observe that loss of autophagy leads to a precocious activation of the Notch pathway in the FCs as monitored by the expression of Cut and Hindsight, two downstream effectors of Notch signaling. Conclusion Our findings point to an unexpected function for autophagy in the modulation of the Notch signaling pathway during Drosophila oogenesis and suggest a function for autophagy in proper receptor activation. Egg development is affected by an imbalance of autophagy between signal sending (germline and signal receiving cell (FC, thus the lack of autophagy in the germline is likely to decrease the amount of active ligand and accordingly compensates for increased signaling in autophagy-defective follicle cells.

  16. Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

    Science.gov (United States)

    House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

    2015-06-01

    Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

  17. Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif)

    Science.gov (United States)

    Ng, Mei Ying; Wang, Mei; Casey, Patrick J.; Gan, Yunn-Hwen; Hagen, Thilo

    2017-01-01

    Cycle inhibiting factors (Cifs) are virulence proteins secreted by the type III secretion system of some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Cif is known to function to deamidate Nedd8, leading to inhibition of Cullin E3 ubiquitin ligases (CRL) and consequently induction of cell cycle arrest. Here we show that Cif can function as a potent activator of MAPK/ERK signaling without significant activation of other signaling pathways downstream of receptor tyrosine kinases. Importantly, we found that the ability of Cif to activate ERK is dependent on its deamidase activity, but independent of Cullin E3 ligase inhibition. This suggests that apart from Nedd8, other cellular targets of Cif-dependent deamidation exist. We provide evidence that the mechanism involved in Cif-mediated ERK activation is dependent on recruitment of the Grb2-SOS1 complex to the plasma membrane. Further investigation revealed that Cif appears to modify the phosphorylation status of SOS1 in a region containing the CDC25-H and proline-rich domains. It is known that prolonged Cullin E3 ligase inhibition leads to cellular apoptosis. Therefore, we hypothesize that ERK activation is an important mechanism to counter the pro-apoptotic effects of Cif. Indeed, we show that Cif dependent ERK activation promotes phosphorylation of the proapoptotic protein Bim, thereby potentially conferring a pro-survival signal. In summary, we identified a novel deamidation-dependent mechanism of action of the B. pseudomallei virulence factor Cif/CHBP to activate MAPK/ERK signaling. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian signaling pathways. PMID:28166272

  18. Genotypic and phenotypic characterization of garlic-fermenting lactic acid bacteria isolated from som-fak, a Thai low-salt fermented fish product

    DEFF Research Database (Denmark)

    Paludan-Müller, Christine; Valyasevi, R.; Huss, Hans Henrik

    2002-01-01

    AIMS: To evaluate the importance of garlic for fermentation of a Thai fish product, and to differentiate among garlic-/inulin-fermenting lactic acid bacteria (LAB) at strain level. METHODS AND RESULTS: Som-fak was prepared by fermentation of a mixture of fish, salt, rice, sucrose and garlic. pH d...

  19. Alternative splicing of MALT1 controls signalling and activation of CD4+ T cells

    OpenAIRE

    Meininger, Isabel; Griesbach, Richard A.; HU, DESHENG; Gehring, Torben; Seeholzer, Thomas; Bertossi, Arianna; Kranich, Jan; Oeckinghaus, Andrea; Eitelhuber, Andrea C.; Greczmiel, Ute; Gewies, Andreas; Schmidt-Supprian, Marc; Ruland, Jürgen; Brocker, Thomas; Heissmeyer, Vigo

    2016-01-01

    MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4+ T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify...

  20. Active Elements for Analog Signal Processing: Classification, Review, and New Proposals

    Directory of Open Access Journals (Sweden)

    Z. Kolka

    2008-12-01

    Full Text Available In the paper, an analysis of the state-of-the-art of active elements for analog signal processing is presented which support – in contrast to the conventional operational amplifiers – not only the voltage-mode but also the current- and mixed-mode operations. Several problems are addressed which are associated with the utilization of these elements in linear applications, particularly in frequency filters. A methodology is proposed which generates a number of fundamentally new active elements with their potential utilization in various areas of signal processing.

  1. PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner.

    Science.gov (United States)

    Sjöqvist, Marika; Antfolk, Daniel; Ferraris, Saima; Rraklli, Vilma; Haga, Cecilia; Antila, Christian; Mutvei, Anders; Imanishi, Susumu Y; Holmberg, Johan; Jin, Shaobo; Eriksson, John E; Lendahl, Urban; Sahlgren, Cecilia

    2014-04-01

    Activation of Notch signaling requires intracellular routing of the receptor, but the mechanisms controlling the distinct steps in the routing process is poorly understood. We identify PKCζ as a key regulator of Notch receptor intracellular routing. When PKCζ was inhibited in the developing chick central nervous system and in cultured myoblasts, Notch-stimulated cells were allowed to undergo differentiation. PKCζ phosphorylates membrane-tethered forms of Notch and regulates two distinct routing steps, depending on the Notch activation state. When Notch is activated, PKCζ promotes re-localization of Notch from late endosomes to the nucleus and enhances production of the Notch intracellular domain, which leads to increased Notch activity. In the non-activated state, PKCζ instead facilitates Notch receptor internalization, accompanied with increased ubiquitylation and interaction with the endosomal sorting protein Hrs. Collectively, these data identify PKCζ as a key regulator of Notch trafficking and demonstrate that distinct steps in intracellular routing are differentially modulated depending on Notch signaling status.

  2. Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

    Science.gov (United States)

    Andrusiak, Matthew G; Jin, Yishi

    2016-04-01

    Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans.

  3. Inhibition on Apoptosis Induced by Elevated Hydrostatic Pressure in Retinal Ganglion Cell-5 via Laminin Upregulating β1-integrin/Focal Adhesion Kinase/Protein Kinase B Signaling Pathway

    Institute of Scientific and Technical Information of China (English)

    Yi Li; Yan-Ming Chen; Ming-Ming Sun; Xiao-Dan Guo; Ya-Chen Wang; Zhong-Zhi Zhang

    2016-01-01

    Background:Glaucoma is a progressive optic neuropathy characterized by degeneration of neurons due to loss of retinal ganglion cells (RGCs).High intraocular pressure (HIOP),the main risk factor,causes the optic nerve damage.However,the precise mechanism of HIOP-induced RGC death is not yet completely understood.This study was conducted to determine apoptosis of RGC-5 cells induced by elevated hydrostatic pressures,explore whether laminin is associated with apoptosis under pressure,whether laminin can protect RGCs from apoptosis and affirm the mechanism that regulates the process of RGCs survival.Methods:RGC-5 cells were exposed to 0,20,40,and 60 mmHg in a pressurized incubator for 6,12,and 24 h,respectively.The effect of elevated hydrostatic pressure on RGC-5 cells was measured by Annexin V-fluorescein isothiocyanate/propidium iodide staining,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,and Western blotting of cleaved caspase-3 protein.Location and expression oflaminin were detected by immunofluorescence.The expression of β 1-integrin,phosphorylation of focal adhesion kinase (FAK) and protein kinase B (PKB,or AKT) were investigated with real-time polymerase chain reaction and Western blotting analysis.Results:Elevated hydrostatic pressure induced apoptosis in cultured RGC-5 cells.Pressure with 40 mmHg for 24 h induced a maximum apoptosis.Laminin was declined in RGC-5 cells after exposing to 40 mmHg for 24 h.After pretreating with laminin,RGC-5 cells survived from elevated pressure.Furthermore,β1-integrin and phosphorylation of FAK and AKT were increased compared to 40 mmHg group.Conclusions:The data show apoptosis tendency of RGC-5 cells with elevated hydrostatic pressure.Laminin can protect RGC-5 cells against high pressure via β 1-integrin/FAK/AKT signaling pathway.These results suggest that the decreased laminin of RGC-5 cells might be responsible for apoptosis induced by elevated hydrostatic pressure,and laminin or activating β1

  4. Receptor Protein Tyrosine Phosphatase α-Mediated Enhancement of Rheumatoid Synovial Fibroblast Signaling and Promotion of Arthritis in Mice

    NARCIS (Netherlands)

    Stanford, Stephanie M; Svensson, Mattias N D; Sacchetti, Cristiano; Pilo, Caila A; Wu, Dennis J; Kiosses, William B; Hellvard, Annelie; Bergum, Brith; Muench, German R Aleman; Elly, Christian; Liu, Yun-Cai; den Hertog, Jeroen; Elson, Ari; Sap, Jan; Mydel, Piotr; Boyle, David L; Corr, Maripat; Firestein, Gary S; Bottini, Nunzio

    2016-01-01

    OBJECTIVE: During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the extracellular matrix of the joint. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to the anomalous behavior of RA FLS.

  5. Clicks, whistles and pulses: Passive and active signal use in dolphin communication

    Science.gov (United States)

    Herzing, Denise L.

    2014-12-01

    The search for signals out of noise is a problem not only with radio signals from the sky but in the study of animal communication. Dolphins use multiple modalities to communicate including body postures, touch, vision, and most elaborately sound. Like SETI radio signal searches, dolphin sound analysis includes the detection, recognition, analysis, and interpretation of signals. Dolphins use both passive listening and active production to communicate. Dolphins use three main types of acoustic signals: frequency modulated whistles (narrowband with harmonics), echolocation (broadband clicks) and burst pulsed sounds (packets of closely spaced broadband clicks). Dolphin sound analysis has focused on frequency-modulated whistles, yet the most commonly used signals are burst-pulsed sounds which, due to their graded and overlapping nature and bimodal inter-click interval (ICI) rates are hard to categorize. We will look at: 1) the mechanism of sound production and categories of sound types, 2) sound analysis techniques and information content, and 3) examples of lessons learned in the study of dolphin acoustics. The goal of this paper is to provide perspective on how animal communication studies might provide insight to both passive and active SETI in the larger context of searching for life signatures.

  6. 粘着斑激酶在前列腺癌中表达的研究%Research on Expression of FAK in Prostate Carcinoma

    Institute of Scientific and Technical Information of China (English)

    廖洪利; 米叶赛尔·阿不都拉

    2016-01-01

    目的:探讨粘着斑激酶(FAK)表达与前列腺癌发病的关系。方法前列腺癌患者60例,经手术切除的癌组织及相应的癌旁组织行免疫组化检测 FAK表达情况。Trizol法提取组织总 RNA,RT-PCR检测 FAK mRNA表达量,并进行统计学分析。结果 FAK 在60例前列腺癌组织中表达明显比癌旁组织高,两者差异有统计学意义(χ2=72.55,P<0.01),FAK的mRNA水平在前列腺癌组织表达显著比癌旁组织高(t=30.51,P<0.01)。淋巴结转移情况看 pN0M0表达阳性例数比pN3M1少,pN0M0的mRNA表达量比pN3M1的表达量低,差异有统计学意义(t=25.43,P<0.01)。结论前列腺癌细胞FAK表达与侵袭转移相关。%Objective To investigate the relationships between expression of FAK and Prostate Carcinoma (PC)morbidity. Methods By surgery,get cancer tissue and para-carcinama tissue from 60 cases PC.To detect FAK expression by immuno-histochemical.To extract total RNA by Trizol.To detect the FAK mRNA by RT-PCR,and analysis these data.Results FAK expression level in cancer tissue was higher than that in para-carcinama tissue.There was statistical difference between them (χ2=72.55,P<0.01).mRNA expression levels of FAK in cancer tissue showed significant higher than the levels in para-carcinama tissue (t=30.51,P<0.01).According to lymphatic metastasis,the expression positive cases of FAK in pN0M0 classification were lower than these in pN3M1 classification,and mRNA expression levels of FAK were the same re-sults.There were clearly statistical distinctive (t=25.43,P<0.01).Conclusion The expression of FAK in PC cells was as-sociated with tumor invasion.

  7. Bz-423 superoxide signals apoptosis via selective activation of JNK, Bak, and Bax.

    Science.gov (United States)

    Blatt, Neal B; Boitano, Anthony E; Lyssiotis, Costas A; Opipari, Anthony W; Glick, Gary D

    2008-11-01

    Bz-423 is a proapoptotic 1,4-benzodiazepine with potent therapeutic properties in murine models of lupus and psoriasis. Bz-423 modulates the F(1)F(0)-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. Herein, we report the signaling pathway activated by Bz-423 in mouse embryonic fibroblasts containing knockouts of key apoptotic proteins. Bz-423-induced superoxide activates cytosolic ASK1 and its release from thioredoxin. A mitogen-activated protein kinase cascade follows, leading to the specific phosphorylation of JNK. JNK signals activation of Bax and Bak which then induces mitochondrial outer membrane permeabilization to cause the release of cytochrome c and a commitment to apoptosis. The response of these cells to Bz-423 is critically dependent on both superoxide and JNK activation as antioxidants and the JNK inhibitor SP600125 prevents Bax translocation, cytochrome c release, and cell death. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a sequential and specific apoptotic response. Collectively, these data suggest that the selectivity of Bz-423 observed in vivo results from cell-type specific differences in redox balance and signaling by ASK1 and Bcl-2 proteins.

  8. Active concentric ring electrode for non-invasive detection of intestinal myoelectric signals.

    Science.gov (United States)

    Prats-Boluda, Gema; Garcia-Casado, Javier; Martinez-de-Juan, Jose L; Ye-Lin, Yiyao

    2011-05-01

    Although the surface electroenterogram (EEnG) is a weak signal contaminated by strong physiological interference, such as ECG and respiration, abdominal surface recordings of the EEnG could provide a non-invasive method of studying intestinal activity. The goal of this work was to develop a modular, active, low-cost and easy-to-use sensor to obtain a direct estimation of the Laplacian of the EEnG on the abdominal surface in order to enhance the quality of bipolar surface monitoring of intestinal activity. The sensor is made up of a set of 3 concentric dry Ag/AgCl ring electrodes and a battery-powered signal-conditioning circuit. Each section is etched on a different printed circuit board (PCB) and the sections are joined to each other by surface mount technology connectors. This means the sensing electrodes can be treated independently for purposes of maintenance and replacement and the signal conditioning circuit can be re-used. A total of ten recording sessions were carried out on humans. The results show that the surface recordings of the EEnG obtained by the active sensor present significantly less ECG and respiration interference than those obtained by bipolar recordings. In addition, bioelectrical sources whose frequency fitted with the slow wave component of the EEnG (SW) were identified by parametric spectral analysis in the surface signals picked up by the active sensors.

  9. Immune signaling pathways activated in response to different pathogenic micro-organisms in Bombyx mori.

    Science.gov (United States)

    Liu, Wei; Liu, Jiabin; Lu, Yahong; Gong, Yongchang; Zhu, Min; Chen, Fei; Liang, Zi; Zhu, Liyuan; Kuang, Sulan; Hu, Xiaolong; Cao, Guangli; Xue, Renyu; Gong, Chengliang

    2015-06-01

    The JAK/STAT, Toll, Imd, and RNAi pathways are the major signaling pathways associated with insect innate immunity. To explore the different immune signaling pathways triggered in response to pathogenic micro-organism infections in the silkworm, Bombyx mori, the expression levels of the signal transducer and activator of transcription (BmSTAT), spatzle-1 (Bmspz-1), peptidoglycan-recognition protein LB (BmPGRP-LB), peptidoglycan-recognition protein LE (BmPGRP-LE), argonaute 2 (Bmago2), and dicer-2 (Bmdcr2) genes after challenge with Escherichia coli (E. coli), Serratiamarcescens (Sm), Bacillus bombyseptieus (Bab), Beauveriabassiana (Beb), nucleopolyhedrovirus (BmNPV), cypovirus (BmCPV), bidensovirus (BmBDV), or Nosemabombycis (Nb) were determined using real-time PCR. We found that the JAK/STAT pathway could be activated by challenge with BmNPV and BmBDV, the Toll pathway could be most robustly induced by challenge with Beb, the Imd pathway was mainly activated in response to infection by E. coli and Sm, and the RNAi pathway was not activated by viral infection, but could be triggered by some bacterial infections. These findings yield insights into the immune signaling pathways activated in response to different pathogenic micro-organisms in the silkworm.

  10. Selenium Deficiency Attenuates Chicken Duodenal Mucosal Immunity via Activation of the NF-κb Signaling Pathway.

    Science.gov (United States)

    Liu, Zhe; Qu, Yanpeng; Wang, Jianfa; Wu, Rui

    2016-08-01

    Selenium (Se) deficiency can cause intestinal mucosal inflammation, which is related to activation of nuclear transcription factor kappa-B (NF-κB) signaling pathway. However, the mechanism of inflammatory response in chicken duodenal mucosa caused by Se deficiency and its relationship with the NF-κB signaling pathway remain elusive. In this study, we firstly obtained Se-deficient chickens bred with 0.01 mg/kg Se and the normal chickens bred with 0.4 mg/kg Se for 35 days. Then, NF-κB signaling pathway, secretory immunoglobulin A (SIgA), inflammatory cytokines, oxidized glutathione, glutathione peroxidase, and glutathione activities were determined. The results showed that Se deficiency obviously enhanced p50, p65, and p65 DNA-binding activities. The phosphorylation of IκB-α and phosphorylation of kappa-B kinase subunit alpha (IKKα) and IKKα were elevated, but IκB-α was decreased (P mucosal immunity via activation of NF-κB signaling pathway regulated by redox activity, which suggested that Se is a crucial host factor involved in regulating inflammation.

  11. Mitogen-activated protein kinase signaling pathways of the tangerine pathotype of Alternaria alternata

    Directory of Open Access Journals (Sweden)

    Kuang-Ren Chung

    2013-06-01

    Full Text Available Mitogen-activated protein kinase (MAPK- mediated signaling pathways have been known to have important functions in eukaryotic organisms. The mechanisms by which the filamentous fungus Alternaria alternata senses and responds to environmental signals have begun to be elucidated. Available data indicate that A. alternata utilizes the Fus3, Hog1 and Slt2 MAPK-mediated signaling pathways, either separately or in a cooperative manner, for conidia formation, resistance to oxidative and osmotic stress, and pathogenesis to citrus. This review provides an overview of our current knowledge of MAPK signaling pathways, in conjunction with the two-component histidine kinase and the Skn7 response regulator, in the tangerine pathotype of A. alternata.

  12. Low-cost detection of RC-IED activation signals in VHF band

    Science.gov (United States)

    Camargo Suarez, Victor Hugo; Marulanda B., Jose Ignacio

    2014-05-01

    The proliferation of Radio Controlled Improvised Explosive Devices (RC-IED) is a growing threat around the world. The ease of construction and low cost of these devices are transforming common things in lethal tramps. The fight against this threats normally involves the use of sophisticated and expensive equipment of Electronic Warfare based on high speed DSP systems, just to detect the presence of detonation signals. In this work is showed how to find activation signals based on the characteristic of the power in a specific band and the previous knowledge about the detonation signals. As proof of concept we have taken the information about the RC-IEDs used in the Colombian conflict and develop an algorithm to find detonation signals based on the measured power in frequencies between 136 MHz and 174 MHz (2 meter civil band)

  13. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling

    Science.gov (United States)

    Holland, Sabrina M.; Collura, Kaitlin M.; Ketschek, Andrea; Noma, Kentaro; Ferguson, Toby A.; Jin, Yishi; Gallo, Gianluca; Thomas, Gareth M.

    2016-01-01

    Dual leucine-zipper kinase (DLK) is critical for axon-to-soma retrograde signaling following nerve injury. However, it is unknown how DLK, a predicted soluble kinase, conveys long-distance signals and why homologous kinases cannot compensate for loss of DLK. Here, we report that DLK, but not homologous kinases, is palmitoylated at a conserved site adjacent to its kinase domain. Using short-hairpin RNA knockdown/rescue, we find that palmitoylation is critical for DLK-dependent retrograde signaling in sensory axons. This functional importance is because of three novel cellular and molecular roles of palmitoylation, which targets DLK to trafficking vesicles, is required to assemble DLK signaling complexes and, unexpectedly, is essential for DLK’s kinase activity. By simultaneously controlling DLK localization, interactions, and activity, palmitoylation ensures that only vesicle-bound DLK is active in neurons. These findings explain how DLK specifically mediates nerve injury responses and reveal a novel cellular mechanism that ensures the specificity of neuronal kinase signaling. PMID:26719418

  14. Activation of the wnt/β-Catenin Signaling Pathway in Polymyositis, Dermatomyositis and Duchenne Muscular Dystrophy

    Science.gov (United States)

    Liu, Fuchen; Liang, Zonglai; Xu, Jingwen; Li, Wei; Zhao, Dandan; Zhao, Yuying

    2016-01-01

    Background and Purpose The wnt/β-catenin signaling pathway plays a critical role in embryonic development and adult-tissue homeostasis. Recent investigations implicate the importance of wnt/β-catenin signaling in normal wound healing and its sustained activation being associated with fibrogenesis. We investigated the immunolocalization and activation of wnt/β-catenin in polymyositis (PM), dermatomyositis (DM), and Duchenne muscular dystrophy (DMD). Methods Immunofluorescence staining and Western blot analysis of β-catenin were performed in muscle specimens from 6 PM, 8 DM, and 6 DMD subjects. The β-catenin/Tcf4 DNA-binding activity in muscle was studied using an electrophoretic mobility shift assay (EMSA), and serum wnt/β-catenin/Tcf transcriptional activity was measured using a luciferase reporter gene assay. Results Immunoreactivity for β-catenin was found in the cytoplasm and nuclei of muscle fibers in PM, DM, and DMD. The protein level of β-catenin was elevated, and EMSA analysis confirmed the activation of wnt/β-catenin signaling. The transcriptional activities of β-catenin/Tcf in the circulation were increased in patients with PM, DM, and DMD, especially in those with interstitial lung disease, and these transcriptional activities decreased when PM or DM patients exhibited obvious clinical improvements. Conclusions Our findings indicate that wnt/β-catenin signaling is activated in PM, DM, and DMD. Its activation in muscle tissue and the circulation may play a role in modulating muscle regeneration and be at least partly involved in the process of muscle and pulmonary fibrosis. PMID:27165423

  15. Circular polarization intrinsic optical signal recording of stimulus-evoked neural activity.

    Science.gov (United States)

    Lu, Rong-Wen; Zhang, Qiu-Xiang; Yao, Xin-Cheng

    2011-05-15

    Linear polarization intrinsic optical signal (LP-IOS) measurement can provide sensitive detection of neural activities in stimulus-activated neural tissues. However, the LP-IOS magnitude and signal-to-noise ratio (SNR) are highly correlated with the nerve orientation relative to the polarization plane of the incident light. Because of the complexity of orientation dependency, LP-IOS optimization and outcome interpretation are time consuming and complicated. In this study, we demonstrate the feasibility of circular polarization intrinsic optical signal (CP-IOS) measurement. Our theoretical modeling and experimental investigation indicate that CP-IOS magnitude and SNR are independent from the nerve orientation. Therefore, CP-IOS promises a practical method for polarization IOS imaging of complex neural systems.

  16. DMPD: Activation of lymphokine genes in T cells: role of cis-acting DNA elements thatrespond to T cell activation signals. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available DNA elements thatrespond to T cell activation signals. PubmedID 1492121 Title Activation of lymphokine genes... in T cells: role of cis-acting DNA elements thatrespond to T cell activation signals. Authors Arai N, Naito...ivation signals. Arai N, Naito Y, Watanabe M, Masuda ES, Yamaguchi-Iwai Y, Tsuboi A...1492121 Activation of lymphokine genes in T cells: role of cis-acting DNA elements thatrespond to T cell act

  17. Role of signaling lymphocytic activation molecule in T helper cell responses

    Directory of Open Access Journals (Sweden)

    Jan E. de Vries

    1998-01-01

    Full Text Available Signaling lymphocytic activation molecule (SLAM; CDw150 is a 70 kDa glycoprotein. Signaling lymphocytic activation molecule is constitutively expressed on memory T cells, CD56+ T cells, a subset of T cell receptor γδ+ cells, immature thymocytes and, at low levels, on a proportion of peripheral blood B cells. Signaling lymphocytic activation molecule is rapidly upregulated on all T and B cells after activation. Engagement of SLAM by F(ab’2 fragments of an anti-SLAM monoclonal antibody (mAb A12 enhances antigen-specific T cell proliferation. In addition, mAb A12 was directly mitogenic for T cell clones and activated T cells. T cell proliferation induced by mAb A12 is independent of interleukin (IL-2, IL-4, IL-12 and IL-15, but is cyclosporin A sensitive. Ligation of SLAM during antigen-specific T cell proliferation resulted in upregulation of interferon (IFN-γ production, even by allergen-specific T helper cell (Th 2 clones, whereas the levels of IL-4 and IL-5 production were only marginally affected. The mAb A12 was unable to induce IL-4 and IL-5 production by Th1 clones. Co-stimulation of skin-derived Der P1-specific Th2 cells from patients with atopic dermatitis via SLAM resulted in the generation of a population of IFN-γ-producing cells, thereby reverting their phenotype to a Th0 pattern. Signaling lymphocytic activation molecule is a high-affinity self ligand mediating homophilic cell interaction. In addition, soluble SLAM enhances both T and B cell proliferation. Collectively, these data indicate that SLAM molecules act both as receptors and ligands that are not only involved in T cell expansion but also drive the expanding T cells during immune responses into the Th0/Th1 pathway. This suggests that signaling through SLAM plays a role in directing Th0/Th1 development.

  18. Signal integration by Ca(2+) regulates intestinal stem-cell activity.

    Science.gov (United States)

    Deng, Hansong; Gerencser, Akos A; Jasper, Heinrich

    2015-12-10

    Somatic stem cells maintain tissue homeostasis by dynamically adjusting proliferation and differentiation in response to stress and metabolic cues. Here we identify Ca(2+) signalling as a central regulator of intestinal stem cell (ISC) activity in Drosophila. We show that dietary L-glutamate stimulates ISC division and gut growth. The metabotropic glutamate receptor (mGluR) is required in ISCs for this response, and for an associated modulation of cytosolic Ca(2+) oscillations that results in sustained high cytosolic Ca(2+) concentrations. High cytosolic Ca(2+) concentrations induce ISC proliferation by regulating Calcineurin and CREB-regulated transcriptional co-activator (Crtc). In response to a wide range of dietary and stress stimuli, ISCs reversibly transition between Ca(2+) oscillation states that represent poised or activated modes of proliferation, respectively. We propose that the dynamic regulation of intracellular Ca(2+) levels allows effective integration of diverse mitogenic signals in ISCs to adapt their proliferative activity to the needs of the tissue.

  19. Detecting stable phase structures in EEG signals to classify brain activity amplitude patterns

    Institute of Scientific and Technical Information of China (English)

    Yusely RUIZ; Guang LI; Walter J. FREEMAN; Eduardo GONZALEZ

    2009-01-01

    Obtaining an electrocorticograms (ECoG) signal requires an invasive procedure in which brain activity is recorded from the cortical surface. In contrast, obtaining electroencephalograms (EEG) recordings requires the non-invasive procedure of recording the brain activity from the scalp surface, which allows EEG recordings to be performed more easily on healthy humans. In this work, a technique previously used to study spatial-temporal patterns of brain activity on animal ECoG was adapted for use on EEG. The main issues are centered on solving the problems introduced by the increment on the interelectrode distance and the procedure to detect stable frames. The results showed that spatial patterns of beta and gamma activity can also be extracted from the EEG signal by using stable frames as time markers for feature extraction. This adapted technique makes it possible to take advantage of the cognitive and phenomenological awareness of a normal healthy subject.

  20. Signal transducer and activator of transcription 6 gene G2964A polymorphism and inflammatory bowel disease.

    NARCIS (Netherlands)

    Xia, B; Crusius, J.B.A.; Wu, J; Zwiers, A.; Bodegraven, van A.A.; Pena, A.S.

    2003-01-01

    Signal transducer and activator of transcription 6 (STAT6) is a key transcription factor involved in interleukin 4 (IL-4) and IL-13-mediated Th2 response. The STAT6 gene is located on chromosome 12q13.3-14.1 (IBD2 region) and is therefore a positional and functional candidate gene for study in infla

  1. Epigenetic regulator Lid maintains germline stem cells through regulating JAK-STAT signaling pathway activity

    Directory of Open Access Journals (Sweden)

    Lama Tarayrah

    2015-11-01

    Full Text Available Signaling pathways and epigenetic mechanisms have both been shown to play essential roles in regulating stem cell activity. While the role of either mechanism in this regulation is well established in multiple stem cell lineages, how the two mechanisms interact to regulate stem cell activity is not as well understood. Here we report that in the Drosophila testis, an H3K4me3-specific histone demethylase encoded by little imaginal discs (lid maintains germline stem cell (GSC mitotic index and prevents GSC premature differentiation. Lid is required in germ cells for proper expression of the Stat92E transcription factor, the downstream effector of the Janus kinase signal transducer and activator of transcription (JAK-STAT signaling pathway. Our findings support a germ cell autonomous role for the JAK-STAT pathway in maintaining GSCs and place Lid as an upstream regulator of this pathway. Our study provides new insights into the biological functions of a histone demethylase in vivo and sheds light on the interaction between epigenetic mechanisms and signaling pathways in regulating stem cell activities.

  2. Characterization of the intracellular signalling capacity of natural FXa mutants with reduced pro-coagulant activity

    NARCIS (Netherlands)

    M. Monti; K.S. Borensztajn; M. Pinotti; A. Canella; A. Branchini; G. Marchetti; P.H. Reitsma; F. Bernardi; C.A. Spek

    2009-01-01

    INTRODUCTION: Factor X (FX) is a serine-protease playing a crucial role in the blood coagulation pathway and triggering intracellular signalling in a variety of cells via protease-activated receptors (PARs). By exploiting naturally occurring variants (V342A and G381D, catalytic domain; E19A, gamma-c

  3. Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor

    OpenAIRE

    Harrison, Hannah; Farnie, Gillian; Howell, Sacha J.; Rock, Rebecca E; Stylianou, Spyros; Brennan, Keith R.; Bundred, Nigel J; Clarke, Robert B.

    2010-01-01

    Notch receptor signaling pathways play an important role not only in normal breast development but also in breast cancer development and progression. We assessed the role of Notch receptors in stem cell activity in breast cancer cell lines and nine primary human tumor samples. Stem cells were enriched by selection of anoikis-resistant cells or cells expressing the membrane phenotype ESA+/CD44+/CD24low. Using these breast cancer stem cell populations, we compared the activation status of Notch...

  4. In vitro analysis of signal peptidase and membrane tannslocation activity in yeast microsomal membranes. Kobomaku kakubun wo mochiiru signal peptidase kassei oyobi makutoka kassei sokuteiho no kento

    Energy Technology Data Exchange (ETDEWEB)

    Nakayama, K.; Machida, M.; Jigami, Y. (The National Chemical Laboratory for Industry, Tsukuba (Japan))

    1991-05-29

    Studies have been pursued on proteins produced using microorganisms and cultured cells. The studies aim at facilitating the purification of the protein by making it secrete outside the cell and improving productivity. Signal peptidase is related to the secretion/translocation process of the protein. An in vitro analysis system for activity of yeast signal peptidase is made to elucidate effects of activity of signal peptidase on velocity and amount of secretion of the protein. As a result, in a combination of wheat germ extract and canine pancreatic membranes, the single peptidase activity and the membrane translocation activity are seen in a colibacillus {beta}-lactamase precursor and a yeast {alpha}-factor precursor, respectively. Moreover, in a combination of yeast lysates and yeast microsomal membranes, as the result of measuring the activity with the {alpha}-factor precursor as a substrate, the membrane translocation activity can be detected in this system. 16 refs., 4 figs., 1 tab.

  5. Cadmium Activates Multiple Signaling Pathways That Coordinately Stimulate Akt Activity to Enhance c-Myc mRNA Stability.

    Directory of Open Access Journals (Sweden)

    Jia-Shiuan Tsai

    Full Text Available Cadmium is a known environmental carcinogen. Exposure of Cd leads to the activation of several proto-oncogenes in cells. We investigated here the mechanism of c-Myc expression in hepatic cells under Cd treatment. The c-Myc protein and mRNA levels increased in dose- and time-dependent manners in HepG2 cells with Cd treatment. This increase was due to an increase in c-Myc mRNA stability. To explore the mechanism involved in enhancing the mRNA stability, several cellular signaling factors that evoked by Cd treatment were analyzed. PI3K, p38, ERK and JNK were activated by Cd. However, ERK did not participate in the Cd-induced c-Myc expression. Further analysis revealed that mTORC2 was a downstream factor of p38. PI3K, JNK and mTORC2 coordinately activated Akt. Akt was phosphorylated at Thr450 in the untreated cells. Cd treatment led to additional phosphorylation at Thr308 and Ser473. Blocking any of the three signaling factors resulted in the reduction of phosphorylation level at all three Akt sites. The activated Akt phosphorylated Foxo1 and allowed the modified protein to translocate into the cytoplasm. We conclude that Cd-induced accumulation of c-Myc requires the activation of several signaling pathways. The signals act coordinately for Akt activation and drive the Foxo1 from the nucleus to the cytoplasm. Reduction of Foxo1 in the nucleus reduces the transcription of its target genes that may affect c-Myc mRNA stability, resulting in a higher accumulation of the c-Myc proteins.

  6. mTOR signaling promotes stem cell activation via counterbalancing BMP-mediated suppression during hair regeneration.

    Science.gov (United States)

    Deng, Zhili; Lei, Xiaohua; Zhang, Xudong; Zhang, Huishan; Liu, Shuang; Chen, Qi; Hu, Huimin; Wang, Xinyue; Ning, Lina; Cao, Yujing; Zhao, Tongbiao; Zhou, Jiaxi; Chen, Ting; Duan, Enkui

    2015-02-01

    Hair follicles (HFs) undergo cycles of degeneration (catagen), rest (telogen), and regeneration (anagen) phases. Anagen begins when the hair follicle stem cells (HFSCs) obtain sufficient activation cues to overcome suppressive signals, mainly the BMP pathway, from their niche cells. Here, we unveil that mTOR complex 1 (mTORC1) signaling is activated in HFSCs, which coincides with the HFSC activation at the telogen-to-anagen transition. By using both an inducible conditional gene targeting strategy and a pharmacological inhibition method to ablate or inhibit mTOR signaling in adult skin epithelium before anagen initiation, we demonstrate that HFs that cannot respond to mTOR signaling display significantly delayed HFSC activation and extended telogen. Unexpectedly, BMP signaling activity is dramatically prolonged in mTOR signaling-deficient HFs. Through both gain- and loss-of-function studies in vitro, we show that mTORC1 signaling negatively affects BMP signaling, which serves as a main mechanism whereby mTORC1 signaling facilitates HFSC activation. Indeed, in vivo suppression of BMP by its antagonist Noggin rescues the HFSC activation defect in mTORC1-null skin. Our findings reveal a critical role for mTOR signaling in regulating stem cell activation through counterbalancing BMP-mediated repression during hair regeneration.

  7. An apodized Kepler periodogram for separating planetary and stellar activity signals

    Science.gov (United States)

    Gregory, Philip C.

    2016-05-01

    A new apodized Keplerian (AK) model is proposed for the analysis of precision radial velocity (RV) data to model both planetary and stellar activity (SA) induced RV signals. A symmetrical Gaussian apodization function with unknown width and centre can distinguish planetary signals from SA signals on the basis of the span of the apodization window. The general model for m AK signals includes a linear regression term between RV and the SA diagnostic log (R'hk), as well as an extra Gaussian noise term with unknown standard deviation. The model parameters are explored using a Bayesian fusion Markov chain Monte Carlo code. A differential version of the generalized Lomb-Scargle periodogram that employs a control diagnostic provides an additional way of distinguishing SA signals and helps guide the choice of new periods. Results are reported for a recent international RV blind challenge which included multiple state-of-the-art simulated data sets supported by a variety of SA diagnostics. In the current implementation, the AK method achieved a reduction in SA noise by a factor of approximately 6. Final parameter estimates for the planetary candidates are derived from fits that include AK signals to model the SA components and simple Keplerians to model the planetary candidates. Preliminary results are also reported for AK models augmented by a moving average component that allows for correlations in the residuals.

  8. Loss of Pancreas upon Activated Wnt Signaling Is Concomitant with Emergence of Gastrointestinal Identity

    Science.gov (United States)

    Herrero-Martin, Griselda; Puri, Sapna; Taketo, Makoto Mark; Rojas, Anabel; Hebrok, Matthias; Cano, David A.

    2016-01-01

    Organ formation is achieved through the complex interplay between signaling pathways and transcriptional cascades. The canonical Wnt signaling pathway plays multiple roles during embryonic development including patterning, proliferation and differentiation in distinct tissues. Previous studies have established the importance of this pathway at multiple stages of pancreas formation as well as in postnatal organ function and homeostasis. In mice, gain-of-function experiments have demonstrated that activation of the canonical Wnt pathway results in pancreatic hypoplasia, a phenomenon whose underlying mechanisms remains to be elucidated. Here, we show that ectopic activation of epithelial canonical Wnt signaling causes aberrant induction of gastric and intestinal markers both in the pancreatic epithelium and mesenchyme, leading to the development of gut-like features. Furthermore, we provide evidence that β -catenin-induced impairment of pancreas formation depends on Hedgehog signaling. Together, our data emphasize the developmental plasticity of pancreatic progenitors and further underscore the key role of precise regulation of signaling pathways to maintain appropriate organ boundaries. PMID:27736991

  9. Quorum activation at a distance: spatiotemporal patterns of gene regulation from diffusion of an autoinducer signal

    Science.gov (United States)

    Dilanji, Gabriel; Langebrake, Jessica; Deleenheer, Patrick; Hagen, Stephen J.

    2012-02-01

    Bacteria in colonies coordinate gene regulation through the exchange of diffusible signal molecules known as autoinducers (AI). This ``quorum signaling'' often occurs in physically heterogeneous and spatially extended environments such as biofilms. Under these conditions the space and time scales for diffusion of the signal limit the range and timing of effective gene regulation. We expect that spatial and temporal patterns of gene expression will reflect physical environmental constraints as well as nonlinear transcriptional activation and feedback within the gene regulatory system. We have combined experiments and modeling to investigate how these spatiotemporal patterns develop. We embed engineered plasmid/GFP quorum sensor strains or wild type strains in a long narrow agar lane, and then introduce AI signal at one terminus of the lane. Diffusion of the AI initiates reporter expression along the length of the lane, extending to macroscopic distances of mm-cm. Resulting patterns are captured quantitatively by a mathematical model that incorporates logistic growth of the population, diffusion of AI, and nonlinear transcriptional activation. Our results show that a diffusing quorum signal can coordinate gene expression over distances of order 1cm on time scales of order 10 hrs.

  10. Brazilian Red Propolis Attenuates Inflammatory Signaling Cascade in LPS-Activated Macrophages

    Science.gov (United States)

    Bueno-Silva, Bruno; Kawamoto, Dione; Ando-Suguimoto, Ellen S.; Alencar, Severino M.; Rosalen, Pedro L.; Mayer, Marcia P. A.

    2015-01-01

    Although previous studies suggested an anti-inflammatory property of Brazilian red propolis (BRP), the mechanisms involved in the anti-inflammatory effects of BRP and its activity on macrophages were still not elucidated. This study aimed to evaluate whether BRP attenuates the inflammatory effect of LPS on macrophages and to investigate its underlying mechanisms. BRP was added to RAW 264.7 murine macrophages after activation with LPS. NO production, cell viability, cytokines profile were evaluated. Activation of inflammatory signaling pathways and macrophage polarization were determined by RT-qPCR and Western blot. BRP at 50 μg/ml inhibited NO production by 78% without affecting cell viability. Cd80 and Cd86 were upregulated whereas mrc1 was down regulated by BRP indicating macrophage polarization at M1. BRP attenuated the production of pro-inflammatory mediators IL-12, GM-CSF, IFN-Ɣ, IL-1β in cell supernatants although levels of TNF- α and IL-6 were slightly increased after BRP treatment. Levels of IL-4, IL-10 and TGF-β were also reduced by BRP. BRP significantly reduced the up-regulation promoted by LPS of transcription of genes in inflammatory signaling (Pdk1, Pak1, Nfkb1, Mtcp1, Gsk3b, Fos and Elk1) and of Il1β and Il1f9 (fold-change rate > 5), which were further confirmed by the inhibition of NF-κB and MAPK signaling pathways. Furthermore, the upstream adaptor MyD88 adaptor-like (Mal), also known as TIRAP, involved in TLR2 and TLR4 signaling, was down- regulated in BRP treated LPS-activated macrophages. Given that BRP inhibited multiple signaling pathways in macrophages involved in the inflammatory process activated by LPS, our data indicated that BRP is a noteworthy food-source for the discovery of new bioactive compounds and a potential candidate to attenuate exhacerbated inflammatory diseases. PMID:26660901

  11. Brazilian Red Propolis Attenuates Inflammatory Signaling Cascade in LPS-Activated Macrophages.

    Directory of Open Access Journals (Sweden)

    Bruno Bueno-Silva

    Full Text Available Although previous studies suggested an anti-inflammatory property of Brazilian red propolis (BRP, the mechanisms involved in the anti-inflammatory effects of BRP and its activity on macrophages were still not elucidated. This study aimed to evaluate whether BRP attenuates the inflammatory effect of LPS on macrophages and to investigate its underlying mechanisms. BRP was added to RAW 264.7 murine macrophages after activation with LPS. NO production, cell viability, cytokines profile were evaluated. Activation of inflammatory signaling pathways and macrophage polarization were determined by RT-qPCR and Western blot. BRP at 50 μg/ml inhibited NO production by 78% without affecting cell viability. Cd80 and Cd86 were upregulated whereas mrc1 was down regulated by BRP indicating macrophage polarization at M1. BRP attenuated the production of pro-inflammatory mediators IL-12, GM-CSF, IFN-Ɣ, IL-1β in cell supernatants although levels of TNF- α and IL-6 were slightly increased after BRP treatment. Levels of IL-4, IL-10 and TGF-β were also reduced by BRP. BRP significantly reduced the up-regulation promoted by LPS of transcription of genes in inflammatory signaling (Pdk1, Pak1, Nfkb1, Mtcp1, Gsk3b, Fos and Elk1 and of Il1β and Il1f9 (fold-change rate > 5, which were further confirmed by the inhibition of NF-κB and MAPK signaling pathways. Furthermore, the upstream adaptor MyD88 adaptor-like (Mal, also known as TIRAP, involved in TLR2 and TLR4 signaling, was down- regulated in BRP treated LPS-activated macrophages. Given that BRP inhibited multiple signaling pathways in macrophages involved in the inflammatory process activated by LPS, our data indicated that BRP is a noteworthy food-source for the discovery of new bioactive compounds and a potential candidate to attenuate exhacerbated inflammatory diseases.

  12. Akt/PKB activation and insulin signaling: a novel insulin signaling pathway in the treatment of type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Mackenzie RWA

    2014-02-01

    Full Text Available Richard WA Mackenzie, Bradley T Elliott Department of Human and Health Sciences, Facility of Science and Technology, University of Westminster, London, UK Abstract: Type 2 diabetes is a metabolic disease categorized primarily by reduced insulin sensitivity, β-cell dysfunction, and elevated hepatic glucose production. Treatments reducing hyperglycemia and the secondary complications that result from these dysfunctions are being sought after. Two distinct pathways encourage glucose transport activity in skeletal muscle, ie, the contraction-stimulated pathway reliant on Ca2+/5′-monophosphate-activated protein kinase (AMPK-dependent mechanisms and an insulin-dependent pathway activated via upregulation of serine/threonine protein kinase Akt/PKB. Metformin is an established treatment for type 2 diabetes due to its ability to increase peripheral glucose uptake while reducing hepatic glucose production in an AMPK-dependent manner. Peripheral insulin action is reduced in type 2 diabetics whereas AMPK signaling remains largely intact. This paper firstly reviews AMPK and its role in glucose uptake and then focuses on a novel mechanism known to operate via an insulin-dependent pathway. Inositol hexakisphosphate (IP6 kinase 1 (IP6K1 produces a pyrophosphate group at the position of IP6 to generate a further inositol pyrophosphate, ie, diphosphoinositol pentakisphosphate (IP7. IP7 binds with Akt/PKB at its pleckstrin homology domain, preventing interaction with phosphatidylinositol 3,4,5-trisphosphate, and therefore reducing Akt/PKB membrane translocation and insulin-stimulated glucose uptake. Novel evidence suggesting a reduction in IP7 production via IP6K1 inhibition represents an exciting therapeutic avenue in the treatment of insulin resistance. Metformin-induced activation of AMPK is a key current intervention in the management of type 2 diabetes. However, this treatment does not seem to improve peripheral insulin resistance. In light of this

  13. Silybin-mediated inhibition of Notch signaling exerts antitumor activity in human hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Song Zhang

    Full Text Available Hepatocellular carcinoma (HCC is a global health burden that is associated with limited treatment options and poor patient prognoses. Silybin (SIL, an antioxidant derived from the milk thistle plant (Silybum marianum, has been reported to exert hepatoprotective and antitumorigenic effects both in vitro and in vivo. While SIL has been shown to have potent antitumor activity against various types of cancer, including HCC, the molecular mechanisms underlying the effects of SIL remain largely unknown. The Notch signaling pathway plays crucial roles in tumorigenesis and immune development. In the present study, we assessed the antitumor activity of SIL in human HCC HepG2 cells in vitro and in vivo and explored the roles of the Notch pathway and of the apoptosis-related signaling pathway on the activity of SIL. SIL treatment resulted in a dose- and time-dependent inhibition of HCC cell viability. Additionally, SIL exhibited strong antitumor activity, as evidenced not only by reductions in tumor cell adhesion, migration, intracellular glutathione (GSH levels and total antioxidant capability (T-AOC but also by increases in the apoptotic index, caspase3 activity, and reactive oxygen species (ROS. Furthermore, SIL treatment decreased the expression of the Notch1 intracellular domain (NICD, RBP-Jκ, and Hes1 proteins, upregulated the apoptosis pathway-related protein Bax, and downregulated Bcl2, survivin, and cyclin D1. Notch1 siRNA (in vitro or DAPT (a known Notch1 inhibitor, in vivo further enhanced the antitumor activity of SIL, and recombinant Jagged1 protein (a known Notch ligand in vitro attenuated the antitumor activity of SIL. Taken together, these data indicate that SIL is a potent inhibitor of HCC cell growth that targets the Notch signaling pathway and suggest that the inhibition of Notch signaling may be a novel therapeutic intervention for HCC.

  14. Calcium signals in the nucleus accumbens: Activation of astrocytes by ATP and succinate

    Directory of Open Access Journals (Sweden)

    Emri Zsuzsa

    2011-10-01

    Full Text Available Abstract Background Accumulating evidence suggests that glial signalling is activated by different brain functions. However, knowledge regarding molecular mechanisms of activation or their relation to neuronal activity is limited. The purpose of the present study is to identify the characteristics of ATP-evoked glial signalling in the brain reward area, the nucleus accumbens (NAc, and thereby to explore the action of citric acid cycle intermediate succinate (SUC. Results We described the burst-like propagation of Ca2+ transients evoked by ATP in acute NAc slices from rat brain. Co-localization of the ATP-evoked Ca2+ signalling with immunoreactivities of the astroglia-specific gap junction forming channel protein connexin43 (Cx43 and the glial fibrillary acidic protein (GFAP indicated that the responsive cells were a subpopulation of Cx43 and GFAP immunoreactive astrocytes. The ATP-evoked Ca2+ transients were present under the blockade of neuronal activity, but were inhibited by Ca2+ store depletion and antagonism of the G protein coupled purinergic P2Y1 receptor subtype-specific antagonist MRS2179. Similarly, Ca2+ transients evoked by the P2Y1 receptor subtype-specific agonist 2-(Methylthioadenosine 5'-diphosphate were also blocked by MRS2179. These characteristics implied that intercellular Ca2+ signalling originated from the release of Ca2+ from internal stores, triggered by the activation of P2Y1 receptors. Inhibition by the gap junction blockers carbenoxolone and flufenamic acid and by an antibody raised against the gating-associated segment of Cx43 suggested that intercellular Ca2+ signalling proceeded through gap junctions. We demonstrated for the first time that extracellular SUC also evoked Ca2+ transients (EC50 = 50-60 μM in about 15% of the ATP-responsive NAc astrocytes. By contrast to glial cells, electrophysiologically identified NAc neurons surrounded by ATP-responsive astrocytes were not activated simultaneously. Conclusions We

  15. 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.

  16. Mechanosensitive Molecular Networks Involved in Transducing Resistance Exercise-Signals into Muscle Protein Accretion.

    Science.gov (United States)

    Rindom, Emil; Vissing, Kristian

    2016-01-01

    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 an 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 2 (TSC2)-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 implicate deformation of Notch to affect Bone Morphogenetic Protein (BMP) signaling through a small mother of decapentaplegic (Smad) axis.

  17. Mechanosensitive Molecular Networks Involved in Transducing Resistance Exercise-Signals into Muscle Protein Accretion

    Science.gov (United States)

    Rindom, Emil; Vissing, Kristian

    2016-01-01

    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 an 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 2 (TSC2)-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 implicate deformation of Notch to affect Bone Morphogenetic Protein (BMP) signaling through a small mother of decapentaplegic (Smad) axis. PMID:27909410

  18. Functional characterization of AMP-activated protein kinase signaling in tumorigenesis.

    Science.gov (United States)

    Cheng, Ji; Zhang, Tao; Ji, Hongbin; Tao, Kaixiong; Guo, Jianping; Wei, Wenyi

    2016-12-01

    AMP-activated protein kinase (AMPK) is a ubiquitously expressed metabolic sensor among various species. Specifically, cellular AMPK is phosphorylated and activated under certain stressful conditions, such as energy deprivation, in turn to activate diversified downstream substrates to modulate the adaptive changes and maintain metabolic homeostasis. Recently, emerging evidences have implicated the potential roles of AMPK signaling in tumor initiation and progression. Nevertheless, a comprehensive description on such topic is still in scarcity, especially in combination of its biochemical features with mouse modeling results to elucidate the physiological role of AMPK signaling in tumorigenesis. Hence, we performed this thorough review by summarizing the tumorigenic role of each component along the AMPK signaling, comprising of both its upstream and downstream effectors. Moreover, their functional interplay with the AMPK heterotrimer and exclusive efficacies in carcinogenesis were chiefly explained among genetically altered mice models. Importantly, the pharmaceutical investigations of AMPK relevant medications have also been highlighted. In summary, in this review, we not only elucidate the potential functions of AMPK signaling pathway in governing tumorigenesis, but also potentiate the future targeted strategy aiming for better treatment of aberrant metabolism-associated diseases, including cancer.

  19. Insulin signaling regulates fatty acid catabolism at the level of CoA activation.

    Directory of Open Access Journals (Sweden)

    Xiaojun Xu

    2012-01-01

    Full Text Available The insulin/IGF signaling pathway is a highly conserved regulator of metabolism in flies and mammals, regulating multiple physiological functions including lipid metabolism. Although insulin signaling is known to regulate the activity of a number of enzymes in metabolic pathways, a comprehensive understanding of how the insulin signaling pathway regulates metabolic pathways is still lacking. Accepted knowledge suggests the key regulated step in triglyceride (TAG catabolism is the release of fatty acids from TAG via the action of lipases. We show here that an additional, important regulated step is the activation of fatty acids for beta-oxidation via Acyl Co-A synthetases (ACS. We identify pudgy as an ACS that is transcriptionally regulated by direct FOXO action in Drosophila. Increasing or reducing pudgy expression in vivo causes a decrease or increase in organismal TAG levels respectively, indicating that pudgy expression levels are important for proper lipid homeostasis. We show that multiple ACSs are also transcriptionally regulated by insulin signaling in mammalian cells. In sum, we identify fatty acid activation onto CoA as an important, regulated step in triglyceride catabolism, and we identify a mechanistic link through which insulin regulates lipid homeostasis.

  20. Linear models of activation cascades: analytical solutions and coarse-graining of delayed signal transduction.

    Science.gov (United States)

    Beguerisse-Díaz, Mariano; Desikan, Radhika; Barahona, Mauricio

    2016-08-01

    Cellular signal transduction usually involves activation cascades, the sequential activation of a series of proteins following the reception of an input signal. Here, we study the classic model of weakly activated cascades and obtain analytical solutions for a variety of inputs. We show that in the special but important case of optimal gain cascades (i.e. when the deactivation rates are identical) the downstream output of the cascade can be represented exactly as a lumped nonlinear module containing an incomplete gamma function with real parameters that depend on the rates and length of the cascade, as well as parameters of the input signal. The expressions obtained can be applied to the non-identical case when the deactivation rates are random to capture the variability in the cascade outputs. We also show that cascades can be rearranged so that blocks with similar rates can be lumped and represented through our nonlinear modules. Our results can be used both to represent cascades in computational models of differential equations and to fit data efficiently, by reducing the number of equations and parameters involved. In particular, the length of the cascade appears as a real-valued parameter and can thus be fitted in the same manner as Hill coefficients. Finally, we show how the obtained nonlinear modules can be used instead of delay differential equations to model delays in signal transduction.

  1. MALT1-ubiquitination triggers non-genomic NF-κB/IKK signaling upon platelet activation.

    Science.gov (United States)

    Karim, Zubair A; Vemana, Hari Priya; Khasawneh, Fadi T

    2015-01-01

    We have recently shown that IKK complex plays an important non-genomic role in platelet function, i.e., regulates SNARE machinery-dependent membrane fusion. In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex. Therefore, the present studies investigated the mechanism by which IKK signaling is regulated in the context of the platelet proteasome. It was found that platelets express a functional proteasome, and form CARMA/MALT1/Bcl10 (CBM) complex when activated. Using a pharmacological inhibitor, the proteasome was found to regulate platelet function (aggregation, integrin activation, secretion, phosphatidylserine exposure and changes in intracellular calcium). It was also found to regulate thrombogenesis and physiologic hemostasis. We also observed, upon platelet activation, that MALT1 is ubiquitinated, and this coincides with the activation of the IKK/NF-κB-signaling pathway. Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex. Thus, our data demonstrate that MALT1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing platelet signals to the NF-κB pathway.

  2. MALT1-ubiquitination triggers non-genomic NF-κB/IKK signaling upon platelet activation.

    Directory of Open Access Journals (Sweden)

    Zubair A Karim

    Full Text Available We have recently shown that IKK complex plays an important non-genomic role in platelet function, i.e., regulates SNARE machinery-dependent membrane fusion. In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex. Therefore, the present studies investigated the mechanism by which IKK signaling is regulated in the context of the platelet proteasome. It was found that platelets express a functional proteasome, and form CARMA/MALT1/Bcl10 (CBM complex when activated. Using a pharmacological inhibitor, the proteasome was found to regulate platelet function (aggregation, integrin activation, secretion, phosphatidylserine exposure and changes in intracellular calcium. It was also found to regulate thrombogenesis and physiologic hemostasis. We also observed, upon platelet activation, that MALT1 is ubiquitinated, and this coincides with the activation of the IKK/NF-κB-signaling pathway. Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex. Thus, our data demonstrate that MALT1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing platelet signals to the NF-κB pathway.

  3. Stress-activated signaling responses leading to apoptosis following photodynamic therapy

    Science.gov (United States)

    Oleinick, Nancy L.; He, Jin; Xue, Liang-yan; Separovic, Duska

    1998-05-01

    Photodynamic treatment with the phthalocyanine Pc 4, a mitochondrially localizing photosensitizer, is an efficient inducer of cell death by apoptosis, a cell suicide pathway that can be triggered by physiological stimuli as well as by various types of cellular damage. Upon exposure of the dye- loaded cells to red light, several stress signalling pathways are rapidly activated. In murine L5178Y-R lymphoblasts, caspase activation and other hallmarks of the final phase of apoptosis are observed within a few minutes post-PDT. In Chinese hamster CHO-K1 cells, the first signs of apoptosis are not observed for 1 - 2 hours. The possible involvement of three parallel mitogen-activated protein kinase (MAPK) signalling pathways has been investigated. The extracellular- regulated kinases (ERK-1 and ERK-2), that are thought to promote cell growth, are not appreciably altered by PDT. However, PDT causes marked activation of the stress-activated protein kinase (SAPK) cascade in both cell types and of the p38/HOG-type kinase in CHO cells. Both of these latter pathways have been demonstrated to be associated with apoptosis. A specific inhibitor of the ERK pathway did not alter PDT-induced apoptosis; however, an inhibitor of the p38 pathway partially blocked PDT-induced apoptosis. Blockage of the SAPK pathway is being pursued by a genetic approach. It appears that the SAPK and p38 pathways may participate in signaling apoptosis in response to PDT with Pc 4.

  4. Neuronal MHC Class I Expression Is Regulated by Activity Driven Calcium Signaling.

    Directory of Open Access Journals (Sweden)

    Dan Lv

    Full Text Available MHC class I (MHC-I molecules are important components of the immune system. Recently MHC-I have been reported to also play important roles in brain development and synaptic plasticity. In this study, we examine the molecular mechanism(s underlying activity-dependent MHC-I expression using hippocampal neurons. Here we report that neuronal expression level of MHC-I is dynamically regulated during hippocampal development after birth in vivo. Kainic acid (KA treatment significantly increases the expression of MHC-I in cultured hippocampal neurons in vitro, suggesting that MHC-I expression is regulated by neuronal activity. In addition, KA stimulation decreased the expression of pre- and post-synaptic proteins. This down-regulation is prevented by addition of an MHC-I antibody to KA treated neurons. Further studies demonstrate that calcium-dependent protein kinase C (PKC is important in relaying KA simulation activation signals to up-regulated MHC-I expression. This signaling cascade relies on activation of the MAPK pathway, which leads to increased phosphorylation of CREB and NF-κB p65 while also enhancing the expression of IRF-1. Together, these results suggest that expression of MHC-I in hippocampal neurons is driven by Ca2+ regulated activation of the MAPK signaling transduction cascade.

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

    Science.gov (United States)

    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.

  6. A switch from low to high Shh activity regulates establishment of limb progenitors and signaling centers.

    Science.gov (United States)

    Zhulyn, Olena; Li, Danyi; Deimling, Steven; Vakili, Niki Alizadeh; Mo, Rong; Puviindran, Vijitha; Chen, Miao-Hsueh; Chuang, Pao-Tien; Hopyan, Sevan; Hui, Chi-chung

    2014-04-28

    The patterning and growth of the embryonic vertebrate limb is dependent on Sonic hedgehog (Shh), a morphogen that regulates the activity of Gli transcription factors. However, Shh expression is not observed during the first 12 hr of limb development. During this phase, the limb bud is prepatterned into anterior and posterior regions through the antagonistic actions of transcription factors Gli3 and Hand2. We demonstrate that precocious activation of Shh signaling during this early phase interferes with the Gli3-dependent specification of anterior progenitors, disturbing establishment of signaling centers and normal outgrowth of the limb. Our findings illustrate that limb development requires a sweet spot in the level and timing of pathway activation that allows for the Shh-dependent expansion of posterior progenitors without interfering with early prepatterning functions of Gli3/Gli3R or specification of anterior progenitors.

  7. Enhanced osteoclastogenesis by mitochondrial retrograde signaling through transcriptional activation of the cathepsin K gene

    Science.gov (United States)

    Guha, Manti; Srinivasan, Satish; Koenigstein, Alexander; Zaidi, Mone; Avadhani, Narayan G.

    2015-01-01

    Mitochondrial dysfunction has emerged as an important factor in wide ranging human pathologies. We have previously defined a retrograde signaling pathway that originates from dysfunctional mitochondria (Mt-RS) and causes a global nuclear transcriptional reprograming as its endpoint. Mitochondrial dysfunction causing disruption of mitochondrial membrane potential and consequent increase in cytosolic calcium [Ca2](c) activates calcineurin and the transcription factors NF-κB, NFAT, CREB, and C/EBPδ. In macrophages this signaling complements receptor activator of nuclear factor kappa-B ligand (RANKL)–induced osteoclastic differentiation. Here, we show that the Mt-RS activated transcriptional coactivator heterogeneous ribonucleoprotein A2 (hnRNP A2) is induced by hypoxia in murine macrophages. We demonstrate that the cathepsin K gene (Cstk), one of the key genes upregulated during osteoclast differentiation, is transcriptionally activated by Mt-RS factors. HnRNP A2 acts as a coactivator with nuclear transcription factors, cRel, and C/EBPδ for Cstk promoter activation under hypoxic conditions. Notably, our study shows that hypoxia-induced activation of the stress target factors mediates effects similar to that of RANKL with regard to Cstk activation. We therefore suggest that mitochondrial dysfunction and activation of Mt-RS, induced by various pathophysiologic conditions, is a potential risk factor for osteoclastogenesis and bone loss. PMID:25800988

  8. Enhanced osteoclastogenesis by mitochondrial retrograde signaling through transcriptional activation of the cathepsin K gene.

    Science.gov (United States)

    Guha, Manti; Srinivasan, Satish; Koenigstein, Alexander; Zaidi, Mone; Avadhani, Narayan G

    2016-01-01

    Mitochondrial dysfunction has emerged as an important factor in wide ranging human pathologies. We have previously defined a retrograde signaling pathway that originates from dysfunctional mitochondria (Mt-RS) and causes a global nuclear transcriptional reprograming as its end point. Mitochondrial dysfunction causing disruption of mitochondrial membrane potential and consequent increase in cytosolic calcium [Ca(2) ](c) activates calcineurin and the transcription factors NF-κB, NFAT, CREB, and C/EBPδ. In macrophages, this signaling complements receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastic differentiation. Here, we show that the Mt-RS activated transcriptional coactivator heterogeneous ribonucleoprotein A2 (hnRNP A2) is induced by hypoxia in murine macrophages. We demonstrate that the cathepsin K gene (Ctsk), one of the key genes upregulated during osteoclast differentiation, is transcriptionally activated by Mt-RS factors. HnRNP A2 acts as a coactivator with nuclear transcription factors, cRel, and C/EBPδ for Ctsk promoter activation under hypoxic conditions. Notably, our study shows that hypoxia-induced activation of the stress target factors mediates effects similar to that of RANKL with regard to Ctsk activation. We therefore suggest that mitochondrial dysfunction and activation of Mt-RS, induced by various pathophysiologic conditions, is a potential risk factor for osteoclastogenesis and bone loss.

  9. Activation and interruption of the reproduction of Varroa destructor is triggered by host signals (Apis mellifera).

    Science.gov (United States)

    Frey, Eva; Odemer, Richard; Blum, Thomas; Rosenkranz, Peter

    2013-05-01

    The reproductive cycle of the parasitic mite Varroa destructor is closely linked to the development of the honey bee host larvae. Using a within colony approach we introduced phoretic Varroa females into brood cells of different age in order to analyze the capacity of certain stages of the honey bee larva to either activate or interrupt the reproduction of Varroa females. Only larvae within 18 h (worker) and 36 h (drones), respectively, after cell capping were able to stimulate the mite's oogenesis. Therewith we could specify for the first time the short time window where honey bee larvae provide the signals for the activation of the Varroa reproduction. Stage specific volatiles of the larval cuticle are at least part of these activation signals. This is confirmed by the successful stimulation of presumably non-reproducing mites to oviposition by the application of a larval extract into the sealed brood cells. According to preliminary quantitative GC-MS analysis we suggest certain fatty acid ethyl esters as candidate compounds. If Varroa females that have just started with egg formation are transferred to brood cells containing host larvae of an elder stage two-thirds of these mites stopped their oogenesis. This confirms the presence of an additional signal in the host larvae allowing the reproducing mites to adjust their own reproductive cycle to the ontogenetic development of the host. From an adaptive point of view that sort of a stop signal enables the female mite to save resources for a next reproductive cycle if the own egg development is not sufficiently synchronized with the development of the host. The results presented here offer the opportunity to analyze exactly those host stages that have the capacity to activate or interrupt the Varroa reproduction in order to identify the crucial host signals.

  10. Activity-dependent survival of developing neocortical neurons depends on PI3K signalling.

    Science.gov (United States)

    Wagner-Golbs, Antje; Luhmann, Heiko J

    2012-02-01

    Spontaneous electrical network activity plays a major role in the control of cell survival in the developing brain. Several intracellular pathways are implicated in transducing electrical activity into gene expression dependent and independent survival signals. These include activation of phosphatidylinositol 3-kinase (PI3K) and its downstream effector Akt, activation of Ras and subsequently MAPK/extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase and signalling via calcium/calmodulin-dependent protein kinase (CaMK). In the present study, we analyzed the role of these pathways for the control of neuronal survival in different extracellular potassium concentrations ([K(+) ](ex) ). Organotypic neocortical slice cultures prepared from newborn mice were kept in 5.3, 8.0 and 25.0mM [K(+) ](ex) and treated with specific inhibitors of PI3K, MEK1, CaMKK and a broad spectrum CaMK inhibitor. After 6h of incubation, slices were immunostained for activated caspase 3 (a-caspase 3) and the number of apoptotic cells was quantified by computer based analysis. We found that in 5.3 and 8.0mM [K(+) ](ex) only PI3K was important for neuronal survival. When [K(+) ](ex) was raised to 25.0mM, a concentration above the depolarization block, we found no influence of PI3K on neuronal survival. Our data demonstrate that only the PI3K pathway, and not the MEK1, CaMKK or CaMKs pathway, plays a central role in the regulation of activity-dependent neuronal survival in the developing cerebral cortex.

  11. Potato signal molecules that activate pectate lyase synthesis in Pectobacterium atrosepticum SCRI1043.

    Science.gov (United States)

    Tarasova, Nadezhda; Gorshkov, Vladimir; Petrova, Olga; Gogolev, Yuri

    2013-07-01

    A new type of plant-derived signal molecules that activate extracellular pectate lyase activity in phytopathogenic bacterium Pectobacterium atrosepticum SCRI1043 was revealed. These compounds were characterized and partially purified by means of several approaches including RT-PCR analysis, luminescence bioassay and HPLC fractionation. They were smaller than 1 kDa, thermoresistant, nonproteinaceous, hydrophilic, and slightly negatively charged molecules. Using gene expression analysis and bacterial biosensor assay the mode of activity of revealed compounds was studied. The possibility of their action through quorum sensing- and KdgR-mediated pathways was analyzed.

  12. Activation of extracellular signal-regulated kinase but not of p38 mitogen-activated protein kinase pathways in lymphocytes requires allosteric activation of SOS.

    Science.gov (United States)

    Jun, Jesse E; Yang, Ming; Chen, Hang; Chakraborty, Arup K; Roose, Jeroen P

    2013-06-01

    Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation.

  13. The Effects of the Active Hypoxia to the Speech Signal Inharmonicity

    Directory of Open Access Journals (Sweden)

    Z. N. Milivojevic

    2014-06-01

    Full Text Available When the people are climbing on the mountain, they are exposed to decreased oxygen concentration in the tissue, which is commonly called the active hypoxia. This paper addressed the problem of an acute hypoxia that affects the speech signal at the altitude up to 2500 m. For the experiment, the speech signal database that contains the articulation of vowels was recorded at different alti¬tudes. This speech signal was processed by the originally developed algorithm, which extracted the fundamental frequency and the inharmonicity coefficient. Then, they were subjected to the analysis in order to derive the effects of the acute hypoxia. The results showed that the hypoxia level can be determined by the change of the inharmonicity coefficient. Accordingly, the degree of hypoxia can be estimated.

  14. Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse.

    Science.gov (United States)

    Soares, Helena; Henriques, Ricardo; Sachse, Martin; Ventimiglia, Leandro; Alonso, Miguel A; Zimmer, Christophe; Thoulouze, Maria-Isabel; Alcover, Andrés

    2013-10-21

    How the vesicular traffic of signaling molecules contributes to T cell receptor (TCR) signal transduction at the immunological synapse remains poorly understood. In this study, we show that the protein tyrosine kinase Lck, the TCRζ subunit, and the adapter LAT traffic through distinct exocytic compartments, which are released at the immunological synapse in a differentially regulated manner. Lck vesicular release depends on MAL protein. Synaptic Lck, in turn, conditions the calcium- and synaptotagmin-7-dependent fusion of LAT and TCRζ containing vesicles. Fusion of vesicles containing TCRζ and LAT at the synaptic membrane determines not only the nanoscale organization of phosphorylated TCRζ, ZAP70, LAT, and SLP76 clusters but also the presence of phosphorylated LAT and SLP76 in interacting signaling nanoterritories. This mechanism is required for priming IL-2 and IFN-γ production and may contribute to fine-tuning T cell activation breadth in response to different stimulatory conditions.

  15. Putamen Activation Represents an Intrinsic Positive Prediction Error Signal for Visual Search in Repeated Configurations.

    Science.gov (United States)

    Sommer, Susanne; Pollmann, Stefan

    2016-01-01

    We investigated fMRI responses to visual search targets appearing at locations that were predicted by the search context. Based on previous work in visual category learning we expected an intrinsic reward prediction error signal in the putamen whenever the target appeared at a location that was predicted with some degree of uncertainty. Comparing target appearance at locations predicted with 50% probability to either locations predicted with 100% probability or unpredicted locations, increased activation was observed in left posterior putamen and adjacent left posterior insula. Thus, our hypothesis of an intrinsic prediction error-like signal was confirmed. This extends the observation of intrinsic prediction error-like signals, driven by intrinsic rather than extrinsic reward, to memory-driven visual search.

  16. Neuropeptidergic Signaling and Active Feeding State Inhibit Nociception in Caenorhabditis elegans.

    Science.gov (United States)

    Ezcurra, Marina; Walker, Denise S; Beets, Isabel; Swoboda, Peter; Schafer, William R

    2016-03-16

    Food availability and nutritional status are important cues affecting behavioral states. Here we report that, in Caenorhabditis elegans, a cascade of dopamine and neuropeptide signaling acts to inhibit nociception in food-poor environments. In the absence of food, animals show decreased sensitivity and increased adaptation to soluble repellents sensed by the polymodal ASH nociceptors. The effects of food on adaptation are affected by dopamine and neuropeptide signaling; dopamine acts via the DOP-1 receptor to decrease adaptation on food, whereas the neuropeptide receptors NPR-1 and NPR-2 act to increase adaptation off food. NPR-1 and NPR-2 function cell autonomously in the ASH neurons to increase adaptation off food, whereas the DOP-1 receptor controls neuropeptide release from interneurons that modulate ASH activity indirectly. These results indicate that feeding state modulates nociception through the interaction of monoamine and neuropeptide signaling pathways.

  17. Neuropeptidergic Signaling and Active Feeding State Inhibit Nociception in Caenorhabditis elegans

    Science.gov (United States)

    Ezcurra, Marina; Walker, Denise S.; Beets, Isabel; Swoboda, Peter

    2016-01-01

    Food availability and nutritional status are important cues affecting behavioral states. Here we report that, in Caenorhabditis elegans, a cascade of dopamine and neuropeptide signaling acts to inhibit nociception in food-poor environments. In the absence of food, animals show decreased sensitivity and increased adaptation to soluble repellents sensed by the polymodal ASH nociceptors. The effects of food on adaptation are affected by dopamine and neuropeptide signaling; dopamine acts via the DOP-1 receptor to decrease adaptation on food, whereas the neuropeptide receptors NPR-1 and NPR-2 act to increase adaptation off food. NPR-1 and NPR-2 function cell autonomously in the ASH neurons to increase adaptation off food, whereas the DOP-1 receptor controls neuropeptide release from interneurons that modulate ASH activity indirectly. These results indicate that feeding state modulates nociception through the interaction of monoamine and neuropeptide signaling pathways. PMID:26985027

  18. Measuring Acoustic Wave Transit Time in Furnace Based on Active Acoustic Source Signal

    Institute of Scientific and Technical Information of China (English)

    Zhen Luo; Feng Tian; Xiao-Ping Sun

    2007-01-01

    Accurate measurement of transit time for acoustic wave between two sensors installed on two sides of a furnace is a key to implementing the temperature field measurement technique based on acoustical method. A new method for measuring transit time of acoustic wave based on active acoustic source signal is proposed in this paper, which includes the followings: the time when the acoustic source signal arrives at the two sensors is measured first; then, the difference of two arriving time arguments is computed, thereby we get the transit time of the acoustic wave between two sensors installed on the two sides of the furnace. Avoiding the restriction on acoustic source signal and background noise, the new method can get the transit time of acoustic wave with higher precision and stronger ability of resisting noise interference.

  19. Putamen Activation Represents an Intrinsic Positive Prediction Error Signal for Visual Search in Repeated Configurations

    Science.gov (United States)

    Sommer, Susanne; Pollmann, Stefan

    2016-01-01

    We investigated fMRI responses to visual search targets appearing at locations that were predicted by the search context. Based on previous work in visual category learning we expected an intrinsic reward prediction error signal in the putamen whenever the target appeared at a location that was predicted with some degree of uncertainty. Comparing target appearance at locations predicted with 50% probability to either locations predicted with 100% probability or unpredicted locations, increased activation was observed in left posterior putamen and adjacent left posterior insula. Thus, our hypothesis of an intrinsic prediction error-like signal was confirmed. This extends the observation of intrinsic prediction error-like signals, driven by intrinsic rather than extrinsic reward, to memory-driven visual search. PMID:27867436

  20. Activation of Signal Transducer and Activator of Transcription 5 (STAT5) in Splenocyte Proliferation of Asthma Mice Induced by Ovalbumin

    Institute of Scientific and Technical Information of China (English)

    Guoping Li; Zhigang Liu; Peixing Ran; Jing Qiu; Nanshan Zhong

    2004-01-01

    To investigate the role of signal transducer and transcriptional activator 5 (STAT5) activated in ovalbumin (OVA)-induced splenocyte proliferation of asthma mice, an asthma mouse model was set up by intraperitoneal injection and aspiration of OVA with nebulizer. The proliferation of splenocytes isolated from the asthma mice was detected by [3H] thymidine incorporation. The phosphorytation of STAT5 was examined by Western blotting and STAT5-DNA binding was measured by electrophoretic mobility shift assay (EMSA). OVA could pronouncedly induce the splenocyte proliferation of asthma mice in a dose-dependent manner compared with control groups. Phosphorylation of STAT5 and STAT5-DNA binding were observed in splenocytes from asthma mice induced by OVA at 1 h and 3 h. These results indicated that STAT5 signal pathway played an important role in lymphocyte proliferation of asthma mice induced by OVA. Cellular & Molecular Immunology.2004;1(6):471-474.

  1. Anti-cancer activity of an osthole derivative, NBM-T-BMX-OS01: targeting vascular endothelial growth factor receptor signaling and angiogenesis.

    Directory of Open Access Journals (Sweden)

    Hung-Yu Yang

    Full Text Available Angiogenesis occurs during tissue growth, development and wound healing. It is also required for tumor progression and represents a rational target for therapeutic intervention. NBM-T-BMX-OS01 (BMX, derived from the semisynthesis of osthole, an active ingredient isolated from Chinese herb Cnidium monnieri (L. Cuss., was recently shown to enhance learning and memory in rats. In this study, we characterized the anti-angiogenic activities of NBM-T-BMX-OS01 (BMX in an effort to develop novel inhibitors to suppress angiogenesis and tumor growth. BMX inhibited vascular endothelial growth factor (VEGF-induced proliferation, migration and endothelial tube formation in human umbilical endothelial cells (HUVECs. BMX also attenuated VEGF-induced microvessel sprouting from aortic rings ex vivo and reduced HCT116 colorectal cancer cells-induced angiogenesis in vivo. Moreover, BMX inhibited the phosphorylation of VEGFR2, FAK, Akt and ERK in HUVECs exposed to VEGF. BMX was also shown to inhibit HCT116 cell proliferation and to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. Taken together, this study provides evidence that BMX modulates vascular endothelial cell remodeling and leads to the inhibition of tumor angiogenesis. These results also support the role of BMX as a potential drug candidate and warrant the clinical development in the treatment of cancer.

  2. Anti-cancer activity of an osthole derivative, NBM-T-BMX-OS01: targeting vascular endothelial growth factor receptor signaling and angiogenesis.

    Science.gov (United States)

    Yang, Hung-Yu; Hsu, Ya-Fen; Chiu, Pei-Ting; Ho, Shiau-Jing; Wang, Chi-Han; Chi, Chih-Chin; Huang, Yu-Han; Lee, Cheng-Feng; Li, Ying-Shiuan; Ou, George; Hsu, Ming-Jen

    2013-01-01

    Angiogenesis occurs during tissue growth, development and wound healing. It is also required for tumor progression and represents a rational target for therapeutic intervention. NBM-T-BMX-OS01 (BMX), derived from the semisynthesis of osthole, an active ingredient isolated from Chinese herb Cnidium monnieri (L.) Cuss., was recently shown to enhance learning and memory in rats. In this study, we characterized the anti-angiogenic activities of NBM-T-BMX-OS01 (BMX) in an effort to develop novel inhibitors to suppress angiogenesis and tumor growth. BMX inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and endothelial tube formation in human umbilical endothelial cells (HUVECs). BMX also attenuated VEGF-induced microvessel sprouting from aortic rings ex vivo and reduced HCT116 colorectal cancer cells-induced angiogenesis in vivo. Moreover, BMX inhibited the phosphorylation of VEGFR2, FAK, Akt and ERK in HUVECs exposed to VEGF. BMX was also shown to inhibit HCT116 cell proliferation and to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. Taken together, this study provides evidence that BMX modulates vascular endothelial cell remodeling and leads to the inhibition of tumor angiogenesis. These results also support the role of BMX as a potential drug candidate and warrant the clinical development in the treatment of cancer.

  3. CD147 promotes Src-dependent activation of Rac1 signaling through STAT3/DOCK8 during the motility of hepatocellular carcinoma cells.

    Science.gov (United States)

    Wang, Shi-Jie; Cui, Hong-Yong; Liu, Yan-Mei; Zhao, Pu; Zhang, Yang; Fu, Zhi-Guang; Chen, Zhi-Nan; Jiang, Jian-Li

    2015-01-01

    Metastasis is considered a dynamic process in tumor development that is related to abnormal migration and invasion. Tumor cells can move as individual cells in two interconvertible modes: mesenchymal-type and amoeboid. Previously, we reported that the interaction between CD147 and Annexin II can inhibit the amoeboid movement in hepatocellular carcinoma (HCC) cells. However, the mechanism of CD147 involved in mesenchymal movement is still unclear. Notably, our results show overexpression of CD147 led to mesenchymal-type movement in HCC cells. Evidence indicated that the mesenchymal-type cell movement induced by CD147 was Src dependent, as observed by confocal microscopy and Rac1 activity assay. The phosphorylation of Src (pY416-Src) can be up-regulated by CD147, and this regulation is mediated by focal adhesion kinase (FAK). Next, we identified DOCK8 as a GEF for Rac1, a key molecule driving mesenchymal-type movement. We also found that Src promotes STAT3 phosphorylation and STAT3 facilitates DOCK8 transcription, thus enhancing DOCK8 expression and Rac1 activation. This study provides a novel mechanism of CD147 regulating mesenchymal-type movement in HCC cells.

  4. Cholesterol selectively regulates IL-5 induced mitogen activated protein kinase signaling in human eosinophils.

    Directory of Open Access Journals (Sweden)

    Mandy E Burnham

    Full Text Available Eosinophils function contributes to human allergic and autoimmune diseases, many of which currently lack curative treatment. Development of more effective treatments for eosinophil-related diseases requires expanded understanding of eosinophil signaling and biology. Cell signaling requires integration of extracellular signals with intracellular responses, and is organized in part by cholesterol rich membrane microdomains (CRMMs, commonly referred to as lipid rafts. Formation of these organizational membrane domains is in turn dependent upon the amount of available cholesterol, which can fluctuate widely with a variety of disease states. We tested the hypothesis that manipulating membrane cholesterol content in primary human peripheral blood eosinophils (PBEos would selectively alter signaling pathways that depend upon membrane-anchored signaling proteins localized within CRMMs (e.g., mitogen activated protein kinase [MAPK] pathway, while not affecting pathways that signal through soluble proteins, like the Janus Kinase/Signal Transducer and Activator of Transcription [JAK/STAT] pathway. Cholesterol levels were increased or decreased utilizing cholesterol-chelating methyl-β-cyclodextrin (MβCD, which can either extract membrane cholesterol or add exogenous membrane cholesterol depending on whether MβCD is preloaded with cholesterol. Human PBEos were pretreated with MβCD (cholesterol removal or MβCD+Cholesterol (MβCD+Chol; cholesterol delivery; subsequent IL-5-stimulated signaling and physiological endpoints were assessed. MβCD reduced membrane cholesterol in PBEos, and attenuated an IL-5-stimulated p38 and extracellular-regulated kinase 1/2 phosphorylation (p-p38, p-ERK1/2, and an IL-5-dependent increase in interleukin-1β (IL-1β mRNA levels. In contrast, MβCD+Chol treatment elevated PBEos membrane cholesterol levels and basal p-p38, but did not alter IL-5-stimulated phosphorylation of ERK1/2, STAT5, or STAT3. Furthermore, M

  5. Chloroquine inhibits human CD4+ T-cell activation by AP-1 signaling modulation

    Science.gov (United States)

    Schmidt, Ralf L. J.; Jutz, Sabrina; Goldhahn, Katrin; Witzeneder, Nadine; Gerner, Marlene C.; Trapin, Doris; Greiner, Georg; Hoermann, Gregor; Steiner, Guenter; Pickl, Winfried F.; Burgmann, Heinz; Steinberger, Peter; Ratzinger, Franz; Schmetterer, Klaus G.

    2017-01-01

    Chloroquine (CQ) is widely used as an anti-inflammatory therapeutic for rheumatic diseases. Although its modes of action on the innate immune system are well described, there is still insufficient knowledge about its direct effects on the adaptive immune system. Thus, we evaluated the influence of CQ on activation parameters of human CD4+ T-cells. CQ directly suppressed proliferation, metabolic activity and cytokine secretion of T-cells following anti-CD3/anti-CD28 activation. In contrast, CQ showed no effect on up-regulation of T-cell activation markers. CQ inhibited activation of all T helper cell subsets, although IL-4 and IL-13 secretion by Th2 cells were less influenced compared to other Th-specific cytokines. Up to 10 μM, CQ did not reduce cell viability, suggesting specific suppressive effects on T-cells. These properties of CQ were fully reversible in re-stimulation experiments. Analyses of intracellular signaling showed that CQ specifically inhibited autophagic flux and additionally activation of AP-1 by reducing phosphorylation of c-JUN. This effect was mediated by inhibition of JNK catalytic activity. In summary, we characterized selective and reversible immunomodulatory effects of CQ on human CD4+ T-cells. These findings provide new insights into the biological actions of JNK/AP-1 signaling in T-cells and may help to expand the therapeutic spectrum of CQ. PMID:28169350

  6. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Satoru, E-mail: smatsuda@cc.nara-wu.ac.jp; Kitagishi, Yasuko [Department of Food Science and Nutrition, Nara Women’s University, Kita-Uoya Nishimachi, Nara 630-8506 (Japan)

    2013-10-21

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  7. Contributions of chaperone and glycosyltransferase activities of O-fucosyltransferase 1 to Notch signaling

    Directory of Open Access Journals (Sweden)

    Irvine Kenneth D

    2008-01-01

    Full Text Available Abstract Background O-fucosyltransferase1 (OFUT1 is a conserved ER protein essential for Notch signaling. OFUT1 glycosylates EGF domains, which can then be further modified by the N-acetylglucosaminyltransferase Fringe. OFUT1 also possesses a chaperone activity that promotes the folding and secretion of Notch. Here, we investigate the respective contributions of these activities to Notch signaling in Drosophila. Results We show that expression of an isoform lacking fucosyltransferase activity, Ofut1R245A, rescues the requirement for Ofut1 in embryonic neurogenesis. Lack of requirement for O-fucosylation is further supported by the absence of embryonic phenotypes in Gmd mutants, which lack all forms of fucosylation. Requirements for O-fucose during imaginal development were evaluated by characterizing clones of cells expressing only Ofut1R245A. These clones phenocopy fringe mutant clones, indicating that the absence of O-fucose is functionally equivalent to the absence of elongated O-fucose. Conclusion Our results establish that Notch does not need to be O-fucosylated for fringe-independent Notch signaling in Drosophila; the chaperone activity of OFUT1 is sufficient for the generation of functional Notch.

  8. Notch Signaling Is Associated With ALDH Activity And An Aggressive Metastatic Phenotype In Murine Osteosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Xiaodong eMu

    2013-06-01

    Full Text Available Osteosarcoma (OS is the most common primary malignancy of bone, and pulmonary metastatic disease accounts for nearly all mortality. However, little is known about the biochemical signaling alterations that drive the progression of metastatic disease. Two murine OS cell populations, K7M2 and K12, are clonally related but differ significantly in their metastatic phenotypes and therefore represent excellent tools for studying metastatic OS molecular biology. K7M2 cells are highly metastatic, whereas K12 cells display limited metastatic potential. Here we report that the expression of Notch genes (Notch1, 2, 4 are up-regulated, including downstream targets Hes1 and Stat3, in the highly metastatic K7M2 cells compared to the less metastatic K12 cells, indicating that the Notch signaling pathway is more active in K7M2 cells. We have previously described that K7M2 cells exhibit higher levels of aldehyde dehydrogenase (ALDH activity. Here we report that K7M2 cell ALDH activity is reduced with Notch inhibition, suggesting that ALDH activity may be regulated in part by the Notch pathway. Notch signaling is also associated with increased resistance to oxidative stress, migration, invasion, and VEGF expression in vitro. However, Notch inhibition did not significantly alter K7M2 cell proliferation. In conclusion, we provide evidence that Notch signaling is associated with ALDH activity and increased metastatic behavior in OS cells. Both Notch and ALDH are putative molecular targets for the treatment and prevention of OS metastasis.

  9. Isoorientin reverts TNF-α-induced insulin resistance in adipocytes activating the insulin signaling pathway.

    Science.gov (United States)

    Alonso-Castro, Angel Josabad; Zapata-Bustos, Rocio; Gómez-Espinoza, Guadalupe; Salazar-Olivo, Luis A

    2012-11-01

    Isoorientin (ISO) is a plant C-glycosylflavonoid with purported antidiabetic effects but unexplored mechanisms of action. To gain insight into its antidiabetic mechanisms, we assayed nontoxic ISO concentrations on the 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxy-d-glucose (2-NBDG) uptake by murine 3T3-F442A and human sc adipocytes. In insulin-sensitive adipocytes, ISO stimulated the 2-NBDG uptake by 210% (murine) and 67% (human), compared with insulin treatment. Notably, ISO also induced 2-NBDG uptake in murine (139%) and human (60%) adipocytes made resistant to insulin by treatment with TNF-α, compared with the incorporation induced in these cells by rosiglitazone. ISO induction of glucose uptake in adipocytes was abolished by inhibitors of the insulin signaling pathway. These inhibitors also blocked the proper phosphorylation of insulin signaling pathway components induced by ISO in both insulin-sensitive and insulin-resistant adipocytes. Additionally, ISO stimulated the transcription of genes encoding components of insulin signaling pathway in murine insulin-sensitive and insulin-resistant adipocytes. In summary, we show here that ISO exerts its antidiabetic effects by activating the insulin signaling pathway in adipocytes, reverts the insulin resistance caused in these cells by TNF-α by stimulating the proper phosphorylation of proteins in this signaling pathway, and induces the expression of genes encoding these proteins.

  10. Redox Signaling as a Therapeutic Target to Inhibit Myofibroblast Activation in Degenerative Fibrotic Disease

    Directory of Open Access Journals (Sweden)

    Natalie Sampson

    2014-01-01

    Full Text Available Degenerative fibrotic diseases encompass numerous systemic and organ-specific disorders. Despite their associated significant morbidity and mortality, there is currently no effective antifibrotic treatment. Fibrosis is characterized by the development and persistence of myofibroblasts, whose unregulated deposition of extracellular matrix components disrupts signaling cascades and normal tissue architecture leading to organ failure and death. The profibrotic cytokine transforming growth factor beta (TGFβ is considered the foremost inducer of fibrosis, driving myofibroblast differentiation in diverse tissues. This review summarizes recent in vitro and in vivo data demonstrating that TGFβ-induced myofibroblast differentiation is driven by a prooxidant shift in redox homeostasis. Elevated NADPH oxidase 4 (NOX4-derived hydrogen peroxide (H2O2 supported by concomitant decreases in nitric oxide (NO signaling and reactive oxygen species scavengers are central factors in the molecular pathogenesis of fibrosis in numerous tissues and organs. Moreover, complex interplay between NOX4-derived H2O2 and NO signaling regulates myofibroblast differentiation. Restoring redox homeostasis via antioxidants or NOX4 inactivation as well as by enhancing NO signaling via activation of soluble guanylyl cyclases or inhibition of phosphodiesterases can inhibit and reverse myofibroblast differentiation. Thus, dysregulated redox signaling represents a potential therapeutic target for the treatment of wide variety of different degenerative fibrotic disorders.

  11. Chronic baclofen desensitizes GABA(B)-mediated G-protein activation and stimulates phosphorylation of kinases in mesocorticolimbic rat brain.

    Science.gov (United States)

    Keegan, Bradley M T; Beveridge, Thomas J R; Pezor, Jeffrey J; Xiao, Ruoyu; Sexton, Tammy; Childers, Steven R; Howlett, Allyn C

    2015-08-01

    The GABAB receptor is a therapeutic target for CNS and neuropathic disorders; however, few preclinical studies have explored effects of chronic stimulation. This study evaluated acute and chronic baclofen treatments on GABAB-activated G-proteins and signaling protein phosphorylation as indicators of GABAB signaling capacity. Brain sections from rats acutely administered baclofen (5 mg/kg, i.p.) showed no significant differences from controls in GABAB-stimulated GTPγS binding in any brain region, but displayed significantly greater phosphorylation/activation of focal adhesion kinase (pFAK(Tyr397)) in mesocorticolimbic regions (caudate putamen, cortex, hippocampus, thalamus) and elevated phosphorylated/activated glycogen synthase kinase 3-β (pGSK3β(Tyr216)) in the prefrontal cortex, cerebral cortex, caudate putamen, nucleus accumbens, thalamus, septum, and globus pallidus. In rats administered chronic baclofen (5 mg/kg, t.i.d. for five days), GABAB-stimulated GTPγS binding was significantly diminished in the prefrontal cortex, septum, amygdala, and parabrachial nucleus compared to controls. This effect was specific to GABAB receptors: there was no effect of chronic baclofen treatment on adenosine A1-stimulated GTPγS binding in any region. Chronically-treated rats also exhibited increases in pFAK(Tyr397) and pGSK3β(Tyr216) compared to controls, and displayed wide-spread elevations in phosphorylated dopamine- and cAMP-regulated phosphoprotein-32 (pDARPP-32(Thr34)) compared to acutely-treated or control rats. We postulate that those neuroadaptive effects of GABAB stimulation mediated by G-proteins and their sequelae correlate with tolerance to several of baclofen's effects, whereas sustained signaling via kinase cascades points to cross-talk between GABAB receptors and alternative mechanisms that are resistant to desensitization. Both desensitized and sustained signaling pathways should be considered in the development of pharmacotherapies targeting the GABA

  12. Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice.

    Science.gov (United States)

    Liang, Hai Po H; Kerschen, Edward J; Basu, Sreemanti; Hernandez, Irene; Zogg, Mark; Jia, Shuang; Hessner, Martin J; Toso, Raffaella; Rezaie, Alireza R; Fernández, José A; Camire, Rodney M; Ruf, Wolfram; Griffin, John H; Weiler, Hartmut

    2015-11-19

    The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection.

  13. Polysaccharides Purified from Wild Cordyceps Activate FGF2/FGFR1c Signaling

    Institute of Scientific and Technical Information of China (English)

    ZENG Yangyang; HAN Zhangrun; YU Guangli; HAO Jiejie; ZHANG Lijuan

    2015-01-01

    Land animals as well as all organisms in ocean synthesize sulfated polysaccharides. Fungi split from animals about 1.5 billion years ago. As fungi make the evolutionary journey from ocean to land, the biggest changes in their living environment may be a sharp decrease in salt concentration. It is established that sulfated polysaccharides interact with hundreds of signaling molecules and facilitate many signaling transduction pathways, including fibroblast growth factor (FGF) and FGF receptor signaling pathway. The disappearance of sulfated polysaccharides in fungi and plants on land might indicate that polysaccharides without sulfation might be sufficient in facilitating protein ligand/receptor interactions in low salinity land. Recently, it was reported that plants on land start to synthesize sulfated polysaccharides in high salt environment, suggesting that fungi might be able to do the same when ex-posed in such environment. Interestingly, Cordyceps, a fungus habituating inside caterpillar body, is the most valued traditional Chi-nese Medicine. One of the important pharmaceutical active ingredients in Cordyceps is polysaccharides. Therefore, we hypothesize that the salty environment inside caterpillar body might allow the fungi to synthesize sulfated polysaccharides. To test the hypothesis, we isolated polysaccharides from both lava and sporophore of wild Cordyceps and also fromCordyceps militaris cultured without or with added salts. We then measured the polysaccharide activity using a FGF2/FGFR1c signaling-dependent BaF3 cell proliferation assay and found that polysaccharides isolated from wild Cordyceps activated FGF2/FGFR signaling, indicating that the polysaccha-rides synthesized by wild Cordyceps are indeed different from those by the cultured mycelium.

  14. Polysaccharides purified from wild Cordyceps activate FGF2/FGFR1c signaling

    Science.gov (United States)

    Zeng, Yangyang; Han, Zhangrun; Yu, Guangli; Hao, Jiejie; Zhang, Lijuan

    2015-02-01

    Land animals as well as all organisms in ocean synthesize sulfated polysaccharides. Fungi split from animals about 1.5 billion years ago. As fungi make the evolutionary journey from ocean to land, the biggest changes in their living environment may be a sharp decrease in salt concentration. It is established that sulfated polysaccharides interact with hundreds of signaling molecules and facilitate many signaling transduction pathways, including fibroblast growth factor (FGF) and FGF receptor signaling pathway. The disappearance of sulfated polysaccharides in fungi and plants on land might indicate that polysaccharides without sulfation might be sufficient in facilitating protein ligand/receptor interactions in low salinity land. Recently, it was reported that plants on land start to synthesize sulfated polysaccharides in high salt environment, suggesting that fungi might be able to do the same when exposed in such environment. Interestingly, Cordyceps, a fungus habituating inside caterpillar body, is the most valued traditional Chinese Medicine. One of the important pharmaceutical active ingredients in Cordyceps is polysaccharides. Therefore, we hypothesize that the salty environment inside caterpillar body might allow the fungi to synthesize sulfated polysaccharides. To test the hypothesis, we isolated polysaccharides from both lava and sporophore of wild Cordyceps and also from Cordyceps militaris cultured without or with added salts. We then measured the polysaccharide activity using a FGF2/FGFR1c signaling-dependent BaF3 cell proliferation assay and found that polysaccharides isolated from wild Cordyceps activated FGF2/FGFR signaling, indicating that the polysaccharides synthesized by wild Cordyceps are indeed different from those by the cultured mycelium.

  15. Bovine lactoferrin counteracts Toll-like receptor mediated activation signals in antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Patrizia Puddu

    Full Text Available Lactoferrin (LF, a key element in mammalian immune system, plays pivotal roles in host defence against infection and excessive inflammation. Its protective effects range from direct antimicrobial activities against a large panel of microbes, including bacteria, viruses, fungi and parasites, to antinflammatory and anticancer activities. In this study, we show that monocyte-derived dendritic cells (MD-DCs generated in the presence of bovine LF (bLF fail to undergo activation by up-modulating CD83, co-stimulatory and major histocompatibility complex molecules, and cytokine/chemokine secretion. Moreover, these cells are weak activators of T cell proliferation and retain antigen uptake activity. Consistent with an impaired maturation, bLF-MD-DC primed T lymphocytes exhibit a functional unresponsiveness characterized by reduced expression of CD154 and impaired expression of IFN-γ and IL-2. The observed imunosuppressive effects correlate with an increased expression of molecules with negative regulatory functions (i.e. immunoglobulin-like transcript 3 and programmed death ligand 1, indoleamine 2,3-dioxygenase, and suppressor of cytokine signaling-3. Interestingly, bLF-MD-DCs produce IL-6 and exhibit constitutive signal transducer and activator of transcription 3 activation. Conversely, bLF exposure of already differentiated MD-DCs completely fails to induce IL-6, and partially inhibits Toll-like receptor (TLR agonist-induced activation. Cell-specific differences in bLF internalization likely account for the distinct response elicited by bLF in monocytes versus immature DCs, providing a mechanistic base for its multiple effects. These results indicate that bLF exerts a potent anti-inflammatory activity by skewing monocyte differentiation into DCs with impaired capacity to undergo activation and to promote Th1 responses. Overall, these bLF-mediated effects may represent a strategy to block excessive DC activation upon TLR-induced inflammation, adding

  16. SLIT2/ROBO2 signaling pathway inhibits nonmuscle myosin IIA activity and destabilizes kidney podocyte adhesion

    Science.gov (United States)

    Fan, Xueping; Yang, Hongying; Kumar, Sudhir; Tumelty, Kathleen E.; Pisarek-Horowitz, Anna; Sharma, Richa; Chan, Stefanie; Tyminski, Edyta; Shamashkin, Michael; Belghasem, Mostafa; Henderson, Joel M.; Coyle, Anthony J.; Berasi, Stephen P.

    2016-01-01

    The repulsive guidance cue SLIT2 and its receptor ROBO2 are required for kidney development and podocyte foot process structure, but the SLIT2/ROBO2 signaling mechanism regulating podocyte function is not known. Here we report that a potentially novel signaling pathway consisting of SLIT/ROBO Rho GTPase activating protein 1 (SRGAP1) and nonmuscle myosin IIA (NMIIA) regulates podocyte adhesion downstream of ROBO2. We found that the myosin II regulatory light chain (MRLC), a subunit of NMIIA, interacts directly with SRGAP1 and forms a complex with ROBO2/SRGAP1/NMIIA in the presence of SLIT2. Immunostaining demonstrated that SRGAP1 is a podocyte protein and is colocalized with ROBO2 on the basal surface of podocytes. In addition, SLIT2 stimulation inhibits NMIIA activity, decreases focal adhesion formation, and reduces podocyte attachment to collagen. In vivo studies further showed that podocyte-specific knockout of Robo2 protects mice from hypertension-induced podocyte detachment and albuminuria and also partially rescues the podocyte-loss phenotype in Myh9 knockout mice. Thus, we have identified SLIT2/ROBO2/SRGAP1/NMIIA as a potentially novel signaling pathway in kidney podocytes, which may play a role in regulating podocyte adhesion and attachment. Our findings also suggest that SLIT2/ROBO2 signaling might be a therapeutic target for kidney diseases associated with podocyte detachment and loss. PMID:27882344

  17. Joint spatial-spectral feature space clustering for speech activity detection from ECoG signals.

    Science.gov (United States)

    Kanas, Vasileios G; Mporas, Iosif; Benz, Heather L; Sgarbas, Kyriakos N; Bezerianos, Anastasios; Crone, Nathan E

    2014-04-01

    Brain-machine interfaces for speech restoration have been extensively studied for more than two decades. The success of such a system will depend in part on selecting the best brain recording sites and signal features corresponding to speech production. The purpose of this study was to detect speech activity automatically from electrocorticographic signals based on joint spatial-frequency clustering of the ECoG feature space. For this study, the ECoG signals were recorded while a subject performed two different syllable repetition tasks. We found that the optimal frequency resolution to detect speech activity from ECoG signals was 8 Hz, achieving 98.8% accuracy by employing support vector machines as a classifier. We also defined the cortical areas that held the most information about the discrimination of speech and nonspeech time intervals. Additionally, the results shed light on the distinct cortical areas associated with the two syllables repetition tasks and may contribute to the development of portable ECoG-based communication.

  18. Overoxidation of chloroplast 2-Cys peroxiredoxins: balancing toxic and signaling activities of hydrogen peroxide

    Directory of Open Access Journals (Sweden)

    Leonor ePuerto-Galán

    2013-08-01

    Full Text Available Photosynthesis, the primary source of biomass and oxygen into the biosphere, involves the transport of electrons in the presence of oxygen and, therefore, chloroplasts constitute an important source of reactive oxygen species (ROS, including hydrogen peroxide. If accumulated at high level, hydrogen peroxide may exert a toxic effect; however, it is as well an important second messenger. In order to balance the toxic and signaling activities of hydrogen peroxide its level has to be tightly controlled. To this end, chloroplasts are equipped with different antioxidant systems such as 2-Cys peroxiredoxins (2-Cys Prxs, thiol-based peroxidases able to reduce hydrogen- and organic peroxides. At high peroxide concentrations the peroxidase function of 2-Cys Prxs may become inactivated through a process of overoxidation. This inactivation has been proposed to explain the signaling function of hydrogen peroxide in eukaryotes, whereas in prokaryotes, the 2-Cys Prxs of which were considered to be insensitive to overoxidation, the signaling activity of hydrogen peroxide is less relevant. Here we discuss the current knowledge about the mechanisms controlling 2-Cys Prx overoxidation in chloroplasts, organelles with an important signaling function in plants. Given the prokaryotic origin of chloroplasts, we discuss the occurrence of 2-Cys Prx overoxidation in cyanobacteria with the aim of identifying similarities between chloroplasts and their ancestors regarding their response to hydrogen peroxide.

  19. Essential Function for PDLIM2 in Cell Polarization in Three-Dimensional Cultures by Feedback Regulation of the β1-Integrin–RhoA Signaling Axis

    Directory of Open Access Journals (Sweden)

    Ravi Kiran Deevi

    2014-05-01

    Full Text Available PDLIM2 is a cytoskeletal and nuclear PDZ-LIM domain protein that regulates the stability of Nuclear Factor kappa-B (NFκB and other transcription factors, and is required for polarized cell migration. PDLIM2 expression is suppressed by methylation in different cancers, but is strongly expressed in invasive breast cancer cells that have undergone an Epithelial Mesenchymal Transition (EMT. PDLIM2 is also expressed in non-transformed breast myoepithelial MCF10A cells and here we asked whether it is important for maintaining the polarized, epithelial phenotype of these cells. Suppression of PDLIM2 in MCF10A cells was sufficient to disrupt cell polarization and acini formation with increased proliferation and reduced apoptosis in the luminal space compared to control acini with hollow lumina. Spheroids with suppressed PDLIM2 exhibited increased expression of cell-cell and cell-matrix adhesion proteins including beta 1 (β1 integrin. Interestingly, levels of the Insulin-like growth factor 1 receptor (IGF-1 R and Receptor of activated protein kinase C 1 (RACK1, which scaffolds IGF-1R to β1 integrin, were also increased, indicating a transformed phenotype. Focal Adhesion Kinase (FAK and cofilin phosphorylation, and RhoA Guanosine Triphosphatase (GTPase activity were all enhanced in these spheroids compared to control acini. Importantly, inhibition of either FAK or Rho Kinase (ROCK was sufficient to rescue the polarity defect. We conclude that PDLIM2 expression is essential for feedback regulation of the β1-integrin-RhoA signalling axis and integration of cellular microenvironment signals with gene expression to control the polarity of breast epithelial acini structures. This is a mechanism by which PDLIM2 could mediate tumour suppression in breast epithelium.

  20. Reactive oxygen species in signalling the transcriptional activation of WIPK expression in tobacco.

    Science.gov (United States)

    Xu, Juan; Yang, Kwang-Yeol; Yoo, Seung Jin; Liu, Yidong; Ren, Dongtao; Zhang, Shuqun

    2014-07-01

    Plant mitogen-activated protein kinases represented by tobacco WIPK (wounding-induced protein kinase) and its orthologs in other species are unique in their regulation at transcriptional level in response to stress and pathogen infection. We previously demonstrated that transcriptional activation of WIPK is essential for induced WIPK activity, and activation of salicylic acid-induced protein kinase (SIPK) by the constitutively active NtMEK2(DD) is sufficient to induce WIPK gene expression. Here, we report that the effect of SIPK on WIPK gene expression is mediated by reactive oxygen species (ROS). Using a combination of pharmacological and gain-of-function transgenic approaches, we studied the relationship among SIPK activation, WIPK gene activation in response to fungal cryptogein, light-dependent ROS generation in chloroplasts, and ROS generated via NADPH oxidase. In the conditional gain-of-function GVG-NtMEK2(DD) transgenic tobacco, induction of WIPK expression is dependent on the ROS generation in chloroplasts. Consistently, methyl viologen, an inducer of ROS generation in chloroplasts, highly activated WIPK expression. In addition to chloroplast-originated ROS, H(2)O(2) generated from the cell-surface NADPH oxidase could also activate WIPK gene expression, and inhibition of cryptogein-induced ROS generation also abolished WIPK gene activation. Our data demonstrate that WIPK gene activation is mediated by ROS, which provides a mechanism by which ROS influence cellular signalling processes in plant stress/defence response.

  1. Commitment to the CD4 lineage mediated by extracellular signal-related kinase mitogen-activated protein kinase and lck signaling.

    Science.gov (United States)

    Sharp, L L; Hedrick, S M

    1999-12-15

    The development of T cells results in a concordance between the specificity of the TCR for MHC class I and class II molecules and the expression of CD8 and CD4 coreceptors. Based on analogy to simple metazoan models of organ development and lineage commitment, we sought to determine whether extracellular signal-related kinase (Erk) mitogen-activated protein (MAP) kinase pathway signaling acts as an inductive signal for the CD4 lineage. Here, we show that, by altering the intracellular signaling involving the Erk/MAP kinase pathway, T cells with specificity for MHC class I can be diverted to express CD4, and, conversely, T cells with specificity for MHC class II can be diverted to express CD8. Furthermore, we find that activation of the src-family tyrosine kinase, p56lck is an upstream mediator of lineage commitment. These results suggest a simple mechanism for lineage commitment in T cell development.

  2. Lifelong physical activity preserves functional sympatholysis and purinergic signalling in the ageing human leg

    DEFF Research Database (Denmark)

    Mortensen, S P; Nyberg, Michael; Winding, K;

    2012-01-01

    and exercise hyperaemia in the leg and whether ATP signalling is altered by ageing and physical activity. Leg haemodynamics, interstitial [ATP] and P2Y(2) receptor content was determined in eight young (23 ± 1 years), eight lifelong sedentary elderly (66 ± 2 years) and eight lifelong active elderly (62 ± 2...... not alter the vasodilator response to ATP infusion in any of the three groups. Plasma [noradrenaline] increased more during tyramine infusion in both elderly groups compared to young (P physically active lifestyle can maintain an intact functional sympatholysis during exercise......Ageing is associated with an impaired ability to modulate sympathetic vasoconstrictor activity (functional sympatholysis) and a reduced exercise hyperaemia. The purpose of this study was to investigate whether a physically active lifestyle can offset the impaired functional sympatholysis...

  3. Nuclear Factor-κB: Activation and Regulation during Toll-like Receptor Signaling

    Institute of Scientific and Technical Information of China (English)

    Ruaidhrí J. Carmody; Youhai H. Chen

    2007-01-01

    Toll-like receptors (TLRs) recognize distinct microbial components to initiate the innate and adaptive immune responses. TLR activation culminates in the expression of appropriate pro-inflammatory and immunomodulatory factors to meet pathogenic challenges. The transcription factor NF-κB is the master regulator of all TLR-induced responses and its activation is the pivotal event in TLR-mediated activation of the innate immune response. Many of the key molecular events required for TLR-induced NF-κB activation have been elucidated. However, much remain to be learned about the ability of TLRs to generate pathogen-specific responses using a limited number of transcription factors. This review will focus on our current understanding of NF-κB activation by TLRs and potential mechanisms for achieving a signal-specific response through NF-κB.

  4. Multifractal analysis of sEMG signal of the complex muscle activity

    CERN Document Server

    Trybek, Paulina; Nowakowski, Michal; Machura, Lukasz

    2014-01-01

    The neuro--muscular activity while working on laparoscopic trainer is the example of the complex (and complicated) movement. This class of problems are still waiting for the proper theory which will be able to describe the actual properties of the muscle performance. Here we consider the signals obtained from three states of muscle activity: at maximum contraction, during complex movements (at actual work) and in the completely relaxed state. In addition the difference between a professional and an amateur is presented. The Multifractal Detrended Fluctuation Analysis was used in description of the properties the kinesiological surface electromyographic signals (sEMG). We demonstrate the dissimilarity between each state of work for the selected group of muscles as well as between trained and untrained individuals.

  5. Dicer-2-dependent activation of Culex Vago occurs via the TRAF-Rel2 signaling pathway.

    Directory of Open Access Journals (Sweden)

    Prasad N Paradkar

    2014-04-01

    Full Text Available Despite their importance as vectors of human and livestock diseases, relatively little is known about innate antiviral immune pathways in mosquitoes and other insects. Previous work has shown that Culex Vago (CxVago, which is induced and secreted from West Nile virus (WNV-infected mosquito cells, acts as a functional homolog of interferon, by activating Jak-STAT pathway and limiting virus replication in neighbouring cells. Here we describe the Dicer-2-dependent pathway leading to WNV-induced CxVago activation. Using a luciferase reporter assay, we show that a NF-κB-like binding site in CxVago promoter region is conserved in mosquito species and is responsible for induction of CxVago expression following WNV infection. Using dsRNA-based gene knockdown, we show that the NF-κB ortholog, Rel2, plays significant role in the signaling pathway that activates CxVago in mosquito cells in vitro and in vivo. Using similar approaches, we also show that TRAF, but not TRAF-3, is involved in activation of Rel2 after viral infection. Overall the study shows that a conserved signaling pathway, which is similar to mammalian interferon activation pathway, is responsible for the induction and antiviral activity of CxVago.

  6. Dicer-2-dependent activation of Culex Vago occurs via the TRAF-Rel2 signaling pathway.

    Science.gov (United States)

    Paradkar, Prasad N; Duchemin, Jean-Bernard; Voysey, Rhonda; Walker, Peter J

    2014-04-01

    Despite their importance as vectors of human and livestock diseases, relatively little is known about innate antiviral immune pathways in mosquitoes and other insects. Previous work has shown that Culex Vago (CxVago), which is induced and secreted from West Nile virus (WNV)-infected mosquito cells, acts as a functional homolog of interferon, by activating Jak-STAT pathway and limiting virus replication in neighbouring cells. Here we describe the Dicer-2-dependent pathway leading to WNV-induced CxVago activation. Using a luciferase reporter assay, we show that a NF-κB-like binding site in CxVago promoter region is conserved in mosquito species and is responsible for induction of CxVago expression following WNV infection. Using dsRNA-based gene knockdown, we show that the NF-κB ortholog, Rel2, plays significant role in the signaling pathway that activates CxVago in mosquito cells in vitro and in vivo. Using similar approaches, we also show that TRAF, but not TRAF-3, is involved in activation of Rel2 after viral infection. Overall the study shows that a conserved signaling pathway, which is similar to mammalian interferon activation pathway, is responsible for the induction and antiviral activity of CxVago.

  7. The Activity of Surface Electromyographic Signal of Selected Muscles during Classic Rehabilitation Exercise

    Directory of Open Access Journals (Sweden)

    Jinzhuang Xiao

    2016-01-01

    Full Text Available Objectives. Prone bridge, unilateral bridge, supine bridge, and bird-dog are classic rehabilitation exercises, which have been advocated as effective ways to improve core stability among healthy individuals and patients with low back pain. The aim of this study was to investigate the activity of seven selected muscles during rehabilitation exercises through the signal of surface electromyographic. Approaches. We measured the surface electromyographic signals of four lower limb muscles, two abdominal muscles, and one back muscle during rehabilitation exercises of 30 healthy students and then analyzed its activity level using the median frequency method. Results. Different levels of muscle activity during the four rehabilitation exercises were observed. The prone bridge and unilateral bridge caused the greatest muscle fatigue; however, the supine bridge generated the lowest muscle activity. There was no significant difference (P>0.05 between left and right body side muscles in the median frequency slope during the four rehabilitation exercises of seven muscles. Conclusions. The prone bridge can affect the low back and lower limb muscles of most people. The unilateral bridge was found to stimulate muscles much more active than the supine bridge. The bird-dog does not cause much fatigue to muscles but can make most selected muscles active.

  8. Activation of NR2A receptors induces ischemic tolerance through CREB signaling.

    Science.gov (United States)

    Terasaki, Yasukazu; Sasaki, Tsutomu; Yagita, Yoshiki; Okazaki, Shuhei; Sugiyama, Yukio; Oyama, Naoki; Omura-Matsuoka, Emi; Sakoda, Saburo; Kitagawa, Kazuo

    2010-08-01

    Previous exposure to a nonlethal ischemic insult protects the brain against subsequent harmful ischemia. N-methyl-D-aspartate (NMDA) receptors are a highly studied target of neuroprotection after ischemia. Recently, NMDA receptor subtypes were implicated in neuronal survival and death. We focused on the contribution of NR2A and cyclic-AMP response element (CRE)-binding protein (CREB) signaling to ischemic tolerance using primary cortical neurons. Ischemia in vitro was modeled by oxygen-glucose deprivation (OGD). Ischemic tolerance was induced by applying 45-mins OGD 24 h before 180-mins OGD. Sublethal OGD also induced cross-tolerance against lethal glutamate and hydrogen peroxide. After sublethal OGD, expression of phosphorylated CREB and CRE transcriptional activity were significantly increased. When CRE activity was inhibited by CREB-S133A, a mutant CREB, ischemic tolerance was abolished. Inhibiting NR2A using NVP-AAM077 attenuated preconditioning-induced neuroprotection and correlated with decreased CRE activity levels. Activating NR2A using bicuculline and 4-aminopiridine induced resistance to lethal ischemia accompanied by elevated CRE activity levels, and this effect was abolished by NVP-AAM077. Elevated brain-derived neurotrophic factor (BDNF) transcriptional activities were observed after sublethal OGD and administration of bicuculline and 4-aminopiridine. NR2A-containing NMDA receptors and CREB signaling have important functions in the induction of ischemic tolerance. This may provide potential novel therapeutic strategies to treat ischemic stroke.

  9. TRAIL-Induced Caspase Activation Is a Prerequisite for Activation of the Endoplasmic Reticulum Stress-Induced Signal Transduction Pathways.

    Science.gov (United States)

    Lee, Dae-Hee; Sung, Ki Sa; Guo, Zong Sheng; Kwon, William Taehyung; Bartlett, David L; Oh, Sang Cheul; Kwon, Yong Tae; Lee, Yong J

    2016-05-01

    It is well known that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis can be initially triggered by surface death receptors (the extrinsic pathway) and subsequently amplified through mitochondrial dysfunction (the intrinsic pathway). However, little is known about signaling pathways activated by the TRAIL-induced endoplasmic reticulum (ER) stress response. In this study, we report that TRAIL-induced apoptosis is associated with the endoplasmic reticulum (ER) stress response. Human colorectal carcinoma HCT116 cells were treated with TRAIL and the ER stress-induced signal transduction pathway was investigated. During TRAIL treatment, expression of ER stress marker genes, in particular the BiP (binding immunoglobulin protein) gene, was increased and activation of the PERK (PKR-like ER kinase)-eIF2α (eukaryotic initiation factor 2α)-ATF4 (activating transcription factor 4)-CHOP (CCAAT-enhancer-binding protein homologous protein) apoptotic signal transduction pathway occurred. Experimental data from use of a siRNA (small interfering RNA) technique, caspase inhibitor, and caspase-3-deficient cell line revealed that TRAIL-induced caspase activation is a prerequisite for the TRAIL-induced ER stress response. TRAIL-induced ER stress was triggered by caspase-8-mediated cleavage of BAP31 (B cell receptor-associated protein 31). The involvement of the proapoptotic PERK-CHOP pathway in TRAIL-induced apoptosis was verified by using a PERK knockout (PERK(-/-)) mouse embryo fibroblast (MEF) cell line and a CHOP(-/-) MEF cell line. These results suggest that TRAIL-induced the activation of ER stress response plays a role in TRAIL-induced apoptotic death.

  10. Functional characterization of protease-activated receptor -1 palmitoylation in receptor signaling and trafficking /

    OpenAIRE

    2014-01-01

    G protein-coupled receptors (GPCRs) are the largest family of signaling receptors that respond to diverse stimuli and regulate many physiological responses. GPCRs elicit their cellular responses by coupling to distinct subtypes of heterotrimeric G-proteins composed of G[alpha] and G[beta][gamma] subunits. Activated GPCRs undergo conformational changes that allow the receptor to exchange GDP for GTP on the G[alpha] subunit, which induces dissociation from the [beta][gamma] subunits and subsequ...

  11. Pleiotropic β-Agonist–Promoted Receptor Conformations and Signals Independent of Intrinsic Activity

    OpenAIRE

    2006-01-01

    β-Agonists used for treatment of obstructive lung disease have a variety of different structures but are typically classified by their intrinsic activities for stimulation of cAMP, and predictions are made concerning other downstream signals based on such a classification. We generated modified β2-adrenergic receptors with insertions of energy donor and acceptor moieties to monitor agonist-promoted conformational changes of the receptor using intramolecular bioluminescence resonance energy tr...

  12. Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells.

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    Dennis J Grab

    Full Text Available BACKGROUND: Using human brain microvascular endothelial cells (HBMECs as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain. In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs known as protease activated receptors (PARs that might be implicated in calcium signaling by African trypanosomes. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA interference (RNAi we found that in vitro PAR-2 gene (F2RL1 expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%-49% and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Galpha(q with Pasteurella multocida toxin (PMT. PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified. CONCLUSIONS/SIGNIFICANCE: Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Galpha(q-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.

  13. Rho signaling in Entamoeba histolytica modulates actomyosin-dependent activities stimulated during invasive behavior.

    Science.gov (United States)

    Franco-Barraza, Janusz; Zamudio-Meza, Horacio; Franco, Elizabeth; del Carmen Domínguez-Robles, M; Villegas-Sepúlveda, Nicolás; Meza, Isaura

    2006-03-01

    Interaction of Entamoeba histolytica trophozoites with target cells and substrates activates signaling pathways in the parasite. Phosphorylation cascades triggered by phospho-inositide and adenyl-cyclase-dependent pathways modulate reorganization of the actin cytoskeleton to form structures that facilitate adhesion. In contrast, little is known about participation of Rho proteins and Rho signaling in actin rearrangements. We report here the in vivo expression of at least one Rho protein in trophozoites, whose activation induced actin reorganization and actin-myosin interaction. Antibodies to EhRhoA1 recombinant protein mainly localized Rho in the cytosol of nonactivated amoebae, but it was translocated to vesicular membranes and to some extent to the plasma membrane after treatment with lysophosphatidic acid (LPA), a specific agonist of Rho activation. Activated Rho was identified in LPA-treated trophozoites. LPA induced striking polymerization of actin into distinct dynamic structures. Disorganization of these structures by inhibition of Rho effector, Rho-kinase (ROCK), and by ML-7, an inhibitor of myosin light chain kinase dependent phosphorylation of myosin light chain, suggested that the actin structures also contained myosin. LPA stimulated concanavalin-A-mediated formation of caps, chemotaxis, invasion of extracellular matrix substrates, and erythrophagocytosis, but not binding to fibronectin. ROCK inhibition impaired LPA-stimulated functions and to some extent adhesion to fibronectin. Similar results were obtained with ML-7. These data suggest the presence and operation of Rho-signaling pathways in E. histolytica, that together with other, already described, signaling routes modulate actomyosin-dependent motile processes, particularly stimulated during invasive behavior.

  14. Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells.

    Science.gov (United States)

    Freund, Jacquelyn; May, Rebecca M; Yang, Enjun; Li, Hongchuan; McCullen, Matthew; Zhang, Bin; Lenvik, Todd; Cichocki, Frank; Anderson, Stephen K; Kambayashi, Taku

    2016-08-01

    It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells.

  15. Actin induction during PMA and cAMP-dependent signal pathway activation in Entamoeba histolytica trophozoites.

    Science.gov (United States)

    Ortiz, D; del Carmen Dominguez-Robles, M; Villegas-Sepúlveda, N; Meza, I

    2000-10-01

    Activation of PKC or cAMP-dependent signalling pathways in Entamoeba histolytica triggers the phosphorylation of proteins involved in actin rearrangements necessary for adhesion and locomotion. Analogous motifs to SRE and CRE sequences--known to respond to PMA and cAMP--were identified within the 5' regulatory region (5'RR) of one of the parasite actin genes. These sequences could be involved in the actin transcriptional upregulation reported during signalling. To test this hypothesis, a plasmid containing the 5'RR of the actin gene fused to the bacterial neomycin gene (neo) was used for stable transfection. Expression of neo and endogenous actin was measured after stimulation of transfected amoebae by PMA and dcAMP. It was found that both compounds induced neo and actin expression and showed a co-operative effect in the induction of neo. Induction by PMA or dcAMP failed if the directing amoebic 5'RR lacked SRE and CRE motifs. Transfection of amoebae with plasmid constructs, containing either progressive deletions of the actin 5'RR or site-directed mutations of the SRE and CRE-like motifs, corroborated that these sequences and a co-ordinated participation of PKC- and PKA-activated transcription factors are responsible for the increments in neo and actin mRNAs. In vivo, these PMA and cAMP-response elements could play an important role in regulating actin expression and organization in signalling processes activated during tissue invasion.

  16. Disintegrins: integrin selective ligands which activate integrin-coupled signaling and modulate leukocyte functions

    Directory of Open Access Journals (Sweden)

    Barja-Fidalgo C.

    2005-01-01

    Full Text Available Extracellular matrix proteins and cell adhesion receptors (integrins play essential roles in the regulation of cell adhesion and migration. Interactions of integrins with the extracellular matrix proteins lead to phosphorylation of several intracellular proteins such as focal adhesion kinase, activating different signaling pathways responsible for the regulation of a variety of cell functions, including cytoskeleton mobilization. Once leukocytes are guided to sites of infection, inflammation, or antigen presentation, integrins can participate in the initiation, maintenance, or termination of the immune and inflammatory responses. The modulation of neutrophil activation through integrin-mediated pathways is important in the homeostatic control of the resolution of inflammatory states. In addition, during recirculation, T lymphocyte movement through distinct microenvironments is mediated by integrins, which are critical for cell cycle, differentiation and gene expression. Disintegrins are a family of low-molecular weight, cysteine-rich peptides first identified in snake venom, usually containing an RGD (Arg-Gly-Asp motif, which confers the ability to selectively bind to integrins, inhibiting integrin-related functions in different cell systems. In this review we show that, depending on the cell type and the microenvironment, disintegrins are able to antagonize the effects of integrins or to act agonistically by activating integrin-mediated signaling. Disintegrins have proven useful as tools to improve the understanding of the molecular events regulated by integrin signaling in leukocytes and prototypes in order to design therapies able to interfere with integrin-mediated effects.

  17. Antitumor Effects of Fucoidan on Human Colon Cancer Cells via Activation of Akt Signaling.

    Science.gov (United States)

    Han, Yong-Seok; Lee, Jun Hee; Lee, Sang Hun

    2015-05-01

    We identified a novel Akt signaling mechanism that mediates fucoidan-induced suppression of human colon cancer cell (HT29) proliferation and anticancer effects. Fucoidan treatment significantly inhibited growth, induced G1-phase-associated upregulation of p21WAF1 expression, and suppressed cyclin and cyclin-dependent kinase expression in HT29 colon cancer cells. Additionally, fucoidan treatment activated the Akt signaling pathway, which was inhibited by treatment with an Akt inhibitor. The inhibition of Akt activation reversed the fucoidan-induced decrease in cell proliferation, the induction of G1-phase-associated p21WAF1 expression, and the reduction in cell cycle regulatory protein expression. Intraperitoneal injection of fucoidan reduced tumor volume; this enhanced antitumor efficacy was associated with induction of apoptosis and decreased angiogenesis. These data suggest that the activation of Akt signaling is involved in the growth inhibition of colon cancer cells treated with fucoidan. Thus, fucoidan may serve as a potential therapeutic agent for colon cancer.

  18. Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells.

    Science.gov (United States)

    te Kamp, Verena; Lindner, Ricco; Jahnke, Heinz-Georg; Krinke, Dana; Kostelnik, Katja B; Beck-Sickinger, Annette G; Robitzki, Andrea A

    2015-05-15

    Label-free and non-invasive monitoring of receptor activation and identification of the involved signal pathways in living cells is an ongoing analytic challenge and a great opportunity for biosensoric systems. In this context, we developed an impedance spectroscopy-based system for the activation monitoring of NPY-receptors in living cells. Using an optimized interdigital electrode array for sensitive detection of cellular alterations, we were able for the first time to quantitatively detect the NPY-receptor activation directly without a secondary or enhancer reaction like cAMP-stimulation by forskolin. More strikingly, we could show that the impedimetric based NPY-receptor activation monitoring is not restricted to the Y1-receptor but also possible for the Y2- and Y5-receptor. Furthermore, we could monitor the NPY-receptor activation in different cell lines that natively express NPY-receptors and proof the specificity of the observed impedimetric effect by agonist/antagonist studies in recombinant NPY-receptor expressing cell lines. To clarify the nature of the observed impedimetric effect we performed an equivalent circuit analysis as well as analyzed the role of cell morphology and receptor internalization. Finally, an antagonist based extensive molecular signal pathway analysis revealed small alterations of the actin cytoskeleton as well as the inhibition of at least L-type calcium channels as major reasons for the observed NPY-induced impedance increase. Taken together, our novel impedance spectroscopy based NPY-receptor activation monitoring system offers the opportunity to identify signal pathways as well as for novel versatile agonist/antagonist screening systems for identification of novel therapeutics in the field of obesity and cancer.

  19. cAMP signaling prevents podocyte apoptosis via activation of protein kinase A and mitochondrial fusion.

    Directory of Open Access Journals (Sweden)

    Xiaoying Li

    Full Text Available Our previous in vitro studies suggested that cyclic AMP (cAMP signaling prevents adriamycin (ADR and puromycin aminonucleoside (PAN-induced apoptosis in podocytes. As cAMP is an important second messenger and plays a key role in cell proliferation, differentiation and cytoskeleton formation via protein kinase A (PKA or exchange protein directly activated by cAMP (Epac pathways, we sought to determine the role of PKA or Epac signaling in cAMP-mediated protection of podocytes. In the ADR nephrosis model, we found that forskolin, a selective activator of adenylate cyclase, attenuated albuminuria and improved the expression of podocyte marker WT-1. When podocytes were treated with pCPT-cAMP (a selective cAMP/PKA activator, PKA activation was increased in a time-dependent manner and prevented PAN-induced podocyte loss and caspase 3 activation, as well as a reduction in mitochondrial membrane potential. We found that PAN and ADR resulted in a decrease in Mfn1 expression and mitochondrial fission in podocytes. pCPT-cAMP restored Mfn1 expression in puromycin or ADR-treated podocytes and induced Drp1 phosphorylation, as well as mitochondrial fusion. Treating podocytes with arachidonic acid resulted in mitochondrial fission, podocyte loss and cleaved caspase 3 production. Arachidonic acid abolished the protective effects of pCPT-cAMP on PAN-treated podocytes. Mdivi, a mitochondrial division inhibitor, prevented PAN-induced cleaved caspase 3 production in podocytes. We conclude that activation of cAMP alleviated murine podocyte caused by ADR. PKA signaling resulted in mitochondrial fusion in podocytes, which at least partially mediated the effects of cAMP.

  20. cAMP signaling prevents podocyte apoptosis via activation of protein kinase A and mitochondrial fusion.

    Science.gov (United States)

    Li, Xiaoying; Tao, Hua; Xie, Kewei; Ni, Zhaohui; Yan, Yucheng; Wei, Kai; Chuang, Peter Y; He, John Cijiang; Gu, Leyi

    2014-01-01

    Our previous in vitro studies suggested that cyclic AMP (cAMP) signaling prevents adriamycin (ADR) and puromycin aminonucleoside (PAN)-induced apoptosis in podocytes. As cAMP is an important second messenger and plays a key role in cell proliferation, differentiation and cytoskeleton formation via protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac) pathways, we sought to determine the role of PKA or Epac signaling in cAMP-mediated protection of podocytes. In the ADR nephrosis model, we found that forskolin, a selective activator of adenylate cyclase, attenuated albuminuria and improved the expression of podocyte marker WT-1. When podocytes were treated with pCPT-cAMP (a selective cAMP/PKA activator), PKA activation was increased in a time-dependent manner and prevented PAN-induced podocyte loss and caspase 3 activation, as well as a reduction in mitochondrial membrane potential. We found that PAN and ADR resulted in a decrease in Mfn1 expression and mitochondrial fission in podocytes. pCPT-cAMP restored Mfn1 expression in puromycin or ADR-treated podocytes and induced Drp1 phosphorylation, as well as mitochondrial fusion. Treating podocytes with arachidonic acid resulted in mitochondrial fission, podocyte loss and cleaved caspase 3 production. Arachidonic acid abolished the protective effects of pCPT-cAMP on PAN-treated podocytes. Mdivi, a mitochondrial division inhibitor, prevented PAN-induced cleaved caspase 3 production in podocytes. We conclude that activation of cAMP alleviated murine podocyte caused by ADR. PKA signaling resulted in mitochondrial fusion in podocytes, which at least partially mediated the effects of cAMP.

  1. CFTR impairment upregulates c-Src activity through IL-1β autocrine signaling.

    Science.gov (United States)

    Massip-Copiz, María Macarena; Clauzure, Mariángeles; Valdivieso, Ángel Gabriel; Santa-Coloma, Tomás Antonio

    2017-02-15

    Cystic Fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Previously, we found several genes showing a differential expression in CFDE cells (epithelial cells derived from a CF patient). One corresponded to c-Src; its expression and activity was found increased in CFDE cells, acting as a signaling molecule between the CFTR activity and MUC1 overexpression. Here we report that bronchial IB3-1 cells (CF cells) also showed increased c-Src activity compared to 'CFTR-corrected' S9 cells. In addition, three different Caco-2 cell lines, each stably transfected with a different CFTR-specific shRNAs, displayed increased c-Src activity. The IL-1β receptor antagonist IL1RN reduced the c-Src activity of Caco-2/pRS26 cells (expressing a CFTR-specific shRNA). In addition, increased mitochondrial and cellular ROS levels were detected in Caco-2/pRS26 cells. ROS levels were partially reduced by incubation with PP2 (c-Src inhibitor) or IL1RN, and further reduced by using the NOX1/4 inhibitor GKT137831. Thus, IL-1β→c-Src and IL-1β→NOX signaling pathways appear to be responsible for the production of cellular and mitochondrial ROS in CFTR-KD cells. In conclusion, IL-1β constitutes a new step in the CFTR signaling pathway, located upstream of c-Src, which is stimulated in cells with impaired CFTR activity.

  2. Signal Transducer and Activator of Transcription 3 (STAT3 and Trophoblast Invasion

    Directory of Open Access Journals (Sweden)

    Fitzgerald JS

    2007-01-01

    Full Text Available Human trophoblast cells have the fascinating property of physiological invasiveness into allogenic tissue. The underlying mechanisms, such as extra- and intracellular signalling, are very similar to those abused by a variety of tumours. The main contrasting feature to cancerous cells is the very fundamental ability of trophoblasts to auto-regulate invasion with respect to time and space. Trophoblast cells start invasion into the decidua very shortly after implantation, which approaches a maximum during the first trimester of gestation period. During this period of time, several cytokines from cells of different maternal origin, including NK cells, dendritic cells, stroma cells and endothelial cells, are present which, analogous to the situation in tumours, have the potential to trigger and enhance invasion, migration and proliferation of trophoblast cells. These mainly include interleukin-6 (IL-6, IL-11, Leukaemia Inhibitory Factor (LIF, Hepatocyte Growth Factor (HGF and Insulin-like Growth Factors (IGF. Cytokines, upon binding to their specific receptors present on the trophoblast cells, trigger several intracellular signalling cascades. One of these signalling pathways is the Janus Kinase (Jak/Signal Transducers and Activators of Transcription (STAT pathway. As recent studies have shown, the tyrosine phosphorylated form of STAT3 is a major inducer of invasiveness which mainly takes place upon binding of LIF to its receptor. For autoregulation of signals, STAT3 induces the transcription of Suppressor of Cytokine Signalling 3 (SOCS3. The balance between STAT3 and SOCS3 may be argued as one of the main tuners of trophoblast invasion for successful implantation. Disturbances in this balance may lead to serious complications like cancer and implantation failure.

  3. Indications for influence of artificial (man-made) activity on radon signals, in simulation experiments

    Science.gov (United States)

    Steinitz, G.; Kotlarsky, P.; Piatibratova, O.

    2016-11-01

    Radon (Rn-222; a radioactive noble gas) is characterized by large temporal variations that differ significantly from variations of (i) other trace elements in geogas (noble gases); (ii) variation patterns of other dynamic geophysical systems (atmospheric, tidal). Consensus exists that there is no simple and straightforward understanding of the phenomena and its behaviour. This lacuna in the understanding of the underlying principles hampers the development of applications-such as radon as a proxy of processes in the seismogenic context. Using results from field investigations and simulation experiments the GSI suggested that an unidentified extraterrestrial component, probably in solar radiation, drives periodic radon signals in the diurnal and annual frequency bands. Recent findings from experimental investigations shed additional perspectives allowing a new evaluation of the issue. Particular transient signals, measured with alpha and gamma detectors, are interpreted to reflect the influence of artificial activity. Criteria are (i) signals lasting several hours that occur around midday on workdays (Sunday-Thursday); (ii) signals composed of a train of around 10 strong pulses, each lasting less than 15 min, occurring within several hours once a week, from Wednesday afternoon/evening to Thursday morning. A first interpretation is that an unidentified artificial activity of some sort (industrial?) generates and emits an unidentified agent that reaches enhanced confined mode experiments at the GSI laboratory, which respond to the incoming agent in the form of radon signals. Developing the capability of identification of such an earth-bound source generating an influencing agent is a key step towards understanding of external influence on radioactivity of radon.

  4. The D. melanogaster capa-1 neuropeptide activates renal NF-kB signaling.

    Science.gov (United States)

    Terhzaz, Selim; Overend, Gayle; Sebastian, Sujith; Dow, Julian A T; Davies, Shireen-A

    2014-03-01

    The capa peptide family exists in a very wide range of insects including species of medical, veterinary and agricultural importance. Capa peptides act via a cognate G-protein coupled receptor (capaR) and have a diuretic action on the Malpighian tubules of Dipteran and Lepidopteran species. Capa signaling is critical for fluid homeostasis and has been associated with desiccation tolerance in the fly, Drosophila melanogaster. The mode of capa signaling is highly complex, affecting calcium, nitric oxide and cyclic GMP pathways. Such complex physiological regulation by cell signaling pathways may occur ultimately for optimal organismal stress tolerance to multiple stressors. Here we show that D. melanogaster capa-1 (Drome-capa-1) acts via the Nuclear Factor kappa B (NF-kB) stress signaling network. Human PCR gene arrays of capaR-transfected Human Embryonic Kidney (HEK) 293 cells showed that Drome-capa-1 increases expression of NF-kB, NF-kB regulated genes including IL8, TNF and PTGS2, and NF-kB pathway-associated transcription factors i.e. EGR1, FOS, cJUN. Furthermore, desiccated HEK293 cells show increased EGR1, EGR3 and PTGS2 - but not IL8, expression. CapaR-transfected NF-kB reporter cells showed that Drome-capa-1 increased NF-kB promoter activity via increased calcium. In Malpighian tubules, both Drome-capa-1 stimulation and desiccation result in increased gene expression of the D. melanogaster NF-kB orthologue, Relish; as well as EGR-like stripe and klumpfuss. Drome-capa-1 also induces Relish translocation in tubule principal cells. Targeted knockdown of Relish in only tubule principal cells reduces desiccation stress tolerance of adult flies. Together, these data suggest that Drome-capa-1 acts in desiccation stress tolerance, by activating NF-kB signaling.

  5. Normalization of voltage-sensitive dye signal with functional activity measures.

    Directory of Open Access Journals (Sweden)

    Kentaroh Takagaki

    Full Text Available In general, signal amplitude in optical imaging is normalized using the well-established DeltaF/F method, where functional activity is divided by the total fluorescent light flux. This measure is used both directly, as a measure of population activity, and indirectly, to quantify spatial and spatiotemporal activity patterns. Despite its ubiquitous use, the stability and accuracy of this measure has not been validated for voltage-sensitive dye imaging of mammalian neocortex in vivo. In this report, we find that this normalization can introduce dynamic biases. In particular, the DeltaF/F is influenced by dye staining quality, and the ratio is also unstable over the course of experiments. As methods to record and analyze optical imaging signals become more precise, such biases can have an increasingly pernicious impact on the accuracy of findings, especially in the comparison of cytoarchitechtonic areas, in area-of-activation measurements, and in plasticity or developmental experiments. These dynamic biases of the DeltaF/F method may, to an extent, be mitigated by a novel method of normalization, DeltaF/DeltaF(epileptiform. This normalization uses as a reference the measured activity of epileptiform spikes elicited by global disinhibition with bicuculline methiodide. Since this normalization is based on a functional measure, i.e. the signal amplitude of "hypersynchronized" bursts of activity in the cortical network, it is less influenced by staining of non-functional elements. We demonstrate that such a functional measure can better represent the amplitude of population mass action, and discuss alternative functional normalizations based on the amplitude of synchronized spontaneous sleep-like activity. These findings demonstrate that the traditional DeltaF/F normalization of voltage-sensitive dye signals can introduce pernicious inaccuracies in the quantification of neural population activity. They further suggest that normalization

  6. Novel angiogenesis inhibitory activity in cinnamon extract blocks VEGFR2 kinase and downstream signaling.

    Science.gov (United States)

    Lu, Jianming; Zhang, Keqiang; Nam, Sangkil; Anderson, Richard A; Jove, Richard; Wen, Wei

    2010-03-01

    As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for anti-angiogenesis treatment. However, the side effects associated with most anti-VEGF agents limit their chronic use. Identification of naturally occurring VEGF inhibitors derived from diet is a potential alternative approach, with the advantage of known safety. To isolate natural inhibitors of VEGF, we established an in vitro tyrosine kinase assay to screen for diet-based agents that suppress VEGFR2 kinase activity. We found that a water-based extract from cinnamon (cinnamon extract, CE), one of the oldest and most popular spices, was a potent inhibitor of VEGFR2 kinase activity, directly inhibiting kinase activity of purified VEGFR2 as well as mitogen-activated protein kinase- and Stat3-mediated signaling pathway in endothelial cells. As a result, CE inhibited VEGF-induced endothelial cell proliferation, migration and tube formation in vitro, sprout formation from aortic ring ex vivo and tumor-induced blood vessel formation in vivo. Depletion of polyphenol from CE with polyvinylpyrrolidone abolished its anti-angiogenesis activity. While cinnamaldehyde, a component responsible for CE aroma, had little effect on VEGFR2 kinase activity, high-performance liquid chromatography-purified components of CE, procyanidin type A trimer (molecular weight, 864) and a tetramer (molecular weight, 1152) were found to inhibit kinase activity of purified VEGFR2 and VEGFR2 signaling, implicating procyanidin oligomers as active components in CE that inhibit angiogenesis. Our data revealed a novel activity in cinnamon and identified a natural VEGF inhibitor that could potentially be useful in cancer prevention and/or treatment.

  7. Regulatory Activity of Polyunsaturated Fatty Acids in T-Cell Signaling

    Science.gov (United States)

    Kim, Wooki; Khan, Naim A.; McMurray, David N.; Prior, Ian A.; Wang, Naisyin; Chapkin, Robert S.

    2010-01-01

    n-3 polyunsaturated fatty acids (PUFA) are considered to be authentic immunosuppressors and appear to exert beneficial effects with respect to certain immune-mediated diseases. In addition to promoting T-helper 1 (Th1) cell to T-helper 2 (Th2) cell effector T-cell differentiation, n-3 PUFA may also exert anti-inflammatory actions by inducing apoptosis in Th1 cells. With respect to mechanisms of action, effects range from the modulation of membrane receptors to gene transcription via perturbation of a number of second messenger cascades. In this review, the putative targets of anti-inflammatory n-3 PUFA, activated during early and late events of T-cell activation will be discussed. Studies have demonstrated that these fatty acids alter plasma membrane micro-organization (lipid rafts) at the immunological synapse, the site where T-cells and antigen presenting cells (APC) form a physical contact for antigen initiated T-cell signaling. In addition, the production of diacylglycerol and the activation of different isoforms of protein kinase C (PKC), mitogen activated protein kinase (MAPK), calcium signaling, and nuclear translocation/activation of transcriptional factors, can be modulated by n-3 PUFA. Advantages and limitations of diverse methodologies to study the membrane lipid raft hypothesis, as well as apparent contradictions regarding the effect of n-3 PUFA on lipid rafts will be critically presented. PMID:20176053

  8. Repetitive Peroxide Exposure Reveals Pleiotropic Mitogen-Activated Protein Kinase Signaling Mechanisms

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    Wayne Chadwick

    2011-01-01

    Full Text Available Oxidative stressors such as hydrogen peroxide control the activation of many interconnected signaling systems and are implicated in neurodegenerative disease etiology. Application of hydrogen peroxide to PC12 cells activated multiple tyrosine kinases (c-Src, epidermal growth factor receptor (EGFR, and Pyk2 and the serine-threonine kinase ERK1/2. Peroxide-induced ERK1/2 activation was sensitive to intracellular calcium chelation and EGFR and c-Src kinase inhibition. Acute application and removal of peroxide allowed ERK1/2 activity levels to rapidly subside to basal serum-deprived levels. Using this protocol, we demonstrated that ERK1/2 activation tachyphylaxis developed upon repeated peroxide exposures. This tachyphylaxis was independent of c-Src/Pyk2 tyrosine phosphorylation but was associated with a progressive reduction of peroxide-induced EGFR tyrosine phosphorylation, EGFR interaction with growth factor receptor binding protein 2, and a redistribution of EGFR from the plasma membrane to the cytoplasm. Our data indicates that components of peroxide-induced ERK1/2 cascades are differentially affected by repeated exposures, indicating that oxidative signaling may be contextually variable.

  9. LOXL2 drives epithelial-mesenchymal transition via activation of IRE1-XBP1 signalling pathway

    Science.gov (United States)

    Cuevas, Eva P.; Eraso, Pilar; Mazón, María J.; Santos, Vanesa; Moreno-Bueno, Gema; Cano, Amparo; Portillo, Francisco

    2017-01-01

    Epithelial-to-Mesenchymal Transition (EMT) is a key process contributing to the aggressiveness of cancer cells. EMT is triggered by activation of different transcription factors collectively known as EMT-TFs. Different cellular cues and cell signalling networks activate EMT at transcriptional and posttranscriptional level in different biological and pathological situations. Among them, overexpression of LOXL2 (lysyl oxidase-like 2) induces EMT independent of its catalytic activity. Remarkably, perinuclear/cytoplasmic accumulation of LOXL2 is a poor prognosis marker of squamous cell carcinomas and is associated to basal breast cancer metastasis by mechanisms no yet fully understood. Here, we report that overexpression of LOXL2 promotes its accumulation in the Endoplasmic Reticulum where it interacts with HSPA5 leading to activation of the IRE1-XBP1 signalling pathway of the ER-stress response. LOXL2-dependent IRE1-XBP1 activation induces the expression of several EMT-TFs: SNAI1, SNAI2, ZEB2 and TCF3 that are direct transcriptional targets of XBP1. Remarkably, inhibition of IRE1 blocks LOXL2-dependent upregulation of EMT-TFs thus hindering EMT induction. PMID:28332555

  10. An epigenetic switch induced by Shh signalling regulates gene activation during development and medulloblastoma growth.

    Science.gov (United States)

    Shi, Xuanming; Zhang, Zilai; Zhan, Xiaoming; Cao, Mou; Satoh, Takashi; Akira, Shizuo; Shpargel, Karl; Magnuson, Terry; Li, Qingtian; Wang, Rongfu; Wang, Chaochen; Ge, Kai; Wu, Jiang

    2014-11-05

    The Sonic hedgehog (Shh) signalling pathway plays important roles during development and in cancer. Here we report a Shh-induced epigenetic switch that cooperates with Gli to control transcription outcomes. Before induction, poised Shh target genes are marked by a bivalent chromatin domain containing a repressive histone H3K27me3 mark and an active H3K4me3 mark. Shh activation induces a local switch of epigenetic cofactors from the H3K27 methyltransferase polycomb repressive complex 2 (PRC2) to an H3K27me3 demethylase Jmjd3/Kdm6b-centred coactivator complex. We also find that non-enzymatic activities of Jmjd3 are important and that Jmjd3 recruits the Set1/MLL H3K4 methyltransferase complexes in a Shh-dependent manner to resolve the bivalent domain. In vivo, changes of the bivalent domain accompanied Shh-activated cerebellar progenitor proliferation. Overall, our results reveal a regulatory mechanism that underlies the activation of Shh target genes and provides insight into the causes of various diseases and cancers exhibiting altered Shh signalling.

  11. An Inside Job: Hacking into Janus Kinase/Signal Transducer and Activator of Transcription Signaling Cascades by the Intracellular Protozoan Toxoplasma gondii

    Science.gov (United States)

    Bzik, David J.; Fox, Barbara A.; Butcher, Barbara A.

    2012-01-01

    The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype. PMID:22104110

  12. An inside job: hacking into Janus kinase/signal transducer and activator of transcription signaling cascades by the intracellular protozoan Toxoplasma gondii.

    Science.gov (United States)

    Denkers, Eric Y; Bzik, David J; Fox, Barbara A; Butcher, Barbara A

    2012-02-01

    The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype.

  13. Sodium butyrate-induced death-associated protein kinase expression promote Raji cell morphological change and apoptosis by reducing FAK protein levels

    Institute of Scientific and Technical Information of China (English)

    Hai-tao ZHANG; Zhe-ling FENG; Jun WU; Ya-jun WANG; Xia GUO; Nian-ci LIANG; Zhen-yu ZHU; Jian-quan MA

    2007-01-01

    Aim:To investigate the role of death-associated protein kinase (DAPK) on the apoptosis of Raji cells induced by sodium butyrate. Methods:The apoptosis of Raji cells were induced by sodium butyrate for 2,4,6,8,and 10 d. Simultaneity,the Raji cells were inhibited to adhere on culture flask by polyHEME. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and the cell apoptosis percentage was estimated by flow cytometry. DAPK and focal adhesion kinase (FAK) expression were measured by Western blotting.Coding sequence on the C-terminal of DAPK,which can suppress the function of DAPK,was tranfected into the Raji cells to investigate whether the C-terminal of DAPK could inhibit the apoptosis of Raji cells induced by sodium butyrate. Results:After being treated with sodium butyrate,the Raji cells expressed DAPK and displayed many protrusions to adhere onto the culture flask. The Raji cells were susceptive to apoptosis when they were inhibited adhesion by polyHEME. At that time,the cell viability decreased,the cell apoptosis percentage increased and the protein levels of total FAK were reduced. The Raji cells,which were transfected with the coding region on the C-terminal of DAPK,sustained apoptosis and the FAK protein level when treated with sodium butyrate. Conclusion:Sodium butyrate induced DAPK expression. It caused the Raji cells to display many protrusions all around the cells and adhere onto the culture flask. DAPK expression prompted apoptosis by reducing the FAK protein level in sodium butyrate induced Raji cells.

  14. Neuronal c-Abl activation leads to induction of cell cycle and interferon signaling pathways

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    Schlatterer Sarah D

    2012-08-01

    Full Text Available Abstract Background Expression of active c-Abl in adult mouse forebrain neurons in the AblPP/tTA mice resulted in severe neurodegeneration, particularly in the CA1 region of the hippocampus. Neuronal loss was preceded and accompanied by substantial microgliosis and astrocytosis. In contrast, expression of constitutively active Arg (Abl-related gene in mouse forebrain neurons (ArgPP/tTA mice caused no detectable neuronal loss or gliosis, although protein expression and kinase activity were at similar levels to those in the AblPP/tTA mice. Methods To begin to elucidate the mechanism of c-Abl-induced neuronal loss and gliosis, gene expression analysis of AblPP/tTA mouse forebrain prior to development of overt pathology was performed. Selected results from gene expression studies were validated with quantitative reverse transcription PCR , immunoblotting and bromodeoxyuridine (BrdU labeling, and by immunocytochemistry. Results Two of the top pathways upregulated in AblPP/tTA mice with c-Abl expression for 2 weeks were cell cycle and interferon signaling. However, only the expression of interferon signaling pathway genes remained elevated at 4 weeks of c-Abl induction. BrdU incorporation studies confirm that, while the cell cycle pathway is upregulated in AblPP/tTA mice at 2 weeks of c-Abl induction, the anatomical localization of the pathway is not consistent with previous pathology seen in the AblPP/tTA mice. Increased expression and activation of STAT1, a known component of interferon signaling and interferon-induced neuronal excitotoxicity, is an early consequence of c-Abl activation in AblPP/tTA mice and occurs in the CA1 region of the hippocampus, the same region that goes on to develop severe neurodegenerative pathology and neuroinflammation. Interestingly, no upregulation of gene expression of interferons themselves was detected. Conclusions Our data suggest that the interferon signaling pathway may play a role in the pathologic processes

  15. Pituitary adenylate cyclase activating peptide (PACAP participates in adipogenesis by activating ERK signaling pathway.

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    Tatjana Arsenijevic

    Full Text Available Pituitary adenylate cyclase activating peptide (PACAP belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP family. Its action can be mediated by three different receptor subtypes: PAC1, which has exclusive affinity for PACAP, and VPAC1 and VPAC2 which have equal affinity for PACAP and VIP. We showed that all three receptors are expressed in 3T3-L1 cells throughout their differentiation into adipocytes. We established the activity of these receptors by cAMP accumulation upon induction by PACAP. Together with insulin and dexamethasone, PACAP induced adipogenesis in 3T3-L1 cell line. PACAP increased cAMP production within 15 min upon stimulation and targeted the expression and phosphorylation of MAPK (ERK1/2, strengthened by the ERK1/2 phosphorylation being partially or completely abolished by different combinations of PACAP receptors antagonists. We therefore speculate that ERK1/2 activation is crucial for the activation of CCAAT/enhancer- binding protein β (C/EBPβ.

  16. Antibody constant region peptides can display immunomodulatory activity through activation of the Dectin-1 signalling pathway.

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    Elena Gabrielli

    Full Text Available We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc of human IgG(1, is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of β-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules.

  17. Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/β-catenin pathway.

    Science.gov (United States)

    Zhang, Qian-Qian; Zhou, Da-Lei; Lei, Yan; Zheng, Li; Chen, Sheng-Xia; Gou, Hong-Ju; Gu, Qu-Liang; He, Xiao-Dong; Lan, Tian; Qi, Cui-Ling; Li, Jiang-Chao; Ding, Yan-Qing; Qiao, Liang; Wang, Li-Jing

    2015-02-20

    Slit2 is often overexpressed in cancers. Slit2 is a secreted protein that binds to Roundabout (Robo) receptors to regulate cell growth and migration. Here, we employed several complementary mouse models of intestinal cancers, including the Slit2 transgenic mice, the ApcMin/+ spontaneous intestinal adenoma mouse model, and the DMH/DSS-induced colorectal carcinoma model to clarify function of Slit2/Robo1 signaling in intestinal tumorigenesis. We showed that Slit2 and Robo1 are overexpressed in intestinal tumors and may contribute to tumor generation. The Slit2/Robo1 signaling can induce precancerous lesions of the intestine and tumor progression. Ectopic expression of Slit2 activated Slit2/Robo1 signaling and promoted tumorigenesis and tumor growth. This was mediated in part through activation of the Src signaling, which then down-regulated E-cadherin, thereby activating Wnt/β-catenin signaling. Thus, Slit2/Robo1 signaling is oncogenic in intestinal tumorigenesis.

  18. Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

    Science.gov (United States)

    Shimizu, M.; Wang, W.; Walch, E. T.; Dunne, P. W.; Epstein, H. F.

    2000-01-01

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

  19. Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice.

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    Gerhard Sengle

    2015-06-01

    Full Text Available Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that

  20. Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice.

    Science.gov (United States)

    Sengle, Gerhard; Carlberg, Valerie; Tufa, Sara F; Charbonneau, Noe L; Smaldone, Silvia; Carlson, Eric J; Ramirez, Francesco; Keene, Douglas R; Sakai, Lynn Y

    2015-06-01

    Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can

  1. Mitogen-activated protein kinase signaling controls basal and oncostatin M-mediated JUNB gene expression.

    Science.gov (United States)

    Hicks, Mellissa J; Hu, Qiuping; Macrae, Erin; DeWille, James

    2015-05-01

    The mitogen-activated protein kinase (MAPK) pathway is aberrantly activated in many human cancers, including breast cancer. Activation of MAPK signaling is associated with the increased expression of a wide range of genes that promote cell survival, proliferation, and migration. This report investigated the influence of MAPK signaling on the regulation and expression of JUNB in human breast cancer cell lines. JUNB has been associated with tumor suppressor and oncogenic functions, with most reports describing JUNB as an oncogene in breast cancer. Our results indicated that JUNB expression is elevated in MCF10A(met), SKBR3, and MDA-MB-231 human breast cancer cell lines compared to nontransformed MCF10A mammary epithelial cells. Increased RAS/MAPK signaling in MCF10A(met) cells correlates with the increased association of RNA polymerase II (Pol II) phosphorylated on serine 5 (Pol IIser5p) with the JUNB proximal promoter. Pol IIser5p is the "transcription initiating" form of Pol II. Treatment with U0126, a MAPK pathway inhibitor, reduces Pol IIser5p association with the JUNB proximal promoter and reduces JUNB expression. Oncostatin M (OSM) enhances MAPK and STAT3 signaling and significantly induces JUNB expression. U0126 treatment reduces OSM-induced Pol IIser5p binding to the JUNB proximal promoter and JUNB expression, but does not reduce pSTAT3 levels or the association of pSTAT3 with the JUNB proximal promoter. These results demonstrate that the MAPK pathway plays a primary role in the control of JUNB gene expression by promoting the association of Pol IIser5p with the JUNB proximal promoter.

  2. Aberrant activation of Sonic hedgehog signaling in chronic cholecystitis and gallbladder carcinoma.

    Science.gov (United States)

    Xie, Fang; Xu, Xiaoping; Xu, Angao; Liu, Cuiping; Liang, Fenfen; Xue, Minmin; Bai, Lan

    2014-03-01

    Sonic hedgehog (Shh) signaling has been extensively studied and is implicated in various inflammatory diseases and malignant tumors. We summarized the clinicopathological features and performed immunohistochemistry assays to examine expression of Shh signaling proteins in 10 normal mucosa, 32 gallbladder carcinoma (GBC), and 95 chronic cholecystitis (CC) specimens. The CC specimens were classified into three groups according to degree of inflammation. Compared with normal mucosa, CC, and GBC specimens exhibited increased expression of Shh. The immunoreactive score of Shh in the GBC group was higher than that in the mild to moderate CC groups but lower than that in the severe CC group (P cholecystitis to malignant tumors. Compared with CC specimens, GBC specimens showed higher cytoplasmic and membranous expression for Ptch (P < .05). Gli1 staining showed cytoplasmic expression of Gli1 in both CC (60% for mild, 77% for moderate, and 84% for severe) and GBC specimens (97%). Nuclear expression of Gli1 was detected in 16% of severe CC specimens with moderate to poor atypical hyperplasia, and in 62.5% of GBC specimens. Shh expression strongly correlated with expression of Ptch and Gli1. Furthermore, patients with strongly positive Gli1 staining had significantly lower survival rates than those with weakly positive staining. Our data indicate that the Shh signaling pathway is aberrantly activated in CC and GBC, and altered Shh signaling may be involved in the course of development from CC to gallbladder carcinogenesis.

  3. Cucurbitacin E Induces Autophagy via Downregulating mTORC1 Signaling and Upregulating AMPK Activity.

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    Qing-Bing Zha

    Full Text Available Cucurbitacins, the natural triterpenoids possessing many biological activities, have been reported to suppress the mTORC1/p70S6K pathway and to induce autophagy. However, the correlation between such activities is largely unknown. In this study, we addressed this issue in human cancer cells in response to cucurbitacin E (CuE treatment. Our results showed that CuE induced autophagy as evidenced by the formation of LC3-II and colocalization of punctate LC3 with the lysosomal marker LAMP2 in HeLa and MCF7 cells. However, CuE induced much lower levels of autophagy in ATG5-knocked down cells and failed to induce autophagy in DU145 cells lacking functional ATG5 expression, suggesting the dependence of CuE-induced autophagy on ATG5. Consistent with autophagy induction, mTORC1 activity (as reflected by p70S6K and ULK1S758 phosphorylation was inhibited by CuE treatment. The suppression of mTORC1 activity was further confirmed by reduced recruitment of mTOR to the lysosome, which is the activation site of mTORC1. In contrast, CuE rapidly activated AMPK leading to increased phosphorylation of its substrates. AMPK activation contributed to CuE-induced suppression of mTORC1/p70S6K signaling and autophagy induction, since AMPK knockdown diminished these effects. Collectively, our data suggested that CuE induced autophagy in human cancer cells at least partly via downregulation of mTORC1 signaling and upregulation of AMPK activity.

  4. Signal Intensities in Preoperative MRI Do Not Reflect Proliferative Activity in Meningioma

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    Stefan Schob

    2016-08-01

    Full Text Available BACKGROUND: Identification of high-grade meningiomas in preoperative magnetic resonance imaging (MRI is important for optimized surgical strategy and best possible resection. Numerous studies investigated subjectively determined morphological features as predictors of tumor biology in meningiomas. The aim of this study was to identify the predictive value of more reliable, quantitatively measured signal intensities in MRI for differentiation of high- and low-grade meningiomas and identification of meningiomas with high proliferation rates, respectively. PATIENTS AND METHODS: Sixty-six patients (56 World Health Organization [WHO] grade I, 9 WHO grade II, and 1 WHO grade I were included in the study. Preoperative MRI signal intensities (fluid-attenuated inversion recovery [FLAIR], T1 precontrast, and T1 postcontrast as genuine and normalized values were correlated with Ki-67 expression in tissue sections of resected meningiomas. Differences between the groups (analysis of variance and Spearman rho correlation were computed using SPSS 22. RESULTS: Mean values of genuine signal intensities of meningiomas in FLAIR, T1 native, and T1 postcontrast were 323.9 ± 59, 332.8 ± 67.9, and 768.5 ± 165.3. Mean values of normalized (to the contralateral white matter signal intensities of meningiomas in FLAIR, T1 native, and T1 postcontrast were 1.5 ± 0.3, 0.8 ± 0.1, and 1.9 ± 0.4. There was no significant correlation between MRI signal intensities and WHO grade or Ki-67 expression. Signal intensities did not differ significantly between WHO grade I and II/III meningiomas. Ki-67 expression was significantly increased in high-grade meningiomas compared with low-grade meningiomas (P < 0.01. Objectively measured values of MRI signal intensities (FLAIR, T1 precontrast, and T1 postcontrast enhancement did not distinguish between high-grade and low-grade meningiomas. Furthermore, there was no association between MRI signal intensities and Ki-67 expression

  5. Functional Near-Infrared Spectroscopy Signals Measure Neuronal Activity in the Cortex

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    Harrivel, Angela; Hearn, Tristan

    2013-01-01

    Functional near infrared spectroscopy (fNIRS) is an emerging optical neuroimaging technology that indirectly measures neuronal activity in the cortex via neurovascular coupling. It quantifies hemoglobin concentration ([Hb]) and thus measures the same hemodynamic response as functional magnetic resonance imaging (fMRI), but is portable, non-confining, relatively inexpensive, and is appropriate for long-duration monitoring and use at the bedside. Like fMRI, it is noninvasive and safe for repeated measurements. Patterns of [Hb] changes are used to classify cognitive state. Thus, fNIRS technology offers much potential for application in operational contexts. For instance, the use of fNIRS to detect the mental state of commercial aircraft operators in near real time could allow intelligent flight decks of the future to optimally support human performance in the interest of safety by responding to hazardous mental states of the operator. However, many opportunities remain for improving robustness and reliability. It is desirable to reduce the impact of motion and poor optical coupling of probes to the skin. Such artifacts degrade signal quality and thus cognitive state classification accuracy. Field application calls for further development of algorithms and filters for the automation of bad channel detection and dynamic artifact removal. This work introduces a novel adaptive filter method for automated real-time fNIRS signal quality detection and improvement. The output signal (after filtering) will have had contributions from motion and poor coupling reduced or removed, thus leaving a signal more indicative of changes due to hemodynamic brain activations of interest. Cognitive state classifications based on these signals reflect brain activity more reliably. The filter has been tested successfully with both synthetic and real human subject data, and requires no auxiliary measurement. This method could be implemented as a real-time filtering option or bad channel

  6. Platelet rich plasma promotes skeletal muscle cell migration in association with up-regulation of FAK, paxillin, and F-Actin formation.

    Science.gov (United States)

    Tsai, Wen-Chung; Yu, Tung-Yang; Lin, Li-Ping; Lin, Mioa-Sui; Tsai, Ting-Ta; Pang, Jong-Hwei S

    2017-02-24

    Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The aim of this study was to investigate the effect and molecular mechanism of PRP on migration of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague-Dawley rats were treated with PRP. The cell migration was evaluated by transwell filter migration assay and electric cell-substrate impedance sensing. The spreading of cells was evaluated microscopically. The formation of filamentous actin (F-actin) cytoskeleton was assessed by immunofluorescence staining. The protein expressions of paxillin and focal adhesion kinase (FAK) were assessed by Western blot analysis. Transfection of paxillin small-interfering RNA (siRNAs) to muscle cells was performed to validate the role of paxillin in PRP-mediated promotion of cell migration. Dose-dependently PRP promotes migration of and spreading and muscle cells. Protein expressions of paxillin and FAK were up-regulated dose-dependently. F-actin formation was also enhanced by PRP treatment. Furthermore, the knockdown of paxillin expression impaired the effect of PRP to promote cell migration. It was concluded that PRP promoting migration of muscle cells is associated with up-regulation of proteins expression of paxillin and FAK as well as increasing F-actin formation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

  7. Sequentially Adapted Parallel Feedforward Active Noise Control of Noisy Sinusoidal Signals

    Directory of Open Access Journals (Sweden)

    Govind Kannan

    2009-01-01

    Full Text Available A large class of acoustic noise sources has an underlying periodic process that generates a periodic noise component, and thus their acoustic noise can in general be modeled as the sum of a periodic signal and a randomly fluctuating signal (usually a broadband background noise. Active control of periodic noise (i.e., for a mixture of sinusoids is more effective than that of random noise. For mixtures of sinusoids in a background broadband random noise, conventional FXLMS-based single filter method does not reach the maximum achievable Noise Attenuation Level (NALmax⁡. In this paper, an alternative approach is taken and the idea of a parallel active noise control (ANC architecture for cancelling mixtures of periodic and random signals is presented. The proposed ANC system separates the noise into periodic and random components and generates corresponding antinoises via separate noise cancelling filters, and tends to reach NALmax⁡ consistently. The derivation of NALmax⁡ is presented. Both the separation and noise cancellation are based on adaptive filtering. Experimental results verify the analytical development by showing superior performance of the proposed method, over the single-filter approach, for several cases of sinusoids in white noise.

  8. Activated WNT signaling in postnatal SOX2-positive dental stem cells can drive odontoma formation.

    Science.gov (United States)

    Xavier, Guilherme M; Patist, Amanda L; Healy, Chris; Pagrut, Ankita; Carreno, Gabriela; Sharpe, Paul T; Martinez-Barbera, Juan Pedro; Thavaraj, Selvam; Cobourne, Martyn T; Andoniadou, Cynthia L

    2015-09-28

    In common with most mammals, humans form only two dentitions during their lifetime. Occasionally, supernumerary teeth develop in addition to the normal complement. Odontoma represent a small group of malformations containing calcified dental tissues of both epithelial and mesenchymal origin, with varying levels of organization, including tooth-like structures. The specific cell type responsible for the induction of odontoma, which retains the capacity to re-initiate de novo tooth development in postnatal tissues, is not known. Here we demonstrate that aberrant activation of WNT signaling by expression of a non-degradable form of β-catenin specifically in SOX2-positive postnatal dental epithelial stem cells is sufficient to generate odontoma containing multiple tooth-like structures complete with all dental tissue layers. Genetic lineage-tracing confirms that odontoma form in a similar manner to normal teeth, derived from both the mutation-sustaining epithelial stem cells and adjacent mesenchymal tissues. Activation of the WNT pathway in embryonic SOX2-positive progenitors results in ectopic expression of secreted signals that promote odontogenesis throughout the oral cavity. Significantly, the inductive potential of epithelial dental stem cells is retained in postnatal tissues, and up-regulation of WNT signaling specifically in these cells is sufficient to promote generation and growth of ectopic malformations faithfully resembling human odontoma.

  9. Saturated fatty acids activate ERK signaling to downregulate hepatic sortilin 1 in obese and diabetic mice.

    Science.gov (United States)

    Bi, Lipeng; Chiang, John Y L; Ding, Wen-Xing; Dunn, Winston; Roberts, Benjamin; Li, Tiangang

    2013-10-01

    Hepatic VLDL overproduction is a characteristic feature of diabetes and an important contributor to diabetic dyslipidemia. Hepatic sortilin 1 (Sort1), a cellular trafficking receptor, is a novel regulator of plasma lipid metabolism and reduces plasma cholesterol and triglycerides by inhibiting hepatic apolipoprotein B production. Elevated circulating free fatty acids play key roles in hepatic VLDL overproduction and the development of dyslipidemia. This study investigated the regulation of hepatic Sort1 in obesity and diabetes and the potential implications in diabetic dyslipidemia. Results showed that hepatic Sort1 protein was markedly decreased in mouse models of type I and type II diabetes and in human individuals with obesity and liver steatosis, whereas increasing hepatic Sort1 expression reduced plasma cholesterol and triglycerides in mice. Mechanistic studies showed that the saturated fatty acid palmitate activated extracellular signal-regulated kinase (ERK) and inhibited Sort1 protein by mechanisms involving Sort1 protein ubiquitination and degradation. Consistently, hepatic ERK signaling was activated in diabetic mice, whereas blocking ERK signaling by an ERK inhibitor increased hepatic Sort1 protein in mice. These results suggest that increased saturated fatty acids downregulate liver Sort1 protein, which may contribute to the development of dyslipidemia in obesity and diabetes.

  10. Extraction of single-trial cortical beta oscillatory activities in EEG signals using empirical mode decomposition

    Directory of Open Access Journals (Sweden)

    Wu Chi-Hsun

    2010-06-01

    Full Text Available Abstract Background Brain oscillatory activities are stochastic and non-linearly dynamic, due to their non-phase-locked nature and inter-trial variability. Non-phase-locked rhythmic signals can vary from trial-to-trial dependent upon variations in a subject's performance and state, which may be linked to fluctuations in expectation, attention, arousal, and task strategy. Therefore, a method that permits the extraction of the oscillatory signal on a single-trial basis is important for the study of subtle brain dynamics, which can be used as probes to study neurophysiology in normal brain and pathophysiology in the diseased. Methods This paper presents an empirical mode decomposition (EMD-based spatiotemporal approach to extract neural oscillatory activities from multi-channel electroencephalograph (EEG data. The efficacy of this approach manifests in extracting single-trial post-movement beta activities when performing a right index-finger lifting task. In each single trial, an EEG epoch recorded at the channel of interest (CI was first separated into a number of intrinsic mode functions (IMFs. Sensorimotor-related oscillatory activities were reconstructed from sensorimotor-related IMFs chosen by a spatial map matching process. Post-movement beta activities were acquired by band-pass filtering the sensorimotor-related oscillatory activities within a trial-specific beta band. Signal envelopes of post-movement beta activities were detected using amplitude modulation (AM method to obtain post-movement beta event-related synchronization (PM-bERS. The maximum amplitude in the PM-bERS within the post-movement period was subtracted by the mean amplitude of the reference period to find the single-trial beta rebound (BR. Results The results showed single-trial BRs computed by the current method were significantly higher than those obtained from conventional average method (P Conclusions The EMD-based method is effective for artefact removal and extracting

  11. Time-dependent regulation of muscle caveolin activation and insulin signalling in response to high-fat diet.

    Science.gov (United States)

    Gómez-Ruiz, Ana; de Miguel, Carlos; Campión, Javier; Martínez, J Alfredo; Milagro, Fermín I

    2009-10-06

    We studied the effect of high-fat diet on the expression and activation of the three caveolins in rat skeletal muscle and their association with the insulin signalling cascade. Initial response was characterized by increased signalling through Cav-1 and Cav-3 phosphorylation, suggesting that both participate in an initial acute response to the calorie surplus. Afterwards, Cav-1 signalling was slightly reduced, whereas Cav-3 remained active. Late chronic phase signalling through both proteins was impaired inducing a prediabetic state. Summarizing, caveolins seem to mediate a time-dependent regulation of insulin cascade in response to high-fat diet in muscle.

  12. Constitutive activation of BMP signalling abrogates experimental metastasis of OVCA429 cells via reduced cell adhesion

    Directory of Open Access Journals (Sweden)

    Shepherd Trevor G

    2010-02-01

    Full Text Available Abstract Background Activation of bone morphogenetic protein (BMP4 signalling in human ovarian cancer cells induces a number of phenotypic changes in vitro, including altered cell morphology, adhesion, motility and invasion, relative to normal human ovarian surface epithelial cells. From these in vitro analyses, we had hypothesized that active BMP signalling promotes the metastatic potential of ovarian cancer. Methods To test this directly, we engineered OVCA429 human ovarian cancer cells possessing doxycycline-inducible expression of a constitutively-active mutant BMP receptor, ALK3QD, and administered these cells to immunocompromised mice. Further characterization was performed in vitro to address the role of activated BMP signalling on the EOC phenotype, with particular emphasis on epithelial-mesenchymal transition (EMT and cell adhesion. Results Unexpectedly, doxycycline-induced ALK3QD expression in OVCA429 cells reduced tumour implantation on peritoneal surfaces and ascites formation when xenografted into immunocompromised mice by intraperitoneal injection. To determine the potential mechanisms controlling this in vivo observation, we followed with several cell culture experiments. Doxycycline-induced ALK3QD expression enhanced the refractile, spindle-shaped morphology of cultured OVCA429 cells eliciting an EMT-like response. Using in vitro wound healing assays, we observed that ALK3QD-expressing cells migrated with long, cytoplasmic projections extending into the wound space. The phenotypic alterations of ALK3QD-expressing cells correlated with changes in specific gene expression patterns of EMT, including increased Snail and Slug and reduced E-cadherin mRNA expression. In addition, ALK3QD signalling reduced β1- and β3-integrin expression, critical molecules involved in ovarian cancer cell adhesion. The combination of reduced E-cadherin and β-integrin expression correlates directly with the reduced EOC cell cohesion in spheroids and

  13. FK866-induced NAMPT inhibition activates AMPK and downregulates mTOR signaling in hepatocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Schuster, Susanne, E-mail: Susanne.Schuster@medizin.uni-leipzig.de [Center for Pediatric Research Leipzig, University Hospital for Children and Adolescents, Faculty of Medicine, University of Leipzig, Liebigstr. 21, 04103 Leipzig (Germany); Penke, Melanie; Gorski, Theresa [Center for Pediatric Research Leipzig, University Hospital for Children and Adolescents, Faculty of Medicine, University of Leipzig, Liebigstr. 21, 04103 Leipzig (Germany); Gebhardt, Rolf [Institute of Biochemistry, Faculty of Medicine, University of Leipzig, Johannisallee 30, 04103 Leipzig (Germany); Weiss, Thomas S. [Children' s University Hospital, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg (Germany); Kiess, Wieland; Garten, Antje [Center for Pediatric Research Leipzig, University Hospital for Children and Adolescents, Faculty of Medicine, University of Leipzig, Liebigstr. 21, 04103 Leipzig (Germany)

    2015-03-06

    Background: Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently, NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here, we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1 (mTORC1) signaling. Results: FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells, Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN), the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hepatocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPKα activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 significantly activated AMPKα and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment. Conclusion: Taken together, these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a potential treatment option for HCC. - Highlights: • FK866 increases cell death in p53-deficient hepatocarcinoma cells. • AMPK/mTOR signaling is dysregulated in hepatocarcinoma cells. • FK866-induced NAMPT inhibition activates AMPK

  14. A superstructure-based electrochemical assay for signal-amplified detection of DNA methyltransferase activity.

    Science.gov (United States)

    Zhang, Hui; Yang, Yin; Dong, Huilei; Cai, Chenxin

    2016-12-15

    DNA methyltransferase (MTase) activity is highly correlated with the occurrence and development of cancer. This work reports a superstructure-based electrochemical assay for signal-amplified detection of DNA MTase activity using M.SssI as an example. First, low-density coverage of DNA duplexes on the surface of the gold electrode was achieved by immobilized mercaptohexanol, followed by immobilization of DNA duplexes. The duplex can be cleaved by BstUI endonuclease in the absence of DNA superstructures. However, the cleavage is blocked after the DNA is methylated by M.SssI. The DNA superstructures are formed with the addition of helper DNA. By using an electroactive complex, RuHex, which can bind to DNA double strands, the activity of M.SssI can be quantitatively detected by differential pulse voltammetry. Due to the high site-specific cleavage by BstUI and signal amplification by the DNA superstructure, the biosensor can achieve ultrasensitive detection of DNA MTase activity down to 0.025U/mL. The method can be used for evaluation and screening of the inhibitors of MTase, and thus has potential in the discovery of methylation-related anticancer drugs.

  15. Malt1 ubiquitination triggers NF-kappaB signaling upon T-cell activation.

    Science.gov (United States)

    Oeckinghaus, Andrea; Wegener, Elmar; Welteke, Verena; Ferch, Uta; Arslan, Seda Cöl; Ruland, Jürgen; Scheidereit, Claus; Krappmann, Daniel

    2007-11-14

    Triggering of antigen receptors on lymphocytes is critical for initiating adaptive immune response against pathogens. T-cell receptor (TCR) engagement induces the formation of the Carma1-Bcl10-Malt1 (CBM) complex that is essential for activation of the IkappaB kinase (IKK)/NF-kappaB pathway. However, the molecular mechanisms that link CBM complex formation to IKK activation remain unclear. Here we report that Malt1 is polyubiquitinated upon T-cell activation. Ubiquitin chains on Malt1 provide a docking surface for the recruitment of the IKK regulatory subunit NEMO/IKKgamma. TRAF6 associates with Malt1 in response to T-cell activation and can function as an E3 ligase for Malt1 in vitro and in vivo, mediating lysine 63-linked ubiquitination of Malt1. Multiple lysine residues in the C-terminus of Malt1 serve as acceptor sites for the assembly of polyubiquitin chains. Malt1 mutants that lack C-terminal ubiquitin acceptor lysines are impaired in rescuing NF-kappaB signaling and IL-2 production in Malt1-/- T cells. Thus, our data demonstrate that induced Malt1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing TCR signals to the canonical NF-kappaB pathway.

  16. Genetic and logic networks with the signal-inhibitor-activator structure are dynamically robust

    Institute of Scientific and Technical Information of China (English)

    LI Fangting; TAN Ning

    2006-01-01

    The proteins, DNA and RNA interaction networks govern various biological functions in living cells, these networks should be dynamically robust in the intracellular and environmental fluctuations. Here, we use Boolean network to study the robust structure of both genetic and logic networks. First, SOS network in bacteria E. coli, which regulates cell survival and repair after DNA damage, is shown to be dynamically robust. Comparing with cell cycle network in budding yeast and flagella network in E. coli, we find the signal-inhibitor-activator (SIA) structure in transcription regulatory networks. Second, under the dynamical rule that inhibition is much stronger than activation, we have searched 3-node non-self-loop logical networks that are dynamically robust, and that if the attractive basin of a final attractor is as large as seven, and the final attractor has only one active node, then the active node acts as inhibitor, and the SIA and signal-inhibitor (SI) structures are fundamental architectures of robust networks. SIA and SI networks with dynamic robustness against environment uncertainties may be selected and maintained over the course of evolution, rather than blind trial-error testing and be ing an accidental consequence of particular evolutionary history. SIA network can perform a more complex process than SI network, andSIA might be used to design robust artificial genetic network. Our results provide dynamical support for why the inhibitors and SIA/SI structures are frequently employed in cellular regulatory networks.

  17. Salidroside Suppresses HUVECs Cell Injury Induced by Oxidative Stress through Activating the Nrf2 Signaling Pathway.

    Science.gov (United States)

    Zhu, Yao; Zhang, Ya-Jie; Liu, Wei-Wei; Shi, Ai-Wu; Gu, Ning

    2016-08-09

    Oxidative stress plays an important role in the pathogenesis of cardiovascular diseases. Salidroside (SAL), one of the main effective constituents of Rhodiola rosea, has been reported to suppress oxidative stress-induced cardiomyocyte injury and necrosis by promoting transcription of nuclear factor E2-related factor 2 (Nrf2)-regulated genes such as heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase (quinone1) (NQO1). However, it has not been indicated whether SAL might ameliorate endothelial injury induced by oxidative stress. Here, our study demonstrated that SAL might suppress HUVEC cell injury induced by oxidative stress through activating the Nrf2 signaling pathway. The results of our study indicated that SAL decreased the levels of intercellular reactive oxygen species (ROS) and malondialdehyde (MDA), and improved the activities of superoxide dismutase (SOD) and catalase (CAT), resulting in protective effects against oxidative stress-induced cell damage in HUVECs. It suppressed oxidative stress damage by inducing Nrf2 nuclear translocation and activating the expression of Nrf2-regulated antioxidant enzyme genes such as HO-1 and NQO1 in HUVECs. Knockdown of Nrf2 with siRNA abolished the cytoprotective effects against oxidative stress, decreased the expression of Nrf2, HO-1, and NQO1, and inhibited the nucleus translocation of Nrf2 in HUVECs. This study is the first to demonstrate that SAL suppresses HUVECs cell injury induced by oxidative stress through activating the Nrf2 signaling pathway.

  18. Salidroside Suppresses HUVECs Cell Injury Induced by Oxidative Stress through Activating the Nrf2 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Yao Zhu

    2016-08-01

    Full Text Available Oxidative stress plays an important role in the pathogenesis of cardiovascular diseases. Salidroside (SAL, one of the main effective constituents of Rhodiola rosea, has been reported to suppress oxidative stress-induced cardiomyocyte injury and necrosis by promoting transcription of nuclear factor E2-related factor 2 (Nrf2-regulated genes such as heme oxygenase-1 (HO-1 and NAD(PH dehydrogenase (quinone1 (NQO1. However, it has not been indicated whether SAL might ameliorate endothelial injury induced by oxidative stress. Here, our study demonstrated that SAL might suppress HUVEC cell injury induced by oxidative stress through activating the Nrf2 signaling pathway. The results of our study indicated that SAL decreased the levels of intercellular reactive oxygen species (ROS and malondialdehyde (MDA, and improved the activities of superoxide dismutase (SOD and catalase (CAT, resulting in protective effects against oxidative stress-induced cell damage in HUVECs. It suppressed oxidative stress damage by inducing Nrf2 nuclear translocation and activating the expression of Nrf2-regulated antioxidant enzyme genes such as HO-1 and NQO1 in HUVECs. Knockdown of Nrf2 with siRNA abolished the cytoprotective effects against oxidative stress, decreased the expression of Nrf2, HO-1, and NQO1, and inhibited the nucleus translocation of Nrf2 in HUVECs. This study is the first to demonstrate that SAL suppresses HUVECs cell injury induced by oxidative stress through activating the Nrf2 signaling pathway.

  19. Ligand Perception, Activation, and Early Signaling of Plant Steroid Receptor Brassinosteroid Insensitive 1

    Institute of Scientific and Technical Information of China (English)

    Jianjun Jiang; Chi Zhang; Xuelu Wang

    2013-01-01

    Leucine-rich repeat receptor-like kinases (LRR-RLKs) belong to a large group of cell surface proteins involved in many aspects of plant development and environmental responses in both monocots and dicots. Brassinosteroid insensitive 1 (BRI1), a member of the LRR X subfamily, was first identified through several forward genetic screenings for mutants insensitive to brassinosteroids (BRs), which are a class of plant-specific steroid hormones. Since its identification, BRI1 and its homologs had been proved as receptors perceiving BRs and initiating BR signaling. The co-receptor BRI1-associated kinase 1 and its homologs, and other BRI1 interacting proteins such as its inhibitor BRI1 kinase inhibitor 1 (BKI1) were identified by genetic and biochemical approaches. The detailed mechanisms of BR perception by BRI1 and the activation of BRI1 receptor complex have also been elucidated. Moreover, several mechanisms for termination of the activated BRI1 signaling were also discovered. In this review, we will focus on the recent advances on the mechanism of BRI1 phosphorylation and activation, the regulation of its receptor complex, the structure basis of BRI1 ectodomain and BR recognition, its direct substrates, and the termination of the activated BRI1 receptor complex.

  20. A model for signal processing and predictive control of semi-active structural control system

    Indian Academy of Sciences (India)

    M-H Shih; W-P Sung; Ching-Jong Wang

    2009-06-01

    The theory for structural control has been well developed and applied to perform excellent energy dissipation using dampers. Both active and semi-active control systems may be used to decide on the optimal switch point of the damper based on the current and past structural responses to the excitation of external forces. However, numerous noises may occur when the control signals are accessed and transported thus causing a delay of the damper. Therefore, a predictive control technique that integrates an improved method of detecting the control signal based on the direction of the structural motion, and a calculator for detecting the velocity using the least-square polynomial regression is proposed in this research. Comparisons of the analytical data and experimental results show that this predictor is effective in switching the moving direction of the semi-active damper. This conclusion is further verified using the component and shaking table test with constant amplitude but various frequencies, and the El Centro earthquake test. All tests confirm that this predictive control technique is effective to alleviate the time delay problem of semi-active dampers. This predictive control technique promotes about 30% to 40% reduction of the structural displacement response and about 35% to 45% reduction of the structural acceleration response.

  1. A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis.

    Science.gov (United States)

    Kular, Jasreen; Scheer, Kaitlin G; Pyne, Natasha T; Allam, Amr H; Pollard, Anthony N; Magenau, Astrid; Wright, Rebecca L; Kolesnikoff, Natasha; Moretti, Paul A; Wullkopf, Lena; Stomski, Frank C; Cowin, Allison J; Woodcock, Joanna M; Grimbaldeston, Michele A; Pitson, Stuart M; Timpson, Paul; Ramshaw, Hayley S; Lopez, Angel F; Samuel, Michael S

    2015-12-21

    ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities.

  2. β3 integrin-mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner.

    Science.gov (United States)

    Fourel, Laure; Valat, Anne; Faurobert, Eva; Guillot, Raphael; Bourrin-Reynard, Ingrid; Ren, Kefeng; Lafanechère, Laurence; Planus, Emmanuelle; Picart, Catherine; Albiges-Rizo, Corinne

    2016-03-14

    Understanding how cells integrate multiple signaling pathways to achieve specific cell differentiation is a challenging question in cell biology. We have explored the physiological presentation of BMP-2 by using a biomaterial that harbors tunable mechanical properties to promote localized BMP-2 signaling. We show that matrix-bound BMP-2 is sufficient to induce β3 integrin-dependent C2C12 cell spreading by overriding the soft signal of the biomaterial and impacting actin organization and adhesion site dynamics. In turn, αvβ3 integrin is required to mediate BMP-2-induced Smad signaling through a Cdc42-Src-FAK-ILK pathway. β3 integrin regulates a multistep process to control first BMP-2 receptor activity and second the inhibitory role of GSK3 on Smad signaling. Overall, our results show that BMP receptors and β3 integrin work together to control Smad signaling and tensional homeostasis, thereby coupling cell adhesion and fate commitment, two fundamental aspects of developmental biology and regenerative medicine.

  3. Chemerin Stimulates Vascular Smooth Muscle Cell Proliferation and Carotid Neointimal Hyperplasia by Activating Mitogen-Activated Protein Kinase Signaling

    Science.gov (United States)

    Xiong, Wei; Luo, Yu; Wu, Lin; Liu, Feng; Liu, Huadong; Li, Jianghua; Liao, Bihong; Dong, Shaohong

    2016-01-01

    Vascular neointimal hyperplasia and remodeling arising from local inflammation are characteristic pathogeneses of proliferative cardiovascular diseases, such as atherosclerosis and post angioplasty restenosis. The molecular mechanisms behind these pathological processes have not been fully determined. The adipokine chemerin is associated with obesity, metabolism, and control of inflammation. Recently, chemerin has gained increased attention as it was found to play a critical role in the development of cardiovascular diseases. In this study, we investigated the effects of chemerin on the regulation of vascular smooth muscle cells and carotid neointimal formation after angioplasty. We found that circulating chemerin levels increased after carotid balloon injury, and that knockdown of chemerin significantly inhibited the proliferative aspects of vascular smooth muscle cells induced by platelet-derived growth factor-BB and pro-inflammatory chemokines in vitro as well as prohibited carotid neointimal hyperplasia and pro-inflammatory chemokines in vivo after angioplasty. Additionally, inhibition of chemerin down-regulated the expression of several proteins, including phosphorylated p38 mitogen-activated protein kinase, phosphorylated extracellular signal regulated kinase 1/2, nuclear factor-kappa B p65, and proliferation cell nuclear antigen. The novel finding of this study is that chemerin stimulated vascular smooth muscle cells proliferation and carotid intimal hyperplasia through activation of the mitogen-activated protein kinase signaling pathway, which may lead to vascular inflammation and remodeling, and is relevant to proliferative cardiovascular diseases. PMID:27792753

  4. Matriptase activation connects tissue factor-dependent coagulation initiation to epithelial proteolysis and signaling.

    Science.gov (United States)

    Le Gall, Sylvain M; Szabo, Roman; Lee, Melody; Kirchhofer, Daniel; Craik, Charles S; Bugge, Thomas H; Camerer, Eric

    2016-06-23

    The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation.

  5. Activation of Phosphotyrosine Phosphatase Activity Attenuates Mitogen-Activated Protein Kinase Signaling and Inhibits c-FOS and Nitric Oxide Synthase Expression in Macrophages Infected with Leishmania donovani

    OpenAIRE

    Nandan, Devki; Lo, Raymond; Reiner, Neil E

    1999-01-01

    Intracellular protozoan parasites of the genus Leishmania antagonize host defense mechanisms by interfering with cell signaling in macrophages. In this report, the impact of Leishmania donovani on mitogen-activated protein (MAP) kinases and nitric oxide synthase (NOS) expression in the macrophage cell line RAW 264 was investigated. Overnight infection of cells with leishmania led to a significant decrease in phorbol-12-myristate-13-acetate (PMA)-stimulated MAP kinase activity and inhibited PM...

  6. PFOS induces adipogenesis and glucose uptake in association with activation of Nrf2 signaling pathway

    Science.gov (United States)

    Xu, Jialin; Shimpi, Prajakta; Armstrong, Laura; Salter, Deanna; Slitt, Angela L.

    2016-01-01

    PFOS is a chemical of nearly ubiquitous exposure in humans. Recent studies have associated PFOS exposure to adipose tissue-related effects. The present study was to determine whether PFOS alters the process of adipogenesis and regulates insulin-stimulated glucose uptake in mouse and human preadipocytes. In murine-derived 3T3-L1 preadipocytes, PFOS enhanced hormone-induced differentiation to adipocytes and adipogenic gene expression, increased insulin-stimulated glucose uptake at concentrations ranging from 10 to 100 µM, and enhanced Glucose transporter type 4 and Insulin receptor substrate-1 expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), NAD(P)H dehydrogenase, quinone 1 and Glutamate-cysteine ligase, catalytic subunit were significantly induced in 3T3-L1 cells treated with PFOS, along with a robust induction of Antioxidant Response Element (ARE) reporter in mouse embryonic fibroblasts isolated from ARE-hPAP transgenic mice by PFOS treatment. Chromatin immunoprecipitation assays further illustrated that PFOS increased Nrf2 binding to ARE sites in mouse Nqo1 promoter, suggesting that PFOS activated Nrf2 signaling in murine-derived preadipocytes. Additionally, PFOS administration in mice (100 µg/kg/day) induced adipogenic gene expression and activated Nrf2 signaling in epididymal white adipose tissue. Moreover, the treatment on human visceral preadipocytes illustrated that PFOS (5 and 50 µM) promoted adipogenesis and increased cellular lipid accumulation. It was observed that PFOS increased Nrf2 binding to ARE sites in association with Nrf2 signaling activation, induction of Peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α expression, and increased adipogenesis. This study points to a potential role PFOS in dysregulation of adipose tissue expandability, and warrants further investigations on the adverse effects of persistent pollutants on human health. PMID:26548598

  7. Generalized proportional integral control for periodic signals under active disturbance rejection approach.

    Science.gov (United States)

    Cortés-Romero, John; Ramos, Germán A; Coral-Enriquez, Horacio

    2014-11-01

    Conventional repetitive control has proven to be an effective strategy to reject/track periodic signals with constant frequency; however, it shows poor performance in varying frequency applications. This paper proposes an active disturbance rejection methodology applied to a large class of uncertain flat systems for the tracking and rejection of periodic signals, in which the possibilities of the generalized proportional integral (GPI) observer-based control to address repetitive control problems are studied. In the proposed scheme, model uncertainties and external disturbances are lumped together in a general additive disturbance input that is estimated and rejected on-line. An illustrative case study of mechatronic nature is considered. Experimental results show that the proposed GPI observer-based control successfully rejects periodic disturbances even under varying speed conditions.

  8. Effects of activated ACM on expression of signal transducers in cerebral cortical neurons of rats.

    Science.gov (United States)

    Wang, Xiaojing; Li, Zhengli; Zhu, Changgeng; Li, Zhongyu

    2007-06-01

    To explore the roles of astrocytes in the epileptogenesis, astrocytes and neurons were isolated, purified and cultured in vitro from cerebral cortex of rats. The astrocytes were activated by ciliary neurotrophic factor (CNTF) and astrocytic conditioned medium (ACM) was collected to treat neurons for 4, 8 and 12 h. By using Western blot, the expression of calmodulin dependent protein kinase II (CaMK II), inducible nitric oxide synthase (iNOS) and adenylate cyclase (AC) was detected in neurons. The results showed that the expression of CaMK II, iNOS and AC was increased significantly in the neurons treated with ACM from 4 h to 12 h (PACM and such signal pathways as NOS-NO-cGMP, Ca2+/CaM-CaMK II and AC-cAMP-PKA might take part in the signal transduction of epileptogenesis.

  9. The cytotoxic and proapoptotic activities of hypnophilin are associated with calcium signaling in UACC-62 cells.

    Science.gov (United States)

    Pinto, Mauro C X; Cota, Betania B; Rodrigues, Michele A; Leite, Maria F; de Souza-Fagundes, Elaine M

    2013-11-01

    Hypnophilin (HNP) is a sesquiterpene that is isolated from Lentinus cf. strigosus and has cytotoxic activities. Here, we studied the calcium signaling and cytotoxic effects of HNP in UACC-62 cells, a human skin melanoma cell line. HNP was able to increase the intracellular calcium concentration in UACC-62 cells, which was blocked in cells stimulated in Ca(2+) -free media. HNP treatment with BAPTA-AM, an intracellular Ca(2+) chelator, caused an increase in calcium signals. HNP showed cytotoxicity against UACC-62 cells in which it induced DNA fragmentation and morphological alterations, including changes in the nuclear chromatin profile and increased cytoplasmatic vacuolization, but it had no effect on the plasma membrane integrity. These data suggest that cytotoxicity in UACC-62 cells, after treatment with HNP, is associated with Ca(2+) influx. Together, these findings suggest that HNP is a relevant tool for the further investigation of new anticancer approaches.

  10. Circadian period integrates network information through activation of the BMP signaling pathway.

    Directory of Open Access Journals (Sweden)

    Esteban J Beckwith

    2013-12-01

    Full Text Available Living organisms use biological clocks to maintain their internal temporal order and anticipate daily environmental changes. In Drosophila, circadian regulation of locomotor behavior is controlled by ∼150 neurons; among them, neurons expressing the PIGMENT DISPERSING FACTOR (PDF set the period of locomotor behavior under free-running conditions. To date, it remains unclear how individual circadian clusters integrate their activity to assemble a distinctive behavioral output. Here we show that the BONE MORPHOGENETIC PROTEIN (BMP signaling pathway plays a crucial role in setting the circadian period in PDF neurons in the adult brain. Acute deregulation of BMP signaling causes period lengthening through regulation of dClock transcription, providing evidence for a novel function of this pathway in the adult brain. We propose that coherence in the circadian network arises from integration in PDF neurons of both the pace of the cell-autonomous molecular clock and information derived from circadian-relevant neurons through release of BMP ligands.

  11. lschemic preconditioning in pigs: a causal role for signal transducer and activator of transcription 3.

    Science.gov (United States)

    Gent, Sabine; Skyschally, Andreas; Kleinbongard, Petra; Heusch, Gerd

    2017-03-01

    Ischemic preconditioning (IPC), i.e., brief episodes of nonlethal myocardial ischemia-reperfusion (I/R) before sustained ischemia with subsequent reperfusion, reduces infarct size in all species tested so far, including humans. In rodents, the cardioprotective signal transduction causally involves an activation of Akt, ERK1/2, and STAT3. However, there are apparent species differences in the signal transduction between rodents and larger mammals such as pigs, where data on IPC's signal transduction are inconsistent for Akt and ERK1/2. The role of STAT3 has not yet been analyzed. Pigs were subjected to 60 min of left anterior descending coronary artery occlusion and 180 min of reperfusion without or with IPC (2 cycles of 3-min occlusion separated by 2 min of reperfusion 15 min before sustained I/R). Infarct size was analyzed by triphenyl tetrazolium chloride staining, and Akt, ERK1/2, and STAT3 phosphorylation was quantified in myocardial biopsies taken at baseline and early reperfusion. AG490 was used to block the STAT3 signaling pathway. IPC reduced infarct size (%area at risk; mean ± SE, I/R, 45 ± 3 vs. IPC, 18 ± 3, P IPC. In contrast, STAT3 phosphorylation at early reperfusion was only increased with IPC (%baseline; mean ± SE, I/R, 126 ± 29 vs. IPC, 408 ± 147, P IPC-related increase of STAT3 phosphorylation at reperfusion (%baseline; mean ± SE, 82 ± 12) and abolished IPC's cardioprotection (%area at risk; mean ± SE, 35 ± 4). In pigs, increased phosphorylation of STAT3 is causally involved, whereas Akt and ERK1/2 seem to play no role in IPC's cardioprotection.NEW & NOTEWORTHY In pig hearts in situ, ischemic preconditioning (IPC) causally involves increased phosphorylation of STAT3, whereas Akt and ERK1/2 play no role for cardioprotection. The cardioprotective signal transduction of IPC is similar to that of ischemic postconditioning and remote IPC in pigs.

  12. Cisplatin selects for stem-like cells in osteosarcoma by activating Notch signaling.

    Science.gov (United States)

    Yu, Ling; Fan, Zhengfu; Fang, Shuo; Yang, Jian; Gao, Tian; Simões, Bruno M; Eyre, Rachel; Guo, Weichun; Clarke, Robert B

    2016-05-31

    Notch signaling regulates normal stem cells and is also thought to regulate cancer stem cells (CSCs). Recent data indicate that Notch signaling plays a role in the development and progression of osteosarcoma, however the regulation of Notch in chemo-resistant stem-like cells has not yet been fully elucidated. In this study we generated cisplatin-resistant osteosarcoma cells by treating them with sub-lethal dose of cisplatin, sufficient to induce DNA damage responses. Cisplatin-resistant osteosarcoma cells exhibited lower proliferation, enhanced spheroid formation and more mesenchymal characteristics than cisplatin-sensitive cells, were enriched for Stro-1+/CD117+ cells and showed increased expression of stem cell-related genes. A similar effect was observed in vivo, and in addition in vivo tumorigenicity was enhanced during serial transplantation. Using several publicly available datasets, we identified that Notch expression was closely associated with osteosarcoma stem cells and chemotherapy resistance. We confirmed that cisplatin-induced enrichment of osteosarcoma stem cells was mediated through Notch signaling in vitro, and immunohistochemistry showed that cleaved Notch1 (NICD1) positive cells were significantly increased in a relapsed xenograft which had received cisplatin treatment. Furthermore, pretreatment with a γ-secretase inhibitor (GSI) to prevent Notch signalling inhibited cisplatin-enriched osteosarcoma stem cell activity in vitro, including Stro-1+/CD117+ double positive cells and spheroid formation capacity. The Notch inhibitor DAPT also prevented tumor recurrence in resistant xenograft tumors. Overall, our results show that cisplatin induces the enrichment of osteosarcoma stem-like cells through Notch signaling, and targeted inactivation of Notch may be useful for the elimination of CSCs and overcoming drug resistance.

  13. HAb18G/CD147 promotes radioresistance in hepatocellular carcinoma cells: a potential role for integrin β1 signaling.

    Science.gov (United States)

    Wu, Jiao; Li, Yong; Dang, Ya-Zheng; Gao, Hong-Xiang; Jiang, Jian-Li; Chen, Zhi-Nan

    2015-02-01

    Radiotherapy has played a limited role in the treatment of hepatocellular carcinoma (HCC) due to the risk of tumor radioresistance. A previous study in our laboratory confirmed that CD147 interacts with integrin β1 and plays an important role in modulating the malignant properties of HCC cells. In this study, we further evaluated the role of CD147 in the radioresistance of HCC and as a potential target for improving radiosensitivity. Upon irradiation, the colony formation, apoptosis, cell-cycle distribution, migration, and invasion of SMMC-7721, CD147-knockout SMMC-7721, HepG2, and CD147-knockdown HepG2 cells were determined. A nude mouse xenograft model and a metastatic model of HCC were used to detect the role of CD147 in radioresistance in vivo. Deletion of HAb18G/CD147 significantly enhanced the radiosensitivity of SMMC-7721 and HepG2 cells, and knocking out HAb18G/CD147 in SMMC-7721 cells attenuated irradiation-enhanced migration and invasion. The knockout and antibody blockade of CD147 decreased the tumor growth and metastatic potentials of HCC cells under irradiation. CD147-deleted SMMC-7721 cells showed diminished levels of calpain, cleaved talin, active integrin β1, and decreased p-FAK (Tyr397) and p-Akt (Ser473) levels. FAK and PI3K inhibitors, as well as integrin β1 antibodies, increased the radiation-induced apoptosis of SMMC-7721 cells. Our data provide evidence for CD147 as an important determinant of radioresistance via the regulation of integrin β1 signaling. Inhibition of the HAb18G/CD147 integrin interaction may improve the efficiency of radiosensitivity and provide a potential new approach for HCC therapy.

  14. Huang Qi Decoction Prevents BDL-Induced Liver Fibrosis Through Inhibition of Notch Signaling Activation.

    Science.gov (United States)

    Zhang, Xiao; Xu, Ying; Chen, Jia-Mei; Liu, Cheng; Du, Guang-Li; Zhang, Hua; Chen, Gao-Feng; Jiang, Shi-Li; Liu, Cheng-Hai; Mu, Yong-Ping; Liu, Ping

    2017-01-01

    Notch signaling has been demonstrated to be involved in ductular reactions and fibrosis. Previous studies have shown that Huang Qi Decoction (HQD) can prevent the progression of cholestatic liver fibrosis (CLF). However, whether HQD affects the Notch signaling pathway is unclear. In this study, CLF was established by common bile duct ligation (BDL) in rats. At the end of the first week, the rats were randomly divided into a model group (i.e., BDL), an HQD group, and a sorafenib positive control group (SORA) and were treated for 3 weeks. Bile duct proliferation and liver fibrosis were determined by tissue staining. Activation of the Notch signaling pathway was evaluated by analyzing expressions of Notch-1, -2, -3, and -4, Jagged (JAG) 1, and Delta like (DLL)-1, -3, and -4. The results showed that HQD significantly reduced the deposition of collagen and the Hyp content of liver tissue and inhibited the activation of HSCs compared with the BDL group. In addition, HQD significantly decreased the protein and mRNA expressions of TGF-[Formula: see text]1 and [Formula: see text]-SMA. In contrast, HQD significantly enhanced expression of the Smad 7 protein. HQD also reduced biliary epithelial cell proliferation, and reduced the mRNA levels of CK7, CK8, CK18, SRY-related high mobility group-box gene (SOX) 9, epithelial cell adhesion molecule (EpCAM) and the positive areas of CK19 and OV6. In addition, the mRNA and protein expressions of Notch-3, -4, JAG1, and DLL-1, -3 were significantly reduced in the HQD compared to the BDL group. These results demonstrated that HQD may prevent biliary liver fibrosis through inhibition of the Notch signaling pathway, and it may be a potential treatment for cholestatic liver disease.

  15. Sam68 Mediates the Activation of Insulin and Leptin Signalling in Breast Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Antonio Pérez-Pérez

    Full Text Available Obesity is a well-known risk factor for breast cancer development in postmenopausal women. High insulin and leptin levels seem to have a role modulating the growth of these tumours. Sam68 is an RNA-binding protein with signalling functions that has been found to be overexpressed in breast cancer. Moreover, Sam68 may be recruited to insulin and leptin signalling pathways, mediating its effects on survival, growth and proliferation in different cellular types. We aimed to study the expression of Sam68 and its phosphorylation level upon insulin and leptin stimulation, and the role of Sam68 in the proliferative effect and signalling pathways that are activated by insulin or leptin in human breast adenocarcinoma cells. In the human breast adenocarcinoma cell lines MCF7, MDA-MB-231 and BT-474, Sam68 protein quantity and gene expression were increased upon leptin or insulin stimulation, as it was checked by qPCR and immunoblot. Moreover, both insulin and leptin stimulation promoted an increase in Sam68 tyrosine phosphorylation and negatively regulated its RNA binding capacity. siRNA was used to downregulate Sam68 expression, which resulted in lower proliferative effects of both insulin and leptin, as well as a lower activation of MAPK and PI3K pathways promoted by both hormones. These effects may be partly explained by the decrease in IRS-1 expression by down-regulation of Sam68. These results suggest the participation of Sam68 in both leptin and insulin receptor signaling in human breast cancer cells, mediating the trophic effects of these hormones in proliferation and cellular growth.

  16. Ciliary transport regulates PDGF-AA/αα signaling via elevated mammalian target of rapamycin signaling and diminished PP2A activity.

    Science.gov (United States)

    Umberger, Nicole L; Caspary, Tamara

    2015-01-15

    Primary cilia are built and maintained by intraflagellar transport (IFT), whereby the two IFT complexes, IFTA and IFTB, carry cargo via kinesin and dynein motors for anterograde and retrograde transport, respectively. Many signaling pathways, including platelet- derived growth factor (PDGF)-AA/αα, are linked to primary cilia. Active PDGF-AA/αα signaling results in phosphorylation of Akt at two residues: P-Akt(T308) and P-Akt(S473), and previous work showed decreased P-Akt(S473) in response to PDGF-AA upon anterograde transport disruption. In this study, we investigated PDGF-AA/αα signaling via P-Akt(T308) and P-Akt(S473) in distinct ciliary transport mutants. We found increased Akt phosphorylation in the absence of PDGF-AA stimulation, which we show is due to impaired dephosphorylation resulting from diminished PP2A activity toward P-Akt(T308). Anterograde transport mutants display low platelet-derived growth factor receptor (PDGFR)α levels, whereas retrograde mutants exhibit normal PDGFRα levels. Despite this, neither shows an increase in P-Akt(S473) or P-Akt(T308) upon PDGF-AA stimulation. Because mammalian target of rapamycin complex 1 (mTORC1) signaling is increased in ciliary transport mutant cells and mTOR signaling inhibits PDGFRα levels, we demonstrate that inhibition of mTORC1 rescues PDGFRα levels as well as PDGF-AA-dependent phosphorylation of Akt(S473) and Akt(T308) in ciliary transport mutant MEFs. Taken together, our data indicate that the regulation of mTORC1 signaling and PP2A activity by ciliary transport plays key roles in PDGF-AA/αα signaling.

  17. Hydrogen peroxide induces activation of insulin signaling pathway via AMP-dependent kinase in podocytes

    Energy Technology Data Exchange (ETDEWEB)

    Piwkowska, Agnieszka, E-mail: apiwkowska@cmdik.pan.pl [Mossakowski Medical Research Centre, Polish Academy of Sciences, Laboratory of Molecular and Cellular Nephrology, Gdansk (Poland); Rogacka, Dorota; Angielski, Stefan [Mossakowski Medical Research Centre, Polish Academy of Sciences, Laboratory of Molecular and Cellular Nephrology, Gdansk (Poland); Jankowski, Maciej [Mossakowski Medical Research Centre, Polish Academy of Sciences, Laboratory of Molecular and Cellular Nephrology, Gdansk (Poland); Medical University of Gdansk, Department of Therapy Monitoring and Pharmacogenetics (Poland)

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer H{sub 2}O{sub 2} activates the insulin signaling pathway and glucose uptake in podocytes. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} induces time-dependent changes in AMPK phosphorylation. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} enhances insulin signaling pathways via AMPK activation. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} stimulation of glucose uptake is AMPK-dependent. -- Abstract: Podocytes are cells that form the glomerular filtration barrier in the kidney. Insulin signaling in podocytes is critical for normal kidney function. Insulin signaling is regulated by oxidative stress and intracellular energy levels. We cultured rat podocytes to investigate the effects of hydrogen peroxide (H{sub 2}O{sub 2}) on the phosphorylation of proximal and distal elements of insulin signaling. We also investigated H{sub 2}O{sub 2}-induced intracellular changes in the distribution of protein kinase B (Akt). Western blots showed that H{sub 2}O{sub 2} (100 {mu}M) induced rapid, transient phosphorylation of the insulin receptor (IR), the IR substrate-1 (IRS1), and Akt with peak activities at 5 min ({Delta} 183%, P < 0.05), 3 min ({Delta} 414%, P < 0.05), and 10 min ({Delta} 35%, P < 0.05), respectively. Immunostaining cells with an Akt-specific antibody showed increased intensity at the plasma membrane after treatment with H{sub 2}O{sub 2}>. Furthermore, H{sub 2}O{sub 2} inhibited phosphorylation of the phosphatase and tensin homologue (PTEN; peak activity at 10 min; {Delta} -32%, P < 0.05) and stimulated phosphorylation of the AMP-dependent kinase alpha subunit (AMPK{alpha}; 78% at 3 min and 244% at 10 min). The stimulation of AMPK was abolished with an AMPK inhibitor, Compound C (100 {mu}M, 2 h). Moreover, Compound C significantly reduced the effect of H{sub 2}O{sub 2} on IR phosphorylation by about 40% (from 2.07 {+-} 0.28 to 1.28 {+-} 0.12, P < 0.05). In addition, H{sub 2}O{sub 2} increased glucose uptake in podocytes

  18. Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

    KAUST Repository

    Irving, Helen R.

    2012-02-01

    Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

  19. Gamma-secretase activity of presenilin 1 regulates acetylcholine muscarinic receptor-mediated signal transduction

    DEFF Research Database (Denmark)

    Popescu, Bogdan O; Cedazo-Minguez, Angel; Benedikz, Eirikur;

    2004-01-01

    causing an exon 9 deletion in PS1 results in enhanced basal phospholipase C (PLC) activity (Cedazo-Minguez, A., Popescu, B. O., Ankarcrona, M., Nishimura, T., and Cowburn, R. F. (2002) J. Biol. Chem. 277, 36646-36655). To further elucidate the mechanisms by which PS1 interferes with PLC-calcium signaling...... by the PLC inhibitor neomycin, the ryanodine receptor antagonist dantrolene, the general aspartyl protease inhibitor pepstatin A, and the specific gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. The cells expressing either PS1 D257A or PS1 D385N had attenuated...... or PS1 D385N dominant negative cells. Our findings suggest that PS1 can regulate PLC activity and that this function is gamma-secretase activity-dependent....

  20. Delaying or advancing higher-order sideband signals with active optomechanics

    CERN Document Server

    Jiao, Yafeng; Qian, Jun; Li, Yong; Jing, H

    2016-01-01

    We study the gain-assisted light transmissions in optomechanical systems, especially the nonlinear higher-order sideband process. We find that: (i) in a single active cavity, the efficiency of the second-order process is considerably enhanced, and the accompanying group delay can surpass that of the probe field, which is unattainable for a lossy cavity (i.e. without any gain); (ii) in an active-passive compound system, the second-order process can be further enhanced by approaching to the gain-loss balance, and hundreds of microsecond of relative delay or advance are achievable between the probe and the second-order signal, indicating an active optomechanical modulator both in frequency and time domains.

  1. A REVIEW ON INFLUENCE OF MUSIC ON BRAIN ACTIVITY USING SIGNAL PROCESSING AND IMAGING SYSTEM

    Directory of Open Access Journals (Sweden)

    Dr. K. ADALARASU,

    2011-04-01

    Full Text Available As per clinical neuroscience, listening to music involves many brain activities and its study has advanced greatly in the last thirty years. Research shows that music has significant effect on our body and mind. Music has a positive effect on the hormone system and allows the brain to concentrate more easily and assimilate more information in less time, thereby boosting learning and information intake and thus augmenting cognitive skills. Studies have found that the silence between two musical notes triggers brain cells and neurons which are responsible for the development of sharp memory. Music at different pitches (for example, Madhyamavati, Sankarabarnam raga and so on elicits exceptionally emotions and is capable ofreliably affecting the mood of individuals, which in turn changes the brain activity. This article provides a brief overview of currently available signal processing and imaging techniques to study the influence of different music on human brain activity.

  2. Characterization of rotavirus RNAs that activate innate immune signaling through the RIG-I-like receptors.

    Directory of Open Access Journals (Sweden)

    Dina Uzri

    Full Text Available In mammalian cells, the first line of defense against viral pathogens is the innate immune response, which is characterized by induction of type I interferons (IFN and other pro-inflammatory cytokines that establish an antiviral milieu both in infected cells and in neighboring uninfected cells. Rotavirus, a double-stranded RNA virus of the Reoviridae family, is the primary etiological agent of severe diarrhea in young children worldwide. Previous studies demonstrated that rotavirus replication induces a MAVS-dependent type I IFN response that involves both RIG-I and MDA5, two cytoplasmic viral RNA sensors. This study reports the isolation and characterization of rotavirus RNAs that activate IFN signaling. Using an in vitro approach with purified rotavirus double-layer particles, nascent single-stranded RNA (ssRNA transcripts (termed in vitro ssRNA were found to be potent IFN inducers. In addition, large RNAs isolated from rotavirus-infected cells six hours post-infection (termed in vivo 6 hr large RNAs, also activated IFN signaling, whereas a comparable large RNA fraction isolated from cells infected for only one hour lacked this stimulatory activity. Experiments using knockout murine embryonic fibroblasts showed that RIG-I is required for and MDA5 partly contributes to innate immune signaling by both in vitro ssRNA and in vivo 6 hr large RNAs. Enzymatic studies demonstrated that in vitro ssRNA and in vivo 6 hr large RNA samples contain uncapped RNAs with exposed 5' phosphate groups. RNAs lacking 2'-O-methylated 5' cap structures were also detected in the in vivo 6 hr large RNA sample. Taken together, our data provide strong evidence that the rotavirus VP3 enzyme, which encodes both guanylyltransferase and methyltransferase activities, is not completely efficient at either 5' capping or 2'-O-methylation of the 5' cap structures of viral transcripts, and in this way produces RNA patterns that activate innate immune signaling through the RIG

  3. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    Directory of Open Access Journals (Sweden)

    Shweta Jain

    Full Text Available Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID, signifying class switch recombination (CSR. Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  4. Synaptic network activity induces neuronal differentiation of adult hippocampal precursor cells through BDNF signaling

    Directory of Open Access Journals (Sweden)

    Harish Babu

    2009-09-01

    Full Text Available Adult hippocampal neurogenesis is regulated by activity. But how do neural precursor cells in the hippocampus respond to surrounding network activity and translate increased neural activity into a developmental program? Here we show that long-term potential (LTP-like synaptic activity within a cellular network of mature hippocampal neurons promotes neuronal differentiation of newly generated cells. In co-cultures of precursor cells with primary hippocampal neurons, LTP-like synaptic plasticity induced by addition of glycine in Mg2+-free media for 5 min, produced synchronous network activity and subsequently increased synaptic strength between neurons. Furthermore, this synchronous network activity led to a significant increase in neuronal differentiation from the co-cultured neural precursor cells. When applied directly to precursor cells, glycine and Mg2+-free solution did not induce neuronal differentiation. Synaptic plasticity-induced neuronal differentiation of precursor cells was observed in the presence of GABAergic neurotransmission blockers but was dependent on NMDA-mediated Ca2+ influx. Most importantly, neuronal differentiation required the release of brain-derived neurotrophic factor (BDNF from the underlying substrate hippocampal neurons as well as TrkB receptor phosphorylation in precursor cells. This suggests that activity-dependent stem cell differentiation within the hippocampal network is mediated via synaptically evoked BDNF signaling.

  5. Activated T cell exosomes promote tumor invasion via Fas signaling pathway.

    Science.gov (United States)

    Cai, Zhijian; Yang, Fei; Yu, Lei; Yu, Zhou; Jiang, Lingling; Wang, Qingqing; Yang, Yunshan; Wang, Lie; Cao, Xuetao; Wang, Jianli

    2012-06-15

    Activated T cells release bioactive Fas ligand (FasL) in exosomes, which subsequently induce self-apoptosis of T cells. However, their potential effects on cell apoptosis in tumors are still unknown. In this study, we purified exosomes expressing FasL from activated CD8(+) T cell from OT-I mice and found that activated T cell exosomes had little effect on apoptosis and proliferation of tumor cells but promoted the invasion of B16 and 3LL cancer cells in vitro via the Fas/FasL pathway. Activated T cell exosomes increased the amount of cellular FLICE inhibitory proteins and subsequently activated the ERK and NF-κB pathways, which subsequently increased MMP9 expression in the B16 murine melanoma cells. In a tumor-invasive model in vivo, we observed that the activated T cell exosomes promoted the migration of B16 tumor cells to lung. Interestingly, pretreatment with FasL mAb significantly reduced the migration of B16 tumor cells to lung. Furthermore, CD8 and FasL double-positive exosomes from tumor mice, but not normal mice, also increased the expression of MMP9 and promoted the invasive ability of B16 murine melanoma and 3LL lung cancer cells. In conclusion, our results indicate that activated T cell exosomes promote melanoma and lung cancer cell metastasis by increasing the expression of MMP9 via Fas signaling, revealing a new mechanism of tumor immune escape.

  6. Methamphetamine-induced neurotoxicity and microglial activation are not mediated by fractalkine receptor signaling.

    Science.gov (United States)

    Thomas, David M; Francescutti-Verbeem, Dina M; Kuhn, Donald M

    2008-07-01

    Methamphetamine (METH) damages dopamine (DA) nerve endings by a process that has been linked to microglial activation but the signaling pathways that mediate this response have not yet been delineated. Cardona et al. [Nat. Neurosci. 9 (2006), 917] recently identified the microglial-specific fractalkine receptor (CX3CR1) as an important mediator of MPTP-induced neurodegeneration of DA neurons. Because the CNS damage caused by METH and MPTP is highly selective for the DA neuronal system in mouse models of neurotoxicity, we hypothesized that the CX3CR1 plays a role in METH-induced neurotoxicity and microglial activation. Mice in which the CX3CR1 gene has been deleted and replaced with a cDNA encoding enhanced green fluorescent protein (eGFP) were treated with METH and examined for striatal neurotoxicity. METH depleted DA, caused microglial activation, and increased body temperature in CX3CR1 knockout mice to the same extent and over the same time course seen in wild-type controls. The effects of METH in CX3CR1 knockout mice were not gender-dependent and did not extend beyond the striatum. Striatal microglia expressing eGFP constitutively show morphological changes after METH that are characteristic of activation. This response was restricted to the striatum and contrasted sharply with unresponsive eGFP-microglia in surrounding brain areas that are not damaged by METH. We conclude from these studies that CX3CR1 signaling does not modulate METH neurotoxicity or microglial activation. Furthermore, it appears that striatal-resident microglia respond to METH with an activation cascade and then return to a surveying state without undergoing apoptosis or migration.

  7. The virulence regulator Sae of Staphylococcus aureus: promoter activities and response to phagocytosis-related signals.

    Science.gov (United States)

    Geiger, Tobias; Goerke, Christiane; Mainiero, Markus; Kraus, Dirk; Wolz, Christiane

    2008-05-01

    The two-component system SaeRS of Staphylococcus aureus is closely involved in the regulation of major virulence factors. However, little is known about the signals leading to saeRS activation. A total of four overlapping transcripts (T1 to T4) from three different transcription starting points are expressed in the sae operon. We used a beta-galactosidase reporter assay to characterize the putative promoter regions within the saeRS upstream region. The main transcript T2 is probably generated by endoribonucleolytic processing of the T1 transcript. Only two distinct promoter elements (P1 and P3) could be detected within the saeRS upstream region. The P3 promoter, upstream of saeRS, generates the T3 transcript, includes a cis-acting enhancer element and is repressed by saeRS. The most distal P1 promoter is strongly autoregulated, activated by agr, and repressed by sigma factor B. In strain Newman a mutation within the histidine kinase SaeS leads to a constitutively activated sae system. Evaluation of different external signals revealed that the P1 promoter in strain ISP479R and strain UAMS-1 is inhibited by low pH and high NaCl concentrations but activated by hydrogen peroxide. The most prominent induction of P1 was observed at subinhibitory concentrations of alpha-defensins in various S. aureus strains, with the exception of strain ISP479R and strain COL. P1 was not activated by the antimicrobial peptides LL37 and daptomycin. In summary, the results indicate that the sensor molecule SaeS is activated by alteration within the membrane allowing the pathogen to react to phagocytosis related effector molecules.

  8. Opposing actions of endocannabinoids on cholangiocarcinoma growth is via the differential activation of Notch signaling

    Energy Technology Data Exchange (ETDEWEB)

    Frampton, Gabriel; Coufal, Monique [Department of Internal Medicine, Texas A and M Health Science Center College of Medicine, Temple, TX (United States); Li, Huang [Department of Internal Medicine, Texas A and M Health Science Center College of Medicine, Temple, TX (United States); Department of Hepatobiliary Surgery, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou (China); Ramirez, Jonathan [Digestive Disease Research Center, Scott and White Hospital, Temple, TX (United States); DeMorrow, Sharon, E-mail: demorrow@medicine.tamhsc.edu [Department of Internal Medicine, Texas A and M Health Science Center College of Medicine, Temple, TX (United States); Digestive Disease Research Center, Scott and White Hospital, Temple, TX (United States)

    2010-05-15

    The endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2-AG) have opposing effects on cholangiocarcinoma growth. Implicated in cancer, Notch signaling requires the {gamma}-secretase complex for activation. The aims of this study were to determine if the opposing effects of endocannabinoids depend on the differential activation of the Notch receptors and to demonstrate that the differential activation of these receptors are due to presenilin 1 containing- and presenilin 2 containing-{gamma}-secretase complexes. Mz-ChA-1 cells were treated with AEA or 2-AG. Notch receptor expression, activation, and nuclear translocation were determined. Specific roles for Notch 1 and 2 on cannabinoid-induced effects were determined by transient transfection of Notch 1 or 2 shRNA vectors before stimulation with AEA or 2-AG. Expression of presenilin 1 and 2 was determined after AEA or 2-AG treatment, and the involvement of presenilin 1 and 2 in the cannabinoid-induced effects was demonstrated in cell lines with low presenilin 1 or 2 expression. Antiproliferative effects of AEA required increased Notch 1 mRNA, activation, and nuclear translocation, whereas the growth-promoting effects induced by 2-AG required increased Notch 2 mRNA expression, activation, and nuclear translocation. AEA increased presenilin 1 expression and recruitment into the {gamma}-secretase complex, whereas 2-AG increased expression and recruitment of presenilin 2. The development of novel therapeutic strategies aimed at modulating the endocannabinoid system or mimicking the mode of action of AEA on Notch signaling pathways would prove beneficial for cholangiocarcinoma management.

  9. Small molecule activators of SIRT1 replicate signaling pathways triggered by calorie restriction in vivo

    Directory of Open Access Journals (Sweden)

    Lavu Siva

    2009-03-01

    Full Text Available Abstract Background Calorie restriction (CR produces a number of health benefits and ameliorates diseases of aging such as type 2 diabetes. The components of the pathways downstream of CR may provide intervention points for developing therapeutics for treating diseases of aging. The NAD+-dependent protein deacetylase SIRT1 has been implicated as one of the key downstream regulators of CR in yeast, rodents, and humans. Small molecule activators of SIRT1 have been identified that exhibit efficacy in animal models of diseases typically associated with aging including type 2 diabetes. To identify molecular processes induced in the liver of mice treated with two structurally distinct SIRT1 activators, SIRT501 (formulated resveratrol and SRT1720, for three days, we utilized a systems biology approach and applied Causal Network Modeling (CNM on gene expression data to elucidate downstream effects of SIRT1 activation. Results Here we demonstrate that SIRT1 activators recapitulate many of the molecular events downstream of CR in vivo, such as enhancing mitochondrial biogenesis, improving metabolic signaling pathways, and blunting pro-inflammatory pathways in mice fed a high fat, high calorie diet. Conclusion CNM of gene expression data from mice treated with SRT501 or SRT1720 in combination with supporting in vitro and in vivo data demonstrates that SRT501 and SRT1720 produce a signaling profile that mirrors CR, improves glucose and insulin homeostasis, and acts via SIRT1 activation in vivo. Taken together these results are encouraging regarding the use of small molecule activators of SIRT1 for therapeutic intervention into type 2 diabetes, a strategy which is currently being investigated in multiple clinical trials.

  10. A novel role of sesamol in inhibiting NF-κB-mediated signaling in platelet activation

    Directory of Open Access Journals (Sweden)

    Chang Chao-Chien

    2011-12-01

    Full Text Available Abstract Background Platelet activation is relevant to a variety of coronary heart diseases. Our previous studies revealed that sesamol possesses potent antiplatelet activity through increasing cyclic AMP formation. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of sesamol in NF-κB-mediated platelet function. Methods Platelet aggregation, Fura 2-AM fluorescence, and immunoblotting analysis were used in this study. Results NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 μg/ml in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 μM. Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 μM-mediated inhibitory effects of IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLCγ2 phosphorylation, protein kinase C (PKC activation, [Ca2+]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca2+]i mobilization. Conclusions Sesamol activates cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca2+]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data demonstrating that inhibition of NF-κB interferes with platelet function may

  11. Retinoic acid signalling is activated in the postischemic heart and may influence remodelling.

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    Dusan Bilbija

    Full Text Available BACKGROUND: All-trans retinoic acid (atRA, an active derivative of vitamin A, regulates cell differentiation, proliferation and cardiac morphogenesis via transcriptional activation of retinoic acid receptors (RARs acting on retinoic acid response elements (RARE. We hypothesized that the retinoic acid (RA signalling pathway is activated in myocardial ischemia and postischemic remodelling. METHODS AND FINDINGS: Myocardial infarction was induced through ligating the left coronary artery in mice. In vivo cardiac activation of the RARs was measured by imaging RARE-luciferase reporter mice, and analysing expression of RAR target genes and proteins by real time RT-PCR and western blot. Endogenous retinoids in postinfarcted hearts were analysed by triple-stage liquid chromatography/tandem mass spectrometry. Cardiomyocytes (CM and cardiofibroblasts (CF were isolated from infarcted and sham operated RARE luciferase reporter hearts and monitored for RAR activity and expression of target genes. The effect of atRA on CF proliferation was evaluated by EdU incorporation. Myocardial infarction increased thoracic RAR activity in vivo (p<0.001, which was ascribed to the heart through ex vivo imaging (p = 0.002 with the largest signal 1 week postinfarct. This was accompanied by increased cardiac gene and protein expression of the RAR target genes retinol binding protein 1 (p = 0.01 for RNA, p = 0,006 for protein and aldehyde dehydrogenase 1A2 (p = 0.04 for RNA, p = 0,014 for protein, while gene expression of cytochrome P450 26B1 was downregulated (p = 0.007. Concomitantly, retinol accumulated in the infarcted zone (p = 0.02. CM and CF isolated from infarcted hearts had higher luminescence than those from sham operated hearts (p = 0.02 and p = 0.008. AtRA inhibited CF proliferation in vitro (p = 0.02. CONCLUSION: The RA signalling pathway is activated in postischemic hearts and may play a role in regulation of damage and

  12. The role of phospholipase D and phosphatidic acid in the mechanical activation of mTOR signaling in skeletal muscle.

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    Hornberger, T A; Chu, W K; Mak, Y W; Hsiung, J W; Huang, S A; Chien, S

    2006-03-21

    Signaling by the mammalian target of rapamycin (mTOR) has been reported to be necessary for mechanical load-induced growth of skeletal muscle. The mechanisms involved in the mechanical activation of mTOR signaling are not known, but several studies indicate that a unique [phosphotidylinositol-3-kinase (PI3K)- and nutrient-independent] mechanism is involved. In this study, we have demonstrated that a regulatory pathway for mTOR signaling that involves phospholipase D (PLD) and the lipid second messenger phosphatidic acid (PA) plays a critical role in the mechanical activation of mTOR signaling. First, an elevation in PA concentration was sufficient for the activation of mTOR signaling. Second, the isozymes of PLD (PLD1 and PLD2) are localized to the z-band in skeletal muscle (a critical site of mechanical force transmission). Third, mechanical stimulation of skeletal muscle with intermittent passive stretch ex vivo induced PLD activation, PA accumulation, and mTOR signaling. Finally, pharmacological inhibition of PLD blocked the mechanically induced increase in PA and the activation of mTOR signaling. Combined, these results indicate that mechanical stimuli activate mTOR signaling through a PLD-dependent increase in PA. Furthermore, we showed that mTOR signaling was partially resistant to rapamycin in muscles subjected to mechanical stimulation. Because rapamycin and PA compete for binding to the FRB domain on mTOR, these results suggest that mechanical stimuli activate mTOR signaling through an enhanced binding of PA to the FRB domain on mTOR.

  13. Kaempferol induces chondrogenesis in ATDC5 cells through activation of ERK/BMP-2 signaling pathway.

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    Nepal, Manoj; Li, Liang; Cho, Hyoung Kwon; Park, Jong Kun; Soh, Yunjo

    2013-12-01

    Endochondral bone formation occurs when mesenchymal cells condense to differentiate into chondrocytes, the primary cell types of cartilage. The aim of the present study was to identify novel factors regulating chondrogenesis. We investigated whether kaempferol induces chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Kaempferol treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. Kaempferol-treated ATDC5 cells stained more intensely with alcian blue staining than control cells, suggesting greater synthesis of matrix proteoglycans in the kaempferol-treated cells. Similarly, kaempferol induced greater activation of alkaline phosphatase activity than control cells, and it enhanced the expression of chondrogenic marker genes, such as collagen type I, collagen type X, OCN, Runx2, and Sox9. Kaempferol induced an acute activation of extracellular signal-regulated kinase (ERK) but not c-jun N-terminal kinase or p38 MAP kinase. PD98059, an inhibitor of MAPK/ERK, decreased in stained cells treated with kaempferol. Furthermore, kaempferol greatly expressed the protein and mRNA levels of BMP-2, suggesting chondrogenesis was stimulated via a BMP-2 pathway. Taken together, our results suggest that kaempferol has chondromodulating effects via an ERK/BMP-2 signaling pathway and could potentially be used as a therapeutic agent for bone growth disorders.

  14. Salmonella Protein AvrA Activates the STAT3 Signaling Pathway in Colon Cancer

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    Rong Lu

    2016-05-01

    Full Text Available Salmonella infection in humans can become chronic, which leads to low-grade persistent inflammation. These chronic infections increase the risk of several gastrointestinal diseases, including cancer. Salmonella AvrA is a multifunctional protein that influences eukaryotic cell pathways by regulating ubiquitination and acetylation. In an animal model, we have demonstrated that infection with AvrA-expressing Salmonella induces beta-catenin signals and enhances colonic tumorigenesis. Beta-catenin signaling is a key player in intestinal proliferation and tumorigenesis. The relative contributions of AvrA-induced proliferation and inflammation on tumorigenesis, however, are unknown. STAT3 is activated in chronically inflamed intestines in human inflammatory bowel diseases and in colitis-associated colon cancer. In the current study, mice were colonized with Salmonella AvrA-sufficient or AvrA-deficient bacterial strains. Then, inflammation-associated colon cancer was induced through the use of azoxymethane/dextran sulfate sodium. We determined that AvrA-expressing bacteria activated the STAT3 pathway, which is predicted to enhance proliferation and promote tumorigenesis. Transcriptional activity of STAT3 and its target genes were upregulated by Salmonella expressing AvrA, thus promoting proliferation and intestinal tumorigenesis. Our findings provide new insights regarding a STAT3-dependent mechanism by which the specific bacterial product AvrA enhances the development of infection-associated colon cancer. These insights might suggest future biomarkers to risk assessment and early detection of infection-related cancer.

  15. Osteogenesis on nanoparticulate mineralized collagen scaffolds via autogenous activation of the canonical BMP receptor signaling pathway.

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    Ren, Xiaoyan; Bischoff, David; Weisgerber, Daniel W; Lewis, Michael S; Tu, Victor; Yamaguchi, Dean T; Miller, Timothy A; Harley, Brendan A C; Lee, Justine C

    2015-05-01

    Skeletal regenerative medicine frequently incorporates deliverable growth factors to stimulate osteogenesis. However, the cost and side effects secondary to supraphysiologic dosages of growth factors warrant investigation of alternative methods of stimulating osteogenesis for clinical utilization. In this work, we describe growth factor independent osteogenic induction of human mesenchymal stem cells (hMSCs) on a novel nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG). hMSCs demonstrated elevated osteogenic gene expression and mineralization on MC-GAG with minimal to no effect upon addition of BMP-2 when compared to non-mineralized scaffolds (Col-GAG). To investigate the intracellular pathways responsible for the increase in osteogenesis, we examined the canonical and non-canonical pathways downstream from BMP receptor activation. Constitutive Smad1/5 phosphorylation with nuclear translocation occurred on MC-GAG independent of BMP-2, whereas Smad1/5 phosphorylation depended on BMP-2 stimulation on Col-GAG. When non-canonical BMPR signaling molecules were examined, ERK1/2 phosphorylation was found to be decreased in MC-GAG but elevated in Col-GAG. No differences in Smad2/3 or p38 activation were detected. Collectively, these results demonstrated that MC-GAG scaffolds induce osteogenesis without exogenous BMP-2 addition via endogenous activation of the canonical BMP receptor signaling pathway.

  16. Five layers of receptor signalling in γδ T cell differentiation and activation

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    Sérgio T. Ribeiro

    2015-01-01

    Full Text Available The contributions of gamma-delta T cells to immunity to infection or tumours critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate gamma-delta T cell physiology. The understanding of how environmental signals are integrated by gamma-delta T cells is critical for their manipulation in clinical settings. Here we discuss how different classes of surface receptors impact on human and murine gamma-delta T cell differentiation, activation and expansion. In particular, we review the role of five receptor types: the T cell receptor (TCR, costimulatory receptors, cytokine receptors, NK receptors and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of gamma-delta T cells; the cytokine receptors IL-2R, IL-7R and IL-15R, which drive functional differentiation and expansion of gamma-delta T cells; the NK receptor NKG2D and its contribution to gamma-delta T cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control gamma-delta T cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signalling in gamma-delta T cell differentiation and activation, and discuss its implications for the manipulation of gamma-delta T cells in immunotherapy.

  17. Epstein-Barr virus LMP2A signaling in statu nascendi mimics a B cell antigen receptor-like activation signal

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    Engels Niklas

    2012-04-01

    Full Text Available Abstract Background The latent membrane protein (LMP 2A of Epstein-Barr virus (EBV is expressed during different latency stages of EBV-infected B cells in which it triggers activation of cytoplasmic protein tyrosine kinases. Early studies revealed that an immunoreceptor tyrosine-based activation motif (ITAM in the cytoplasmic N-terminus of LMP2A can trigger a transient increase of the cytosolic Ca2+ concentration similar to that observed in antigen-activated B cells when expressed as a chimeric transmembrane receptor. Even so, LMP2A was subsequently ascribed an inhibitory rather than an activating function because its expression seemed to partially inhibit B cell antigen receptor (BCR signaling in EBV-transformed B cell lines. However, the analysis of LMP2A signaling has been hampered by the lack of cellular model systems in which LMP2A can be studied without the influence of other EBV-encoded factors. Results We have reanalyzed LMP2A signaling using B cells in which LMP2A is expressed in an inducible manner in the absence of any other EBV signaling protein. This allowed us for the first time to monitor LMP2A signaling in statu nascendi as it occurs during the EBV life cycle in vivo. We show that mere expression of LMP2A not only stimulated protein tyrosine kinases but also induced phospholipase C-γ2-mediated Ca2+ oscillations followed by activation of the extracellular signal-regulated kinase (Erk mitogen-activated protein kinase pathway and induction of the lytic EBV gene bzlf1. Furthermore, expression of the constitutively phosphorylated LMP2A ITAM modulated rather than inhibited BCR-induced Ca2+ mobilization. Conclusion Our data establish that LMP2A expression has a function beyond the putative inhibition of the BCR by generating a ligand-independent cellular activation signal that may provide a molecular switch for different EBV life cycle stages and most probably contributes to EBV-associated lymphoproliferative disorders.

  18. Lightening up Light Therapy: Activation of Retrograde Signaling Pathway by Photobiomodulation.

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    Kim, Hong Pyo

    2014-11-01

    Photobiomodulation utilizes monochromatic (or quasimonochromatic) light in the electromagnetic region of 600∼1000 nm for the treatment of soft tissues in a nondestructive and nonthermal mode. It is conceivable that photobiomodulation is based upon the ability of the light to alter cell metabolism as it is absorbed by general hemoproteins and cytochrome c oxidase (COX) in particular. Recently it has been suggested radiation of visible and infrared (IR) activates retrograde signaling pathway from mitochondria to nucleus. In this review, the role of COX in the photobiomodulation will be discussed. Further a possible role of water as a photoreceptor will be suggested.

  19. Activation of Sonic Hedgehog Signaling Pathway in S-type Neuroblastoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    周昱男; 戴若连; 毛玲; 夏远鹏; 姚玉芳; 杨雪; 胡波

    2010-01-01

    The effects of Sonic hedgehog(Shh) signaling pathway activation on S-type neuroblastoma(NB) cell lines and its role in NB tumorigenesis were investigated.Immunohistochemistry was used to detect the expression of Shh pathway components- Patched1(PTCH1) and Gli1 in 40 human primary NB samples.Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Gli1 in three kinds of S-type NB cell lines(SK-N-AS,SK-N-SH and SHEP1),respectively.Exogenous Shh was administrated to ...

  20. Signal frequency based self-tuning fuzzy controller for semi-active suspension system

    Institute of Scientific and Technical Information of China (English)

    孙涛; 黄震宇; 陈大跃; 汤磊

    2003-01-01

    A new kind of fuzzy control scheme, based on the identification of the signal's main frequency and the behavior of the ER damper, is proposed to control the semi-active suspension system. This method adjusts the fuzzy controller to achieve the best isolation effect by analyzing the main frequency's characters and inspecting the change of system parameters. The input of the fuzzy controller is the main frequency and the optimal damping ratio is the output. Simulation results indicated that the proposed control method is very effective in isolating the vibration.

  1. Signal frequency based self-tuning fuzzy controller for semi-active suspension system

    Institute of Scientific and Technical Information of China (English)

    孙涛; 黄震宇; 陈大跃; 汤磊

    2003-01-01

    A new kind of fuzzy control scheme, based on the identification of the signal' s main frequency and the behavior of the ER damper, is proposed to control the semi-active suspension system. This method ad-justs the fuzzy controller to achieve the best isolation effect by analyzing the main frequency' s characters and inspecting the change of system parameters. The input of the fuzzy controller is the main frequency and the op-timal damping ratio is the output. Simulation results indicated that the proposed control method is very effec-tive in isolating the vibration.

  2. ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization.

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    Sun, Wenxiang; Li, Yang; Chen, Lu; Chen, Huihui; You, Fuping; Zhou, Xiang; Zhou, Yi; Zhai, Zhonghe; Chen, Danying; Jiang, Zhengfan

    2009-05-26

    We report here the identification and characterization of a protein, ERIS, an endoplasmic reticulum (ER) IFN stimulator, which is a strong type I IFN stimulator and plays a pivotal role in response to both non-self-cytosolic RNA and dsDNA. ERIS (also known as STING or MITA) resided exclusively on ER membrane. The ER retention/retrieval sequence RIR was found to be critical to retain the protein on ER membrane and to maintain its integrity. ERIS was dimerized on innate immune challenges. Coumermycin-induced ERIS dimerization led to strong and fast IFN induction, suggesting that dimerization of ERIS was critical for self-activation and subsequent downstream signaling.

  3. Expanding the neuron's calcium signaling repertoire: intracellular calcium release via voltage-induced PLC and IP3R activation.

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    Stefanie Ryglewski

    2007-04-01

    Full Text Available Neuronal calcium acts as a charge carrier during information processing and as a ubiquitous intracellular messenger. Calcium signals are fundamental to numerous aspects of neuronal development and plasticity. Specific and independent regulation of these vital cellular processes is achieved by a rich bouquet of different calcium signaling mechanisms within the neuron, which either can operate independently or may act in concert. This study demonstrates the existence of a novel calcium signaling mechanism by simultaneous patch clamping and calcium imaging from acutely isolated central neurons. These neurons possess a membrane voltage sensor that, independent of calcium influx, causes G-protein activation, which subsequently leads to calcium release from intracellular stores via phospholipase C and inositol 1,4,5-trisphosphate receptor activation. This allows neurons to monitor activity by intracellular calcium release without relying on calcium as the input signal and opens up new insights into intracellular signaling, developmental regulation, and information processing in neuronal compartments lacking calcium channels.

  4. When the brain simulates stopping: Neural activity recorded during real and imagined stop-signal tasks.

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    González-Villar, Alberto J; Bonilla, F Mauricio; Carrillo-de-la-Peña, María T

    2016-10-01

    It has been suggested that mental rehearsal activates brain areas similar to those activated by real performance. Although inhibition is a key function of human behavior, there are no previous reports of brain activity during imagined response cancellation. We analyzed event-related potentials (ERPs) and time-frequency data associated with motor execution and inhibition during real and imagined performance of a stop-signal task. The ERPs characteristic of stop trials-that is, the stop-N2 and stop-P3-were also observed during covert performance of the task. Imagined stop (IS) trials yielded smaller stop-N2 amplitudes than did successful stop (SS) and unsuccessful stop (US) trials, but midfrontal theta power similar to that in SS trials. The stop-P3 amplitude for IS was intermediate between those observed for SS and US. The results may be explained by the absence of error-processing and correction processes during imagined performance. For go trials, real execution was associated with higher mu and beta desynchronization over motor areas, which confirms previous reports of lower motor activation during imagined execution and also with larger P3b amplitudes, probably indicating increased top-down attention to the real task. The similar patterns of activity observed for imagined and real performance suggest that imagination tasks may be useful for training inhibitory processes. Nevertheless, brain activation was generally weaker during mental rehearsal, probably as a result of the reduced engagement of top-down mechanisms and l