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Sample records for synthase kinase-3 gsk-3

  1. Nuclear glycogen synthase kinase-3 {beta} (GSK-3) in Rhipicephalus (Boophilus) microplus tick embryogenesis

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    Mentzingen, Leticia; Andrade, Josiana G. de; Logullo, Carlos [Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, RJ (Brazil). Centro de Biociencias e Biotecnologia. Lab. de Quimica e Funcao de Proteinas e Peptideos (LQFPP); Andrade, Caroline P. de; Vaz Junior, Itabajara [Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS (Brazil). Centro de Biotecnologia

    2008-07-01

    Full text: Glycogen synthase kinase-3 (GSK3) is recognized as a key component of a large number of cellular processes and diseases. Several mechanisms play a part in controlling the actions of GSK3, including phosphorylation, protein complex formation, and subcellular distribution. Recent observations point to functions for phosphorylases several transcription factors in the nucleus. Also, GSK3b participate of the canonical W nt signalling pathway, which has been studied intensively in embryonic and cancer cells. Like in many other signaling pathways, most components in W nt signal transduction were highly conserved during the evolution. More than 40 proteins have been reported to be phosphorylated by GSK3, including over a dozen transcription factors. Although the mechanisms regulating GSK3 are not fully understood, precise control appears to be achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins. Although GSK3 is traditionally considered a cytosolic protein, it is also present in nuclei. Nuclear GSK3 is particularly interesting because of the many transcription factors that it regulates enabling GSK3 to influence many signaling pathways that converge on these transcription factors, thereby regulating the expression of many genes. Our group identified that GSK-3 {beta} could be detected in different stage eggs of R. micro plus. In this work we detected the GSK-3 in isolated nuclear fraction from the egg homogenates of R. micro plus by western-blot analysis, using anti-GSK- 3 {beta} antibodies. The enzyme activity was also detected radiochemically throughout embryogenesis in same fraction. The GSK-3 activity was inhibiting by using SB 216763 (selective molecule inhibitors of GSK-3). Taken together our results suggest that GSK-3 {beta} isoform probably is involved in gene transcription factors during R. micro plus embryo development.

  2. Inactivation of glycogen synthase kinase-3β (GSK-3β) enhances skeletal muscle oxidative metabolism.

    Science.gov (United States)

    Theeuwes, W F; Gosker, H R; Langen, R C J; Verhees, K J P; Pansters, N A M; Schols, A M W J; Remels, A H V

    2017-12-01

    Aberrant skeletal muscle mitochondrial oxidative metabolism is a debilitating feature of chronic diseases such as chronic obstructive pulmonary disease, type 2 diabetes and chronic heart failure. Evidence in non-muscle cells suggests that glycogen synthase kinase-3β (GSK-3β) represses mitochondrial biogenesis and inhibits PPAR-γ co-activator 1 (PGC-1), a master regulator of cellular oxidative metabolism. The role of GSK-3β in the regulation of skeletal muscle oxidative metabolism is unknown. We hypothesized that inactivation of GSK-3β stimulates muscle oxidative metabolism by activating PGC-1 signaling and explored if GSK-3β inactivation could protect against physical inactivity-induced alterations in skeletal muscle oxidative metabolism. GSK-3β was modulated genetically and pharmacologically in C2C12 myotubes in vitro and in skeletal muscle in vivo. Wild-type and muscle-specific GSK-3β knock-out (KO) mice were subjected to hind limb suspension for 14days. Key constituents of oxidative metabolism and PGC-1 signaling were investigated. In vitro, knock-down of GSK-3β increased mitochondrial DNA copy number, protein and mRNA abundance of oxidative phosphorylation (OXPHOS) complexes and activity of oxidative metabolic enzymes but also enhanced protein and mRNA abundance of key PGC-1 signaling constituents. Similarly, pharmacological inhibition of GSK-3β increased transcript and protein abundance of key constituents and regulators of mitochondrial energy metabolism. Furthermore, GSK-3β KO animals were protected against unloading-induced decrements in expression levels of these constituents. Inactivation of GSK-3β up-regulates skeletal muscle mitochondrial metabolism and increases expression levels of PGC-1 signaling constituents. In vivo, GSK-3β KO protects against inactivity-induced reductions in muscle metabolic gene expression. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Regulation of glycogen synthase kinase-3{beta} (GSK-3{beta}) after ionizing radiation; Regulation der Glykogen Synthase Kinase-3{beta} (GSK-3{beta}) nach ionisierender Strahlung

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    Boehme, K.A.

    2006-12-15

    Glycogen Synthase Kinase-3{beta} (GSK-3{beta}) phosphorylates the Mdm2 protein in the central domain. This phosphorylation is absolutely required for p53 degradation. Ionizing radiation inactivates GSK-3{beta} by phosphorylation at serine 9 and in consequence prevents Mdm2 mediated p53 degradation. During the work for my PhD I identified Akt/PKB as the kinase that phosphorylates GSK-3{beta} at serine 9 after ionizing radiation. Ionizing radiation leads to phosphorylation of Akt/PKB at threonine 308 and serine 473. The PI3 Kinase inhibitor LY294002 completely abolished Akt/PKB serine 473 phosphorylation and prevented the induction of GSK-3{beta} serine 9 phosphorylation after ionizing radiation. Interestingly, the most significant activation of Akt/PKB after ionizing radiation occurred in the nucleus while cytoplasmic Akt/PKB was only weakly activated after radiation. By using siRNA, I showed that Akt1/PKBa, but not Akt2/PKB{beta}, is required for phosphorylation of GSK- 3{beta} at serine 9 after ionizing radiation. Phosphorylation and activation of Akt/PKB after ionizing radiation depends on the DNA dependent protein kinase (DNA-PK), a member of the PI3 Kinase family, that is activated by free DNA ends. Both, in cells from SCID mice and after knockdown of the catalytic subunit of DNA-PK by siRNA in osteosarcoma cells, phosphorylation of Akt/PKB at serine 473 and of GSK-3{beta} at serine 9 was completely abolished. Consistent with the principle that phosphorylation of GSK-3 at serine 9 contributes to p53 stabilization after radiation, the accumulation of p53 in response to ionizing radiation was largely prevented by downregulation of DNA-PK. From these results I conclude, that ionizing radiation induces a signaling cascade that leads to Akt1/PKBa activation mediated by DNA-PK dependent phosphorylation of serine 473. After activation Akt1/PKBa phosphorylates and inhibits GSK-3{beta} in the nucleus. The resulting hypophosphorylated form of Mdm2 protein is no longer

  4. Regulation of glycogen synthase kinase-3β (GSK-3β) after ionizing radiation

    International Nuclear Information System (INIS)

    Boehme, K.A.

    2006-12-01

    Glycogen Synthase Kinase-3β (GSK-3β) phosphorylates the Mdm2 protein in the central domain. This phosphorylation is absolutely required for p53 degradation. Ionizing radiation inactivates GSK-3β by phosphorylation at serine 9 and in consequence prevents Mdm2 mediated p53 degradation. During the work for my PhD I identified Akt/PKB as the kinase that phosphorylates GSK-3β at serine 9 after ionizing radiation. Ionizing radiation leads to phosphorylation of Akt/PKB at threonine 308 and serine 473. The PI3 Kinase inhibitor LY294002 completely abolished Akt/PKB serine 473 phosphorylation and prevented the induction of GSK-3β serine 9 phosphorylation after ionizing radiation. Interestingly, the most significant activation of Akt/PKB after ionizing radiation occurred in the nucleus while cytoplasmic Akt/PKB was only weakly activated after radiation. By using siRNA, I showed that Akt1/PKBa, but not Akt2/PKBβ, is required for phosphorylation of GSK- 3β at serine 9 after ionizing radiation. Phosphorylation and activation of Akt/PKB after ionizing radiation depends on the DNA dependent protein kinase (DNA-PK), a member of the PI3 Kinase family, that is activated by free DNA ends. Both, in cells from SCID mice and after knockdown of the catalytic subunit of DNA-PK by siRNA in osteosarcoma cells, phosphorylation of Akt/PKB at serine 473 and of GSK-3β at serine 9 was completely abolished. Consistent with the principle that phosphorylation of GSK-3 at serine 9 contributes to p53 stabilization after radiation, the accumulation of p53 in response to ionizing radiation was largely prevented by downregulation of DNA-PK. From these results I conclude, that ionizing radiation induces a signaling cascade that leads to Akt1/PKBa activation mediated by DNA-PK dependent phosphorylation of serine 473. After activation Akt1/PKBa phosphorylates and inhibits GSK-3β in the nucleus. The resulting hypophosphorylated form of Mdm2 protein is no longer able to degrade p53 which in

  5. Brain derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3β (GSK3β) signalling

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    Gupta, Vivek, E-mail: vivek.gupta@mq.edu.au [Australian School of Advanced Medicine, Macquarie University (Australia); Chitranshi, Nitin; You, Yuyi [Australian School of Advanced Medicine, Macquarie University (Australia); Gupta, Veer [School of Medical Sciences, Edith Cowan University, Perth (Australia); Klistorner, Alexander; Graham, Stuart [Australian School of Advanced Medicine, Macquarie University (Australia); Save Sight Institute, Sydney University, Sydney (Australia)

    2014-11-21

    Highlights: • BDNF knockdown leads to activation of GSK3β in the neuronal cells. • BDNF knockdown can induce GSK3β activation beyond TrkB mediated effects. • BDNF impairment in vivo leads to age dependent activation of GSK3β in the retina. • Systemic treatment with TrkB agonist induces inhibition of retinal GSK3β. - Abstract: Glycogen synthase kinase 3β (GSK3β) is involved in several biochemical processes in neurons regulating cellular survival, gene expression, cell fate determination, metabolism and proliferation. GSK3β activity is inhibited through the phosphorylation of its Ser-9 residue. In this study we sought to investigate the role of BDNF/TrkB signalling in the modulation of GSK3β activity. BDNF/TrkB signalling regulates the GSK3β activity both in vivo in the retinal tissue as well as in the neuronal cells under culture conditions. We report here for the first time that BDNF can also regulate GSK3β activity independent of its effects through the TrkB receptor signalling. Knockdown of BDNF lead to a decline in GSK3β phosphorylation without having a detectable effect on the TrkB activity or its downstream effectors Akt and Erk1/2. Treatment with TrkB receptor agonist had a stimulating effect on the GSK3β phosphorylation, but the effect was significantly less pronounced in the cells in which BDNF was knocked down. The use of TrkB receptor antagonist similarly, manifested itself in the form of downregulation of GSK3β phosphorylation, but a combined TrkB inhibition and BDNF knockdown exhibited a much stronger negative effect. In vivo, we observed reduced levels of GSK3β phosphorylation in the retinal tissues of the BDNF{sup +/−} animals implicating critical role of BDNF in the regulation of the GSK3β activity. Concluding, BDNF/TrkB axis strongly regulates the GSK3β activity and BDNF also exhibits GSK3β regulatory effect independent of its actions through the TrkB receptor signalling.

  6. Brain derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3β (GSK3β) signalling

    International Nuclear Information System (INIS)

    Gupta, Vivek; Chitranshi, Nitin; You, Yuyi; Gupta, Veer; Klistorner, Alexander; Graham, Stuart

    2014-01-01

    Highlights: • BDNF knockdown leads to activation of GSK3β in the neuronal cells. • BDNF knockdown can induce GSK3β activation beyond TrkB mediated effects. • BDNF impairment in vivo leads to age dependent activation of GSK3β in the retina. • Systemic treatment with TrkB agonist induces inhibition of retinal GSK3β. - Abstract: Glycogen synthase kinase 3β (GSK3β) is involved in several biochemical processes in neurons regulating cellular survival, gene expression, cell fate determination, metabolism and proliferation. GSK3β activity is inhibited through the phosphorylation of its Ser-9 residue. In this study we sought to investigate the role of BDNF/TrkB signalling in the modulation of GSK3β activity. BDNF/TrkB signalling regulates the GSK3β activity both in vivo in the retinal tissue as well as in the neuronal cells under culture conditions. We report here for the first time that BDNF can also regulate GSK3β activity independent of its effects through the TrkB receptor signalling. Knockdown of BDNF lead to a decline in GSK3β phosphorylation without having a detectable effect on the TrkB activity or its downstream effectors Akt and Erk1/2. Treatment with TrkB receptor agonist had a stimulating effect on the GSK3β phosphorylation, but the effect was significantly less pronounced in the cells in which BDNF was knocked down. The use of TrkB receptor antagonist similarly, manifested itself in the form of downregulation of GSK3β phosphorylation, but a combined TrkB inhibition and BDNF knockdown exhibited a much stronger negative effect. In vivo, we observed reduced levels of GSK3β phosphorylation in the retinal tissues of the BDNF +/− animals implicating critical role of BDNF in the regulation of the GSK3β activity. Concluding, BDNF/TrkB axis strongly regulates the GSK3β activity and BDNF also exhibits GSK3β regulatory effect independent of its actions through the TrkB receptor signalling

  7. Tissue-specific analysis of glycogen synthase kinase-3α (GSK-3α in glucose metabolism: effect of strain variation.

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    Satish Patel

    Full Text Available BACKGROUND: Over-activity and elevated expression of glycogen synthase kinase-3 (GSK-3 has been implicated in the etiology of insulin resistance and Type 2 diabetes. Administration of specific GSK-3 inhibitors to diabetic or obese rodent models improves glycaemic control and insulin sensitivity. However, due to the indiscriminatory nature of these inhibitors, the relative contribution of the two isoforms of GSK-3 (GSK-3α and GSK-3β is not known. Recently, we demonstrated that an out-bred strain of mice (ICR lacking expression of GSK-3α in all tissues displayed improved insulin sensitivity and enhanced hepatic glucose metabolism. We also found that muscle (but not liver inactivation of GSK-3β conferred insulin and glucose sensitization in an in-bred strain of mice (C57BL/6. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have employed tissue-specific deletion of GSK-3α, to examine the relative contribution of two insulin-sensitive tissues, muscle and liver, towards the insulin sensitization phenotype originally observed in the global GSK-3α KO animals. We found that mice in which GSK-3α has been inactivated in either skeletal-muscle or liver displayed no differences in glucose tolerance or insulin sensitivity compared to wild type littermates. Given the strain differences in our original analyses, we examined the insulin and glucose sensitivity of global GSK-3α KO animals bred onto a C57BL/6 background. These animals also revealed no significant differences in glucose metabolism/insulin sensitivity compared to their wild type littermates. Furthermore, deletion of hepatic GSK-3α on the out-bred, ICR background failed to reproduce the insulin sensitivity manifested by the global deletion of this isoform. CONCLUSIONS/SIGNIFICANCE: From these data we conclude that the improved insulin sensitivity and hepatic glucose homeostasis phenotype observed upon global inactivation of GSK-3α is strain-specific. We surmise that the insulin

  8. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    International Nuclear Information System (INIS)

    Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.; Parkos, Charles A.; Nusrat, Asma

    2010-01-01

    The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

  9. Glycogen synthase kinase-3beta (GSK3beta) negatively regulates PTTG1/human securin protein stability, and GSK3beta inactivation correlates with securin accumulation in breast tumors.

    Science.gov (United States)

    Mora-Santos, Mar; Limón-Mortés, M Cristina; Giráldez, Servando; Herrero-Ruiz, Joaquín; Sáez, Carmen; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

    2011-08-26

    PTTG1, also known as securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for securin in tumor progression. Stability of securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting securin for proteolysis have not been identified, and mechanistic insight into the cause of securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3β (GSK3β) phosphorylates securin to promote its proteolysis via SCF(βTrCP) E3 ubiquitin ligase. Importantly, a strong correlation between securin accumulation and GSK3β inactivation was observed in breast cancer tissues, indicating that GSK3β inactivation may account for securin accumulation in breast cancers.

  10. Glycogen Synthase Kinase-3β (GSK-3β) and Nuclear Factor Kappa-B (NFKB) in Childhood Acute Lymphoblastic Leukemia.

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    Tovar, Cristian Fabian Layton; Zerón, Hugo Mendieta; Romero, Maria Del Socorro Camarillo; Sánchez, Yanko V Fabila; Romero, Isidoro Tejocote

    2016-01-01

    Acute lymphocytic leukemia (ALL) is the most common hematologic malignancy in early childhood. In children with acute lymphoblastic leukemia (ALL), the activity of glycogen synthase kinase (GSK-3β) has been associated with changes in the transcriptional activity and expression of nuclear factor kappa beta (NFKB) in the mononuclear cells of bone marrow. The aim of the study was to determine the possible role of glycogen synthase kinase 3beta (GSK-3β) and nuclear factor kappa beta (NFKB) as prognostic variables in pediatric patients with ALL. This was a descriptive, transversal, and observational study. Bone marrow and blood samples were obtained from 30 children with newly-diagnosed ALL, who were seen at the Hematology-Oncology Service, Hospital para el Niño (HPN), Toluca, Mexico, from 2014‒2015. Anthropometric variables, clinical lab results, immunophenotype and cytogenetic abnormalities were registered. GSK-3β was evaluated through immunohistochemistry, and NFKB messenger RNA (mRNA) with real-time polymerase chain reaction (qPCR). The cases of ALL were classified into two groups of risk: high and habitual. Thirty patients were included in this study, with a mean age of 7.1 years (range 2‒13 years). Twenty-one were male and 9 female. Employing the morphological classification, 26 patients had type L1 ALL and the remaining 4 patients had type L2 ALL. Abnormal genes were found in 7 (23.33%) patients, ETV-RUNX1 in 3, followed by TCF3-PBX1 (two), STL1-TAL1 (one), and BCR-ABL1 (one). NFKB relative expression levels, in comparison to the GSK-3β immunohistochemistry results of the bone marrow samples, showed significant differences between positive and negative cases (p = 0.001) and between weak-positive and negative cases (p = 0.002). These results suggest that GSK-3β may be a prognostic biomarker in childhood ALL.

  11. The role of glycogen synthase kinase-3β (GSK-3β) in endometrial carcinoma: A carcinogenesis, progression, prognosis, and target therapy marker.

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    Chen, Shuo; Sun, Kai-Xuan; Liu, Bo-Liang; Zong, Zhi-Hong; Zhao, Yang

    2016-05-10

    Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase involved in cancer development. Herein, we demonstrated the role of GSK-3β in endometrial cancer (EC) and identified new therapeutic targets. GSK-3β was overexpressed in EC tissues, and was positively correlated with International Federation of Gynecology and Obstetrics (FIGO) staging, dedifferentiation, and myometrial infiltration depth. Besides, GSK-3β overexpression predicted lower cumulative and relapse-free survival rate. si-GSK-3β transfection suppressed cell proliferation, migration, invasion, and promoted cell apoptosis through downregulating NF-kB, Cyclin D1 and MMP9 expression whereas upregulating P21 expression. Bioinformatic predictions and dual-luciferase reporter assays showed that GSK-3β was a possible target of miR-129. MiR-129 transfection reduced GSK-3β expression, and exhibited the same trend as si-GSK-3β transfection in cell function experiments. The nude mouse xenograft assay showed that miR-129 overexpression may suppress tumor growth through downregulating GSK-3β expression. Further studies showed that AZD1080, a GSK-3β inhibitor, could also inhibit EC cell proliferation, migration and invasion, while induced cell apoptosis through modulating relevant genes downstream of GSK-3β signaling. GSK-3β expression was determined in EC tissue and normal endometrial tissues by immunohistochemistry. After GSK-3β down-regulation by si-GSK-3β, microRNA-129 mimic transfection or GSK-3β inhibitor exposure, EC cell phenotypes and related molecules were examined. Our results demonstrate for the first time that GSK-3β may be a novel and important therapeutic target for the treatment of endometrial carcinoma. GSK-3β inhibitor AZD1080 may be an effective drug for treating endometrial carcinoma.

  12. Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure-activity relationships with Trypanosoma brucei GSK-3

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    Ojo, Kayode K; Arakaki, Tracy L; Napuli, Alberto J; Inampudi, Krishna K; Keyloun, Katelyn R; Zhang, Li; Hol, Wim G.J.; Verlind, Christophe L.M.J.; Merritt, Ethan A; Van Voorhis, Wesley C [UWASH

    2012-04-24

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

  13. Glycogen Synthase Kinase-3β

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    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie

    2016-01-01

    Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation...

  14. High-frequency electrical stimulation (HFES data lean and obese Zucker rat tibialis anterior muscle: Regulation of glycogen synthase kinase 3 beta (GSK3B associated proteins

    Directory of Open Access Journals (Sweden)

    Gautam K. Ginjupalli

    2018-02-01

    Full Text Available Anaerobic exercise has been advocated as a prescribed treatment for the management of diabetes: however, alterations in exercise-induced signaling remain largely unexplored in the diabetic muscle. Here, we compare the basal and the in situ contraction-induced phosphorylation of the AMPK, GSK3beta, and Shp2 in the lean and obese (fa/fa Zucker rat tibialis anterior (TA muscle following a single bout of contractile stimuli. This article represents data associated with prior publications from our lab (Katta et al., 2009; Katta et al., 2009; Tullgren et al., 1991 [1–3] and concurrent Data in Brief articles (Ginjupalli et al., 2017; Rice et al., 2017; Rice et al., 2017; Rice et al., 2017 [4–7].

  15. A Novel Approach for Overcoming Drug Resistance in Breast Cancer Chemotherapy by Targeting new Synthetic Curcumin Analogues Against Aldehyde Dehydrogenase 1 (ALDH1A1) and Glycogen Synthase Kinase-3 β (GSK-3β).

    Science.gov (United States)

    Kesharwani, Rajesh Kumar; Srivastava, Vandana; Singh, Prabhakar; Rizvi, Syed Ibrahim; Adeppa, Kuruba; Misra, Krishna

    2015-08-01

    Breast cancer stem cells are well known to resist the traditional methods like chemo and radio therapy. Aldehyde dehydrogenase 1 (ALDHIA1) and glycogen synthase kinase-3 β (GSK-3β) are the two selected proteins for study, due to their overexpression and upregulation in breast cancer cells. Curcumin, the yellow pigment of the spice turmeric, is widely reported as an antioxidant and acts as a synergist along with traditional drugs. Under hypoxic conditions, it gets converted to free radical which causes apoptosis. Three naturally occurring curcuminoids, i.e. curcumin, demethoxycurcumin, and bisdemethoxycurcumin along with five derivatives/analogues of curcumin, viz. 4,4'-di-O-(carboxy-methyl)-curcumin, 4-O-(2-hydroxyethyl)curcumin, 4,4'-di-O-allyl-curcumin, 4,4'-di-O-(acetyl)-curcumin, and 3,3'-bisdemethylcurcumin were synthesized and evaluated for their anti-breast cancer potential by docking simulation and assessment of their antioxidant character, studied via 2, 2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(·+)) radical cation scavenging assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH·) radical, and ferric reducing ability potential (FRAP) assay. A co-relation between structure and activity of curcuminoids/its analogues and derivatives has been worked out.

  16. Glycogen synthase kinase-3β activity and cognitive functioning in patients with bipolar I disorder

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Miskowiak, Kamilla Woznica; Jacoby, Anne Sophie

    2018-01-01

    Cognitive deficits are common in patients with bipolar disorder (BD) in remission and may be associated with glycogen synthase kinase-3 (GSK-3) activity, which is inhibited by lithium. GSK-3 may be a relevant treatment target for interventions tailored at cognitive disturbances in BD...... ratio (serine-9-pGSK-3β /total GSK-3β), was negatively associated with sustained attention (p = 0.009 and p = 0.042, respectively), but not with other cognitive domains or global cognition. A crossover interaction between lithium treatment and the GSK activity was observed, indicating that lower pGSK-3β...... but the relation between GSK-3 activity, cognition and lithium treatment is unknown. We therefore investigated the possible association between GSK-3 activity and cognition and whether lithium treatment moderates this association in patients with BD. In a prospective 6-12 month follow-up study, GSK- 3β activity...

  17. Glycogen synthase kinase-3β in patients with bipolar I disorder

    DEFF Research Database (Denmark)

    Jacoby, Anne S; Munkholm, Klaus; Vinberg, Maj

    2016-01-01

    OBJECTIVES: The enzyme glycogen synthase kinase-3β (GSK3β) is involved in the mechanisms of action of lithium and may play a role in relation to affective states in bipolar disorder. The objectives of the present study were to compare the activity of GSK-3β (measured as levels of phosphorylated GSK...

  18. Structural Injury after Lithium Treatment in Human and Rat Kidney involves Glycogen Synthase Kinase-3β Positive Epithelium

    DEFF Research Database (Denmark)

    Kjærsgaard, Gitte; Madsen, Kirsten; Marcussen, Niels

    2011-01-01

    of glycogen synthase kinase-3β (GSK-3β). GSK-3β and pGSK-3β was investigated in a developing series of rat kidney cortex and medulla. Li+ was given to female wistar rats with litters through food pellets at postnatal (P) days 7-28. In human fetal and adult kidney the expression of GSK-3β was examined and also...

  19. Lithium Impairs Kidney Development and Inhibits Glycogen Synthase Kinase-3β in Collecting Duct Principal Cells

    DEFF Research Database (Denmark)

    Kjærsgaard, Gitte; Madsen, Kirsten; Marcussen, Niels

    on serine9 (pGSK-3β)and subsequent epithelial to mesenchymal dedifferentiation (EMT). GSK-3β immunoreactive protein was associated with collecting ducts in developing and adult human and rat kidney. Total GSK-3β protein abundance was stable in medulla while it decreased in cortex in the postnatal period......GSK-3β abundance in collecting duct. The data are compatible with the notion that increased GSK-3β activity in the postnatal kidney medulla is necessary for kidney development.......The postnatal rat kidney is highly susceptible to Lithium (Li+), which leads to significant tissue injury. We hypothesized that Li+ impairs development of the kidney through entry into epithelial cells of the distal nephron, inhibition of Glycogen Synthase Kinase-3β (GSK-3β) through phosphorylation...

  20. Inhibition of Glycogen Synthase Kinase-3ß Enhances Cognitive Recovery after Stroke: The Role of TAK1

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    Venna, Venugopal Reddy; Benashski, Sharon E.; Chauhan, Anjali; McCullough, Louise D.

    2015-01-01

    Memory deficits are common among stroke survivors. Identifying neuroprotective agents that can prevent memory impairment or improve memory recovery is a vital area of research. Glycogen synthase kinase-3ß (GSK-3ß) is involved in several essential intracellular signaling pathways. Unlike many other kinases, GSK-3ß is active only when…

  1. Glycogen synthase kinase 3 (GSK3) inhibitor 6-bromoindirubin-3 ...

    African Journals Online (AJOL)

    KSR) medium, we got a relatively optimal feeder- and serum-free system for mGSCs in vitro. After continuous culturing, the proliferation efficiency of undifferentiated mGSCs and differentiation capacity of mGSC-induced embryoid bodies (EBs) ...

  2. Enantioselective synthesis of the novel chiral sulfoxide derivative as a glycogen synthase kinase 3beta inhibitor.

    Science.gov (United States)

    Saitoh, Morihisa; Kunitomo, Jun; Kimura, Eiji; Yamano, Toru; Itoh, Fumio; Kori, Masakuni

    2010-09-01

    Glycogen synthase kinase 3beta (GSK-3beta) inhibitors are expected to be attractive therapeutic agents for the treatment of Alzheimer's disease (AD). Recently we discovered sulfoxides (S)-1 as a novel GSK-3beta inhibitor having in vivo efficacy. We investigated practical asymmetric preparation methods for the scale-up synthesis of (S)-1. The highly enantioselective synthesis of (S)-1 (94% ee) was achieved by titanium-mediated oxidation with D-(-)-diethyl tartrate on gram scale.

  3. Glycogen Synthase Kinase-3 is involved in glycogen metabolism control and embryogenesis of Rhodnius prolixus.

    Science.gov (United States)

    Mury, Flávia B; Lugon, Magda D; DA Fonseca, Rodrigo Nunes; Silva, Jose R; Berni, Mateus; Araujo, Helena M; Fontenele, Marcio Ribeiro; Abreu, Leonardo Araujo DE; Dansa, Marílvia; Braz, Glória; Masuda, Hatisaburo; Logullo, Carlos

    2016-10-01

    Rhodnius prolixus is a blood-feeding insect that transmits Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Rhodnius prolixus is also a classical model in insect physiology, and the recent availability of R. prolixus genome has opened new avenues on triatomine research. Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism, also acting as a downstream component of the Wnt pathway during embryogenesis. GSK-3 has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. Meanwhile, the role of GSK-3 during R. prolixus embryogenesis or glycogen metabolism has not been investigated. Here we show that chemical inhibition of GSK-3 by alsterpaullone, an ATP-competitive inhibitor of GSK3, does not affect adult survival rate, though it alters oviposition and egg hatching. Specific GSK-3 gene silencing by dsRNA injection in adult females showed a similar phenotype. Furthermore, bright field and 4'-6-diamidino-2-phenylindole (DAPI) staining analysis revealed that ovaries and eggs from dsGSK-3 injected females exhibited specific morphological defects. We also demonstrate that glycogen content was inversely related to activity and transcription levels of GSK-3 during embryogenesis. Lastly, after GSK-3 knockdown, we observed changes in the expression of the Wingless (Wnt) downstream target β-catenin as well as in members of other pathways such as the receptor Notch. Taken together, our results show that GSK-3 regulation is essential for R. prolixus oogenesis and embryogenesis.

  4. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    Directory of Open Access Journals (Sweden)

    Hoseok Choi

    2016-04-01

    Full Text Available Assaying the glycogen synthase kinase-3 (GSK3 activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

  5. Distinct Molecular Regulation of Glycogen Synthase Kinase-3α Isozyme Controlled by Its N-terminal Region

    Science.gov (United States)

    Azoulay-Alfaguter, Inbar; Yaffe, Yakey; Licht-Murava, Avital; Urbanska, Malgorzata; Jaworski, Jacek; Pietrokovski, Shmuel; Hirschberg, Koret; Eldar-Finkelman, Hagit

    2011-01-01

    Glycogen synthase kinase-3 (GSK-3) is expressed as two isozymes α and β. They share high similarity in their catalytic domains but differ in their N- and C-terminal regions, with GSK-3α having an extended glycine-rich N terminus. Here, we undertook live cell imaging combined with molecular and bioinformatic studies to understand the distinct functions of the GSK-3 isozymes focusing on GSK-3α N-terminal region. We found that unlike GSK-3β, which shuttles between the nucleus and cytoplasm, GSK-3α was excluded from the nucleus. Deletion of the N-terminal region of GSK-3α resulted in nuclear localization, and treatment with leptomycin B resulted in GSK-3α accumulation in the nucleus. GSK-3α rapidly accumulated in the nucleus in response to calcium or serum deprivation, and accumulation was strongly inhibited by the calpain inhibitor calpeptin. This nuclear accumulation was not mediated by cleavage of the N-terminal region or phosphorylation of GSK-3α. Rather, we show that calcium-induced GSK-3α nuclear accumulation was governed by GSK-3α binding with as yet unknown calpain-sensitive protein or proteins; this binding was mediated by the N-terminal region. Bioinformatic and experimental analyses indicated that nuclear exclusion of GSK-3α was likely an exclusive characteristic of mammalian GSK-3α. Finally, we show that nuclear localization of GSK-3α reduced the nuclear pool of β-catenin and its target cyclin D1. Taken together, these data suggest that the N-terminal region of GSK-3α is responsible for its nuclear exclusion and that binding with a calcium/calpain-sensitive product enables GSK-3α nuclear retention. We further uncovered a novel link between calcium and nuclear GSK-3α-mediated inhibition of the canonical Wnt/β-catenin pathway. PMID:21266584

  6. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    Science.gov (United States)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  7. Light and electron microscopy study of glycogen synthase kinase-3beta in the mouse brain.

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    Emma Perez-Costas

    Full Text Available Glycogen synthase kinase-3beta (GSK3beta is highly abundant in the brain. Various biochemical analyses have indicated that GSK3beta is localized to different intracellular compartments within brain cells. However, ultrastructural visualization of this kinase in various brain regions and in different brain cell types has not been reported. The goal of the present study was to examine GSK3beta distribution and subcellular localization in the brain using immunohistochemistry combined with light and electron microscopy. Initial examination by light microscopy revealed that GSK3beta is expressed in brain neurons and their dendrites throughout all the rostrocaudal extent of the adult mouse brain, and abundant GSK3beta staining was found in the cortex, hippocampus, basal ganglia, the cerebellum, and some brainstem nuclei. Examination by transmission electron microscopy revealed highly specific subcellular localization of GSK3beta in neurons and astrocytes. At the subcellular level, GSK3beta was present in the rough endoplasmic reticulum, free ribosomes, and mitochondria of neurons and astrocytes. In addition GSK3beta was also present in dendrites and dendritic spines, with some postsynaptic densities clearly labeled for GSK3beta. Phosphorylation at serine-9 of GSK3beta (pSer9GSK3beta reduces kinase activity. pSer9GSK3beta labeling was present in all brain regions, but the pattern of staining was clearly different, with an abundance of labeling in microglia cells in all regions analyzed and much less neuronal staining in the subcortical regions. At the subcellular level pSer9GSK3beta labeling was located in the endoplasmic reticulum, free ribosomes and in some of the nuclei. Overall, in normal brains constitutively active GSK3beta is predominantly present in neurons while pSer9GSK3beta is more evident in resting microglia cells. This visual assessment of GSK3beta localization within the subcellular structures of various brain cells may help in

  8. Glycogen synthase kinase 3 controls migration of the neural crest lineage in mouse and Xenopus.

    Science.gov (United States)

    Gonzalez Malagon, Sandra G; Lopez Muñoz, Anna M; Doro, Daniel; Bolger, Triòna G; Poon, Evon; Tucker, Elizabeth R; Adel Al-Lami, Hadeel; Krause, Matthias; Phiel, Christopher J; Chesler, Louis; Liu, Karen J

    2018-03-19

    Neural crest migration is critical to its physiological function. Mechanisms controlling mammalian neural crest migration are comparatively unknown, due to difficulties accessing this cell population in vivo. Here we report requirements of glycogen synthase kinase 3 (GSK3) in regulating the neural crest in Xenopus and mouse models. We demonstrate that GSK3 is tyrosine phosphorylated (pY) in mouse neural crest cells and that loss of GSK3 leads to increased pFAK and misregulation of Rac1 and lamellipodin, key regulators of cell migration. Genetic reduction of GSK3 results in failure of migration. We find that pY-GSK3 phosphorylation depends on anaplastic lymphoma kinase (ALK), a protein associated with neuroblastoma. Consistent with this, neuroblastoma cells with increased ALK activity express high levels of pY-GSK3, and blockade of GSK3 or ALK can affect migration of these cells. Altogether, this work identifies a role for GSK3 in cell migration during neural crest development and cancer.

  9. Glycogen synthase kinase 3beta contributes to proliferation of arterial smooth muscle cells in pulmonary hypertension.

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    Piotr Sklepkiewicz

    2011-04-01

    Full Text Available Pulmonary arterial hypertension (PAH is a rare progressive pulmonary vascular disorder associated with vascular remodeling and right heart failure. Vascular remodeling involves numerous signaling cascades governing pulmonary arterial smooth muscle cell (PASMC proliferation, migration and differentiation. Glycogen synthase kinase 3beta (GSK3ß is a serine/threonine kinase and can act as a downstream regulatory switch for numerous signaling pathways. Hence, we hypothesized that GSK3ß plays a crucial role in pulmonary vascular remodeling.All experiments were done with lung tissue or isolated PASMCs in a well-established monocrotaline (MCT-induced PAH rat model. The mRNA expression of Wnt ligands (Wnt1, Wnt3a, Wnt5a, upstream Wnt signaling regulator genes (Frizzled Receptors 1, 2 and secreted Frizzled related protein sFRP-1 and canonical Wnt intracellular effectors (GSK3ß, Axin1 were assessed by real-time polymerase chain reaction and protein levels of GSK3ß, phospho-GSK3ß (ser 9 by western blotting and localization by immunohistochemistry. The role of GSK3ß in PASMCs proliferation was assessed by overexpression of wild-type GSK3ß (WT and constitutively active GSK3ß S9A by [(3H]-thymidine incorporation assay.Increased levels of total and phosphorylated GSK3ß (inhibitory phosphorylation were observed in lungs and PASMCs isolated from MCT-induced PAH rats compared to controls. Further, stimulation of MCT-PASMCs with growth factors induced GSK3ß inactivation. Most importantly, treatment with the PDGFR inhibitor, Imatinib, attenuated PDGF-BB and FCS induced GSK3ß phosphorylation. Increased expression of GSK3ß observed in lungs and PASMC isolated from MCT-induced PAH rats was confirmed to be clinically relevant as the same observation was identified in human iPAH lung explants. Overexpression of GSK3ß significantly increased MCT-PASMCs proliferation by regulating ERK phosphorylation. Constitutive activation of GSK3ß (GSK3ß S9A, 9th serine

  10. The Effects of Glycogen Synthase Kinase-3beta in Serotonin Neurons

    Science.gov (United States)

    Zhou, Wenjun; Chen, Ligong; Paul, Jodi; Yang, Sufen; Li, Fuzeng; Sampson, Karen; Woodgett, Jim R.; Beaulieu, Jean Martin; Gamble, Karen L.; Li, Xiaohua

    2012-01-01

    Glycogen synthase kinase-3 (GSK3) is a constitutively active protein kinase in brain. Increasing evidence has shown that GSK3 acts as a modulator in the serotonin neurotransmission system, including direct interaction with serotonin 1B (5-HT1B) receptors in a highly selective manner and prominent modulating effect on 5-HT1B receptor activity. In this study, we utilized the serotonin neuron-selective GSK3β knockout (snGSK3β-KO) mice to test if GSK3β in serotonin neurons selectively modulates 5-HT1B autoreceptor activity and function. The snGSK3β-KO mice were generated by crossbreeding GSK3β-floxed mice and ePet1-Cre mice. These mice had normal growth and physiological characteristics, similar numbers of tryptophan hydroxylase-2 (TpH2)-expressing serotonin neurons, and the same brain serotonin content as in littermate wild type mice. However, the expression of GSK3β in snGSK3β-KO mice was diminished in TpH2-expressing serotonin neurons. Compared to littermate wild type mice, snGSK3β-KO mice had a reduced response to the 5-HT1B receptor agonist anpirtoline in the regulation of serotonergic neuron firing, cAMP production, and serotonin release, whereas these animals displayed a normal response to the 5-HT1A receptor agonist 8-OH-DPAT. The effect of anpirtoline on the horizontal, center, and vertical activities in the open field test was differentially affected by GSK3β depletion in serotonin neurons, wherein vertical activity, but not horizontal activity, was significantly altered in snGSK3β-KO mice. In addition, there was an enhanced anti-immobility response to anpirtoline in the tail suspension test in snGSK3β-KO mice. Therefore, results of this study demonstrated a serotonin neuron-targeting function of GSK3β by regulating 5-HT1B autoreceptors, which impacts serotonergic neuron firing, serotonin release, and serotonin-regulated behaviors. PMID:22912839

  11. Glycogen Synthase Kinase-3β as a Putative Therapeutic Target for Bipolar Disorder.

    Science.gov (United States)

    Dandekar, Manoj P; Valvassori, Samira S; Dal-Pont, Gustavo Colombo; Quevedo, Joao

    2017-12-27

    Bipolar disorder (BD) is a debilitating mental ailment characterized by recurrent episodes of mania and depression. Primary mood-stabilizing drugs like lithium and valproate alleviate the hypomanic or mild to moderate manic episodes in patients with BD. One of the extensively studied underlying mechanisms for these pharmacological interventions is inhibition of intracellular signaling cascades associated with glycogen synthase kinase-3 beta (GSK-3β), a multi-functional serine-threonine kinase. To summarize the different mechanistic aspects associated with GSK-3β signaling involved in the pathophysiology of BD and highlights drug discovery approaches pursued for the development of GSK-3β inhibition with detailed strength, weakness, opportunity, and threat (SWOT) analysis. In this review, we endeavor to establish the correlation between neuronal GSK-3β inhibition and anti-manic response of different therapeutics used for the treatment of patients with BD. The gene depletion or pharmacological inhibition of GSK-3β reproduces some of the behavioral effects of lithium including reduction of depression- and manic-like behaviors in rodents, which attested the intracellular GSK-3β inhibition as one of the critical steps in mediating behavioral effect of mood-stabilizers. Furthermore, converging evidence supported the participation of GSK-3β in the regulation of various neurobehavioral functions governed by neurotransmitters dopamine and serotonin. Apart from its crucial involvement in the mechanism of action of mood stabilizers, GSK-3β signaling pathways have also received attention for their role in the effects of psychoactive therapies like antidepressants, antipsychotics, and neurotrophic factors. We anticipate that the GSK-3β could be a druggable target for several incurable neuropsychiatric disorders including BD. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. The Nuclear Localization of Glycogen Synthase Kinase 3β Is Required Its Putative PY-Nuclear Localization Sequences

    OpenAIRE

    Shin, Sung Hwa; Lee, Eun Jeoung; Chun, Jaesun; Hyun, Sunghee; Kim, Youg Il; Kang, Sang Sun

    2012-01-01

    Glycogen synthase kinase-3β (GSK-3β), which is a member of the serine/threonine kinase family, has been shown to be crucial for cellular survival, differentiation, and metabolism. Here, we present evidence that GSK-3β is associated with the karyopherin β2 (Kap β2) (102-kDa), which functions as a substrate for transportation into the nucleus. A potential PY-NLS motif (109IVRLRYFFY117) was observed, which is similar with the consensus PY NLS motif (R/K/H)X2–5PY in the GSK-3β catalytic domain. U...

  13. Novel reporter alleles of GSK-3α and GSK-3β.

    Directory of Open Access Journals (Sweden)

    William B Barrell

    Full Text Available Glycogen Synthase Kinase 3 (GSK-3 is a key player in development, physiology and disease. Because of this, GSK-3 inhibitors are increasingly being explored for a variety of applications. In addition most analyses focus on GSK-3β and overlook the closely related protein GSK-3α. Here, we describe novel GSK-3α and GSK-3β mouse alleles that allow us to visualise expression of their respective mRNAs by tracking β-galactosidase activity. We used these new lacZ alleles to compare expression in the palate and cranial sutures and found that there was indeed differential expression. Furthermore, both are loss of function alleles and can be used to generate homozygous mutant mice; in addition, excision of the lacZ cassette from GSK-3α creates a Cre-dependent tissue-specific knockout. As expected, GSK3α mutants were viable, while GSK3β mutants died after birth with a complete cleft palate. We also assessed the GSK-3α mutants for cranial and sternal phenotypes and found that they were essentially normal. Finally, we observed gestational lethality in compound GSK-3β(-/-; GSK3α(+/- mutants, suggesting that GSK-3 dosage is critical during embryonic development.

  14. Distinct molecular regulation of glycogen synthase kinase-3alpha isozyme controlled by its N-terminal region: functional role in calcium/calpain signaling.

    Science.gov (United States)

    Azoulay-Alfaguter, Inbar; Yaffe, Yakey; Licht-Murava, Avital; Urbanska, Malgorzata; Jaworski, Jacek; Pietrokovski, Shmuel; Hirschberg, Koret; Eldar-Finkelman, Hagit

    2011-04-15

    Glycogen synthase kinase-3 (GSK-3) is expressed as two isozymes α and β. They share high similarity in their catalytic domains but differ in their N- and C-terminal regions, with GSK-3α having an extended glycine-rich N terminus. Here, we undertook live cell imaging combined with molecular and bioinformatic studies to understand the distinct functions of the GSK-3 isozymes focusing on GSK-3α N-terminal region. We found that unlike GSK-3β, which shuttles between the nucleus and cytoplasm, GSK-3α was excluded from the nucleus. Deletion of the N-terminal region of GSK-3α resulted in nuclear localization, and treatment with leptomycin B resulted in GSK-3α accumulation in the nucleus. GSK-3α rapidly accumulated in the nucleus in response to calcium or serum deprivation, and accumulation was strongly inhibited by the calpain inhibitor calpeptin. This nuclear accumulation was not mediated by cleavage of the N-terminal region or phosphorylation of GSK-3α. Rather, we show that calcium-induced GSK-3α nuclear accumulation was governed by GSK-3α binding with as yet unknown calpain-sensitive protein or proteins; this binding was mediated by the N-terminal region. Bioinformatic and experimental analyses indicated that nuclear exclusion of GSK-3α was likely an exclusive characteristic of mammalian GSK-3α. Finally, we show that nuclear localization of GSK-3α reduced the nuclear pool of β-catenin and its target cyclin D1. Taken together, these data suggest that the N-terminal region of GSK-3α is responsible for its nuclear exclusion and that binding with a calcium/calpain-sensitive product enables GSK-3α nuclear retention. We further uncovered a novel link between calcium and nuclear GSK-3α-mediated inhibition of the canonical Wnt/β-catenin pathway.

  15. Tissue injury after lithium treatment in human and rat postnatal kidney involves glycogen synthase kinase 3β-positive epithelium

    DEFF Research Database (Denmark)

    Kjaersgaard, Gitte; Madsen, Kirsten; Marcussen, Niels

    2012-01-01

    It was hypothesized that lithium causes accelerated and permanent injury to the postnatally developing kidney through entry into epithelial cells of the distal nephron and inhibition of glycogen synthase kinase-3β (GSK-3β). GSK-3β immunoreactivity was associated with glomeruli, thick ascending limb...... of Henle's loop and collecting ducts in developing and adult human and rat kidney. In rats, the abundance of inactive, phosphorylated GSK-3β (pGSK-3β) protein decreased during postnatal development. After feeding dams with litters lithium (50 mmol Li/kg chow, postnatal (P) day 7-28), the offspring showed......-immunopositive epithelium. The postnatal rat kidney may serve as an experimental model for the study of lithium-induced human kidney injury. The data are compatible with a causal relation between epithelial entry of lithium into cells of the aldosterone-sensitive distal nephron, inactivation of GSK-3β, proliferation...

  16. Clinicopathological and biological significance of aberrant activation of glycogen synthase kinase-3 in ovarian cancer

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    Fu Y

    2014-06-01

    Full Text Available Yunfeng Fu,1 Xinyu Wang,1 Xiaodong Cheng,1 Feng Ye,2 Xing Xie,1,2 Weiguo Lu1,2 1Department of Gynecologic Oncology, Women's Hospital, School of Medicine, Zhejiang University, 2Women's Reproduction and Health Laboratory of Zhejiang Province, Hangzhou, People's Republic of China Background: Glycogen synthase kinase-3 (GSK-3 plays an important role in human cancer. The aim of this study is to evaluate the clinicopathological significance of expression of GSK-3α/β and pGSK-3α/βTyr279/216 in patients with epithelial ovarian cancer and to investigate whether GSK-3 inhibition can influence cell viability and tumor growth of ovarian cancer. Methods: Immunohistochemistry was used to examine expression of GSK-3α/β and pGSK-3α/βTyr279/216 in 71 human epithelial ovarian cancer tissues and correlations between protein expression, and clinicopathological factors were analyzed. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay following exposure of ovarian carcinoma cells to pharmacological inhibitors of GSK-3 or GSK-3 small interfering RNA. In vivo validation of tumor growth inhibition was performed with xenograft mice. Results: The expression levels of GSK-3α/β and pGSK-3α/βTyr279/216 in ovarian cancers were significantly higher than those in benign tumors. High expression of GSK-3α/β was more likely to be found in patients with advanced International Federation of Gynecology and Obstetrics (FIGO stages and high serum cancer antigen 125. Higher expression of pGSK-3α/βTyr279/216 was associated with advanced FIGO stages, residual tumor mass, high serum cancer antigen 125, and poor chemoresponse. Worse overall survival was revealed by Kaplan–Meier survival curves in patients with high expression of GSK-3α/β or pGSK-3α/βTyr279/216. Multivariate analysis indicated that FIGO stage, GSK-3α/β expression, and pGSK-3α/βTyr279/216 expression were independent prognostic factors for overall

  17. Glycogen synthase kinase-3 inhibitors reverse deficits in long-term potentiation and cognition in Fragile X mice

    Science.gov (United States)

    Franklin, Aimee V.; King, Margaret K.; Palomo, Valle; Martinez, Ana; McMahon, Lori L.; Jope, Richard S.

    2013-01-01

    Background Identifying feasible therapeutic interventions is crucial for ameliorating the intellectual disability and other afflictions of Fragile X Syndrome (FXS), the most common inherited cause of intellectual disability and autism. Hippocampal glycogen synthase kinase-3 (GSK3) is hyperactive in the mouse model of FXS (FX mice), and hyperactive GSK3 promotes locomotor hyperactivity and audiogenic seizure susceptibility in FX mice, raising the possibility that specific GSK3 inhibitors may improve cognitive processes. Methods We tested if specific GSK3 inhibitors improve deficits in N-methyl-D-aspartate receptor (NMDAR)-dependent long term potentiation (LTP) at medial perforant path synapses onto dentate granule cells (MPP-DGC) and dentate gyrus-dependent cognitive behavioral tasks. Results GSK3 inhibitors completely rescued deficits in LTP at MPP-DGC synapses in FX mice. Furthermore, synaptosomes from the dentate gyrus of FX mice displayed decreased inhibitory serine-phosphorylation of GSK3β compared with wild-type littermates. The potential therapeutic utility of GSK3 inhibitors was further tested on dentate gyrus-dependent congnitive behaviors. In vivo administration of GSK3 inhibitors completely reversed impairments in several cognitive tasks in FX mice, including novel object detection, coordinate and categorical spatial processing, and temporal ordering for visual objects. Conclusions These findings establish that synaptic plasticity and cognitive deficits in FX mice can be improved by intervention with inhibitors of GSK3, which may prove therapeutically beneficial in FXS. PMID:24041505

  18. Glycogen synthase kinase-3β ablation limits pancreatitis-induced acinar-to-ductal metaplasia.

    Science.gov (United States)

    Ding, Li; Liou, Geou-Yarh; Schmitt, Daniel M; Storz, Peter; Zhang, Jin-San; Billadeau, Daniel D

    2017-09-01

    Acinar-to-ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre-malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase-3beta (GSK-3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK-3β promotes TGF-α-induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK-3β attenuates caerulein-induced ADM formation and PanIN progression in Kras G12D transgenic mice. Furthermore, we demonstrate that GSK-3β ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas-driven cell proliferation. Mechanistically, we show that GSK-3β regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK-3β participates in early pancreatitis-induced ADM and thus could be a target for the treatment of chronic pancreatitis and the prevention of PDAC progression. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  19. Glycogen synthase kinase 3 beta: can it be a target for oral cancer

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    Mishra Rajakishore

    2010-06-01

    Full Text Available Abstract Despite progress in treatment approaches for oral cancer, there has been only modest improvement in patient outcomes in the past three decades. The frequent treatment failure is due to the failure to control tumor recurrence and metastasis. These failures suggest that new targets should be identified to reverse oral epithelial dysplastic lesions. Recent developments suggest an active role of glycogen synthase kinase 3 beta (GSK3 β in various human cancers either as a tumor suppressor or as a tumor promoter. GSK3β is a Ser/Thr protein kinase, and there is emerging evidence that it is a tumor suppressor in oral cancer. The evidence suggests a link between key players in oral cancer that control transcription, accelerated cell cycle progression, activation of invasion/metastasis and anti-apoptosis, and regulation of these factors by GSK3β. Moreover, the major upstream kinases of GSK3β and their oncogenic activation by several etiological agents of oral cancer support this hypothesis. In spite of all this evidence, a detailed analysis of the role of GSK3β in oral cancer and of its therapeutic potential has yet to be conducted by the scientific community. The focus of this review is to discuss the multitude of roles of GSK3β, its possible role in controlling different oncogenic events and how it can be targeted in oral cancer.

  20. Critical role of glycogen synthase kinase-3ß in regulating the avian heterophil response to Salmonella enterica serovar Enteritidis

    Science.gov (United States)

    A microarray-assisted gene expression screen of chicken heterophils revealed glycogen synthase kinase-3ß (GSK-3ß), a multifunctional Ser/Thr kinase, to be consistently up-regulated 30-180 min following stimulation with Salmonella enterica serovar Enteritidis (S. Enteritidis). The present study was ...

  1. Hypoxic inactivation of glycogen synthase kinase-3β promotes gastric tumor growth and angiogenesis by facilitating hypoxia-inducible factor-1 signaling.

    Science.gov (United States)

    Ko, Young San; Cho, Sung Jin; Park, Jinju; Choi, Yiseul; Lee, Jae-Seon; Youn, Hong-Duk; Kim, Woo Ho; Kim, Min A; Park, Jong-Wan; Lee, Byung Lan

    2016-09-01

    Since the molecular mechanism of hypoxic adaptation in cancer cells is cell-type specific, we investigated whether glycogen synthase kinase-3β (GSK-3β) activation is involved in hypoxia-induced gastric tumor promotion. Stable gastric cancer cell lines (SNU-638, SNU-484, MKN1, and MKN45) were cultured under hypoxic conditions. Cells overexpressing wild-type GSK-3β (WT-GSK-3β) or kinase-dead mutant of GSK-3β (KD-GSK-3β) were generated and used for cell culture and animal studies. In cell culture experiments, hypoxia decreased GSK-3β activation in gastric cancer cells. Cell viability and the expressions of HIF-1α protein and VEGF mRNA in gastric cancer cells were higher in KD-GSK-3β transfectants than in WT-GSK-3β transfectants under hypoxic conditions, but not under normoxic conditions. Gastric cancer xenografts showed that tumor growth, microvessel area, HIF-1α activation, and VEGF expression were higher in KD-GSK-3β tumors than in WT-GSK-3β tumors in vivo. In addition, the expression of hypoxia-induced HIF-1α protein was regulated by GSK-3β at the translational level. Our data suggest that GSK-3β is involved in hypoxic adaptation of gastric cancer cells as an inhibitory upstream regulator of the HIF-1α/VEGF signaling pathway. © 2016 APMIS. Published by John Wiley & Sons Ltd.

  2. Glycogen synthase kinase-3β haploinsufficiency lengthens the circadian locomotor activity period in mice.

    Science.gov (United States)

    Lavoie, Joëlle; Hébert, Marc; Beaulieu, Jean-Martin

    2013-09-15

    The mood stabiliser drug lithium has been reported to impact circadian rhythms in vertebrates. Among several putative therapeutic molecular targets, direct inhibition of glycogen synthase kinase-3 beta (GSK3β) by lithium has been proposed to underlie its effects on circadian physiology. Here we study the effect of GSK3β haploinsufficiency on the circadian locomotor activity in mice during a free-running period in comparison to wildtype littermates (WT). Mice were housed individually to record their circadian wheel running activity and were entrained to a 12h light/12h dark cycle for 14 days and then placed under constant darkness for 14 days to allow free-running. During the free-running phase, the circadian locomotor activity period of GSK3β(+/-) was significantly lengthened (23.83±0.05h) when compared to the WT mice (23.54±0.10h; p=0.0374). No significant difference in locomotor activity was observed. Knowing that GSK3β interacts with most of the core clock components, these data suggest that GSK3β acts as a critical intrinsic regulator of the circadian clock and plays an important role in regulating its period in response to lithium treatment. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Glycogen Synthase Kinase 3β Inhibition as a Therapeutic Approach in the Treatment of Endometrial Cancer

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    Liang Ma

    2013-08-01

    Full Text Available Alternative strategies beyond current chemotherapy and radiation therapy regimens are needed in the treatment of advanced stage and recurrent endometrial cancers. There is considerable promise for biologic agents targeting the extracellular signal-regulated kinase (ERK pathway for treatment of these cancers. Many downstream substrates of the ERK signaling pathway, such as glycogen synthase kinase 3β (GSK3β, and their roles in endometrial carcinogenesis have not yet been investigated. In this study, we tested the importance of GSK3β inhibition in endometrial cancer cell lines and in vivo models. Inhibition of GSK3β by either lithium chloride (LiCl or specific GSK3β inhibitor VIII showed cytostatic and cytotoxic effects on multiple endometrial cancer cell lines, with little effect on the immortalized normal endometrial cell line. Flow cytometry and immunofluorescence revealed a G2/M cell cycle arrest in both type I (AN3CA, KLE, and RL952 and type II (ARK1 endometrial cancer cell lines. In addition, LiCl pre-treatment sensitized AN3CA cells to the chemotherapy agent paclitaxel. Administration of LiCl to AN3CA tumor-bearing mice resulted in partial or complete regression of some tumors. Thus, GSK3β activity is associated with endometrial cancer tumorigenesis and its pharmacologic inhibition reduces cell proliferation and tumor growth.

  4. Glycogen synthase kinase 3α regulates urine concentrating mechanism in mice

    DEFF Research Database (Denmark)

    Nørregaard, Rikke; Tao, Shixin; Nilsson, Line

    2015-01-01

    In mammals, glycogen synthase kinase (GSK)3 comprises GSK3α and GSK3β isoforms. GSK3β has been shown to play a role in the ability of kidneys to concentrate urine by regulating vasopressin-mediated water permeability of collecting ducts, whereas the role of GSK3α has yet to be discerned. To inves......In mammals, glycogen synthase kinase (GSK)3 comprises GSK3α and GSK3β isoforms. GSK3β has been shown to play a role in the ability of kidneys to concentrate urine by regulating vasopressin-mediated water permeability of collecting ducts, whereas the role of GSK3α has yet to be discerned...

  5. Glycogen synthase kinase-3 inhibition attenuates fibroblast activation and development of fibrosis following renal ischemia-reperfusion in mice

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    Shailendra P. Singh

    2015-08-01

    Full Text Available Glycogen synthase kinase-3β (GSK3β is a serine/threonine protein kinase that plays an important role in renal tubular injury and regeneration in acute kidney injury. However, its role in the development of renal fibrosis, often a long-term consequence of acute kidney injury, is unknown. Using a mouse model of renal fibrosis induced by ischemia-reperfusion injury, we demonstrate increased GSK3β expression and activity in fibrotic kidneys, and its presence in myofibroblasts in addition to tubular epithelial cells. Pharmacological inhibition of GSK3 using TDZD-8 starting before or after ischemia-reperfusion significantly suppressed renal fibrosis by reducing the myofibroblast population, collagen-1 and fibronectin deposition, inflammatory cytokines, and macrophage infiltration. GSK3 inhibition in vivo reduced TGF-β1, SMAD3 activation and plasminogen activator inhibitor-1 levels. Consistently in vitro, TGF-β1 treatment increased GSK3β expression and GSK3 inhibition abolished TGF-β1-induced SMAD3 activation and α-smooth muscle actin (α-SMA expression in cultured renal fibroblasts. Importantly, overexpression of constitutively active GSK3β stimulated α-SMA expression even in the absence of TGF-β1 treatment. These results suggest that TGF-β regulates GSK3β, which in turn is important for TGF-β–SMAD3 signaling and fibroblast-to-myofibroblast differentiation. Overall, these studies demonstrate that GSK3 could promote renal fibrosis by activation of TGF-β signaling and the use of GSK3 inhibitors might represent a novel therapeutic approach for progressive renal fibrosis that develops as a consequence of acute kidney injury.

  6. Glycogen synthase kinase 3 regulates expression of nuclear factor-erythroid-2 related transcription factor-1 (Nrf1) and inhibits pro-survival function of Nrf1

    Energy Technology Data Exchange (ETDEWEB)

    Biswas, Madhurima; Kwong, Erick K.; Park, Eujean; Nagra, Parminder; Chan, Jefferson Y., E-mail: jchan@uci.edu

    2013-08-01

    Nuclear factor E2-related factor-1 (Nrf1) is a basic leucine zipper transcription factor that is known to regulate antioxidant and cytoprotective gene expression. It was recently shown that Nrf1 is regulated by SCF–Fbw7 ubiquitin ligase. However our knowledge of upstream signals that targets Nrf1 for degradation by the UPS is not known. We report here that Nrf1 expression is negatively regulated by glycogen synthase kinase 3 (GSK3) in Fbw7-dependent manner. We show that GSK3 interacts with Nrf1 and phosphorylates the Cdc4 phosphodegron domain (CPD) in Nrf1. Mutation of serine residue in the CPD of Nrf1 to alanine (S350A), blocks Nrf1 from phosphorylation by GSK3, and stabilizes Nrf1. Knockdown of Nrf1 and expression of a constitutively active form of GSK3 results in increased apoptosis in neuronal cells in response to ER stress, while expression of the GSK3 phosphorylation resistant S350A–Nrf1 attenuates apoptotic cell death. Together these data suggest that GSK3 regulates Nrf1 expression and cell survival function in response to stress activation. Highlights: • The effect of GSK3 on Nrf1 expression was examined. • GSK3 destabilizes Nrf1 protein via Fbw7 ubiquitin ligase. • GSK3 binds and phosphorylates Nrf1. • Protection from stress-induced apoptosis by Nrf1 is inhibited by GSK3.

  7. Glycogen synthase kinase-3: A promising therapeutic target for Fragile X Syndrome

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    Marjelo M. Mines

    2011-11-01

    Full Text Available Recent advances in understanding the pathophysiological mechanisms contributing to Fragile X Syndrome (FXS have increased optimism that drug interventions can provide significant therapeutic benefits. FXS results from inadequate expression of functional fragile X mental retardation protein (FMRP. FMRP may have several functions, but it is most well-established as an RNA-binding protein that regulates translation, and it is by this mechanism that FMRP is capable of affecting numerous cellular processes by selectively regulating protein levels. The multiple cellular functions regulated by FMRP suggest that multiple interventions may be required for reversing the effects of deficient FMRP. Evidence that inhibitors of glycogen synthase kinase-3 (GSK3 may contribute to the therapeutic treatment of FXS is reviewed here. In the mouse model of FXS, which lacks FMRP expression (FX mice, GSK3 is hyperactive in several brain regions. Furthermore, significant improvements in several FX-related phenotypes have been obtained in FX mice following the administration of lithium, and in some case other GSK3 inhibitors. These responses include normalization of heightened audiogenic seizure susceptibility and of hyperactive locomotor behavior, enhancement of passive avoidance learning retention and of sociability behaviors, and corrections of macroorchidism, neuronal spine density, and neural plasticity measured electrophysiologically as long term depression. A pilot clinical trial of lithium in FXS patients also found improvements in several measures of behavior. Taken together, these findings indicate that lithium and other inhibitors of GSK3 are promising candidate therapeutic agents for treating FXS.

  8. Exploiting an Asp-Glu "switch" in glycogen synthase kinase 3 to design paralog-selective inhibitors for use in acute myeloid leukemia.

    Science.gov (United States)

    Wagner, Florence F; Benajiba, Lina; Campbell, Arthur J; Weïwer, Michel; Sacher, Joshua R; Gale, Jennifer P; Ross, Linda; Puissant, Alexandre; Alexe, Gabriela; Conway, Amy; Back, Morgan; Pikman, Yana; Galinsky, Ilene; DeAngelo, Daniel J; Stone, Richard M; Kaya, Taner; Shi, Xi; Robers, Matthew B; Machleidt, Thomas; Wilkinson, Jennifer; Hermine, Olivier; Kung, Andrew; Stein, Adam J; Lakshminarasimhan, Damodharan; Hemann, Michael T; Scolnick, Edward; Zhang, Yan-Ling; Pan, Jen Q; Stegmaier, Kimberly; Holson, Edward B

    2018-03-07

    Glycogen synthase kinase 3 (GSK3), a key regulatory kinase in the wingless-type MMTV integration site family (WNT) pathway, is a therapeutic target of interest in many diseases. Although dual GSK3α/β inhibitors have entered clinical trials, none has successfully translated to clinical application. Mechanism-based toxicities, driven in part by the inhibition of both GSK3 paralogs and subsequent β-catenin stabilization, are a concern in the translation of this target class because mutations and overexpression of β-catenin are associated with many cancers. Knockdown of GSK3α or GSK3β individually does not increase β-catenin and offers a conceptual resolution to targeting GSK3: paralog-selective inhibition. However, inadequate chemical tools exist. The design of selective adenosine triphosphate (ATP)-competitive inhibitors poses a drug discovery challenge due to the high homology (95% identity and 100% similarity) in this binding domain. Taking advantage of an Asp 133 →Glu 196 "switch" in their kinase hinge, we present a rational design strategy toward the discovery of paralog-selective GSK3 inhibitors. These GSK3α- and GSK3β-selective inhibitors provide insights into GSK3 targeting in acute myeloid leukemia (AML), where GSK3α was identified as a therapeutic target using genetic approaches. The GSK3α-selective compound BRD0705 inhibits kinase function and does not stabilize β-catenin, mitigating potential neoplastic concerns. BRD0705 induces myeloid differentiation and impairs colony formation in AML cells, with no apparent effect on normal hematopoietic cells. Moreover, BRD0705 impairs leukemia initiation and prolongs survival in AML mouse models. These studies demonstrate feasibility of paralog-selective GSK3α inhibition, offering a promising therapeutic approach in AML. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  9. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    International Nuclear Information System (INIS)

    Manceur, Aziza P.; Tseng, Michael; Holowacz, Tamara; Witterick, Ian; Weksberg, Rosanna; McCurdy, Richard D.; Warsh, Jerry J.; Audet, Julie

    2011-01-01

    The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

  10. Glycogen synthase kinase-3β regulates leucine-309 demethylation of protein phosphatase-2A via PPMT1 and PME-1.

    Science.gov (United States)

    Yao, Xiu-Qing; Li, Xia-Chun; Zhang, Xiao-Xue; Yin, Yang-Yang; Liu, Bin; Luo, Dan-Ju; Wang, Qun; Wang, Jian-Zhi; Liu, Gong-Ping

    2012-07-30

    Protein phosphatase-2A (PP2A) activity is significantly suppressed in Alzheimer's disease. We have reported that glycogen synthase kinase-3β (GSK-3β) inhibits PP2A via upregulating the phosphorylation of PP2A catalytic subunit (PP2A(C)). Here we studied the effects of GSK-3β on the inhibitory demethylation of PP2A at leucine-309 (dmL309-PP2A(C)). We found that GSK-3β regulates dmL309-PP2A(C) level by regulating PME-1 and PPMT1. Knockdown of PME-1 or PPMT1 eliminated the effects of GSK-3β on PP2A(C). GSK-3 could negatively regulate PP2A regulatory subunit protein level. We conclude that GSK-3β can inhibit PP2A by increasing the inhibitory L309-demethylation involving upregulation of PME-1 and inhibition of PPMT1. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  11. Identification of a Glycogen Synthase Kinase-3[beta] Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice

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    Kozikowski, Alan P.; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N.; Walter, Richard L.; Ketcherside, Ariel; McClung, Colleen A.; Mesecar, Andrew D.; Caldarone, Barbara (Psychogenics); (Purdue); (UIC); (UTSMC)

    2012-05-02

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called 'mood-stabilizing drugs', such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3{beta} (GSK-3{beta}) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3{beta}. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC{sub 50} values in the range of 4 to 680 nM against human GSK-3{beta}. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mg kg{sup -1} resulted in the attenuation of hyperactivity in amphetamine/chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mg kg{sup -1}) and the antipsychotic haloperidol (1 mg kg{sup -1}). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3{beta} in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3{beta} as a relevant therapeutic target in the identification of new therapies for bipolar patients.

  12. Differential expression of GSK3β and pS9GSK3β in normal human tissues: can pS9GSK3β be an epithelial marker?

    Science.gov (United States)

    Lee, Hojung; Ro, Jae Y

    2015-01-01

    Glycogen synthase kinase 3β (GSK3β) and phosphorylated GSK3β at Ser9 (pS9GSK3β) are crucial in cellular proliferation and metabolism. GSK3β and pS9GSK3β are deregulated in many diseases including tumors. Data on altered expression of GSK3β and pS9GSK3β are mainly limited to tumor tissues, thus the expression of GSK3β and pS9GSK3β in normal human tissue has been largely unknown. Thus, we examined the immunohistochemical localization of GSK3β and pS9GSK3β in human fetal and adult tissues, and also compared the expression pattern of GSK3β and pS9GSK3β with that of the CK7 and CK20. We found GSK3β expression in neurons of brain, myenteric plexus in gastrointestinal tract, squamous epithelium of skin, and mammary gland. The expression of pS9GSK3β was restricted to the epithelial cells of breast and pancreaticobiliary duct, distal nephron of kidney, gastrointestinal tract, fallopian tube, epididymis, secretory cell of prostatic gland, and umbrella cell of urinary tract. The staining pattern of pS9GSK3β and CK7 was overlapped in most organs except for gastrointestinal tract where CK7 was negative and CK20 was positive. Our results show that the expression of GSK3β may be associated with differentiation of ectodermal derived tissues and pS9GSK3β with that of epithelial cells of endodermal derived tissues in human. In addition, the expression of pS9GSK3β in the selective epithelial cells may indicate its association with secretory or barrier function of specific cells and may serve as another immunohistochemical marker for epithelial cells.

  13. Glycogen synthase kinase 3β in the basolateral amygdala is critical for the reconsolidation of cocaine reward memory.

    Science.gov (United States)

    Wu, Ping; Xue, Yan-Xue; Ding, Zeng-Bo; Xue, Li-Fen; Xu, Chun-Mei; Lu, Lin

    2011-07-01

    Exposure to cocaine-associated conditioned stimuli elicits craving and increases the probability of cocaine relapse in cocaine users even after extended periods of abstinence. Recent evidence indicates that cocaine seeking can be inhibited by disrupting the reconsolidation of the cocaine cue memories and that basolateral amygdala (BLA) neuronal activity plays a role in this effect. Previous studies demonstrated that glycogen synthase kinase 3β (GSK-3β) plays a role in the reconsolidation of fear memory. Here, we used a conditioned place preference procedure to examine the role of GSK-3β in the BLA in the reconsolidation of cocaine cue memories. GSK-3β activity in the BLA, but not central amygdala (CeA), in rats that acquired cocaine (10 mg/kg)-induced conditioned place preference increased after re-exposure to a previously cocaine-paired chamber (i.e., a memory reactivation procedure). Systemic injections of the GSK-3β inhibitor lithium chloride after memory reactivation impaired the reconsolidation of cocaine cue memories and inhibited subsequent cue-induced GSK-3β activity in the BLA. Basolateral amygdala, but not central amygdala, injections of SB216763, a selective inhibitor of GSK-3β, immediately after the reactivation of cocaine cue memories also disrupted cocaine cue memory reconsolidation and prevented cue-induced increases in GSK-3β activity in the BLA. The effect of SB216763 on the reconsolidation of cocaine cue memories lasted at least 2 weeks and was not recovered by a cocaine priming injection. These results indicate that GSK-3β activity in the BLA mediates the reconsolidation of cocaine cue memories. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  14. Glycogen Synthase Kinase-3 regulates IGFBP-1 gene transcription through the Thymine-rich Insulin Response Element

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    Marquez Rodolfo

    2004-09-01

    Full Text Available Abstract Background Hepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK, glucose-6-phosphatase (G6Pase and insulin-like growth factor binding protein-1 (IGFBP-1, is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the Thymine-rich Insulin Response Element (TIRE. The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase. However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3 is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase, whose products are required for gluconeogenesis. Results In this report we demonstrate that in H4IIE-C3 cells, four distinct classes of GSK-3 inhibitor mimic the effect of insulin on a third TIRE-containing gene, IGFBP-1. We identify the TIRE as the minimum requirement for inhibition by these agents, and demonstrate that the target of GSK-3 is unlikely to be the postulated TIRE-binding protein FOXO-1. Importantly, overexpression of GSK-3 in cells reduces the insulin regulation of TIRE activity as well as endogenous IGFBP-1 expression. Conclusions These results implicate GSK-3 as an intermediate in the pathway from the insulin receptor to the TIRE. Indeed, this is the first demonstration of an absolute requirement for GSK-3 inhibition in insulin regulation of gene transcription. These data support the potential use of GSK-3 inhibitors in the treatment of insulin resistant states such as Type 2 diabetes mellitus, but suggest that it will be important to identify all TIRE-containing genes to assess potential side effects of these agents.

  15. Pharmacological inhibition of glycogen synthase kinase 3 regulates T cell development in vitro.

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    Jan-Hendrik Schroeder

    Full Text Available The development of functional T cells requires receptor-mediated transition through multiple checkpoints in the thymus. Double negative 3 (DN3 thymocytes are selected for the presence of a rearranged TCR beta chain in a process termed β-selection which requires signalling via the pre-TCR, Notch1 and CXCL12. Signal integration by these receptors converges on core pathways including the Phosphatidylinositol-3-kinase (PI3K pathway. Glycogen Synthase Kinase 3 (GSK3 is generally thought to be negatively regulated by the PI3K pathway but its role in β-selection has not been characterised. Here we show that developmental progression of DN3 thymocytes is promoted following inhibition of GSK3 by the synthetic compound CHIR99021. CHIR99021 allows differentiation in the absence of pre-TCR-, Notch1- or CXCL12-mediated signalling. It antagonizes IL-7-mediated inhibition of DP thymocyte differentiation and increases IL-7-promoted cell recovery. These data indicate a potentially important role for inactivation of GSK3 during β-selection. They might help to establish an in vitro stromal cell-free culture system of thymocyte development and offer a new platform for screening regulators of proliferation, differentiation and apoptosis.

  16. Glycogen Synthase Kinase-3 Regulates Production of Amyloid-β Peptides and Tau Phosphorylation in Diabetic Rat Brain

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    Zhong-Sen Qu

    2014-01-01

    Full Text Available The pathogenesis of diabetic neurological complications is not fully understood. Diabetes mellitus (DM and Alzheimer’s disease (AD are characterized by amyloid deposits. Glycogen synthase kinase-3 (GSK-3 plays an important role in the pathogenesis of AD and DM. Here we tried to investigate the production of amyloid-β peptides (Aβ and phosphorylation of microtubule-associated protein tau in DM rats and elucidate the role of GSK-3 and Akt (protein kinase B, PKB in these processes. Streptozotocin injection-induced DM rats displayed an increased GSK-3 activity, decreased activity and expression of Akt. And Aβ40 and Aβ42 were found overproduced and the microtubule-associated protein tau was hyperphosphorylated in the hippocampus. Furthermore, selective inhibition of GSK-3 by lithium could attenuate the conditions of Aβ overproduction and tau hyperphosphorylation. Taken together, our studies suggest that GSK-3 regulates both the production of Aβ and the phosphorylation of tau in rat brain and may therefore contribute to DM caused AD-like neurological defects.

  17. GSK-3α is a central regulator of age-related pathologies in mice

    OpenAIRE

    Zhou, Jibin; Freeman, Theresa A.; Ahmad, Firdos; Shang, Xiying; Mangano, Emily; Gao, Erhe; Farber, John; Wang, Yajing; Ma, Xin-Liang; Woodgett, James; Vagnozzi, Ronald J.; Lal, Hind; Force, Thomas

    2013-01-01

    Aging is regulated by conserved signaling pathways. The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases regulates several of these pathways, but the role of GSK-3 in aging is unknown. Herein, we demonstrate premature death and acceleration of age-related pathologies in the Gsk3a global KO mouse. KO mice developed cardiac hypertrophy and contractile dysfunction as well as sarcomere disruption and striking sarcopenia in cardiac and skeletal muscle, a classical finding in a...

  18. A pivotal role of GSK-3 in synaptic plasticity

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    Clarrisa A Bradley

    2012-02-01

    Full Text Available Glycogen synthase kinase-3 (GSK-3 has many cellular functions. Recent evidence suggests that it plays a key role in certain types of synaptic plasticity, in particular a form of long-term depression (LTD that is induced by the synaptic activation of N-methyl-D-aspartate (NMDA receptors. In the present article we summarise what is currently known concerning the roles of GSK-3 in synaptic plasticity at both glutamatergic and GABAergic synapses. We summarise its role in cognition and speculate on how alterations in the synaptic functioning of GSK-3 may be a major factor in certain neurodegenerative disorders.

  19. A pivotal role of GSK-3 in synaptic plasticity

    Science.gov (United States)

    Bradley, Clarrisa A.; Peineau, Stéphane; Taghibiglou, Changiz; Nicolas, Celine S.; Whitcomb, Daniel J.; Bortolotto, Zuner A.; Kaang, Bong-Kiun; Cho, Kwangwook; Wang, Yu Tian; Collingridge, Graham L.

    2012-01-01

    Glycogen synthase kinase-3 (GSK-3) has many cellular functions. Recent evidence suggests that it plays a key role in certain types of synaptic plasticity, in particular a form of long-term depression (LTD) that is induced by the synaptic activation of N-methyl-D-aspartate receptors (NMDARs). In the present article we summarize what is currently known concerning the roles of GSK-3 in synaptic plasticity at both glutamatergic and GABAergic synapses. We summarize its role in cognition and speculate on how alterations in the synaptic functioning of GSK-3 may be a major factor in certain neurodegenerative disorders. PMID:22363262

  20. Saturated free fatty acid sodium palmitate-induced lipoapoptosis by targeting glycogen synthase kinase-3β activation in human liver cells.

    Science.gov (United States)

    Cao, Jie; Feng, Xiao-Xia; Yao, Long; Ning, Bo; Yang, Zhao-Xia; Fang, Dian-Liang; Shen, Wei

    2014-02-01

    Elevated serum saturated fatty acid levels and hepatocyte lipoapoptosis are features of nonalcoholic fatty liver disease (NAFLD). The purpose of this study was to investigate saturated fatty acid induction of lipoapoptosis in human liver cells and the underlying mechanisms. Human liver L02 and HepG2 cells were treated with sodium palmitate, a saturated fatty acid, for up to 48 h with or without lithium chloride, a glycogen synthase kinase-3β (GSK-3β) inhibitor, or GSK-3β shRNA transfection. Transmission electron microscopy was used to detect morphological changes, flow cytometry was used to detect apoptosis, a colorimetric assay was used to detect caspase-3 activity, and western blot analysis was used to detect protein expression. The data showed that sodium palmitate was able to induce lipoapoptosis in L02 and HepG2 cells. Western blot analysis showed that sodium palmitate activated GSK-3β protein, which was indicated by dephosphorylation of GSK-3β at Ser-9. However, inhibition of GSK-3β activity with lithium chloride treatment or knockdown of GSK-3β expression with shRNA suppressed sodium palmitate-induced lipoapoptosis in L02 and HepG2 cells. On a molecular level, inhibition of GSK-3β expression or activity suppressed sodium palmitate-induced c-Jun-N-terminal kinase (JNK) phosphorylation and Bax upregulation, whereas GSK-3β inhibition did not affect endoplasmic reticulum stress-induced activation of unfolded protein response. The present data demonstrated that saturated fatty acid sodium palmitate-induced lipoapoptosis in human liver L02 and HepG2 cells was regulated by GSK-3β activation, which led to JNK activation and Bax upregulation. This finding indicates that GSK-3β inhibition may be a potential therapeutic target to control NAFLD.

  1. Planarian GSK3s are involved in neural regeneration.

    Science.gov (United States)

    Adell, Teresa; Marsal, Maria; Saló, Emili

    2008-02-01

    Glycogen synthase kinase-3 (GSK3) is a key element in several signaling cascades that is known to be involved in both patterning and neuronal organization. It is, therefore, a good candidate to play a role in neural regeneration in planarians. We report the characterization of three GSK3 genes in Schmidtea mediterranea. Phylogenetic analysis shows that Smed-GSK3.1 is highly conserved compared to GSK3 sequences from other species, whereas Smed-GSK3.2 and Smed-GSK3.3 are more divergent. Treatment of regenerating planarians with 1-azakenpaullone, a synthetic GSK3 inhibitor, suggests that planarian GSK3s are essential for normal differentiation and morphogenesis of the nervous system. Cephalic ganglia appear smaller and disconnected in 1-azakenpaullone-treated animals, whereas visual axons are ectopically projected, and the pharynx does not regenerate properly. This phenotype is consistent with a role for Smed-GSK3s in neuronal polarization and axonal growth.

  2. Glycogen Synthase Kinase 3 Inhibitors in the Next Horizon for Alzheimer's Disease Treatment

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    Ana Martinez

    2011-01-01

    Full Text Available Glycogen synthase kinase 3 (GSK-3, a proline/serine protein kinase ubiquitously expressed and involved in many cellular signaling pathways, plays a key role in the pathogenesis of Alzheimer's disease (AD being probably the link between β-amyloid and tau pathology. A great effort has recently been done in the discovery and development of different new molecules, of synthetic and natural origin, able to inhibit this enzyme, and several kinetics mechanisms of binding have been described. The small molecule called tideglusib belonging to the thiadiazolidindione family is currently on phase IIb clinical trials for AD. The potential risks and benefits of this new kind of disease modifying drugs for the future therapy of AD are discussed in this paper.

  3. Possible Role of the Glycogen Synthase Kinase-3 Signaling Pathway in Trimethyltin-Induced Hippocampal Neurodegeneration in Mice

    Science.gov (United States)

    Kim, Sung-Ho; Kim, Jong-Choon; Wang, Hongbing; Shin, Taekyun; Moon, Changjong

    2013-01-01

    Trimethyltin (TMT) is an organotin compound with potent neurotoxic effects characterized by neuronal destruction in selective regions, including the hippocampus. Glycogen synthase kinase-3 (GSK-3) regulates many cellular processes, and is implicated in several neurodegenerative disorders. In this study, we evaluated the therapeutic effect of lithium, a selective GSK-3 inhibitor, on the hippocampus of adult C57BL/6 mice with TMT treatment (2.6 mg/kg, intraperitoneal [i.p.]) and on cultured hippocampal neurons (12 days in vitro) with TMT treatment (5 µM). Lithium (50 mg/kg, i.p., 0 and 24 h after TMT injection) significantly attenuated TMT-induced hippocampal cell degeneration, seizure, and memory deficits in mice. In cultured hippocampal neurons, lithium treatment (0–10 mM; 1 h before TMT application) significantly reduced TMT-induced cytotoxicity in a dose-dependent manner. Additionally, the dynamic changes in GSK-3/β-catenin signaling were observed in the mouse hippocampus and cultured hippocampal neurons after TMT treatment with or without lithium. Therefore, lithium inhibited the detrimental effects of TMT on the hippocampal neurons in vivo and in vitro, suggesting involvement of the GSK-3/β-catenin signaling pathway in TMT-induced hippocampal cell degeneration and dysfunction. PMID:23940567

  4. Inhibition of glycogen synthase kinase-3β by falcarindiol isolated from Japanese Parsley (Oenanthe javanica).

    Science.gov (United States)

    Yoshida, Jun; Seino, Hiroko; Ito, Yoshiaki; Nakano, Toshimitsu; Satoh, Takumi; Ogane, Yoshiko; Suwa, Saori; Koshino, Hiroyuki; Kimura, Ken-Ichi

    2013-08-07

    A new biological activity of falcarindiol isolated from Japanese parsley (Oenanthe javanica) using the mutant yeast YNS17 strain (zds1Δ erg3Δ pdr1Δ pdr3Δ) was discovered as an inhibitor of glycogen synthase kinase-3β (GSK-3β). Falcarindiol inhibited GSK-3β in an ATP noncompetitive manner with a Ki value of 86.9 μM using a human enzyme and luminescent kinase assay platform. Falcarindiol also both suppressed gene expression of glucose-6-phosphatase (G6Pase) in rat hepatoma H4IIE cells and protected mouse neuroblastoma HT22 cells from glutamate-induced oxidative cell death at 10 μM. During an oral glucose tolerance test (OGTT), the blood glucose level was significantly decreased in the rats treated with oral administration of O. javanica extract containing falcarindiol (15 mg/kg). These findings indicate that Japanese parsley could be a useful food ingredient against type-2 diabetes and Alzheimer's disease.

  5. Dynamic interplay between O-linked N-acetylglucosaminylation and glycogen synthase kinase-3-dependent phosphorylation.

    Science.gov (United States)

    Wang, Zihao; Pandey, Akhilesh; Hart, Gerald W

    2007-08-01

    O-GlcNAcylation on serine and threonine side chains of nuclear and cytoplasmic proteins is dynamically regulated in response to various environmental and biological stimuli. O-GlcNAcylation is remarkably similar to O-phosphorylation and appears to have a dynamic interplay with O-phosphate in cellular regulation. A systematic glycoproteomics analysis of the affects of inhibiting specific kinases on O-GlcNAcylation should help reveal both the global and specific dynamic relationships between these two abundant post-translational modifications. Here we report the O-GlcNAc perturbations in response to inhibition of glycogen synthase kinase-3 (GSK-3), a pivotal kinase involved in many signaling pathways. By combining immunoaffinity chromatography and SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative mass spectrometry, we identified 45 potentially O-GlcNAcylated proteins. Quantitative measurements indicated that at least 10 proteins had an apparent increase of O-GlcNAcylation upon GSK-3 inhibition by lithium, whereas surprisingly 19 other proteins showed decreases. O-GlcNAcylation changes on a subset of the proteins were confirmed by follow-up experiments. By combining a new O-GlcNAc peptide enrichment method and beta-elimination followed by Michael addition with DTT, we also mapped the O-GlcNAc site (Ser-55) of vimentin, which showed an apparent increase of O-GlcNAcylation upon GSK-3 inhibition. Based on the MS data, we further investigated potential roles of O-GlcNAc on host cell factor-1, a transcription co-activator, and showed that dynamic regulation of O-GlcNAcylation on host cell factor-1 influenced its subcellular distribution. Taken together, these data indicated the complex interplay between phosphorylation and O-GlcNAcylation that occurs within signaling networks.

  6. Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

    Directory of Open Access Journals (Sweden)

    Chen Wu-Fu

    2010-12-01

    Full Text Available Abstract Background Deciphering the mechanisms that modulate the inflammatory response induced by microglial activation not only improves our insight into neuroinflammation but also provides avenues for designing novel therapies that could halt inflammation-induced neuronal degeneration. Decreasing glycogen synthase kinase-3β (GSK-3β activity has therapeutic benefits in inflammatory diseases. However, the exact molecular mechanisms underlying GSK-3β inactivation-mediated suppression of the inflammatory response induced by microglial activation have not been completely clarified. Tumor necrosis factor-α (TNF-α plays a central role in injury caused by neuroinflammation. We investigated the regulatory effect of GSK-3β on TNF-α production by microglia to discern the molecular mechanisms of this modulation. Methods Lipopolysaccharide (LPS was used to induce an inflammatory response in cultured primary microglia or murine BV-2 microglial cells. Release of TNF-α was measured by ELISA. Signaling molecules were analyzed by western blotting, and activation of NF-κB and AP-1 was measured by ELISA-based DNA binding analysis and luciferase reporter assay. Protein interaction was examined by coimmunoprecipitation. Results Inhibition of GSK-3β by selective GSK-3β inhibitors or by RNA interference attenuated LPS-induced TNF-α production in cultured microglia. Exploration of the mechanisms by which GSK-3β positively regulates inflammatory response showed that LPS-induced IκB-α degradation, NF-κBp65 nuclear translocation, and p65 DNA binding activity were not affected by inhibition of GSK-3β activity. However, GSK-3β inactivation inhibited transactivation activity of p65 by deacetylating p65 at lysine 310. Furthermore, we also demonstrated a functional interaction between mixed lineage kinase 3 (MLK3 and GSK-3β during LPS-induced TNF-α production in microglia. The phosphorylated levels of MLK3, MKK4, and JNK were increased upon LPS treatment

  7. Glycogen synthase kinase-3 inhibition disrupts nuclear factor-kappaB activity in pancreatic cancer, but fails to sensitize to gemcitabine chemotherapy

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    Mamaghani Shadi

    2009-04-01

    Full Text Available Abstract Background Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine. Methods GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition. Results GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-XL, and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic. Conclusion GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard

  8. Protective Effects of Kaempferol against Myocardial Ischemia/Reperfusion Injury in Isolated Rat Heart via Antioxidant Activity and Inhibition of Glycogen Synthase Kinase-3β

    Science.gov (United States)

    Zhou, Mingjie; Ren, Huanhuan; Wang, Wenjuan; Zheng, Qiusheng; Wang, Dong

    2015-01-01

    Objective. This study aimed to evaluate the protective effect of kaempferol against myocardial ischemia/reperfusion (I/R) injury in rats. Method. Left ventricular developed pressure (LVDP) and its maximum up/down rate (±dp/dt max) were recorded as myocardial function. Infarct size was detected with 2,3,5-triphenyltetrazolium chloride staining. Cardiomyocyte apoptosis was determined using terminal deoxynucleotidyl nick-end labeling (TUNEL). The levels of creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione/glutathione disulfide (GSH/GSSG) ratio, and tumor necrosis factor-alpha (TNF-α) were determined using enzyme linked immunosorbent assay (ELISA). Moreover, total glycogen synthase kinase-3β (GSK-3β), phospho-GSK-3β (P-GSK-3β), precaspase-3, cleaved caspase-3, and cytoplasm cytochrome C were assayed using Western blot analysis. Results. Pretreatment with kaempferol significantly improved the recovery of LVDP and ±dp/dt max, as well as increased the levels of SOD and P-GSK-3β and GSH/GSSG ratio. However, the pretreatment reduced myocardial infarct size and TUNEL-positive cell rate, as well as decreased the levels of cleaved caspase-3, cytoplasm cytochrome C, CK, LDH, MDA, and TNF-α. Conclusion. These results suggested that kaempferol provides cardioprotection via antioxidant activity and inhibition of GSK-3β activity in rats with I/R. PMID:26265983

  9. Phylogenetic diversification of glycogen synthase kinase 3/SHAGGY-like kinase genes in plants

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    Soltis Pamela S

    2006-02-01

    Full Text Available Abstract Background The glycogen synthase kinase 3 (GSK3/SHAGGY-like kinases (GSKs are non-receptor serine/threonine protein kinases that are involved in a variety of biological processes. In contrast to the two members of the GSK3 family in mammals, plants appear to have a much larger set of divergent GSK genes. Plant GSKs are encoded by a multigene family; analysis of the Arabidopsis genome revealed the existence of 10 GSK genes that fall into four major groups. Here we characterized the structure of Arabidopsis and rice GSK genes and conducted the first broad phylogenetic analysis of the plant GSK gene family, covering a taxonomically diverse array of algal and land plant sequences. Results We found that the structure of GSK genes is generally conserved in Arabidopsis and rice, although we documented examples of exon expansion and intron loss. Our phylogenetic analyses of 139 sequences revealed four major clades of GSK genes that correspond to the four subgroups initially recognized in Arabidopsis. ESTs from basal angiosperms were represented in all four major clades; GSK homologs from the basal angiosperm Persea americana (avocado appeared in all four clades. Gymnosperm sequences occurred in clades I, III, and IV, and a sequence of the red alga Porphyra was sister to all green plant sequences. Conclusion Our results indicate that (1 the plant-specific GSK gene lineage was established early in the history of green plants, (2 plant GSKs began to diversify prior to the origin of extant seed plants, (3 three of the four major clades of GSKs present in Arabidopsis and rice were established early in the evolutionary history of extant seed plants, and (4 diversification into four major clades (as initially reported in Arabidopsis occurred either just prior to the origin of the angiosperms or very early in angiosperm history.

  10. Up-Regulation of Excitatory Amino Acid Transporters EAAT3 and EAAT4 by Lithium Sensitive Glycogen Synthase Kinase GSK3ß

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    Abeer Abousaab

    2016-12-01

    Full Text Available Background: Cellular uptake of glutamate by the excitatory amino-acid transporters (EAATs decreases excitation and thus participates in the regulation of neuroexcitability. Kinases impacting on neuronal function include Lithium-sensitive glycogen synthase kinase GSK3ß. The present study thus explored whether the activities of EAAT3 and/or EAAT4 isoforms are sensitive to GSK3ß. Methods: cRNA encoding wild type EAAT3 (SLC1A1 or EAAT4 (SLC1A6 was injected into Xenopus oocytes without or with additional injection of cRNA encoding wild type GSK3ß or the inactive mutant K85AGSK3ß. Dual electrode voltage clamp was performed in order to determine glutamate-induced current (IEAAT. Results: Appreciable IEAAT was observed in EAAT3 or EAAT4 expressing but not in water injected oocytes. IEAAT was significantly increased by coexpression of GSK3ß but not by coexpression of K85AGSK3ß. Coexpression of GSK3ß increased significantly the maximal IEAAT in EAAT3 or EAAT4 expressing oocytes, without significantly modifying apparent affinity of the carriers. Lithium (1 mM exposure for 24 hours decreased IEAAT in EAAT3 and GSK3ß expressing oocytes to values similar to IEAAT in oocytes expressing EAAT3 alone. Lithium did not significantly modify IEAAT in oocytes expressing EAAT3 without GSK3ß. Conclusions: Lithium-sensitive GSK3ß is a powerful regulator of excitatory amino acid transporters EAAT3 and EAAT4.

  11. Recent advances in understanding the cellular roles of GSK-3.

    Science.gov (United States)

    Cormier, Kevin W; Woodgett, James R

    2017-01-01

    Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed protein kinase that sits at the nexus of multiple signaling pathways. Its deep integration into cellular control circuits is consummate to its implication in diseases ranging from mood disorders to diabetes to neurodegenerative diseases and cancers. The selectivity and insulation of such a promiscuous kinase from unwanted crosstalk between pathways, while orchestrating a multifaceted response to cellular stimuli, offer key insights into more general mechanisms of cell regulation. Here, we review recent advances that have contributed to the understanding of GSK-3 and its role in driving appreciation of intracellular signal coordination.

  12. Studying the mechanism that enables paullones to selectively inhibit glycogen synthase kinase 3 rather than cyclin-dependent kinase 5 by molecular dynamics simulations and free-energy calculations.

    Science.gov (United States)

    Chen, Quan; Cui, Wei; Cheng, Yuanhua; Zhang, Fushi; Ji, Mingjuan

    2011-04-01

    Glycogen synthase kinase 3 (GSK-3) is an attractive target for the treatment of diabetes, and paullones have been reported to be effective inhibitors of GSK-3. However, it is still a challenging task to improve selectivity among protein kinases, especially cyclin-dependent kinases (CDKs). Here we investigated the mechanism that enables paullones to selectively inhibit GSK-3 rather than cyclin-dependent kinase 5 (CDK5) using sequence alignment, molecular dynamics simulations, free-energy calculations and free-energy decomposition analysis. The results indicate that the interaction between paullones and Val135 of GSK-3 is obviously stronger than that between paullones and Cys83 of CDK5, suggesting that paullones could be utilized as potent selective inhibitors. Meanwhile, we observed that the decrease in the interaction between paullones and the Asp86 of CDK5 favors their selectivity towards GSK-3 rather than CDK5, as demonstrated using 1-azakenpaullone as an example. Although substitution at position 9 and replacement at position 2 may influence the activity of GSK-3, they only have a minor effect on the selectivity. We expect that the information obtained here could prove useful for developing specific paullone inhibitors of GSK-3.

  13. Cancer cells become susceptible to natural killer cell killing after exposure to histone deacetylase inhibitors due to glycogen synthase kinase-3-dependent expression of MHC class I-related chain A and B

    DEFF Research Database (Denmark)

    Skov, Søren; Pedersen, Marianne Terndrup; Andresen, Lars

    2005-01-01

    apoptosis or oxidative stress caused by HDAC inhibitor treatment did not affect MICA/B expression, suggesting involvement of a separate signal pathway not directly coupled to induction of cell death. HDAC inhibitor treatment induced glycogen synthase kinase-3 (GSK-3) activity and down-regulation of GSK-3......We show that histone deacetylase (HDAC) inhibitors lead to functional expression of MHC class I-related chain A and B (MICA/B) on cancer cells, making them potent targets for natural killer (NK) cell-mediated killing through a NK group 2, member D (NKG2D) restricted mechanism. Blocking either...

  14. 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. Copyright © 2015 by The American Association of Immunologists, Inc.

  15. Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus

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    Octavio Silva-García

    2018-01-01

    Full Text Available Glycogen synthase kinase 3 (GSK3 is a constitutive enzyme implicated in the regulation of cytokine expression and the inflammatory response during bacterial infections. Mammals have two GSK3 isoforms named GSK3α and GSK3β that plays different but often overlapping functions. Although the role of GSK3β in cytokine regulation during the inflammatory response caused by bacteria is well described, GSK3α has not been found to participate in this process. Therefore, we tested if GSK3α may act as a regulatory isoform in the cytokine expression by bovine endothelial cells infected with Staphylococcus aureus because this bacterium is one of the major pathogens that cause tissue damage associated with inflammatory dysfunction. Interestingly, although both isoforms were phosphorylated–inactivated, we consistently observed a higher phosphorylation of GSK3α at Ser21 than that of GSK3β at Ser9 after bacterial challenge. During a temporal course of infection, we characterized a molecular switch from pro-inflammatory cytokine expression (IL-8, promoted by nuclear factor-kappa B (NF-κB, at an early stage (2 h to an anti-inflammatory cytokine expression (IL-10, promoted by cAMP response element binding (CREB, at a later stage (6 h. We observed an indirect effect of GSK3α activity on NF-κB activation that resulted in a low phosphorylation of CREB at Ser133, a decreased interaction between CREB and the co-activator CREB-binding protein (CBP, and a lower expression level of IL-10. Gene silencing of GSK3α and GSK3β with siRNA indicated that GSK3α knockout promoted the interaction between CREB and CBP that, in turn, increased the expression of IL-10, reduced the interaction of NF-κB with CBP, and reduced the expression of IL-8. These results indicate that GSK3α functions as the primary isoform that regulates the expression of IL-10 in endothelial cells infected with S. aureus.

  16. Hypercholesterolemia abrogates the cardioprotection of ischemic postconditioning in isolated rat heart: roles of glycogen synthase kinase-3β and the mitochondrial permeability transition pore.

    Science.gov (United States)

    Wu, Nan; Zhang, Xiaowen; Guan, Yuee; Shu, Wenqi; Jia, Pengyu; Jia, Dalin

    2014-05-01

    Ischemic postconditioning (IPO) reduces lethal reperfusion injury under normal conditions, but its effectiveness in hypercholesterolemia (HC) is disputed. We measured the cardioprotection of IPO in hypercholesterolemic rats and determined the roles of glycogen synthase kinase-3β (GSK-3β) and the mitochondrial permeability transition pore (mPTP). Isolated rat hearts underwent 30-min global ischemia and 120-min reperfusion. Postconditioning protocol induced six cycles of 10s ischemia and 10s reperfusion at the onset of the reperfusion. Myocardial infarct size was estimated by triphenyltetrazolium chloride staining and cardiomyocyte apoptosis was assessed by TUNEL staining. GSK-3β phosphorylation was measured by immunoblotting. The opening of mPTP was measured by NAD(+) content in myocardium. In normocholesterolemia (NC) groups, infarct size and cardiomyocyte apoptosis were significantly reduced after IPO. These reductions were completely abolished by HC, as evidenced by a similar infarct size and cardiomyocyte apoptosis observed between the IPO-HC and IR (ischemia-reperfusion)-HC groups. GSK-3β phosphorylation was significantly higher in the IPO-NC than the IPO-HC group. In addition, NAD(+) content in myocardium, a marker of mPTP opening, was higher in the IPO-NC group than the IPO-HC group. In conclusion, cardioprotection of IPO is blocked by hypercholesterolemia. This might be due to the impairment of phosphorylation of GSK-3β and attenuation of mPTP opening.

  17. Insulin like growth factor-1 prevents 1-mentyl-4-phenylphyridinium-induced apoptosis in PC12 cells through activation of glycogen synthase kinase-3beta

    International Nuclear Information System (INIS)

    Sun, Xin; Huang, Luqi; Zhang, Min; Sun, Shenggang; Wu, Yan

    2010-01-01

    Dopaminergic neurons are lost mainly through apoptosis in Parkinson's disease. Insulin like growth factor-1 (IGF-1) inhibits apoptosis in a wide variety of tissues. Here we have shown that IGF-1 protects PC12 cells from toxic effects of 1-methyl-4-phenylpyridiniumion (MPP + ). Treatment of PC12 cells with recombinant human IGF-1 significantly decreased apoptosis caused by MPP + as measured by acridine orange/ethidium bromide staining. IGF-1 treatment induced sustained phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) as shown by western blot analysis. The anti-apoptotic effect of IGF-1 was abrogated by LY294002, which indirectly inhibits phosphorylation of GSK-3beta. Lithium chloride (LiCl), a known inhibitor of GSK-3beta, also blocked MPP + -induced apoptosis. Finally, although IGF-1 enhanced phosphorylation of extracellular signal-regulated kinases ERK1 and 2 (ERK1/2), PD98059, a specific inhibitor of ERK1/2, did not alter the survival effect of IGF-1. Thus, our findings indicate that IGF-1 protects PC12 cells exposed to MPP + from apoptosis via the GSK-3beta signaling pathway.

  18. Treadmill exercise decreases incidence of Alzheimer’s disease by suppressing glycogen synthase kinase-3β expression in streptozotocin-induced diabetic rats

    Science.gov (United States)

    Kim, Dae-Young; Jung, Sun-Young; Kim, Tae-Woon; Lee, Kwang-Sik; Kim, Kijeong

    2015-01-01

    Diabetes is a metabolic disorder, and it is considered as a major risk factor for Alzheimer’s disease (AD). In the present study, we evaluated whether treadmill exercise ameliorates progression of AD in relation with glycogen synthase kinase-3β (GSK-3β) activity using streptozotocin (STZ)-induced diabetic rats. For this study, step-down avoidance task, immunohistochemistry for glycogen synthase kinase-3β (GSK-3β) and tau, and western blot for phosphor-phosphoinositide 3 kinase (p-PI3K)/PI3K and phosphor-Akt (p-Akt)/Akt were performed. Diabetes mellitus was induced by intraperitoneal injection of STZ. The rats in the exercise groups were made to run on the treadmill for 30 min per one day, five times a week, during 12 weeks. The present results showed that short-term and long-term latencies in the step-down avoidance task were decreased by induction of diabetes, and treadmill exercise inhibited these latencies in the diabetic rats. Induction of diabetes suppressed the ratio of p-PI3K to PI3K and the ratio of p-Akt to Akt, and treadmill exercise increased these ratios in the diabetic rats. The numbers of GSK-3β-positive and tau-positive cells in the hippocampal dentate gyrus was higher in the diabetes-induction group than that in the control group, and treadmill exercise inhibited these numbers in the diabetic rats. In the present study, treadmill exercise suppressed hyperphosphorylation of tau in the hippocampus by decreased GSK-3β activity through PI3K/Akt pathway activation in the diabetic rats. Based on the present results, treadmill exercise may helpful to prevent diabetes-associated AD occurrence. PMID:25960981

  19. Ursolic acid and luteolin-7-glucoside improve lipid profiles and increase liver glycogen content through glycogen synthase kinase-3.

    Science.gov (United States)

    Azevedo, Marisa F; Camsari, Cagri; Sá, Carla M; Lima, Cristovao F; Fernandes-Ferreira, Manuel; Pereira-Wilson, Cristina

    2010-06-01

    In the present study, two phytochemicals - ursolic acid (UA) and luteolin-7-glucoside (L7G) - were assessed in vivo in healthy rats regarding effects on plasma glucose and lipid profile (total cholesterol, HDL and LDL), as well as liver glycogen content, in view of their importance in the aetiology of diabetes and associated complications. Both UA and L7G significantly decreased plasma glucose concentration. UA also significantly increased liver glycogen levels accompanied by phosphorylation of glycogen synthase kinase-3 (GSK3). The increase in glycogen deposition induced by UA (mediated by GSK3) could have contributed to the lower plasma glucose levels observed. Both compounds significantly lowered total plasma cholesterol and low-density lipoprotein levels, and, in addition, UA increased plasma high-density lipoprotein levels. Our results show that UA particularly may be useful in preventable strategies for people at risk of developing diabetes and associated cardiovascular complications by improving plasma glucose levels and lipid profile, as well as by promoting liver glycogen deposition.

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

    OpenAIRE

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

    2014-01-01

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. 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. Here, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates S...

  1. One-class classification as a novel method of ligand-based virtual screening: the case of glycogen synthase kinase 3β inhibitors.

    Science.gov (United States)

    Karpov, Pavel V; Osolodkin, Dmitry I; Baskin, Igor I; Palyulin, Vladimir A; Zefirov, Nikolay S

    2011-11-15

    A virtual screening system based on one-class classification with molecular fingerprints as descriptors is developed and tested on a series of 1226 inhibitors and 209 noninhibitors of glycogen synthase kinase 3β (GSK-3β). The suggested system outperforms the ones based on pharmacophore hypothesis and molecular docking in a retrospective study. However, in a prospective study it should not be used as a sole classifier. The system is exceptionally useful for the identification of new scaffolds among the virtual screening results obtained with other methods. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Regulation of skeletal muscle plasticity by glycogen synthase kinase-3β: a potential target for the treatment of muscle wasting.

    Science.gov (United States)

    Verhees, Koen J P; Pansters, Nicholas A M; Schols, Annemie M W J; Langen, Ramon C J

    2013-01-01

    Muscle wasting is a prevalent and disabling condition in chronic disease and cancer and has been associated with increased mortality and impaired efficacy of surgical and medical interventions. Pharmacological therapies to combat muscle wasting are currently limited but considered as an important unmet medical need. Muscle wasting has been attributed to increased muscle proteolysis, and in particular ubiquitin 26S-proteasome system (UPS)-dependent protein breakdown. However, rates of muscle protein synthesis are also subject to extensive (patho) physiological regulation, and the balance between synthesis and degradation ultimately determines net muscle protein turnover. As multinucleated muscle fibers accommodate threshold changes in muscle protein content by the accretion and loss of muscle nuclei, myonuclear turnover may additionally determine muscle mass. Current insights in the mechanisms dictating muscle mass plasticity not only reveal intricate interactions and crosstalk between these processes, but imply the existence of signaling molecules that act as molecular switchboards, which coordinate and integrate cellular responses upon conditions that evoke changes in muscle mass. These "master regulators" of skeletal muscle mass plasticity are preferred targets for pharmacological modulation of skeletal muscle wasting. In this review Glycogen synthase kinase-3β (GSK-3β) is highlighted as a master regulator of muscle mass plasticity since, in addition to its role in UPS-mediated muscle protein degradation, it also controls protein synthesis, and influences myonuclear accretion and cell death. Moreover, the regulation of GSK-3β activity as well as currently available pharmacological inhibitors are described and discussed in the context of multimodal treatment strategies aimed at the inhibition of GSK-3β, and optimal exploitation of its potential role as a central regulator of skeletal muscle mass plasticity for the treatment of muscle wasting.

  3. Cooperative regulation of GSK-3 by muscarinic and PDGF receptors is associated with airway myocyte proliferation

    NARCIS (Netherlands)

    Gosens, Reinoud; Dueck, Gordon; Rector, Edward; Nunes, Raquel O.; Gerthoffer, William T.; Unruh, Helmut; Zaagsma, Johan; Meurs, Herman; Halayko, Andrew J.

    2007-01-01

    Muscarinic receptors and platelet-derived growth factor (PDGF) receptors synergistically induce proliferation of airway smooth muscle (ASM), but the pathways that regulate these effects are not yet completely identified. We hypothesized that glycogen synthase kinase-3 (GSK-3), a kinase that

  4. Cell Migration: GSK3β Steers the Cytoskeleton’s Tip

    OpenAIRE

    Yucel, Gozde; Oro, Anthony E.

    2011-01-01

    Directed cell migration polarizes the cytoskeleton, allowing the cell to move toward migratory cues. In this issue, Wu et al. (2011) demonstrate that the glycogen synthase kinase 3β (GSK3β) controls microtubule architecture and polarized movement of skin stem cells during wound healing in mammals by regulating the microtubule crosslinking protein ACF7.

  5. Phosphorylations of Serines 21/9 in Glycogen Synthase Kinase 3α/β Are Not Required for Cell Lineage Commitment or WNT Signaling in the Normal Mouse Intestine.

    Directory of Open Access Journals (Sweden)

    Fiona Hey

    Full Text Available The WNT signalling pathway controls many developmental processes and plays a key role in maintenance of intestine renewal and homeostasis. Glycogen Synthase Kinase 3 (GSK3 is an important component of the WNT pathway and is involved in regulating β-catenin stability and expression of WNT target genes. The mechanisms underpinning GSK3 regulation in this context are not completely understood, with some evidence suggesting this occurs through inhibitory N-terminal serine phosphorylation in a similar way to GSK3 inactivation in insulin signaling. To investigate this in a physiologically relevant context, we have analysed the intestinal phenotype of GSK3 knockin mice in which N-terminal serines 21/9 of GSK3α/β have been mutated to non-phosphorylatable alanine residues. We show that these knockin mutations have very little effect on overall intestinal integrity, cell lineage commitment, β-catenin localization or WNT target gene expression although a small increase in apoptosis at villi tips is observed. Our results provide in vivo evidence that GSK3 is regulated through mechanisms independent of N-terminal serine phosphorylation in order for β-catenin to be stabilised.

  6. CG0009, a novel glycogen synthase kinase 3 inhibitor, induces cell death through cyclin D1 depletion in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Hyun Mi Kim

    Full Text Available Glycogen synthase kinase 3α/β (GSK3α/β is a constitutively active serine/threonine kinase involved in multiple physiological processes, such as protein synthesis, stem cell maintenance and apoptosis, and acts as a key suppressor of the Wnt-β-catenin pathway. In the present study, we examined the therapeutic potential of a novel GSK3 inhibitor, CG0009, in the breast cancer cell lines, BT549, HS578T, MDA-MB-231, NCI/ADR-RES, T47D, MCF7 and MDA-MB-435, from the NCI-60 cancer cell line panel. Assessment of cytotoxicity, apoptosis and changes in estrogen-signaling proteins was performed using cell viability assays, Western blotting and quantitative real-time PCR. CG0009 enhanced the inactivating phosphorylation of GSK3α at Ser21 and GSK3β at Ser9 and simultaneously decreased activating phosphorylation of GSK3β at Tyr216, and induced caspase-dependent apoptosis independently of estrogen receptor α (ERα expression status, which was not observed with the other GSK3 inhibitors examined, including SB216763, kenpaullone and LiCl. CG0009 treatment (1 µmol/L completely ablated cyclin D1 expression in a time-dependent manner in all the cell lines examined, except T47D. CG0009 alone significantly activated p53, leading to relocation of p53 and Bax to the mitochondria. GSK3 inhibition by CG0009 led to slight upregulation of the β-catenin target genes, c-Jun and c-Myc, but not cyclin D1, indicating that CG0009-mediated cyclin D1 depletion overwhelms the pro-survival signal of β-catenin, resulting in cell death. Our findings suggest that the novel GSK3 inhibitor, CG0009, inhibits breast cancer cell growth through cyclin D1 depletion and p53 activation, and may thus offer an innovative therapeutic approach for breast cancers resistant to hormone-based therapy.

  7. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    International Nuclear Information System (INIS)

    Gilmour, Peter S.; O'Shea, Patrick J.; Fagura, Malbinder; Pilling, James E.; Sanganee, Hitesh; Wada, Hiroki; Courtney, Paul F.; Kavanagh, Stefan; Hall, Peter A.; Escott, K. Jane

    2013-01-01

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH 1–34 or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis and

  8. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    Energy Technology Data Exchange (ETDEWEB)

    Gilmour, Peter S., E-mail: Peter.Gilmour@astrazeneca.com [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); O' Shea, Patrick J.; Fagura, Malbinder [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Pilling, James E. [Discovery Sciences, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Sanganee, Hitesh [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Wada, Hiroki [R and I IMed, AstraZeneca R and D, Molndal (Sweden); Courtney, Paul F. [DMPK, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Kavanagh, Stefan; Hall, Peter A. [Safety Assessment, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Escott, K. Jane [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom)

    2013-10-15

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH{sub 1–34} or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis

  9. Voluntary exercise increases adult hippocampal neurogenesis by increasing GSK-3β activity in mice.

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    Zang, Jiankun; Liu, Yinghua; Li, Wei; Xiao, Di; Zhang, Yingcheng; Luo, Yuxiang; Liang, Wanying; Liu, Fei; Wei, Wei

    2017-06-23

    Exercise has been proven to promote learning and memory, and is closely related to increased adult neurogenesis in the hippocampus. In our study, the β subunit of Glycogen synthase kinase-3 (GSK3β) can be significantly regulated by exercise, and the modulation of GSK3β activity can enhance adult neurogenesis and memory. To explore the mechanism by which exercise can improve cognitive function and adult neurogenesis, and the role GSK3β plays in this process, we established a mouse model of voluntary exercise to examine the expression and activity of GSK3β, and its associated signaling pathways, in the hippocampus dentate gyrus. The results showed an obvious increase in adult neurogenesis and cognitive functions, and the up-regulation of GSK3β, after exercise. The activity of the insulin pathway, which negatively regulates GSK3β, was also increased. Moreover, our results showed that the dopamine D 1 receptor (DARP D 1 ) pathway and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) were also activated, which indicates a relationship between GSK3β and neurogenesis. Overall, our findings demonstrated that voluntary exercise promotes cognition and neurogenesis in the adult mouse dentate gyrus by the regulation of GSK3β expression and activity, which may be implemented through the DARP D 1 receptor-signaling pathway. Copyright © 2017 IBRO. All rights reserved.

  10. GSK3β and β-Catenin Modulate Radiation Cytotoxicity in Pancreatic Cancer

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    Richard L. Watson

    2010-05-01

    Full Text Available BACKGROUND: Knowledge of factors and mechanisms contributing to the inherent radioresistance of pancreatic cancer may improve cancer treatment. Irradiation inhibits glycogen synthase kinase 3β (GSK3β by phosphorylation at serine 9. In turn, release of cytosolic membrane β-catenin with subsequent nuclear translocation promotes survival. Both GSK3β and β-catenin have been implicated in cancer cell proliferation and resistance to death. METHODS: We investigated pancreatic cancer cell survival after radiation in vitro and in vivo, with a particular focus on the role of the function of the GSK3β/β-catenin axis. RESULTS: Lithium chloride, RNAi-medicated silencing of GSK3β, or the expression of a kinase dead mutant GSK3β resulted in radioresistance of Panc1 and BxPC3 pancreatic cancer cells. Conversely, ectopic expression of a constitutively active form of GSK3β resulted in radiosensitization of Panc1 cells. GSK3β silencing increased radiation-induced β-catenin target gene expression asmeasured by studies of AXIN2 and LEF1 transcript levels. Western blot analysis of total and phosphorylated levels of GSK3β and β-catenin showed that GSK3β inhibition resulted in stabilization of β-catenin. Xenografts of both BxPC3 and Panc1 with targeted silencing of GSK3β exhibited radioresistance in vivo. Silencing of β-catenin resulted in radiosensitization, whereas a nondegradable β-catenin construct induced radioresistance. CONCLUSIONS: These data support the hypothesis that GSK3β modulates the cellular response to radiation in a β-catenin-dependent mechanism. Further understanding of this pathway may enhance the development of clinical trials combining drugs inhibiting β-catenin activation with radiation and chemotherapy in locally advanced pancreatic cancer.

  11. Elucidating the functions of brain GSK3α: Possible synergy with GSK3β upregulation and reversal by antidepressant treatment in a mouse model of depressive-like behaviour.

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    Pavlov, Dmitrii; Markova, Nataliia; Bettendorff, Lucien; Chekhonin, Vladimir; Pomytkin, Igor; Lioudyno, Viktoria; Svistunov, Andrei; Ponomarev, Eugene; Lesch, Klaus-Peter; Strekalova, Tatyana

    2017-09-29

    Glycogen synthase kinase 3 (GSK3) has been linked to the mechanisms of stress, mood regulation, and the effects of antidepressants. The functions of the GSK3β isoform have been extensively investigated, but little is known about the α-isoform, although they may functionally related. In a recently established modified swim test with a third delayed swim exposure, brain GSK3β mRNA expression positively correlated with floating behaviour on the third test. A two-week-long pretreatment regime with imipramine (7.5mg/kg/day) or thiamine (200mg/kg/day), which is known to have antidepressant properties, reduced the GSK3β over-expression and decreased floating behaviour on Day 5. GSK3α mRNA levels were measured in the hippocampus and prefrontal cortex on Days 1, 2 and 5. GSK3α expression was decreased in the prefrontal cortex on Day 2 and increased on Day 5. In this model, GSK3α mRNA changes were prevented by imipramine or thiamine treatment. There was a significant correlation between the expression of the two isoforms in the prefrontal cortex on Day 2 in untreated group. These results provide the first evidence for the potential involvement of GSK3α in depressive-like behaviours and as a target of anti-depressant therapy. Furthermore, the correlations suggest some cross-talk may exist between the two GSK3 isoforms. Copyright © 2017. Published by Elsevier B.V.

  12. Discovery of new acylaminopyridines as GSK-3 inhibitors by a structure guided in-depth exploration of chemical space around a pyrrolopyridinone core.

    Science.gov (United States)

    Sivaprakasam, Prasanna; Han, Xiaojun; Civiello, Rita L; Jacutin-Porte, Swanee; Kish, Kevin; Pokross, Matt; Lewis, Hal A; Ahmed, Nazia; Szapiel, Nicolas; Newitt, John A; Baldwin, Eric T; Xiao, Hong; Krause, Carol M; Park, Hyunsoo; Nophsker, Michelle; Lippy, Jonathan S; Burton, Catherine R; Langley, David R; Macor, John E; Dubowchik, Gene M

    2015-05-01

    Glycogen synthase kinase-3 (GSK-3) has been proposed to play a crucial role in the pathogenesis of many diseases including cancer, stroke, bipolar disorders, diabetes and neurodegenerative diseases. GSK-3 inhibition has been a major area of pharmaceutical interest over the last two decades. A plethora of reports appeared recently on selective inhibitors and their co-crystal structures in GSK-3β. We identified several series of promising new GSK-3β inhibitors from a coherent design around a pyrrolopyridinone core structure. A systematic exploration of the chemical space around the central spacer led to potent single digit and sub-nanomolar GSK-3β inhibitors. When dosed orally in a transgenic mouse model of Alzheimer's disease (AD), an exemplary compound showed significant lowering of Tau phosphorylation at one of the GSK-3 phosphorylating sites, Ser396. X-ray crystallography greatly aided in validating the binding hypotheses. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. A novel role for GSK3 in the regulation of the processes of human labour.

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    Lim, Ratana; Lappas, Martha

    2015-02-01

    Preterm birth remains the largest single cause of neonatal death and morbidity. Infection and/or inflammation are strongly associated with preterm delivery. Glycogen synthase kinase 3 (GSK3) is known to be a crucial mediator of inflammation homeostasis. The aims of this study were to determine the effect of spontaneous human labour in foetal membranes and myometrium on GSK3α/β expression, and the effect of inhibition of GSK3α/β on pro-labour mediators in foetal membranes and myometrium stimulated with Toll-like receptor (TLR) ligands and pro-inflammatory cytokines. Term and preterm labour in foetal membranes was associated with significantly decreased serine phosphorylated GSK3α and β expression, and thus increased GSK3 activity. There was no effect of term labour on serine phosphorylated GSK3β expression in myometrium. The specific GSK3α/β inhibitor CHIR99021 significantly decreased lipopolysaccharide (ligand to TLR4)-stimulated pro-inflammatory cytokine gene expression and release; COX2 gene expression and prostaglandin release; and MMP9 gene expression and pro MMP9 release in foetal membranes and/or myometrium. CHIR99021 also decreased FSL1 (TLR2 ligand) and flagellin (TLR5 ligand)-induced pro-inflammatory cytokine gene expression and release and COX2 mRNA expression and prostaglandin release. GSK3β siRNA knockdown in primary myometrial cells was associated with a significant decrease in IL1β and TNFα-induced pro-inflammatory cytokine and prostaglandin release. In conclusion, GSK3α/β activity is increased in foetal membranes after term and preterm labour. Pharmacological blockade of the kinase GSK3 markedly reduced pro-inflammatory and pro-labour mediators in human foetal membranes and myometrium, providing a possible therapeutics for the management of preterm labour. © 2015 Society for Reproduction and Fertility.

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

    Science.gov (United States)

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

    2014-08-01

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

  15. GSK3 as a sensor determining cell fate in the brain

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    Adam R Cole

    2012-02-01

    Full Text Available Glycogen synthase kinase 3 (GSK3 is an unusual serine/threonine kinase that controls many neuronal functions, including neurite outgrowth, synapse formation, neurotransmission and neurogenesis. It mediates these functions by phosphorylating a wide range of substrates involved in gene transcription, metabolism, apoptosis, cytoskeletal dynamics, signal transduction, lipid membrane dynamics and trafficking, amongst others. This complicated list of diverse substrates generally follow a more simple pattern: substrates negatively regulated by GSK3-mediated phosphorylation favour a proliferative/survival state, while substrates positively regulated by GSK3 favour a more differentiated/functional state. Accordingly, GSK3 activity is higher in differentiated cells than undifferentiated cells and physiological (Wnt, growth factors and pharmacological inhibitors of GSK3 promote the proliferative capacity of embryonic stem cells. In the brain, the level of GSK3 activity influences neural progenitor cell proliferation/differentiation in neuroplasticity and repair, as well as efficient neurotransmission in differentiated adult neurons. While defects in GSK3 activity are unlikely to be the primary cause of neurodegenerative diseases, therapeutic regulation of its activity to promote a proliferative/survival versus differentiated/mature functional environment in the brain could be a powerful strategy for treatment of neurodegenerative and other mental disorders.

  16. GSK-3α is a central regulator of age-related pathologies in mice.

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    Zhou, Jibin; Freeman, Theresa A; Ahmad, Firdos; Shang, Xiying; Mangano, Emily; Gao, Erhe; Farber, John; Wang, Yajing; Ma, Xin-Liang; Woodgett, James; Vagnozzi, Ronald J; Lal, Hind; Force, Thomas

    2013-04-01

    Aging is regulated by conserved signaling pathways. The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases regulates several of these pathways, but the role of GSK-3 in aging is unknown. Herein, we demonstrate premature death and acceleration of age-related pathologies in the Gsk3a global KO mouse. KO mice developed cardiac hypertrophy and contractile dysfunction as well as sarcomere disruption and striking sarcopenia in cardiac and skeletal muscle, a classical finding in aging. We also observed severe vacuolar degeneration of myofibers and large tubular aggregates in skeletal muscle, consistent with impaired clearance of insoluble cellular debris. Other organ systems, including gut, liver, and the skeletal system, also demonstrated age-related pathologies. Mechanistically, we found marked activation of mTORC1 and associated suppression of autophagy markers in KO mice. Loss of GSK-3α, either by pharmacologic inhibition or Gsk3a gene deletion, suppressed autophagy in fibroblasts. mTOR inhibition rescued this effect and reversed the established pathologies in the striated muscle of the KO mouse. Thus, GSK-3α is a critical regulator of mTORC1, autophagy, and aging. In its absence, aging/senescence is accelerated in multiple tissues. Strategies to maintain GSK-3α activity and/or inhibit mTOR in the elderly could retard the appearance of age-related pathologies.

  17. Neurotensin Phosphorylates GSK-3α/β through the Activation of PKC in Human Colon Cancer Cells

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    Qingding Wang

    2006-09-01

    Full Text Available Neurotensin (NT, a gastrointestinal hormone, binds its receptor [neurotensin receptor (NTR] to regulate the growth of normal and neoplastic intestinal cells; molecular mechanisms remain largely undefined. Glycogen synthase kinase-3 (GSK-3 regulates diverse cellular processes, including cell growth and apoptosis. Here, we show that NT induces the phosphorylation of GSK-3α/β in the human colon cancer cell line HT29, HCT116, or SW480, which possesses high-affinity NTR. The effect of NT was blocked by inhibitors of protein kinase C (PKC, but not by inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK1 or phosphatidylinositol-3 kinase, suggesting a predominant role for PKC in GSK-3β phosphorylation by NT. Pretreatment with Gö6976 (which inhibits PKCα and PKCβ1 or downregulation of endogenous PKCα or PKCβ1 blocked NT-mediated GSK-3β (but not GSK-3α phosphorylation. Moreover, a selective PKCβ inhibitor, LY379196, reduced NT-mediated GSK-3β (but not GSK-3α phosphorylation, suggesting a role for PKCbβ in the NT-mediated phosphorylation of GSK-3β and an undefined kinase in the NT-mediated phosphorylation of GSK-3α. Treatment with NT or the GSK-3 inhibitor SB216763 increased the expression of cyclin D1, a downstream effector protein of GSK-3 and a critical protein for the proliferation of various cells. Our results indicate that NT uses PKC-dependent pathways to modulate GSK-3, which may play a role in the NT regulation of intestinal cell growth.

  18. The Mechanisms of Pharmacological Preconditioning of the Brain and the Comparative Efficacy of the Drugs — Direct- and Indirect-Acting Glycogen Synthase Kinase-3β Inhibitors: Experimental Study

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    V. V. Likhvantsev

    2012-01-01

    Full Text Available Objective: to investigate the activity of sevoflurane, dalargin, and lithium chloride in protecting the rat brain from total ischemia/reperfusion and to define whether the GSK=3^ deposphorylation contributes to the mechanism of pharmacological preconditioning. Materials and methods. Experiments were carried out on 80 male albino rats in which temporary circulatory arrest (CA was simulated by ligating the cardiovascular fascicle for 10 and 20 minutes. The animals were revived by mechanical ventilation external cardiac massage, and the intratracheal injection of adrenaline (epinephrine, Moscow Endocrinology Plant at a dose of 0.1 mg/kg. Animals were divided into 9 groups and sevorane (sevoflurane, Abbott Laboratories, dalargin (Microgen Research-and-Production Association, or lithium chloride (Sigma Chemical Co. were separately given with and without CA. Brain tissue homogenate specimens were obtained from euthanized animals. The concentration of total glycogen synthase kinase-3^ (GSK-3^ was colorimetrically determined using a Hitachi-557 spectrophotometer (Hitachi Ltd., Japan. The content of phosphorylated GSK-3/3 (pGSK-3^ in brain homogenate was estimated by Western blotting. Results. The total level of GSK-3^ in each group was similar (80—90 relative units and remained unchanged throughout each experiment. Twenty-minute ischemia maximally activated GSK-30 through dephosphorylation. Ten-minute ischemia elevated pGSK-3^ levels by more than 5 times as compared to the baseline value revealing the «training» effect. The quantity of pGSK-3^ was unchanged in the ischemia/perfusion group during sevoflurane insufflation and was decreased by 27% during dalargin administration. Conclusion. The experimental model of total ischemia provided evidence that the test drugs had a pharmacological preconditioning effect on brain neurons. According to their increasing effect, the drugs were arranged in the following order: dalargin < sevoflurane < lithium

  19. Glycogen synthase kinase-3β inhibition in the medial prefrontal cortex mediates paradoxical amphetamine action in a mouse model of ADHD

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    Yi-Chun eYen

    2015-03-01

    Full Text Available Psychostimulants show therapeutic efficacy in the treatment of attention-deficit hyperactivity disorder (ADHD. It is generally assumed that they ameliorate ADHD symptoms via interfering with monoaminergic signaling. We combined behavioral pharmacology, neurochemistry and molecular analyses to identify mechanisms underlying the paradoxical calming effect of amphetamine in low trait anxiety behavior (LAB mice, a novel multigenetic animal model of ADHD. Amphetamine (1 mg/kg and methylphenidate (10 mg/kg elicited similar dopamine and norepinephrine release in the medial prefrontal cortex (mPFC and in the striatum of LAB mice. In contrast, amphetamine decreased, while methylphenidate increased locomotor activity. This argues against changes in dopamine and/or norepinephrine release as mediators of amphetamine paradoxical effects. Instead, the calming activity of amphetamine corresponded to the inhibition of glycogen synthase kinase3β (GSK3β activity, specifically in the mPFC. Accordingly, not only systemic administration of the GSK3β inhibitor TDZD-8 (20 mg/kg, but also local microinjections of TDZD-8 and amphetamine into the mPFC, but not into the striatum, decreased locomotor activity in LAB mice. Amphetamine effects seem to depend on NMDA receptor signaling, since pre- or co-treatment with MK-801 (0.3 mg/kg abolished the effects of amphetamine (1 mg/kg on the locomotion and on the phosphorylation of GSK3β at the level of the mPFC. Taken together, the paradoxical calming effect of amphetamine in hyperactive LAB mice concurs with a decreased GSK3β activity in the mPFC. This effect appears to be independent of dopamine or norepinephrine release, but contingent on NMDA receptor signaling.

  20. GSK3 influences social preference and anxiety-related behaviors during social interaction in a mouse model of fragile X syndrome and autism.

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    Marjelo A Mines

    Full Text Available BACKGROUND: Nearly 1% of children in the United States exhibit autism spectrum disorders, but causes and treatments remain to be identified. Mice with deletion of the fragile X mental retardation 1 (Fmr1 gene are used to model autism because loss of Fmr1 gene function causes Fragile X Syndrome (FXS and many people with FXS exhibit autistic-like behaviors. Glycogen synthase kinase-3 (GSK3 is hyperactive in brains of Fmr1 knockout mice, and inhibition of GSK3 by lithium administration ameliorates some behavioral impairment in these mice. We extended our studies of this association by testing whether GSK3 contributes to socialization behaviors. This used two mouse models with disrupted regulation of GSK3, Fmr1 knockout mice and GSK3 knockin mice, in which inhibitory serines of the two isoforms of GSK3, GSK3alpha and GSK3beta, are mutated to alanines, leaving GSK3 fully active. METHODOLOGY/PRINCIPAL FINDINGS: To assess sociability, test mice were introduced to a restrained stimulus mouse (S1 for 10 min, followed by introduction of a second restrained stimulus mouse (S2 for 10 min, which assesses social preference. Fmr1 knockout and GSK3 knockin mice displayed no deficit in sociability with the S1 mouse, but unlike wild-type mice neither demonstrated social preference for the novel S2 mouse. Fmr1 knockout mice displayed more anxiety-related behaviors during social interaction (grooming, rearing, and digging than wild-type mice, which was ameliorated by inhibition of GSK3 with chronic lithium treatment. CONCLUSIONS/SIGNIFICANCE: These results indicate that impaired inhibitory regulation of GSK3 in Fmr1 knockout mice may contribute to some socialization deficits and that lithium treatment can ameliorate certain socialization impairments. As discussed in the present work, these results suggest a role for GSK3 in social behaviors and implicate inhibition of GSK3 as a potential therapeutic.

  1. Pivotal role of glycogen synthase kinase-3: A therapeutic target for Alzheimer's disease.

    Science.gov (United States)

    Maqbool, Mudasir; Mobashir, Mohammad; Hoda, Nasimul

    2016-01-01

    Neurodegenerative diseases are among the most challenging diseases with poorly known mechanism of cause and paucity of complete cure. Out of all the neurodegenerative diseases, Alzheimer's disease is the most devastating and loosening of thinking and judging ability disease that occurs in the old age people. Many hypotheses came forth in order to explain its causes. In this review, we have enlightened Glycogen Synthase Kinase-3 which has been considered as a concrete cause for Alzheimer's disease. Plaques and Tangles (abnormal structures) are the basic suspects in damaging and killing of nerve cells wherein Glycogen Synthase Kinase-3 has a key role in the formation of these fatal accumulations. Various Glycogen Synthase Kinase-3 inhibitors have been reported to reduce the amount of amyloid-beta as well as the tau hyperphosphorylation in both neuronal and nonneuronal cells. Additionally, Glycogen Synthase Kinase-3 inhibitors have been reported to enhance the adult hippocampal neurogenesis in vivo as well as in vitro. Keeping the chemotype of the reported Glycogen Synthase Kinase-3 inhibitors in consideration, they may be grouped into natural inhibitors, inorganic metal ions, organo-synthetic, and peptide like inhibitors. On the basis of their mode of binding to the constituent enzyme, they may also be grouped as ATP, nonATP, and allosteric binding sites competitive inhibitors. ATP competitive inhibitors were known earlier inhibitors but they lack efficient selectivity. This led to find the new ways for the enzyme inhibition. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  2. GSK-3β/NFAT Signaling Is Involved in Testosterone-Induced Cardiac Myocyte Hypertrophy.

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    Javier Duran

    Full Text Available Testosterone induces cardiac hypertrophy through a mechanism that involves a concerted crosstalk between cytosolic and nuclear signaling pathways. Nuclear factor of activated T-cells (NFAT is associated with the promotion of cardiac hypertrophy, glycogen synthase kinase-3β (GSK-3β is considered to function as a negative regulator, mainly by modulating NFAT activity. However, the role played by calcineurin-NFAT and GSK-3β signaling in testosterone-induced cardiac hypertrophy has remained unknown. Here, we determined that testosterone stimulates cardiac myocyte hypertrophy through NFAT activation and GSK-3β inhibition. Testosterone increased the activity of NFAT-luciferase (NFAT-Luc in a time- and dose-dependent manner, with the activity peaking after 24 h of stimulation with 100 nM testosterone. NFAT-Luc activity induced by testosterone was blocked by the calcineurin inhibitors FK506 and cyclosporine A and by 11R-VIVIT, a specific peptide inhibitor of NFAT. Conversely, testosterone inhibited GSK-3β activity as determined by increased GSK-3β phosphorylation at Ser9 and β-catenin protein accumulation, and also by reduction in β-catenin phosphorylation at residues Ser33, Ser37, and Thr41. GSK-3β inhibition with 1-azakenpaullone or a GSK-3β-targeting siRNA increased NFAT-Luc activity, whereas overexpression of a constitutively active GSK-3β mutant (GSK-3βS9A inhibited NFAT-Luc activation mediated by testosterone. Testosterone-induced cardiac myocyte hypertrophy was established by increased cardiac myocyte size and [3H]-leucine incorporation (as a measurement of cellular protein synthesis. Calcineurin-NFAT inhibition abolished and GSK-3β inhibition promoted the hypertrophy stimulated by testosterone. GSK-3β activation by GSK-3βS9A blocked the increase of hypertrophic markers induced by testosterone. Moreover, inhibition of intracellular androgen receptor prevented testosterone-induced NFAT-Luc activation. Collectively, these results

  3. Binding Energy calculation of GSK-3 protein of Human against some anti-diabetic compounds of Momordica charantia linn (Bitter melon).

    Science.gov (United States)

    Hazarika, Ridip; Parida, Pratap; Neog, Bijoy; Yadav, Raj Narain Singh

    2012-01-01

    Diabetes is one of the major life threatening diseases worldwide. It creates major health problems in urban India. Glycogen Synthase Kinase-3 (GSK-3) protein of human is known for phosphorylating and inactivating glycogen synthase which also acts as a negative regulator in the hormonal control of glucose homeostasis. In traditional medicine, Momordica charantia is used as antidiabetic plant because of its hypoglycemic effect. Hence to block the active site of the GSK-3 protein three anti-diabetic compounds namely, charantin, momordenol & momordicilin were taken from Momordica charantia for docking study and calculation of binding energy. The aim of present investigation is to find the binding energy of three major insulin-like active compounds against glycogen synthase kinase-3 (GSK-3), one of the key proteins involved in carbohydrate metabolism, with the help of molecular docking using ExomeTM Horizon suite. The study recorded minimum binding energy by momordicilin in comparison to the others.

  4. Glycogen synthase kinase-3 inhibitors suppress the AR-V7-mediated transcription and selectively inhibit cell growth in AR-V7-positive prostate cancer cells.

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    Nakata, Daisuke; Koyama, Ryokichi; Nakayama, Kazuhide; Kitazawa, Satoshi; Watanabe, Tatsuya; Hara, Takahito

    2017-06-01

    Recent evidence suggests that androgen receptor (AR) splice variants, including AR-V7, play a pivotal role in resistance to androgen blockade in prostate cancer treatment. The development of new therapeutic agents that can suppress the transcriptional activities of AR splice variants has been anticipated as the next generation treatment of castration-resistant prostate cancer. High-throughput screening of AR-V7 signaling inhibitors was performed using an AR-V7 reporter system. The effects of a glycogen synthase kinase-3 (GSK3) inhibitor, LY-2090314, on endogenous AR-V7 signaling were evaluated in an AR-V7-positive cell line, JDCaP-hr, by quantitative reverse transcription polymerase chain reaction. The relationship between AR-V7 signaling and β-catenin signaling was assessed using RNA interference. The effect of LY-2090314 on cell growth in various prostate cancer cell lines was also evaluated. We identified GSK3 inhibitors as transcriptional suppressors of AR-V7 using a high-throughput screen with an AR-V7 reporter system. LY-2090314 suppressed the reporter activity and endogenous AR-V7 activity in JDCaP-hr cells. Because silencing of β-catenin partly rescued the suppression, it was evident that the suppression was mediated, at least partially, via the activation of β-catenin signaling. AR-V7 signaling and β-catenin signaling reciprocally regulate each other in JDCaP-hr cells, and therefore, GSK3 inhibition can repress AR-V7 transcriptional activity by accumulating intracellular β-catenin. Notably, LY-2090314 selectively inhibited the growth of AR-V7-positive prostate cancer cells in vitro. Our findings demonstrate the potential of GSK3 inhibitors in treating advanced prostate cancer driven by AR splice variants. In vivo evaluation of AR splice variant-positive prostate cancer models will help illustrate the overall significance of GSK3 inhibitors in treating prostate cancer. © 2017 Wiley Periodicals, Inc.

  5. Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β.

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    Wang, Shuxin; Li, Zheyi; Shen, Hongyan; Zhang, Zhong; Yin, Yuxin; Wang, Qingsong; Zhao, Xuyang; Ji, Jianguo

    2016-08-01

    Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3β (Gsk3β). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3β, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101. © 2016 AlphaMed Press.

  6. GSK3 inactivation is involved in mitochondrial complex IV defect in transforming growth factor (TGF) {beta}1-induced senescence

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    Byun, Hae-Ok; Jung, Hyun-Jung; Seo, Yong-Hak; Lee, Young-Kyoung [Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon 443-721 (Korea, Republic of); Department of Molecular Science and Technology, The Graduate School, Ajou University, Suwon 443-721 (Korea, Republic of); Hwang, Sung-Chul [Department of Pulmonary and Critical Care Medicine, Ajou University School of Medicine, Suwon 443-721 (Korea, Republic of); Seong Hwang, Eun [Department of Life Science, University of Seoul, Seoul 130-743 (Korea, Republic of); Yoon, Gyesoon, E-mail: ypeace@ajou.ac.kr [Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon 443-721 (Korea, Republic of); Department of Molecular Science and Technology, The Graduate School, Ajou University, Suwon 443-721 (Korea, Republic of)

    2012-09-10

    Transforming growth factor {beta}1 (TGF {beta}1) induces Mv1Lu cell senescence by persistently producing mitochondrial reactive oxygen species (ROS) through decreased complex IV activity. Here, we investigated the molecular mechanism underlying the effect of TGF {beta}1 on mitochondrial complex IV activity. TGF {beta}1 progressively phosphorylated the negative regulatory sites of both glycogen synthase kinase 3 (GSK3) {alpha} and {beta}, corresponding well to the intracellular ROS generation profile. Pre-treatment of N-acetyl cysteine, an antioxidant, did not alter this GSK3 phosphorylation (inactivation), whereas pharmacological inhibition of GSK3 by SB415286 significantly increased mitochondrial ROS, implying that GSK3 phosphorylation is an upstream event of the ROS generation. GSK3 inhibition by SB415286 decreased complex IV activity and cellular O{sub 2} consumption rate and eventually induced senescence of Mv1Lu cell. Similar results were obtained with siRNA-mediated knockdown of GSK3. Moreover, we found that GSK3 not only exists in cytosol but also in mitochondria of Mv1Lu cell and the mitochondrial GSK3 binds complex IV subunit 6b which has no electron carrier and is topologically located in the mitochondrial intermembrane space. Involvement of subunit 6b in controlling complex IV activity and overall respiration rate was proved with siRNA-mediated knockdown of subunit 6b. Finally, TGF {beta}1 treatment decreased the binding of the subunit 6b to GSK3 and subunit 6b phosphorylation. Taken together, our results suggest that GSK3 inactivation is importantly involved in TGF {beta}1-induced complex IV defects through decreasing phosphorylation of the subunit 6b, thereby contributing to senescence-associated mitochondrial ROS generation.

  7. Decrease of GSK3β phosphorylation in the rat nucleus accumbens core enhances cocaine-induced hyper-locomotor activity.

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    Kim, Wha Y; Jang, Ju K; Lee, Jung W; Jang, Hyunduk; Kim, Jeong-Hoon

    2013-06-01

    Glycogen synthase kinase 3β (GSK3β), which is abundantly present in the brain, is known to contribute to psychomotor stimulant-induced locomotor behaviors. However, most studies have been focused in showing that GSK3β is able to attenuate psychomotor stimulants-induced hyperactivity by increasing its phosphorylation levels in the nucleus accumbens (NAcc). So, here we examined in the opposite direction about the effects of decreased phosphorylation of GSK3β in the NAcc core on both basal and cocaine-induced locomotor activity by a bilateral microinjection into this site of an artificially synthesized peptide, S9 (0.5 or 5.0 μg/μL), which contains sequences around N-terminal serine 9 residue of GSK3β. We found that decreased levels of GSK3β phosphorylation in the NAcc core enhance cocaine-induced hyper-locomotor activity, while leaving basal locomotor activity unchanged. This is the first demonstration, to our knowledge, that the selective decrease of GSK3β phosphorylation levels in the NAcc core may contribute positively to cocaine-induced locomotor activity, while this is not sufficient for the generation of locomotor behavior by itself without cocaine. Taken together, these findings importantly suggest that GSK3β may need other molecular targets which are co-activated (or deactivated) by psychomotor stimulants like cocaine to contribute to generation of locomotor behaviors. © 2013 International Society for Neurochemistry.

  8. Issues associated with the use of phosphospecific antibodies to localise active and inactive pools of GSK-3 in cells

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    Kypta Robert M

    2011-01-01

    Full Text Available Abstract Background Glycogen synthase kinase-3 (GSK-3 is a ubiquitously expressed serine/threonine (Ser/Thr kinase comprising two isoforms, GSK-3α and GSK-3β. Both enzymes are similarly inactivated by serine phosphorylation (GSK-3α at Ser21 and GSK-3β at Ser9 and activated by tyrosine phosphorylation (GSK-3α at Tyr279 and GSK-3β at Tyr216. Antibodies raised to phosphopeptides containing the sequences around these phosphorylation sites are frequently used to provide an indication of the activation state of GSK-3 in cell and tissue extracts. These antibodies have further been used to determine the subcellular localisation of active and inactive forms of GSK-3, and the results of those studies support roles for GSK-3 phosphorylation in diverse cellular processes. However, the specificity of these antibodies in immunocytochemistry has not been addressed in any detail. Results Taking advantage of gene silencing technology, we examined the specificity of several commercially available anti-phosphorylated GSK-3 antibodies. We show that antibodies raised to peptides containing the phosphorylated Ser21/9 epitope crossreact with unidentified antigens that are highly expressed by mitotic cells and that mainly localise to spindle poles. In addition, two antibodies raised to peptides containing the phosphorylated Tyr279/216 epitope recognise an unidentified protein at focal contacts, and a third antibody recognises a protein found in Ki-67-positive cell nuclei. While the phosphorylated Ser9/21 GSK-3 antibodies also recognise other proteins whose levels increase in mitotic cells in western blots, the phosphorylated Tyr279/216 antibodies appear to be specific in western blotting. However, we cannot rule out the posssibility that they recognise very large or very small proteins that might not be detected using a standard western blotting approach. Conclusions Our findings indicate that care should be taken when examining the subcellular localisation of

  9. Involvement of Glycogen Synthase Kinase-3 in the Mechanisms of Conditioned Food Aversion Memory Reconsolidation.

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    Nikitin, V P; Solntseva, S V; Kozyrev, S A

    2017-02-01

    Experiments were performed on the snails trained in conditioned food aversion for 3 days. Injection of TDZD-8 (glycogen synthase kinase-3 inhibitor, 2 mg/kg) in combination with reminder (presentation of a conditioned food stimulus) led to memory impairment developing 3 days after inhibitor/reminder exposure and followed by spontaneous recovery in 10 days. Injections of TDZD-8 in a dose of 4 or 20 mg/kg before reminder were shown to cause amnesia that persisted for more than 10 days. Memory recovery during repeated training was observed at the earlier period than after initial training. The impairment of memory reconsolidation by TDZD-8 after training of snails for 1 day was less pronounced than under standard training conditions (3 days). The effect of a glycogen synthase kinase-3 inhibitor during memory reconsolidation is probably followed by impairment of memory retrieval and/or partial loss, which can be compensated spontaneously or after repeated training.

  10. Extracellular α-synuclein leads to microtubule destabilization via GSK-3β-dependent Tau phosphorylation in PC12 cells.

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    Magdalena Gąssowska

    Full Text Available α-Synuclein (ASN plays an important role in pathogenesis of Parkinson's disease (PD and other neurodegenerative disorders. Novel and most interesting data showed elevated tauopathy in PD and suggested relationship between ASN and Tau protein. However, the mechanism of ASN-evoked Tau protein modification is not fully elucidated. In this study we investigated the role of extracellular ASN in Tau hyperphosphorylation in rat pheochromocytoma (PC12 cells and the involvement of glycogen synthase kinase-3β (GSK-3β and cyclin-dependent kinase 5 (CDK5 in ASN-dependent Tau modification. Our results indicated that exogenously added ASN increases Tau phosphorylation at Ser396. Accordingly, the GSK-3β inhibitor (SB-216763 prevented ASN-evoked Tau hyperphosphorylation, but the CDK5 inhibitor had no effect. Moreover, western blot analysis showed that ASN affected GSK-3β via increasing of protein level and activation of this enzyme. GSK-3β activity evaluated by its phosphorylation status assay showed that ASN significantly increased the phosphorylation of this enzyme at Tyr216 with parallel decrease in phosphorylation at Ser9, indicative of stimulation of GSK-3β activity. Moreover, the effect of ASN on microtubule (MT destabilization and cell death with simultaneous the involvement of GSK-3β in these processes were analyzed. ASN treatment increased the amount of free tubulin and concomitantly reduced the amount of polymerized tubulin and SB-216763 suppressed these ASN-induced changes in tubulin, indicating that GSK-3β is involved in ASN-evoked MT destabilization. ASN-induced apoptotic processes lead to decrease in PC12 cells viability and SB-216763 protected those cells against ASN-evoked cytotoxicity. Concluding, extracellular ASN is involved in GSK-3β-dependent Tau hyperphosphorylation, which leads to microtubule destabilization. GSK-3β inhibition may be an effective strategy for protecting against ASN-induced cytotoxicity.

  11. GSK-3beta inhibition enhances sorafenib-induced apoptosis in melanoma cell lines.

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    Panka, David J; Cho, Daniel C; Atkins, Michael B; Mier, James W

    2008-01-11

    Glycogen synthase kinase-3beta (GSK-3beta) can participate in the induction of apoptosis or, alternatively, provide a survival signal that minimizes cellular injury. We previously demonstrated that the multikinase inhibitor sorafenib induces apoptosis in melanoma cell lines. In this report, we show that sorafenib activates GSK-3beta in multiple subcellular compartments and that this activation undermines the lethality of the drug. Pharmacologic inhibition and/or down-modulation of the kinase enhances sorafenib-induced apoptosis as determined by propidium iodide staining and by assessing the mitochondrial release of apoptosis-inducing factor and Smac/DIABLO. Conversely, the forced expression of a constitutively active form of the enzyme (GSK-3beta(S9A)) protects the cells from the apoptotic effects of the drug. This protective effect is associated with a marked increase in basal levels of Bcl-2, Bcl-x(L), and survivin and a diminution in the degree to which these anti-apoptotic proteins are down-modulated by sorafenib exposure. Sorafenib down-modulates the pro-apoptotic Bcl-2 family member Noxa in cells with high constitutive GSK-3beta activity. Pharmacologic inhibition of GSK-3beta prevents the disappearance of Noxa induced by sorafenib and enhances the down-modulation of Mcl-1. Down-modulation of Noxa largely eliminates the enhancing effect of GSK-3 inhibition on sorafenib-induced apoptosis. These data provide a strong rationale for the use of GSK-3beta inhibitors as adjuncts to sorafenib treatment and suggest that preservation of Noxa may contribute to their efficacy.

  12. Leptin promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells by inhibiting glycogen synthase kinase (GSK)-3{beta}

    Energy Technology Data Exchange (ETDEWEB)

    Zeadin, Melec G.; Butcher, Martin K.; Shaughnessy, Stephen G. [Department of Medicine, McMaster University, Hamilton, ON (Canada); Thrombosis and Atherosclerosis Research Institute, Hamilton, ON (Canada); Werstuck, Geoff H., E-mail: Geoff.Werstuck@taari.ca [Department of Medicine, McMaster University, Hamilton, ON (Canada); Thrombosis and Atherosclerosis Research Institute, Hamilton, ON (Canada)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Leptin promotes osteoblast differentiation of primary smooth muscle cells. Black-Right-Pointing-Pointer Leptin regulates the expression of genes involved in osteoblast differentiation. Black-Right-Pointing-Pointer Constitutively active GSK-3{beta} attenuates leptin-induced osteoblast differentiation. Black-Right-Pointing-Pointer This suggests that leptin signals through GSK-3{beta} to promote osteoblast differentiation. -- Abstract: In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 {mu}g/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3{beta} on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of {beta}-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3{beta} (Ad-GSK-3{beta} S9A) resulted in a >2-fold increase in GSK-3{beta} activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3{beta} activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.

  13. GSK-3β suppresses HCC cell dissociation in vitro by upregulating epithelial junction proteins and inhibiting Wnt/β-catenin signaling pathway.

    Science.gov (United States)

    Zhang, Jing-Hua; Jiao, Li-Yan; Li, Tie-Jun; Zhu, York Yuanyuan; Zhou, Jian-Wei; Tian, Jian

    2017-01-01

    Glycogen synthase kinase-3β (GSK-3β) is required in the expression of epithelial junction proteins. It was found downregulated in hepatocellular carcinoma (HCC) tissues. The purpose of this study was to investigate the role of GSK-3β in modulating the metastatic behaviors of human HCC cell lines in vitro . In this study, the expression level of GSK-3β was measured in 4 human HCC cell lines, and the small interfering RNA (siRNA) vectors against or plasmids encoding GSK-3β were used to evaluate the responses of target cells to the knockdown or overexpression of this kinase, respectively. Our results showed that GSK-3β expression was significantly lower in human HCC cell lines with high metastatic potential than that in HCC cell lines without metastatic characteristics or in a normal human liver cell line. The knockdown of GSK-3β by siRNA led to a decreased expression of the epithelial junction molecules (ZO-1, E-cadherin) and an increase in the expression of a mesenchymal cell marker (α-SMA) and a gene transcription factor (β-catenin), resulting in enhanced tumor cell dissemination. In contrast, gain-of-function studies revealed that ectopic expression of GSK-3β reduced invasive and migratory abilities of HCC cells accompanied by decreased HCC cell proliferation and induced apoptosis. More importantly, downregulation of GSK-3β led to an increase in the expression and accumulation of β-catenin in the nuclei, promoting gene transcription. In conclusion, GSK-3β might play a vital role in suppressing HCC dissociation by preventing the disassembly of cancer cell epithelial junctional complex via the GSK-3β/β-catenin pathway.

  14. GSK3β is increased in adipose tissue and skeletal muscle from women with gestational diabetes where it regulates the inflammatory response.

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    Martha Lappas

    Full Text Available Infection and inflammation, through their ability to increase pro-inflammatory cytokines and chemokines and adhesion molecules, are thought to play a central role in the pathophysiology of insulin resistance and type 2 diabetes. Recent studies have shown that glycogen synthase kinase 3 (GSK3 plays a central role in regulating this inflammation. There are, however, no studies on the role of GSK3 in pregnancies complicated by gestational diabetes mellitus (GDM. Thus, the aims of this study were (i to determine whether GSK3 is increased in adipose tissue and skeletal muscle from women with GDM; and (ii to investigate the effect of GSK3 inhibition on inflammation in the presence of inflammation induced by bacterial endotoxin lipopolysaccharide (LPS or the pro-inflammatory cytokine IL-1β. Human omental adipose tissue and skeletal muscle were obtained from normal glucose tolerant (NGT women and BMI-matched women with diet-control GDM at the time of Caesarean section. Western blotting was performed to determine GSK3 protein expression. Tissue explants were performed to determine the effect of the GSK3 inhibitor CHIR99021 on markers of inflammation. When compared to women with NGT, omental adipose tissue and skeletal muscle obtained from women with diet-controlled GDM had significantly higher GSK3β activity as evidenced by a decrease in the expression of GSK3β phosphorylated at serine 9. The GSK3 inhibitor CHIR99021 significantly reduced the gene expression and secretion of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6; the pro-inflammatory chemokines IL-8 and MCP-1; and the adhesion molecules ICAM-1 and VCAM-1 in tissues stimulated with LPS or IL-1β. In conclusion, GSK3 activity is increased in GDM adipose tissue and skeletal muscle and regulates infection- and inflammation-induced pro-inflammatory mediators.

  15. Theoretical study of GSK-3α: neural networks QSAR studies for the design of new inhibitors using 2D descriptors.

    Science.gov (United States)

    García, Isela; Fall, Yagamare; García-Mera, Xerardo; Prado-Prado, Francisco

    2011-11-01

    Glycogen synthase kinase-3 (GSK-3) targets encompass proteins implicated in AD and neurological disorders. The functions of GSK-3 and its implication in various human diseases have triggered an active search for potent and selective GSK-3 inhibitors. In this sense, QSAR could play an important role in studying these GSK-3 inhibitors. For this reason, we developed QSAR models for GSK-3α, linear discriminant analysis (LDA), and artificial neural networks (ANNs) from nearly 50,000 cases with more than 700 different GSK-3α inhibitors obtained from ChEMBL database server; in total we used more than 20,000 different molecules to develop the QSAR models. The model correctly classified 237 out of 275 active compounds (86.2%) and 14,870 out of 15,970 non-active compounds (93.2%) in the training series. The overall training performance was 93.0%. Validation of the model was carried out using an external predicting series. In these series, the model classified correctly 458 out of 549 (83.4%) compounds and 29,637 out of 31,927 non-active compounds (83.4%). The overall predictability performance was 92.7%. In this study, we propose three types of non-linear ANN as alternative to already existing models, such as LDA. Linear neural network: LNN: 236:236-1-1:1 which had an overall training performance of 96% proved to be the best model. In addition, we did a study of the different fragments of the molecules of the database to see which fragments had more influence in the activity. This can help design new inhibitors of GSK-3α. This study reports the attempts to calculate, within a unified framework probabilities of GSK-3α inhibitors against different molecules found in the literature.

  16. Inhibition of GSK-3 ameliorates Abeta pathology in an adult-onset Drosophila model of Alzheimer's disease.

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    Oyinkan Sofola

    2010-09-01

    Full Text Available Abeta peptide accumulation is thought to be the primary event in the pathogenesis of Alzheimer's disease (AD, with downstream neurotoxic effects including the hyperphosphorylation of tau protein. Glycogen synthase kinase-3 (GSK-3 is increasingly implicated as playing a pivotal role in this amyloid cascade. We have developed an adult-onset Drosophila model of AD, using an inducible gene expression system to express Arctic mutant Abeta42 specifically in adult neurons, to avoid developmental effects. Abeta42 accumulated with age in these flies and they displayed increased mortality together with progressive neuronal dysfunction, but in the apparent absence of neuronal loss. This fly model can thus be used to examine the role of events during adulthood and early AD aetiology. Expression of Abeta42 in adult neurons increased GSK-3 activity, and inhibition of GSK-3 (either genetically or pharmacologically by lithium treatment rescued Abeta42 toxicity. Abeta42 pathogenesis was also reduced by removal of endogenous fly tau; but, within the limits of detection of available methods, tau phosphorylation did not appear to be altered in flies expressing Abeta42. The GSK-3-mediated effects on Abeta42 toxicity appear to be at least in part mediated by tau-independent mechanisms, because the protective effect of lithium alone was greater than that of the removal of tau alone. Finally, Abeta42 levels were reduced upon GSK-3 inhibition, pointing to a direct role of GSK-3 in the regulation of Abeta42 peptide level, in the absence of APP processing. Our study points to the need both to identify the mechanisms by which GSK-3 modulates Abeta42 levels in the fly and to determine if similar mechanisms are present in mammals, and it supports the potential therapeutic use of GSK-3 inhibitors in AD.

  17. Muscle-specific GSK-3β ablation accelerates regeneration of disuse-atrophied skeletal muscle.

    Science.gov (United States)

    Pansters, Nicholas A M; Schols, Annemie M W J; Verhees, Koen J P; de Theije, Chiel C; Snepvangers, Frank J; Kelders, Marco C J M; Ubags, Niki D J; Haegens, Astrid; Langen, Ramon C J

    2015-03-01

    Muscle wasting impairs physical performance, increases mortality and reduces medical intervention efficacy in chronic diseases and cancer. Developing proficient intervention strategies requires improved understanding of the molecular mechanisms governing muscle mass wasting and recovery. Involvement of muscle protein- and myonuclear turnover during recovery from muscle atrophy has received limited attention. The insulin-like growth factor (IGF)-I signaling pathway has been implicated in muscle mass regulation. As glycogen synthase kinase 3 (GSK-3) is inhibited by IGF-I signaling, we hypothesized that muscle-specific GSK-3β deletion facilitates the recovery of disuse-atrophied skeletal muscle. Wild-type mice and mice lacking muscle GSK-3β (MGSK-3β KO) were subjected to a hindlimb suspension model of reversible disuse-induced muscle atrophy and followed during recovery. Indices of muscle mass, protein synthesis and proteolysis, and post-natal myogenesis which contribute to myonuclear accretion, were monitored during the reloading of atrophied muscle. Early muscle mass recovery occurred more rapidly in MGSK-3β KO muscle. Reloading-associated changes in muscle protein turnover were not affected by GSK-3β ablation. However, coherent effects were observed in the extent and kinetics of satellite cell activation, proliferation and myogenic differentiation observed during reloading, suggestive of increased myonuclear accretion in regenerating skeletal muscle lacking GSK-3β. This study demonstrates that muscle mass recovery and post-natal myogenesis from disuse-atrophy are accelerated in the absence of GSK-3β. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Glycogen synthase kinase-3beta heterozygote knockout mice as a model of findings in postmortem schizophrenia brain or as a model of behaviors mimicking lithium action: negative results.

    Science.gov (United States)

    Bersudsky, Yuly; Shaldubina, Alona; Kozlovsky, Nitzan; Woodgett, James R; Agam, Galila; Belmaker, R H

    2008-05-01

    In mice glycogen synthase kinase (GSK)-3beta heterozygote knockout status was reported to cause reduced immobility in the Porsolt forced swim test and reduced amphetamine-induced hyperactivity, behaviors that mimic the effects of lithium. GSK-3beta protein and mRNA level and activity have been reported to be reduced in the postmortem brain of schizophrenia patients and this could suggest the involvement of GSK-3beta in the etiology of schizophrenia. However, apomorphine-induced stereotyping was reported to be unchanged in GSK-3beta heterozygote (HZ) knockout (KO) mice. As such behaviors are not always robust, study in another laboratory seemed indicated. Motor activity and coordination were assessed in the rotarod test. Behavior was studied in the following tests: pilocarpine-induced seizures model for lithium action, Porsolt forced swim test, tail suspension test, elevated plus-maze, large open field, startle response and prepulse inhibition of acoustic startle response, amphetamine-induced hyperactivity, and apomorphine-induced stereotypic climbing. We could not confirm the report that GSK-3beta HZ KO mice exhibit reduced immobility in the Porsolt forced swim or reduced amphetamine-induced hyperactivity in a manner mimicking the behavioral effects of lithium. We did not find increased apomorphine-induced stereotypic climbing or disruption of prepulse inhibition, suggesting that human postmortem findings regarding GSK-3beta in schizophrenia are not mediated by changes in dopamine receptors and are not the cause of prepulse inhibition deficits in schizophrenia. These data do not support the role of GSK-3beta in schizophrenia or in the mechanism of therapeutic action of lithium. Although differences in the genetic background of the GSK-3beta HZ KOs used in the present study compared with that of the previous study could be responsible, such results could suggest that the previously reported effects of GSK-3beta knockout on behavior are not robust.

  19. Gsk3 Signalling and Redox Status in Bipolar Disorder: Evidence from Lithium Efficacy

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    Antonina Luca

    2016-01-01

    Full Text Available Objective. To discuss the link between glycogen synthase kinase-3 (GSK3 and the main biological alterations demonstrated in bipolar disorder (BD, with special attention to the redox status and the evidence supporting the efficacy of lithium (a GSK3 inhibitor in the treatment of BD. Methods. A literature research on the discussed topics, using Pubmed and Google Scholar, has been conducted. Moreover, a manual selection of interesting references from the identified articles has been performed. Results. The main biological alterations of BD, pertaining to inflammation, oxidative stress, membrane ion channels, and circadian system, seem to be intertwined. The dysfunction of the GSK3 signalling pathway is involved in all the aforementioned “biological causes” of BD. In a complex scenario, it can be seen as the common denominator linking them all. Lithium inhibition of GSK3 could, at least in part, explain its positive effect on these biological dysfunctions and its superiority in terms of clinical efficacy. Conclusions. Deepening the knowledge on the molecular bases of BD is fundamental to identifying the biochemical pathways that must be targeted in order to provide patients with increasingly effective therapeutic tools against an invalidating disorder such as BD.

  20. TLR signalling affects sperm mitochondrial function and motility via phosphatidylinositol 3-kinase and glycogen synthase kinase-3α.

    Science.gov (United States)

    Zhu, Xingxing; Shi, Dongyan; Li, Xiaoqian; Gong, Weijuan; Wu, Fengjiao; Guo, Xuejiang; Xiao, Hui; Liu, Lixin; Zhou, Hong

    2016-03-01

    Infection in male and female genital tracts can lead to infertility. The underlying mechanisms of this process remain unclear. Toll-like receptors (TLRs) recognize conserved structures and respond to pathogens by initiating signals that activate inflammatory gene transcription. Here, we demonstrate that TLR activation in sperm reduces sperm motility via signalling through myeloid differentiation factor 88 (MyD88), phosphatidylinositol 3-kinase (PI3K), and glycogen synthase kinase (GSK)-3α. Upon TLR activation, phosphorylated forms of PI3K and GSK3α were detected in the mitochondria, and the mitochondrial membrane potential was impaired in sperm. In addition, mitochondrial ATP levels were decreased after TLR agonist stimulation. Furthermore, blocking PI3K or GSK3α activation abrogated these effects and reversed the TLR-induced reduction in sperm motility. These results identify a previously unrecognized TLR signalling pathway that leads to dysfunctional sperm mitochondria, which reduce sperm motility. Our study reveals a novel mechanism by which pathogenic infection affects sperm motility and possibly leads to infertility. Copyright © 2015. Published by Elsevier Inc.

  1. Synaptic Wnt/GSK3β Signaling Hub in Autism

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    Caracci, Mario O.; Ávila, Miguel E.; De Ferrari, Giancarlo V.

    2016-01-01

    Hundreds of genes have been associated with autism spectrum disorders (ASDs) and the interaction of weak and de novo variants derive from distinct autistic phenotypes thus making up the “spectrum.” The convergence of these variants in networks of genes associated with synaptic function warrants the study of cell signaling pathways involved in the regulation of the synapse. The Wnt/β-catenin signaling pathway plays a central role in the development and regulation of the central nervous system and several genes belonging to the cascade have been genetically associated with ASDs. In the present paper, we review basic information regarding the role of Wnt/β-catenin signaling in excitatory/inhibitory balance (E/I balance) through the regulation of pre- and postsynaptic compartments. Furthermore, we integrate information supporting the role of the glycogen synthase kinase 3β (GSK3β) in the onset/development of ASDs through direct modulation of Wnt/β-catenin signaling. Finally, given GSK3β activity as key modulator of synaptic plasticity, we explore the potential of this kinase as a therapeutic target for ASD. PMID:26881141

  2. Lithium Enhances Axonal Regeneration in Peripheral Nerve by Inhibiting Glycogen Synthase Kinase 3β Activation

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    Huanxing Su

    2014-01-01

    Full Text Available Brachial plexus injury often involves traumatic root avulsion resulting in permanent paralysis of the innervated muscles. The lack of sufficient regeneration from spinal motoneurons to the peripheral nerve (PN is considered to be one of the major causes of the unsatisfactory outcome of various surgical interventions for repair of the devastating injury. The present study was undertaken to investigate potential inhibitory signals which influence axonal regeneration after root avulsion injury. The results of the study showed that root avulsion triggered GSK-3β activation in the injured motoneurons and remaining axons in the ventral funiculus. Systemic application of a clinical dose of lithium suppressed activated GSK-3β in the lesioned spinal cord to the normal level and induced extensive axonal regeneration into replanted ventral roots. Our study suggests that GSK-3β activity is involved in negative regulation for axonal elongation and regeneration and lithium, the specific GSK-3β inhibitor, enhances motoneuron regeneration from CNS to PNS.

  3. Application of an ESI-QTOF method for the detailed characterization of GSK-3β inhibitors.

    Science.gov (United States)

    De Simone, Angela; Fiori, Jessica; Naldi, Marina; D'Urzo, Annalisa; Tumiatti, Vincenzo; Milelli, Andrea; Andrisano, Vincenza

    2017-09-10

    The crucial role of Glycogen Synthase Kinase 3 (GSK-3β) as a pivotal player in Alzheimer's Disease (AD) has recently inspired significant attempts to design and synthesize potent kinase inhibitors. In fact GSK-3β is considered the main kinase which catalyzes the microtubule-associated protein tau hyper-phosphorylation and the neurofibrillary tangles (NFT) in vitro and in vivo, The first classes of GSK-3β inhibitors were classified as ATP-competitive and, therefore, they lack of an efficient degree of selectivity over other kinases. In light of this consideration, many efforts are devoted to characterize new non ATP-competitive GSK-3β inhibitors, endowed with high selectivity. In parallel, there is an urgent need to develop new analytical methodologies for the hit selection (highthroughput screening) and ligand binding characterization in terms of potency, affinity and mechanism of action. The new methodology for GSK-3β enzymatic activity determination can be adopted as a realistic alternative to the currently used radioactive, luminescence and fluorescence detection methods, each showing limitations in terms of safety and interferences. Herein, we propose an alternative and selective electrospray ionization quadrupole time-of-flight (ESI-QTOF) method, based on the direct quantification of phosphorylated substrate muscle glycogen synthase GSM, a peptide resembling the high affinity sequence of natural substrate muscle glycogen synthase 1, for the detailed characterization of GSK-3β inhibitors. The method was validated in terms of accuracy and reproducibility of GSM signal intensity with a relative standard deviation RSD% value of 3.55%; Limit of Detection (LOD): 0.006μM; Lower Limit of Quantification (LLOQ): 0.02μM; linearity r 2 0.9951. The kinetic constants (K M and v max ) of the GSK-3β catalyzed kinase reaction and the inhibitory potency of known ligands (IC 50 ), were determined. All the obtained results were in agreement with those reported in

  4. GSK-3 as a Target for Lithium-induced Neuroprotection against Excitotoxicity in Neuronal Cultures and Animal Models of Ischemic Stroke

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    De-Maw eChuang

    2011-08-01

    Full Text Available The mood stabilizer lithium inhibits glycogen synthase kinase-3 (GSK-3 directly or indirectly by enhancing serine phosphorylation of both alpha and beta isoforms. Lithium robustly protected primary brain neurons from glutamate-induced excitotoxicity; these actions were mimicked by other GSK-3 inhibitors or silencing/inhibiting GSK-3alpha and/or beta isoforms. Lithium rapidly activated Akt to enhance GSK-3 serine phosphorylation and to block glutamate-induced Akt inactivation. Lithium also upregulated Bcl-2 and suppressed glutamate-induced p53 and Bax. Induction of BDNF was required for lithium’s neuroprotection to occur. BDNF promoter IV was activated by GSK-3 inhibition using lithium or other drugs, or through gene silencing/inactivation of either isoform. Further, lithium’s neuroprotective effects were associated with inhibition of NMDA receptor-mediated calcium influx and downstream signaling. In rodent ischemic models, post-insult treatment with lithium decreased infarct volume, ameliorated neurological deficits and improved functional recovery. Upregulation of heat shock protein 70 (HSP70 and Bcl-2 as well as downregulation of p53 likely contributed to lithium’s protective effects. Delayed treatment with lithium improved functional MRI responses, which was accompanied by enhanced angiogenesis. Two GSK-3-regulated pro-angiogenic factors, matrix metalloproteinase-9 (MMP-9 and vascular endothelial growth factor were induced by lithium. Finally, lithium promoted migration of mesenchymal stem cells (MSCs by upregulation of MMP-9 through GSK-3beta inhibition. Notably, transplantation of lithium-primed MSCs into ischemic rats enhanced MSC migration to the injured brain regions and improved the neurological performance. Several other GSK-3 inhibitors have also been reported to be beneficial in rodent ischemic models. Together, GSK-3 inhibition is a rational strategy to combat ischemic stroke and other excitotoxicity-related brain disorders.

  5. Structural Plasticity of Dendritic Spines Requires GSK3α and GSK3β.

    Science.gov (United States)

    Cymerman, Iwona A; Gozdz, Agata; Urbanska, Malgorzata; Milek, Jacek; Dziembowska, Magdalena; Jaworski, Jacek

    2015-01-01

    Although memories appear to be elusive phenomena, they are stored in the network of physical connections between neurons. Dendritic spines, which are actin-rich dendritic protrusions, serve as the contact points between networked neurons. The spines' shape contributes to the strength of signal transmission. To acquire and store information, dendritic spines must remain plastic, i.e., able to respond to signals, by changing their shape. We asked whether glycogen synthase kinase (GSK) 3α and GSK3β, which are implicated in diseases with neuropsychiatric symptoms, such as Alzheimer's disease, bipolar disease and schizophrenia, play a role in a spine structural plasticity. We used Latrunculin B, an actin polymerization inhibitor, and chemically induced Long-Term Depression to trigger fast spine shape remodeling in cultured hippocampal neurons. Spine shrinkage induced by either stimulus required GSK3α activity. GSK3β activity was only important for spine structural changes after treatment with Latrunculin B. Our results indicate that GSK3α is an essential component for short-term spine structural plasticity. This specific function should be considered in future studies of neurodegenerative diseases and neuropsychiatric conditions that originate from suboptimal levels of GSK3α/β activity.

  6. Structural Plasticity of Dendritic Spines Requires GSK3α and GSK3β.

    Directory of Open Access Journals (Sweden)

    Iwona A Cymerman

    Full Text Available Although memories appear to be elusive phenomena, they are stored in the network of physical connections between neurons. Dendritic spines, which are actin-rich dendritic protrusions, serve as the contact points between networked neurons. The spines' shape contributes to the strength of signal transmission. To acquire and store information, dendritic spines must remain plastic, i.e., able to respond to signals, by changing their shape. We asked whether glycogen synthase kinase (GSK 3α and GSK3β, which are implicated in diseases with neuropsychiatric symptoms, such as Alzheimer's disease, bipolar disease and schizophrenia, play a role in a spine structural plasticity. We used Latrunculin B, an actin polymerization inhibitor, and chemically induced Long-Term Depression to trigger fast spine shape remodeling in cultured hippocampal neurons. Spine shrinkage induced by either stimulus required GSK3α activity. GSK3β activity was only important for spine structural changes after treatment with Latrunculin B. Our results indicate that GSK3α is an essential component for short-term spine structural plasticity. This specific function should be considered in future studies of neurodegenerative diseases and neuropsychiatric conditions that originate from suboptimal levels of GSK3α/β activity.

  7. Correction of GSK3β at young age prevents muscle pathology in mice with myotonic dystrophy type 1.

    Science.gov (United States)

    Wei, Christina; Stock, Lauren; Valanejad, Leila; Zalewski, Zachary A; Karns, Rebekah; Puymirat, Jack; Nelson, David; Witte, David; Woodgett, Jim; Timchenko, Nikolai A; Timchenko, Lubov

    2018-01-05

    Myotonic dystrophy type 1 (DM1) is a progressive neuromuscular disease caused by expanded CUG repeats, which misregulate RNA metabolism through several RNA-binding proteins, including CUG-binding protein/CUGBP1 elav-like factor 1 (CUGBP1/CELF1) and muscleblind 1 protein. Mutant CUG repeats elevate CUGBP1 and alter CUGBP1 activity via a glycogen synthase kinase 3β (GSK3β)-cyclin D3-cyclin D-dependent kinase 4 (CDK4) signaling pathway. Inhibition of GSK3β corrects abnormal activity of CUGBP1 in DM1 mice [human skeletal actin mRNA, containing long repeats ( HSA LR ) model]. Here, we show that the inhibition of GSK3β in young HSA LR mice prevents development of DM1 muscle pathology. Skeletal muscle in 1-yr-old HSA LR mice, treated at 1.5 mo for 6 wk with the inhibitors of GSK3, exhibits high fiber density, corrected atrophy, normal fiber size, with reduced central nuclei and normalized grip strength. Because CUG-GSK3β-cyclin D3-CDK4 converts the active form of CUGBP1 into a form of translational repressor, we examined the contribution of CUGBP1 in myogenesis using Celf1 knockout mice. We found that a loss of CUGBP1 disrupts myogenesis, affecting genes that regulate differentiation and the extracellular matrix. Proteins of those pathways are also misregulated in young HSA LR mice and in muscle biopsies of patients with congenital DM1. These findings suggest that the correction of GSK3β-CUGBP1 pathway in young HSA LR mice might have a positive effect on the myogenesis over time.-Wei, C., Stock, L., Valanejad, L., Zalewski, Z. A., Karns, R., Puymirat, J., Nelson, D., Witte, D., Woodgett, J., Timchenko, N. A., Timchenko, L. Correction of GSK3β at young age prevents muscle pathology in mice with myotonic dystrophy type 1.

  8. Binding free-energy calculation is a powerful tool for drug optimization: calculation and measurement of binding free energy for 7-azaindole derivatives to glycogen synthase kinase-3β.

    Science.gov (United States)

    Kitamura, Kunihiro; Tamura, Yunoshin; Ueki, Tomokazu; Ogata, Koji; Noda, Shigeho; Himeno, Ryutaro; Chuman, Hiroshi

    2014-06-23

    Present computational lead (drug)-optimization is lacking in thermodynamic tactics. To examine whether calculation of binding free-energy change (ΔG) is effective for the lead-optimization process, binding ΔGs of 7-azaindole derivatives to the ATP binding site of glycogen synthase kinase-3β (GSK-3β) were calculated. The result was a significant correlation coefficient of r = 0.895 between calculated and observed ΔGs. This indicates that calculated ΔG reflects the inhibitory activities of 7-azaindole derivatives. In addition to quantitative estimation of activity, ΔG calculation characterizes the thermodynamic behavior of 7-azaindole derivatives, providing also useful information for inhibitor optimization on affinity to water molecules.

  9. Redox-sensitive glycogen synthase kinase 3β-directed control of mitochondrial permeability transition: rheostatic regulation of acute kidney injury.

    Science.gov (United States)

    Wang, Zhen; Ge, Yan; Bao, Hui; Dworkin, Lance; Peng, Ai; Gong, Rujun

    2013-12-01

    Mitochondrial dysfunction plays a pivotal role in necroapoptotic cell death and in the development of acute kidney injury (AKI). Evidence suggests that glycogen synthase kinase (GSK) 3β resides at the nexus of multiple signaling pathways implicated in the regulation of mitochondrial permeability transition (MPT). In cultured renal tubular epithelial cells, a discrete pool of GSK3β was detected in mitochondria. Coimmunoprecipitation assay confirmed that GSK3β physically interacts with cyclophilin F and voltage-dependent anion channel (VDAC), key MPT regulators that possess multiple GSK3β phosphorylation consensus motifs, suggesting that GSK3β has a direct control of MPT. Upon a strong burst of reactive oxygen species elicited by the pro-oxidant herbicide paraquat, the activity of the redox-sensitive GSK3β was drastically enhanced. This was accompanied by augmented phosphorylation of cyclophilin F and VDAC, associated with MPT and cell death. Inhibition of GSK3β by either the selective inhibitor 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) or forced expression of a kinase-dead mutant obliterated paraquat-induced phosphorylation of cyclophilin F and VDAC, prevented MPT, and improved cellular viability. Conversely, ectopic expression of a constitutively active GSK3β amplified the effect of paraquat on cyclophilin F and VDAC phosphorylation and sensitized cells to paraquat-induced MPT and death. In vivo, paraquat injection elicited marked oxidant stress in the kidney and resulted in acute kidney dysfunction and massive tubular apoptosis and necrosis. Consistent with in vitro findings, the activity of GSK3β was augmented in the kidney after paraquat injury, associated with increased phosphorylation of cyclophilin F and VDAC and sensitized MPT. TDZD-8 blocked GSK3β activity in the kidney, intercepted cyclophilin F and VDAC phosphorylation, prevented MPT, attenuated tubular cell death, and ameliorated paraquat-induced AKI. Our data suggest that the

  10. Cardioprotection by GSK-3 inhibition: role of enhanced glycogen synthesis and attenuation of calcium overload.

    Science.gov (United States)

    Omar, Mohamed A; Wang, Lianguo; Clanachan, Alexander S

    2010-06-01

    Glycogen synthase kinase-3 (GSK-3) is a multi-functional kinase that regulates signalling pathways affecting glycogen metabolism, protein synthesis, mitosis, and apoptosis. GSK-3 inhibition limits cardiac ischaemia-reperfusion (IR) injury, but mechanisms are not clearly defined. This study tested the hypothesis that acute GSK-3 inhibition stimulates glycogen synthesis, repartitions glucose away from glycolysis, reduces proton (H+) production from glucose metabolism, and attenuates intracellular Ca2+ (Ca2+(i)) overload. In isolated perfused working rat hearts subjected to global ischaemia and reperfusion, the selective GSK-3 inhibitor, SB-216763 (SB, 3 micromol/L), when added either prior to ischaemia or at the onset of reperfusion, improved recovery of left-ventricular (LV) work. SB increased glycogen synthesis during reperfusion while glycolysis and H+ production were reduced. Rates of glucose and palmitate oxidation were improved by SB. Measurement of Ca2+(i) concentration by rapid acquisition indo-1 fluorescence imaging showed that SB, when added either prior to ischaemia or at the onset of reperfusion, reduced diastolic Ca2+(i) overload during reperfusion. In aerobic hearts depleted of glycogen by substrate-free perfusion to a level similar to that measured at the onset of reperfusion, SB accelerated glycogen synthesis and reduced glycolysis and H+ production independent of changes in LV work. Our study indicates that reduction in H+ production by GSK-3 inhibition is an early and upstream event that lessens Ca2+(i) overload during ischaemia and early reperfusion independent of LV work which enhances the recovery of post-ischaemic LV function and that may ultimately contribute to previously observed reductions in cell death and infarction.

  11. Hippocampal GSK3β as a Molecular Link Between Obesity and Depression.

    Science.gov (United States)

    Papazoglou, Ioannis K; Jean, Arnaud; Gertler, Arieh; Taouis, Mohammed; Vacher, Claire-Marie

    2015-08-01

    Obesity is considered as a risk factor for mood disorders including depression. Nevertheless, the mechanisms underlying this association are not clearly understood. To address this issue, we investigated the impact of high-fat (HF)-diet-induced obesity on depressive-like behavior and on serotonin (5-HT)-dependent Akt/glycogen synthase kinase 3β (GSK3β) signaling in the dentate gyrus (DG) of the hippocampus, which has been associated with mood regulation. We first showed that a HF diet induced significant overweight and hyperglycemia as well as a depressive-like behavior in adult Wistar rats. By using an ex vivo approach on brain slices, we demonstrated that 5-HT activates the Akt/GSK3β cascade in the DG of control chow (C) diet-fed animals and that a 16-week HF diet feeding abolishes this activation, concurrently with a desensitization of leptin and insulin signaling in the same region. Furthermore, depressive-like behavior inversely correlated with 5-HT-induced phosphorylation of GSK3β in the subgranular neurons of the DG. Interestingly, a substitution of HF with C diet for 6 weeks induced a total loss of depressive symptoms, whereas body weight and glycemia remained significantly higher compared to control rats. In addition, food restoration led to a recovery of the Akt/GSK3β signaling pathway activation in the DG. In parallel, we observed a negative correlation between body weight and cell proliferation in the subgranular zone of the DG. To conclude, we provide evidence for a desensitization of 5-HT-induced Akt/GSK3β signaling and an impaired cell proliferation in the DG by HF diet, suggesting novel molecular mechanisms linking obesity to depression.

  12. Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy.

    Science.gov (United States)

    Verhees, Koen J P; Pansters, Nicholas A M; Baarsma, Hoeke A; Remels, Alexander H V; Haegens, Astrid; de Theije, Chiel C; Schols, Annemie M W J; Gosens, Reinoud; Langen, Ramon C J

    2013-11-01

    Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy. Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors. Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors. In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.

  13. Effect of pressure overload on cardioprotection of mitochondrial KATP channels and GSK-3beta: interaction with the MPT pore.

    Science.gov (United States)

    Mozaffari, Mahmood S; Schaffer, Stephen W

    2008-05-01

    The mitochondrial permeability transition (MPT) pore may serve as the end-effector of cardioprotective mechanisms, namely the mitochondrial K(ATP) channels and glycogen synthase kinase-3beta (GSK-3beta). We recently showed that augmented MPT pore induction contributes to pressure overload-induced exacerbation of infarct size. This study tests the hypotheses that (i) elevation in perfusion pressure attenuates cardioprotection associated with activation of mitochondrial KATP channels or inhibition of GSK-3beta and (ii) perfusion pressure modulates the regulation of the MPT pore by mitochondrial KATP channels and/or GSK-3beta. Langendorff-perfused hearts were subjected to a regional ischemia-reperfusion insult at a perfusion pressure of either 80 or 160 cm H2O. The perfusion medium contained no drug, diazoxide (80 micromol/l; mitochondrial KATP channel opener), lithium chloride (LiCl, 1 mmol/l; nonselective inhibitor of GSK-3beta), SB-216763 (3 micromol/l; selective inhibitor of GSK-3beta), cyclosporine A (0.2 micromol/l; inhibitor of MPT pore induction), glibenclamide (50 micromol/l; inhibitor of KATP channels), and the combination of cyclosporine A and glibenclamide or the combination of glibenclamide and LiCl. The increase in perfusion pressure in the absence of a drug caused larger infarcts, an effect associated with poorer recovery of function following ischemia reperfusion. Treatment with either diazoxide or cyclosporine A reduced infarct size at both perfusion pressures but in contrast to diazoxide, cyclosporine A was more protective at the higher pressure. On the other hand, LiCl and SB-216763 reduced infarct size at both pressures, with the effect more marked at the higher perfusion pressure. Glibenclamide did not affect infarct size but eliminated the cardioprotective effect of cyclosporine A while having no effect on LiCl-induced cardioprotection. Perfusion pressure primarily affects GSK-3beta-mediated regulation of MPT pore formation in the ischemic

  14. Leptin Inhibits Glycogen Synthase Kinase-3β to Prevent Tau Phosphorylation in Neuronal Cells

    OpenAIRE

    Greco, Steven J.; Sarkar, Sraboni; Casadesus, Gemma; Zhu, Xiongwei; Smith, Mark A.; Ashford, J. Wesson; Johnston, Jane M.; Tezapsidis, Nikolaos

    2009-01-01

    We have previously demonstrated that Leptin reduces extracellular amyloid β (Aβ) protein both in vitro and in vivo, and intracellular tau phosphorylation in vitro. Further, we have shown that these effects are dependent on activation of AMP-activated protein kinase (AMPK) in vitro. Herein, we investigated downstream effectors of AMPK signaling directly linked to tau phosphorylation. One such target, of relevance to Alzheimer’s disease (AD), may be GSK-3β, which has been shown to be inactivate...

  15. NMDA Reduces Tau Phosphorylation in Rat Hippocampal Slices by Targeting NR2A Receptors, GSK3β, and PKC Activities

    Science.gov (United States)

    Elhiri, Ismaël; Allyson, Julie; Cyr, Michel; Massicotte, Guy

    2013-01-01

    The molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. In the present study, pharmacological inhibitors were deployed to investigate potential processes by which the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors modulates Tau phosphorylation in rat hippocampal slices. Our results demonstrated that Tau phosphorylation at Ser199-202 residues was decreased in NMDA-treated hippocampal slices, an effect that was not reproduced at Ser262 and Ser404 epitopes. NMDA-induced reduction of Tau phosphorylation at Ser199-202 was further promoted when NR2A-containing receptors were pharmacologically isolated and were completely abrogated by the NR2A receptor antagonist NVP-AAM077. Compared with nontreated slices, we observed that NMDA receptor activation was reflected in high Ser9 and low Tyr216 phosphorylation of glycogen synthase kinase-3 beta (GSK3β), suggesting that NMDA receptor activation might diminish Tau phosphorylation via a pathway involving GSK3β inhibition. Accordingly, we found that GSK3β inactivation by a protein kinase C- (PKC-) dependent mechanism is involved in the NMDA-induced reduction of Tau phosphorylation at Ser199-202 epitopes. Taken together, these data indicate that NR2A receptor activation may be important in limiting Tau phosphorylation by a PKC/GSK3β pathway and strengthen the idea that these receptors might act as an important molecular device counteracting neuronal cell death mechanisms in various pathological conditions. PMID:24349798

  16. Phosphorylation of Tip60 by GSK-3 determines the induction of PUMA and apoptosis by p53

    Science.gov (United States)

    Charvet, Céline; Wissler, Manuela; Brauns-Schubert, Prisca; Wang, Shang-Jui; Tang, Yi; Sigloch, Florian C.; Mellert, Hestia; Brandenburg, Martin; Lindner, Silke E.; Breit, Bernhard; Green, Douglas R.; McMahon, Steven B.; Borner, Christoph; Gu, Wei; Maurer, Ulrich

    2011-01-01

    Summary Activation of p53 by DNA damage results in either cell cycle arrest, allowing DNA repair and cell survival, or induction of apoptosis. As these opposite outcomes are both mediated by p53 stabilization, additional mechanisms to determine this decision must exist. Here we show that glycogen synthase kinase-3 (GSK-3) is required for the p53-mediated induction of the pro-apoptotic BH3 only-protein PUMA, an essential mediator of p53-induced apoptosis. Inhibition of GSK-3 protected from cell death induced by DNA damage and promoted increased long-term cell survival. We demonstrate that GSK-3 phosphorylates serine 86 of the p53-acetyltransferase Tip60. A Tip60S86A mutant was less active to induce p53 K120 acetylation, Histone 4 acetylation and expression of PUMA. Our data suggest that GSK-3 mediated Tip60S86-phosphorylation provides a link between PI3K signaling and the choice for or against apoptosis induction by p53. PMID:21658600

  17. Platelet-derived growth factor-BB restores HIV Tat -mediated impairment of neurogenesis: role of GSK-3β/β-catenin.

    Science.gov (United States)

    Chao, Jie; Yang, Lu; Yao, Honghong; Buch, Shilpa

    2014-03-01

    Our previous study demonstrated that platelet-derived growth factor-BB (PDGF-BB) increased the cell proliferation of primary rat neuronal progenitor cells (NPCs). However, whether PDGF-BB regulates neurogenesis in HIV-associated neurological disorder (HAND) remains largely unknown. In this study we demonstrated that pre-treatment of NPCs with PDGF-BB restored Tat-mediated impairment of cell proliferation via activation of p38 and JNK MAPK pathways. Moreover, treatment with PDGF-BB induced inactivation of glycogen synthase kinase-3β (GSK-3β), evidenced by its phosphorylation at Ser9, this effect was significantly inhibited by the p38 and JNK inhibitors. Level of nuclear β-catenin, the primary substrate of GSK-3β, was also concomitantly increased following PDGF-BB treatment, suggesting that PDGF-BB stimulates NPC proliferation via acting on GSK-3β to promote nuclear accumulation of β-catenin. This was further validated by gain and loss of function studies using cells transfected with either the wild type or mutant GSK-3β constructs. Together these data underpin the role of GSK-3β/β-catenin as a novel target that regulates NPC proliferation mediated by PDGF-BB with implications for therapeutic intervention for reversal of impaired neurogenesis inflicted by Tat.

  18. Ferulic acid regulates the AKT/GSK-3β/CRMP-2 signaling pathway in a middle cerebral artery occlusion animal model

    Science.gov (United States)

    Gim, Sang-A; Sung, Jin-Hee; Shah, Fawad-Ali; Kim, Myeong-Ok

    2013-01-01

    Ferulic acid, a component of the plants Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort, exerts a neuroprotective effect by regulating various signaling pathways. This study showed that ferulic acid treatment prevents the injury-induced increase of collapsin response mediator protein 2 (CRMP-2) in focal cerebral ischemia. Glycogen synthase kinase-3β (GSK-3β) regulates CRMP-2 function through phosphorylation of CRMP-2. Moreover, the pro-apoptotic activity of GSK-3β is inactivated by phosphorylation by Akt. This study investigated whether ferulic acid modulates the expression of CRMP-2 and its upstream targets, Akt and GSK-3β, in focal cerebral ischemia. Male rats were treated immediately with ferulic acid (100 mg/kg, i.v.) or vehicle after middle cerebral artery occlusion (MCAO), and then cerebral cortices were collected 24 hr after MCAO. MCAO resulted in decreased levels of phospho-Akt and phospho-GSK-3β, while ferulic acid treatment prevented the decrease in the levels of these proteins. Moreover, phospho-CRMP-2 and CRMP-2 levels increased during MCAO, whereas ferulic acid attenuated these injury-induced increases. These results demonstrate that ferulic acid regulates the Akt/GSK-3β/CRMP-2 signaling pathway in focal cerebral ischemic injury, thereby protecting against brain injury. PMID:23825478

  19. The primary defect in glycogen synthase activity is not based on increased glycogen synthase kinase-3a activity in diabetic myotubes

    DEFF Research Database (Denmark)

    Gaster, Michael; Brusgaard, Klaus; Handberg, Aa.

    2004-01-01

    in diabetic subjects, whereas the corresponding GSK-3alpha but not GSK-3beta activity was significantly reduced by acute insulin treatment in both groups. However, in myotubes precultured at high insulin concentrations the effect of insulin on GS and GSK-3alpha activity was blunted in both groups. The protein...

  20. Prohibitin confers cytoprotection against ISO-induced hypertrophy in H9c2 cells via attenuation of oxidative stress and modulation of Akt/Gsk-3β signaling.

    Science.gov (United States)

    Chowdhury, Debabrata; Kumar, Dinesh; Bhadra, Utpal; Devi, Tangutur Anjana; Bhadra, Manika Pal

    2017-01-01

    Numerous hypertrophic stimuli, including β-adrenergic agonists such as isoproterenol (ISO), result in generation of reactive oxygen species (ROS) and alteration in the mitochondrial membrane potential (Δψ) leading to oxidative stress. This process is well associated with phosphorylation of thymoma viral proto-oncogene Akt (Ser473) and glycogen synthase kinase-3β (Gsk-3β) (Ser9), with resultant inactivation of Gsk-3β. In the present study, we found that the protective defensive role of prohibitin (PHB) against ISO-induced hypertrophic response in rat H9c2 cells is via attenuation of oxidative stress-dependent signaling pathways. The intracellular levels of mitochondrial membrane potential along with cellular ROS levels and mitochondrial superoxide generation were determined. In order to understand the regulation of Akt/Gsk-3β signaling pathway, we carried out immmunoblotting for key proteins of the pathway such as PTEN, PI3K, phosphorylated, and unphosphorylated forms of Akt, Gsk-3β, and immunofluorescence experiments of p-Gsk-3β. Enforced expression of PHB in ISO-treated H9c2 cells suppressed cellular ROS production with mitochondrial superoxide generation and enhanced the mitochondrial membrane potential resulting in suppression of oxidative stress which likely offered potent cellular protection, led to the availability of more healthy cells, and also, significant constitutive activation of Gsk-3β via inactivation of Akt was observed. Knockdown of PHB expression using PHB siRNA in control H9c2 cells reversed these effects. Overall, our results demonstrate that PHB confers cytoprotection against oxidative stress in ISO-induced hypertrophy and this process is associated with modulation of Akt/Gsk-3β signaling mechanisms as evident from our PHB overexpression and knockdown experiments.

  1. MiR-26a enhances invasive capacity by suppressing GSK3β in human lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Gaoyang; Liu, Boning [Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052 (China); Meng, Zhaowei [Department of Nuclear Medicine, Tianjin Medical University General Hospital, Tianjin 300052 (China); Liu, Yunde [School of Laboratory Medicine, Tianjin Medical University, Tianjin 300052 (China); Li, Xuebing [Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052 (China); Wu, Xiang [Core Facility Center, Tianjin Medical University General Hospital, Tianjin 300052 (China); Zhou, Qinghua [Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052 (China); Xu, Ke, E-mail: ke_xu@hotmail.com [Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052 (China)

    2017-03-15

    Lung cancer is the common cause of death from cancer, and most lung cancer patients die of metastasis. MicroRNAs (miRNAs) function as either oncogenes or tumor suppressors, playing crucial role not only in tumorigenesis, but also in tumor invasion and metastasis. There are several studies showed that miR-26a is involved in carcinogenesis, however, its role in tumor metastasis need to be elucidated. In this study, we showed that ectopic expression of miR-26a enhanced migration and invasion of lung cancer cells. Glycogen synthase kinase-3β (GSK3β) was identified as a direct target of miR-26a. GSK3β expression negatively correlated with miR-26a expression in lung cancer tissues. Silencing of GSK3β achieved similar effect as miR-26a over-expression; over-expression of GSK3β reversed the enhanced effect of miR-26a on lung cancer cell migration and invasion. Further study indicated that miR-26a increased β-catenin expression and nuclear translocation. C-myc and cyclin D1, the downstream genes of β-catenin, were also up-regulated by miR-26a. Furthermore, xenograft study showed that miR-26a promoted lung cancer cell growth in vivo, and suppressed GSK3β expression. Collectively, our results demonstrated that miR-26a enhanced metastatic potential of lung cancer cells via activation of β-catenin pathway by targeting GSK3β, suggesting the potential applicability of miR-26a as a target for cancer treatment. - Highlights: • miR-26a enhances migration and invasion of lung cancer cells. • GSK3β is identified as a direct target of miR-26a. • miR-26a activates β-catenin pathway by targeting GSK3β. • miR-26a promotes lung cancer cell growth in vivo.

  2. COP1 and GSK3β Cooperate to Promote c-Jun Degradation and Inhibit Breast Cancer Cell Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Jing Shao

    2013-09-01

    Full Text Available High abundance of c-Jun is detected in invasive breast cancer cells and aggressive breast tumor malignancies. Here, we demonstrate that a major cause of high c-Jun abundance in invasive breast cancer cells is prolonged c-Jun protein stability owing to poor poly-ubiquitination of c-Jun. Among the known c-Jun-targeting E3 ligases, we identified constitutive photomorphogenesis protein 1 (COP1 as an E3 ligase responsible for c-Jun degradation in less invasive breast cancer cells because depletion of COP1 reduced c-Jun poly-ubiquitination leading to the stabilization of c-Jun protein. In a panel of breast cancer cell lines, we observed an inverse association between the levels of COP1 and c-Jun. However, overexpressing COP1 alone was unable to decrease c-Jun level in invasive breast cancer cells, indicating that efficient c-Jun protein degradation necessitates an additional event. Indeed, we found that glycogen synthase kinase 3 (GSK3 inhibitors elevated c-Jun abundance in less invasive breast cancer cells and that GSK3β nonphosphorylable c-Jun-T239A mutant displayed greater protein stability and poorer poly-ubiquitination compared to the wild-type c-Jun. The ability of simultaneously enforced expression of COP1 and constitutively active GSK3β to decrease c-Jun abundance in invasive breast cancer cells allowed us to conclude that c-Jun is negatively regulated through the coordinated action of COP1 and GSK3β. Importantly, co-expressing COP1 and active GSK3β blocked in vitro cell growth/migration and in vivo metastasis of invasive breast cancer cells. Gene expression profiling of breast tumor specimens further revealed that higher COP1 expression correlated with better recurrence-free survival. Our study supports the notion that COP1 is a suppressor of breast cancer progression.

  3. GSK-3β Overexpression Alters the Dendritic Spines of Developmentally Generated Granule Neurons in the Mouse Hippocampal Dentate Gyrus

    Science.gov (United States)

    Pallas-Bazarra, Noemí; Kastanauskaite, Asta; Avila, Jesús; DeFelipe, Javier; Llorens-Martín, María

    2017-01-01

    The dentate gyrus (DG) plays a crucial role in hippocampal-related memory. The most abundant cellular type in the DG, namely granule neurons, are developmentally generated around postnatal day P6 in mice. Moreover, a unique feature of the DG is the occurrence of adult hippocampal neurogenesis, a process that gives rise to newborn granule neurons throughout life. Adult-born and developmentally generated granule neurons share some maturational aspects but differ in others, such as in their positioning within the granule cell layer. Adult hippocampal neurogenesis encompasses a series of plastic changes that modify the function of the hippocampal trisynaptic network. In this regard, it is known that glycogen synthase kinase 3β (GSK-3β) regulates both synaptic plasticity and memory. By using a transgenic mouse overexpressing GSK-3β in hippocampal neurons, we previously demonstrated that the overexpression of this kinase has deleterious effects on the maturation of newborn granule neurons. In the present study, we addressed the effects of GSK-3β overexpression on the morphology and number of dendritic spines of developmentally generated granule neurons. To this end, we performed intracellular injections of Lucifer Yellow in developmentally generated granule neurons of wild-type and GSK-3β-overexpressing mice and analyzed the number and morphologies of dendritic spines (namely, stubby, thin and mushroom). GSK-3β overexpression led to a general reduction in the number of dendritic spines. In addition, it caused a slight reduction in the percentage, head diameter and length of thin spines, whereas the head diameter of mushroom spines was increased. PMID:28344548

  4. Electron Transport Chain Remodeling by GSK3 during Oogenesis Connects Nutrient State to Reproduction.

    Science.gov (United States)

    Sieber, Matthew H; Thomsen, Michael B; Spradling, Allan C

    2016-01-28

    Reproduction is heavily influenced by nutrition and metabolic state. Many common reproductive disorders in humans are associated with diabetes and metabolic syndrome. We characterized the metabolic mechanisms that support oogenesis and found that mitochondria in mature Drosophila oocytes enter a low-activity state of respiratory quiescence by remodeling the electron transport chain (ETC). This shift in mitochondrial function leads to extensive glycogen accumulation late in oogenesis and is required for the developmental competence of the oocyte. Decreased insulin signaling initiates ETC remodeling and mitochondrial respiratory quiescence through glycogen synthase kinase 3 (GSK3). Intriguingly, we observed similar ETC remodeling and glycogen uptake in maturing Xenopus oocytes, suggesting that these processes are evolutionarily conserved aspects of oocyte development. Our studies reveal an important link between metabolism and oocyte maturation. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. The role of glycogen synthase kinase 3 beta in brain injury induced by myocardial ischemia/reperfusion injury in a rat model of diabetes mellitus.

    Science.gov (United States)

    Zhao, Bo; Gao, Wen-Wei; Liu, Ya-Jing; Jiang, Meng; Liu, Lian; Yuan, Quan; Hou, Jia-Bao; Xia, Zhong-Yuan

    2017-10-01

    Myocardial ischemia/reperfusion injury can lead to severe brain injury. Glycogen synthase kinase 3 beta is known to be involved in myo-cardial ischemia/reperfusion injury and diabetes mellitus. However, the precise role of glycogen synthase kinase 3 beta in myocardial ischemia/reperfusion injury-induced brain injury is unclear. In this study, we observed the effects of glycogen synthase kinase 3 beta on brain injury induced by myocardial ischemia/reperfusion injury in diabetic rats. Rat models of diabetes mellitus were generated via intraperitoneal injection of streptozotocin. Models of myocardial ischemia/reperfusion injury were generated by occluding the anterior descending branch of the left coronary artery. Post-conditioning comprised three cycles of ischemia/reperfusion. Immunohistochemical staining and western blot assays demonstrated that after 48 hours of reperfusion, the structure of the brain was seriously damaged in the experimental rats compared with normal controls. Expression of Bax, interleukin-6, interleukin-8, terminal deoxynucleotidyl transferase dUTP nick end labeling, and cleaved caspase-3 in the brain was significantly increased, while expression of Bcl-2, interleukin-10, and phospho-glycogen synthase kinase 3 beta was decreased. Diabetes mellitus can aggravate inflammatory reactions and apoptosis. Ischemic post-conditioning with glycogen synthase kinase 3 beta inhibitor lithium chloride can effectively reverse these changes. Our results showed that myocardial ischemic post-conditioning attenuated myocardial ischemia/reperfusion injury-induced brain injury by activating glyco-gen synthase kinase 3 beta. According to these results, glycogen synthase kinase 3 beta appears to be an important factor in brain injury induced by myocardial ischemia/reperfusion injury.

  6. Primary Blast-Induced Changes in Akt and GSK3β Phosphorylation in Rat Hippocampus

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    Yushan Wang

    2017-08-01

    Full Text Available Traumatic brain injury (TBI due to blast from improvised explosive devices has been a leading cause of morbidity and mortality in recent conflicts in Iraq and Afghanistan. However, the mechanisms of primary blast-induced TBI are not well understood. The Akt signal transduction pathway has been implicated in various brain pathologies including TBI. In the present study, the effects of simulated primary blast waves on the phosphorylation status of Akt and its downstream effector kinase, glycogen synthase kinase 3β (GSK3β, in rat hippocampus, were investigated. Male Sprague-Dawley (SD rats (350–400 g were exposed to a single pulse shock wave (25 psi; ~7 ms duration and sacrificed 1 day, 1 week, or 6 weeks after exposure. Total and phosphorylated Akt, as well as phosphorylation of its downstream effector kinase GSK3β (at serine 9, were detected with western blot analysis and immunohistochemistry. Results showed that Akt phosphorylation at both serine 473 and threonine 308 was increased 1 day after blast on the ipsilateral side of the hippocampus, and this elevation persisted until at least 6 weeks postexposure. Similarly, phosphorylation of GSK3β at serine 9, which inhibits GSK3β activity, was also increased starting at 1 day and persisted until at least 6 weeks after primary blast on the ipsilateral side. In contrast, p-Akt was increased at 1 and 6 weeks on the contralateral side, while p-GSK3β was increased 1 day and 1 week after primary blast exposure. No significant changes in total protein levels of Akt and GSK were observed on either side of the hippocampus at any time points. Immunohistochemical results showed that increased p-Akt was mainly of neuronal origin in the CA1 region of the hippocampus and once phosphorylated, the majority was translocated to the dendritic and plasma membranes. Finally, electrophysiological data showed that evoked synaptic N-methyl-d-aspartate (NMDA receptor activity was

  7. Enhanced TLR4 Expression on Colon Cancer Cells After Chemotherapy Promotes Cell Survival and Epithelial-Mesenchymal Transition Through Phosphorylation of GSK3β.

    Science.gov (United States)

    Chung, Yoon Hee; Kim, Daejin

    2016-07-01

    Phosphorylation of glycogen synthase kinase 3β (GSK3β) by phosphatidyl-inositide 3-kinase (PI3K)/protein kinase B (AKT) or inhibition of GSK3β with small-molecule inhibitor attenuates cell survival and proliferation and increases apoptosis in most cancer cell lines. In this study, we investigated the role of phosphorylated GSK3β activated by enhanced toll-like receptor 4 (TLR4) expression in drug-treated colon cancer cells as a model of post-chemotherapy cancer cells. The effect of TLR4 stimulation on metastasis and apoptosis in drug-exposed colon cancer cells was determined by real-time polymerase chain reaction (PCR) and immunoblotting. Despite the induction of apoptosis after treatment with oxaliplatin and 5-fluorouracil, lipopolysaccharide (LPS) stimulation via increased TLR4 in drug-treated cancer cells effectively inhibited apoptosis through up-regulation of expression of anti-apoptosis-related B-cell lymphoma 2 (BCL2) family proteins [X-linked inhibitor of apoptosis protein (XIAP), BCL2, and survivin] and drug-resistance proteins [multidrug-resistance protein 1 (MDR1), multidrug resistance-associated protein (MRP)1/2/3]. LPS-mediated signaling in drug-treated cancer cells elevated the expression of phosphorylated GSK3β, extracellular signal-regulated kinase (ERK), and the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB). Pharmacological inhibition of GSK3β (using SB216763) reduced phosphorylation of GSK3β, re-activated caspase-dependent apoptosis, and blocked the expression of cancer stem cell markers and invasive characteristics in LPS-stimulated drug-treated cells. In addition, the ERK-specific inhibitor, PD98059, triggered the apoptosis of TLR4-activated drug-exposed colon cancer cells, whereas there was no effect on the expression of epithelial-mesenchymal transition markers or GSK3β phosphorylation. These results suggest that TLR4-induced GSK3β and ERK phosphorylation independently controls cancer cell

  8. GSK3 Inhibitors in the Therapeutic Development of Diabetes, Cancer and Neurodegeneration: Past, Present and Future.

    Science.gov (United States)

    Maqbool, Mudasir; Hoda, Nasimul

    2017-11-16

    GSK3 has gained a considerable attention of researchers in the late 1970s as an inevitable drug target to treat diabetes. Furthermore, it was found to have a key role in the development of diseases like cancer and neurodegeneration (ND). A broad spectrum of GSK3 inhibitors have been discovered from time to time in order to curb these diseases. Inhibition of GSK3 by insulin boosts the dephosphorylation of glycogen synthase, hence its activation to convert UDP glucose into glycogen. Lack of insulin and insulin-resistance is supposed to be the cause of type 2 diabetes (Diabetes mellitus). Additionally, GSK3 stabilizes the components of beta-catenin complex, hence promotes oncogenesis. Phosphorylation of GSK3 by Akt and some other kinases also favours the carcinogenesis. However, in some cases GSK3 has tumor supressing character. GSK3 has been found to have a prominent role in the formation of amyloid plaques and neurofibrillary tangles (abnormal protein accumulations) which are the main suspects of Alzheimer's disease (AD). GSK3 inhibitors have been reported to have amyloidbeta disaggregation property and have been found to promote the adult hippocampal neurogenesis in vivo as well as in vitro. This manuscript thoroughly reviews the involvement of GSK3 in diabetes, cancer and ND. Furthermore, development of GSK3 inhibitors as antidiabetes, anticancer and antineurodegenerative agents focusing mainly on lead optimization has been discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  9. Long-lasting antidepressant action of ketamine, but not glycogen synthase kinase-3 inhibitor SB216763, in the chronic mild stress model of mice.

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    Xian-Cang Ma

    Full Text Available BACKGROUND: Clinical studies demonstrate that the N-methyl-D-aspartate (NMDA receptor antagonist, ketamine, induces rapid antidepressant effects in patients with refractive major depressive disorder and bipolar depression. This rapid onset of action makes ketamine a highly attractive drug for patients, particularly those who do not typically respond to therapy. A recent study suggested that glycogen synthase kinase (GSK-3 may underlie the rapid antidepressant action of ketamine, although the precise mechanisms are unclear. In this study, we examined the effects of ketamine and GSK-3 inhibitor SB216763 in the unpredictable, chronic mild stress (CMS mouse model of mice. METHODOLOGY/PRINCIPAL FINDINGS: Adult C57/B6 male mice were divided into 2 groups, a non-stressed control group and the unpredictable CMS (35 days group. Then, either vehicle, ketamine (10 mg/kg, or the established GSK-3 inhibitor, SB216763 (10 mg/kg, were administered into mice in the CMS group, while vehicle was administered to controls. In the open field test, there was no difference between the four groups (control+vehicle, CMS+vehicle, CMS+ketamine, CMS+SB216763. In the sucrose intake test, a 1% sucrose intake drop, seen in CMS mice, was significantly attenuated after a single dose of ketamine, but not SB216763. In the tail suspension test (TST and forced swimming test (FST, the increased immobility time seen in CMS mice was significantly attenuated by a single dose of ketamine, but not SB216763. Interestingly, the ketamine-induced increase in the sucrose intake test persisted for 8 days after a single dose of ketamine. Furthermore, a single administration of ketamine, but not SB216763, significantly attenuated the immobility time of the TST and FST in the control (non-stressed mice. CONCLUSIONS/SIGNIFICANCE: These findings suggest that a single administration of ketamine, but not GSK-3 inhibitor SB216763, produces a long-lasting antidepressant action in CMS model mice.

  10. Prostaglandin E1 protects coronary microvascular function via the glycogen synthase kinase 3β-mitochondrial permeability transition pore pathway in rat hearts subjected to sodium laurate-induced coronary microembolization.

    Science.gov (United States)

    Zhu, Houyong; Ding, Yu; Xu, Xiaoqun; Li, Meiya; Fang, Yangliang; Gao, Beibei; Mao, Hengyi; Tong, Guoxin; Zhou, Liang; Huang, Jinyu

    2017-01-01

    Prostaglandin E1 (PGE1) is used as a pretreatment for ischemia reperfusion injury in many biological systems. However, its value as a pretreatment for coronary microembolization (CME) is unknown. The goal of this study was to determine whether PGE1 would protect against CME. In a CME rat model, we observed microthrombi and early myocardial ischemia, with endothelium appearing exfoliated and mitochondria having irregular morphology and decreased internal complexity. The level of fibrinogen-like protein 2 prothrombinase was increased and superoxide dismutase and catalase levels were decreased. Moreover, mitochondria copy number and mitochondrial permeability transition pore (mPTP) opening were increased. Pretreatment with PGE1 (1 or 2 μg/kg) significantly improved these cardiological deficits, acting via the glycogen synthase kinase 3β (GSK-3β)-mPTP pathway. Unexpectedly, the phosphorylation of Akt at Ser473 decreased in the PGE1 at high dose. Overall, our findings suggested an important role for PGE1 in pretreatment of coronary microvascular dysfunction.

  11. Increased iPLA2 activity and levels of phosphorylated GSK3B in platelets are associated with donepezil treatment in Alzheimer's disease patients.

    Science.gov (United States)

    Talib, L L; Hototian, S R; Joaquim, H P G; Forlenza, O V; Gattaz, W F

    2015-12-01

    Reduced phospholipase A2 (PLA2) activity and increased phosphorylation of glycogen synthase kinase 3B (GSK3B) participate in the production of beta-amyloid plaques and of neurofibrillary tangles, which are two neuropathological hallmarks of Alzheimer's disease (AD). Experimental evidences suggest a neuroprotective effect of the cholinesterase inhibitor donepezil in the treatment the disease. The aims of the present study were to evaluate in AD patients the effects of treatment with donepezil on PLA2 activity and GSK3B level. Thirty patients with AD were treated during 6 months with 10 mg daily of donepezil. Radio-enzymatic assays were used to measure PLA2 activity and Elisa assays for GSK3B level, both in platelets. Before treatment and after 3 and 6 months on donepezil, AD patients underwent a cognitive assessment and platelet samples were collected. Values were compared to a healthy control group of 42 sex- and age-matched elderly individuals. Before treatment, iPLA2 activity was lower in patients with AD as compared to controls (p donepezil delays the progression of AD. The increment of iPLA2 activity may reduce the production of beta-amyloid plaques, whereas the phosphorylation of GSK3B inactivates the enzyme, reducing thus the phosphorylation of tau protein.

  12. Indirubin-3-Oxime Prevents H2O2-Induced Neuronal Apoptosis via Concurrently Inhibiting GSK3β and the ERK Pathway.

    Science.gov (United States)

    Yu, Jie; Zheng, Jiacheng; Lin, Jiajia; Jin, Linlu; Yu, Rui; Mak, Shinghung; Hu, Shengquan; Sun, Hongya; Wu, Xiang; Zhang, Zaijun; Lee, Mingyuen; Tsim, Wahkeung; Su, Wei; Zhou, Wenhua; Cui, Wei; Han, Yifan; Wang, Qinwen

    2017-05-01

    Oxidative stress-induced neuronal apoptosis plays an important role in many neurodegenerative disorders. In this study, we have shown that indirubin-3-oxime, a derivative of indirubin originally designed for leukemia therapy, could prevent hydrogen peroxide (H 2 O 2 )-induced apoptosis in both SH-SY5Y cells and primary cerebellar granule neurons. H 2 O 2 exposure led to the increased activities of glycogen synthase kinase 3β (GSK3β) and extracellular signal-regulated kinase (ERK) in SH-SY5Y cells. Indirubin-3-oxime treatment significantly reversed the altered activity of both the PI3-K/Akt/GSK3β cascade and the ERK pathway induced by H 2 O 2 . In addition, both GSK3β and mitogen-activated protein kinase inhibitors significantly prevented H 2 O 2 -induced neuronal apoptosis. Moreover, specific inhibitors of the phosphoinositide 3-kinase (PI3-K) abolished the neuroprotective effects of indirubin-3-oxime against H 2 O 2 -induced neuronal apoptosis. These results strongly suggest that indirubin-3-oxime prevents H 2 O 2 -induced apoptosis via concurrent inhibiting GSK3β and the ERK pathway in SH-SY5Y cells, providing support for the use of indirubin-3-oxime to treat neurodegenerative disorders caused or exacerbated by oxidative stress.

  13. Pioglitazone Protected against Cardiac Hypertrophy via Inhibiting AKT/GSK3β and MAPK Signaling Pathways

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    Wen-Ying Wei

    2016-01-01

    Full Text Available Peroxisome proliferator activated receptor γ (PPARγ has been closely involved in the process of cardiovascular diseases. This study was to investigate whether pioglitazone (PIO, a PPARγ agonist, could protect against pressure overload-induced cardiac hypertrophy. Mice were orally given PIO (2.5 mg/kg from 1 week after aortic banding and continuing for 7 weeks. The morphological examination and biochemical analysis were used to evaluate the effects of PIO. Neonatal rat ventricular cardiomyocytes were also used to verify the protection of PIO against hypertrophy in vitro. The results in our study demonstrated that PIO remarkably inhibited hypertrophic response induced by aortic banding in vivo. Besides, PIO also suppressed cardiac fibrosis in vivo. PIO treatment also inhibited the activation of protein kinase B (AKT/glycogen synthase kinase-3β (GSK3β and mitogen-activated protein kinase (MAPK in the heart. In addition, PIO alleviated angiotensin II-induced hypertrophic response in vitro. In conclusion, PIO could inhibit cardiac hypertrophy via attenuation of AKT/GSK3β and MAPK pathways.

  14. Andrographolide suppresses melanin synthesis through Akt/GSK3β/β-catenin signal pathway.

    Science.gov (United States)

    Zhu, Ping-Ya; Yin, Wei-Han; Wang, Meng-Ran; Dang, Yong-Yan; Ye, Xi-Yun

    2015-07-01

    Tyrosinase (TYR) is the key enzyme controlling the production of melanin. Very few papers have reported that andrographolide can inhibit melanin content. To investigate the effects of andrographolide on melanin synthesis. Cell viability, melanin content, TYR activity, transcriptional and protein expression levels of TYR family and other kinds of proteins involved in melanogenesis were measured after the treatments of andrographolide. It was found that andrographolide decreased melanin content, TYR activity and transcriptional and protein expression of TYR family and microphthalmia-associated transcription factor (MITF) in B16F10 melanoma cells. Data showed andrographolide also decreased melanin content and TYR content in ultraviolet B (UVB) irradiation induced brown guinea pigs. Moreover, we found that melanin content and TYR activity were effectively inhibited in Human Epidermis Melanocyte (HEM) treated with andrographolide at the medium concentrations without apparent effect on cell viability. Results in experiments treated with MG-132 or cycloheximide (CHX) showed that andrographolide lowered the content of β-catenin in cell nucleus resulting from accelerating the degradation of β-catenin. Phosphorylation of glycogen synthase kinase 3β (GSK3β) and Akt decreased simultaneously. 6-Bromoindirubin-3'-oxime (BIO, inhibitor of GSK3β) and insulin-like growth factors-1 (IGF-1, activator of Akt) could reverse the decline of β-catenin in B16F10 cells induced by andrographolide. These results demonstrate that andrographolide can effectively suppress melanin content and TYR activity in B16F10 cells, HEM cells and UVB-induced brown guinea pig skin by decreasing phosphorylation of GSK3β dependent on Akt, promoting the degradation of β-catenin, inhibiting β-catenin into the nucleus and decreasing the expression of MITF and TYR family. Data indicate that andrographolide may be a potential whiting agent which can have great market in cosmetics and in clinical such as

  15. Lithium attenuates cannabinoid-induced dependence in the animal model: involvement of phosphorylated ERK1/2 and GSK-3β signaling pathways.

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    Hamid Reza Rahimi

    2014-09-01

    Full Text Available Cannabis is one of the most banned drugs in the world. Cannabinoid-induced dependence or withdrawal signs are indicated by the result of complex molecular mechanisms including upstream protein kinases (PKs, such as an extracellular signal regulated kinase1/2 (ERK1/2 and downstream glycogen synthase kinase-3β (GSK-3β, which lead to neuronal plasticity. In this study, we examined the protective effect of lithium (Li as a potent ERK1/2 and GSK-3β modulator to prevent the development of dependence on cannabinoids. For this purpose, rats were treated twice daily with increasing doses of WIN 55,212-2 (WIN, 2-8 mg/kg, intraperitoneally (i.p., for five consecutive days. AM251 (AM, 2 mg/kg, a cannabinoid antagonist, was injected i.p to induce manifestations of abstinence in rat dependency on WIN, and the subsequent withdrawal signs were recorded. To evaluate the preventive effect of Li, the rats were pre-treated with Li (10 mg/kg, i.p. twice daily, 30 minutes before every injection of WIN. SL327, as an ERK1/2 inhibitor, was also injected (SL, 50 mg/kg, i.p. 30 minutes before the last doses of WIN in separate groups. The p-ERK1/2, total ERK1/2, p-GSK-3β and total GSK-3β expressions were determined with Western blot method after 60 minutes, prior to the Li, WIN or AM injections. Li and SL pre-treatment attenuated the global withdrawal signs in regarding their modulation effect on the up-regulation of p-ERK1/2 cascade enhanced by AM injection. Furthermore, the p-GSK-3β expression was up-regulated with SL and Li pre-treatment against AM injection, without alteration on the total contents of ERK1/2 and GSK-3β level. Therefore, p-ERK1/2 and p-GSK-3β pathways are involved in the cannabinoid-induced dependence. However, no crosstalk was indicated between these two pathways. In conclusion, Li neuroprotectionwith regard to cannabinoid abstinence may occur through the regulation of the p-ERK1/2 cascade inconsequent of p-GSK-3β signaling pathways in rats.

  16. Chromosomal mapping and mutational analysis of the coding region of the glycogen synthase kinase-3alpha and beta isoforms in patients with NIDDM

    DEFF Research Database (Denmark)

    Hansen, L; Arden, K C; Rasmussen, S B

    1997-01-01

    isoforms of GSK-3alpha and GSK-3beta in 72 NIDDM patients and 12 control subjects. No structural changes were detected apart from a few silent mutations. Mapping of the GSK-3alpha to chromosome 19q13.1-13.2 and the GSK-3beta to chromosome 3q13.3-q21 outside known genetic loci linked to NIDDM further makes...

  17. GSK-3 inhibitors induce chromosome instability

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    Staples Oliver D

    2007-08-01

    Full Text Available Abstract Background Several mechanisms operate during mitosis to ensure accurate chromosome segregation. However, during tumour evolution these mechanisms go awry resulting in chromosome instability. While several lines of evidence suggest that mutations in adenomatous polyposis coli (APC may promote chromosome instability, at least in colon cancer, the underlying mechanisms remain unclear. Here, we turn our attention to GSK-3 – a protein kinase, which in concert with APC, targets β-catenin for proteolysis – and ask whether GSK-3 is required for accurate chromosome segregation. Results To probe the role of GSK-3 in mitosis, we inhibited GSK-3 kinase activity in cells using a panel of small molecule inhibitors, including SB-415286, AR-A014418, 1-Azakenpaullone and CHIR99021. Analysis of synchronised HeLa cells shows that GSK-3 inhibitors do not prevent G1/S progression or cell division. They do, however, significantly delay mitotic exit, largely because inhibitor-treated cells have difficulty aligning all their chromosomes. Although bipolar spindles form and the majority of chromosomes biorient, one or more chromosomes often remain mono-oriented near the spindle poles. Despite a prolonged mitotic delay, anaphase frequently initiates without the last chromosome aligning, resulting in chromosome non-disjunction. To rule out the possibility of "off-target" effects, we also used RNA interference to selectively repress GSK-3β. Cells deficient for GSK-3β exhibit a similar chromosome alignment defect, with chromosomes clustered near the spindle poles. GSK-3β repression also results in cells accumulating micronuclei, a hallmark of chromosome missegregation. Conclusion Thus, not only do our observations indicate a role for GSK-3 in accurate chromosome segregation, but they also raise the possibility that, if used as therapeutic agents, GSK-3 inhibitors may induce unwanted side effects by inducing chromosome instability.

  18. Breast cancer cell line MCF7 escapes from G1/S arrest induced by proteasome inhibition through a GSK-3β dependent mechanism

    Science.gov (United States)

    Gavilán, Elena; Giráldez, Servando; Sánchez-Aguayo, Inmaculada; Romero, Francisco; Ruano, Diego; Daza, Paula

    2015-01-01

    Targeting the ubiquitin proteasome pathway has emerged as a rational approach in the treatment of human cancers. Autophagy has been described as a cytoprotective mechanism to increase tumor cell survival under stress conditions. Here, we have focused on the role of proteasome inhibition in cell cycle progression and the role of autophagy in the proliferation recovery. The study was performed in the breast cancer cell line MCF7 compared to the normal mammary cell line MCF10A. We found that the proteasome inhibitor MG132 induced G1/S arrest in MCF10A, but G2/M arrest in MCF7 cells. The effect of MG132 on MCF7 was reproduced on MCF10A cells in the presence of the glycogen synthase kinase 3β (GSK-3β) inhibitor VII. Similarly, MCF7 cells overexpressing constitutively active GSK-3β behaved like MCF10A cells. On the other hand, MCF10A cells remained arrested after MG132 removal while MCF7 recovered the proliferative capacity. Importantly, this recovery was abolished in the presence of the autophagy inhibitor 3-methyladenine (3-MA). Thus, our results support the relevance of GSK-3β and autophagy as two targets for controlling cell cycle progression and proliferative capacity in MCF7, highlighting the co-treatment of breast cancer cells with 3-MA to synergize the effect of the proteasome inhibition. PMID:25941117

  19. Human cardiac stem cells exhibit mesenchymal features and are maintained through Akt/GSK-3β signaling

    International Nuclear Information System (INIS)

    Tateishi, Kento; Ashihara, Eishi; Honsho, Shoken; Takehara, Naofumi; Nomura, Tetsuya; Takahashi, Tomosaburo; Ueyama, Tomomi; Yamagishi, Masaaki; Yaku, Hitoshi; Matsubara, Hiroaki; Oh, Hidemasa

    2007-01-01

    Recent evidence suggested that human cardiac stem cells (hCSCs) may have the clinical application for cardiac repair; however, their characteristics and the regulatory mechanisms of their growth have not been fully investigated. Here, we show the novel property of hCSCs with respect to their origin and tissue distribution in human heart, and demonstrate the signaling pathway that regulates their growth and survival. Telomerase-active hCSCs were predominantly present in the right atrium and outflow tract of the heart (infant > adult) and had a mesenchymal cell-like phenotype. These hCSCs expressed the embryonic stem cell markers and differentiated into cardiomyocytes to support cardiac function when transplanted them into ischemic myocardium. Inhibition of Akt pathway impaired the hCSC proliferation and induced apoptosis, whereas inhibition of glycogen synthase kinase-3 (GSK-3) enhanced their growth and survival. We conclude that hCSCs exhibit mesenchymal features and that Akt/GSK-3β may be crucial modulators for hCSC maintenance in human heart

  20. First dual AK/GSK-3β inhibitors endowed with antioxidant properties as multifunctional, potential neuroprotective agents.

    Science.gov (United States)

    Brogi, Simone; Ramunno, Anna; Savi, Lida; Chemi, Giulia; Alfano, Gloria; Pecorelli, Alessandra; Pambianchi, Erika; Galatello, Paola; Compagnoni, Giulia; Focher, Federico; Biamonti, Giuseppe; Valacchi, Giuseppe; Butini, Stefania; Gemma, Sandra; Campiani, Giuseppe; Brindisi, Margherita

    2017-09-29

    The manuscript deals with the design, synthesis and biological evaluation of novel benzoxazinone-based and indole-based compounds as multifunctional neuroprotective agents. These compounds inhibit human adenosine kinase (hAK) and human glycogen synthase kinase 3 beta (hGSK-3β) enzymes. Computational analysis based on a molecular docking approach underlined the potential structural requirements for simultaneously targeting both proteins' allosteric sites. In silico hints drove the synthesis of appropriately decorated benzoxazinones and indoles (5a-s, and 6a-c) and biochemical analysis revealed their behavior as allosteric inhibitors of hGSK-3β. For both our hit 4 and the best compounds of the series (5c,l and 6b) the potential antioxidant profile was assessed in human neuroblastoma cell lines (IMR 32, undifferentiated and neuronal differentiated), by evaluating the protective effect of selected compounds against H 2 O 2 cytotoxicity and reactive oxygen species (ROS) production. Results showed a strong efficacy of the tested compounds, even at the lower doses, in counteracting the induced oxidative stress (50 μM of H 2 O 2 ) and in preventing ROS formation. In addition, the tested compounds did not show any cytotoxic effect determined by the LDH release, at the concentration range analyzed (from 0.1 to 50 μM). This study allowed the identification of compound 5l, as the first dual hAK/hGSK-3β inhibitor reported to date. Compound 5l, which behaves as an effective antioxidant, holds promise for the development of new series of potential therapeutic agents for the treatment of neurodegenerative diseases characterized by an innovative pharmacological profile. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  1. [Gly14]-Humanin offers neuroprotection through glycogen synthase kinase-3β inhibition in a mouse model of intracerebral hemorrhage.

    Science.gov (United States)

    Wang, Tao; Huang, Ya; Zhang, Mingyang; Wang, Long; Wang, Yaoqi; Zhang, Lu; Dong, Wenwen; Chang, Pan; Wang, Zufeng; Chen, Xiping; Tao, Luyang

    2013-06-15

    Perihematomal brain edema formation and consequent cell death contribute to second brain injury resulting in severe neurological deficits and sometimes delayed fatality after intracerebral hemorrhage (ICH). [Gly14]-Humanin (HNG), a variant of Humanin (HN) in which the 14th amino acid serine is replaced with glycine, reduced Alzheimer's disease-relevant insults and improved neurological deficits in an ischemia stroke model. In the study, we aimed to evaluate whether HNG posttreatment attenuated early brain injury after ICH and whether the protective effect was associated with regulation of apoptosis via phosphatidylinositol 3-kinase (PI3K)-Akt/GSK-3β signaling. Male ICR mice were subjected to infusion of Type IV collagenase (to induce ICH) of saline (for shams) into the left striatum. ICH animals received vehicle, HNG (1 or 2.5 μg in 100 μl saline) administration intraperitoneally 1h post injury. Compared with vehicle, HNG-2.5 μg treatment improved neurological outcome and reduced brain edema at 24 and 72 h after surgery (P<0.05), but wortmannin (15 μg/kg, 90 min before HNG-2.5 μg, intravenously) obliterated the effect. HNG-2.5 μg also reduced cell insults and injury volume at 24 and 72 h after surgery (P<0.05, vs. vehicle). Furthermore, HNG-2.5 μg treatment increased p-Akt and Bcl-2 and decreased p-GSK-3β, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase expressions in the ipsilateral hemisphere (P<0.05, vs. vehicle), however, the effect was reversed by wortmannin. In conclusion, HNG treatment improved functional and morphological outcomes after experimental ICH in mice and the protective effect was associated with suppressing apoptosis through PI3K-Akt/GSK-3β signaling pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Platelet-derived growth factor CC-mediated neuroprotection against HIV Tat involves TRPC-mediated inactivation of GSK 3beta.

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    Fuwang Peng

    Full Text Available Platelet-derived growth factor-CC (PDGF-CC is the third member of the PDGF family, and has been implicated both in embryogenesis and development of the CNS. The biological function of this isoform however, remains largely unexplored in the context of HIV-associated dementia (HAD. In the present study, we demonstrate that exposure of human neuroblastoma cells SH-SY5Y to HIV transactivator protein Tat resulted in decreased intrinsic expression of PDGF-CC as evidenced by RT-PCR and western blot assays. Reciprocally, pretreatment of SH-SY5Y cells with PDGF-CC abrogated Tat-mediated neurotoxicity by mitigating apoptosis and neurite & MAP-2 loss. Using pharmacological and loss of function approaches we identified the role of phosphatidylinositol 3-kinase (PI3K/Akt signaling in PDGF-CC-mediated neuroprotection. We report herein a novel role about the involvement of transient receptor potential canonical (TRPC channel 1 in modulation of calcium transients in PDGF-CC-mediated neuroprotection. Furthermore we also demonstrated PDGF-CC-mediated inactivation of the downstream mediator--glycogen synthase kinase 3β (GSK3β evidenced by its phosphorylation at Ser-9. This was further validated by gain and loss of function studies using cells transfected with either the wild type or mutant GSK3β constructs. Intriguingly, pretreatment of cells with either the PI3K inhibitor or TRPC blocker resulted in failure of PDGF-CC to inactivate GSK3β, thereby suggesting the intersection of PI3K and TRPC signaling at GSK3β. Taken together our findings lead to the suggestion that PDGF-CC could be developed as a therapeutic target to reverse Tat-mediated neurotoxicity with implications for HAD.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  4. Role of glycogen synthase kinase following myocardial infarction and ischemia-reperfusion.

    Science.gov (United States)

    Ghaderi, S; Alidadiani, N; Dilaver, N; Heidari, H R; Parvizi, R; Rahbarghazi, R; Soleimani-Rad, J; Baradaran, B

    2017-07-01

    Glycogen synthase kinase-3 beta (GSK3β) is principally is a glycogen synthase phosphorylating enzyme that is well known for its role in muscle metabolism. GSK3β is a serine/threonine protein Kinase, which is responsible for several essential roles in mammalian cells. This enzyme is implicated in the pathophysiology of many conditions involved in homeostasis and cellular immigration. GSK3β is involved in several pathways leading to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Increasing evidence has shown the potential importance of GSK3β in ischemic heart disease and ischemia-reperfusion pathologies. Reperfusion injury may occur in tissues after prolonged ischemia following reperfusion. Reperfusion injury can be life threatening. Reperfusion injury occurs due to a change in ionic homeostasis, excess free radical production, mitochondrial damage and cell death. There are however clear, cardiac-protective signals; although the molecular pathophysiology is not clearly understood. In normal physiology, GSK3β has a critical role in the cytoprotective pathway. However, it`s controversial role in ischemia and ischemia-reperfusion is a topic of current interest. In this review, we have opted to focus on GSK3β interactions with mitochondria in ischemic heart disease and expand on the therapeutic interventions.

  5. Lithium Chloride Increases COX-2 Expression and PGE2 Production in a Human Granulosa-Lutein SVOG Cell Line Via a GSK-3β/β-Catenin Signaling Pathway.

    Science.gov (United States)

    Bai, Long; Chang, Hsun-Ming; Cheng, Jung-Chien; Chu, Guiyan; Leung, Peter C K; Yang, Gongshe

    2017-09-01

    Lithium chloride (LiCl) is widely prescribed for the treatment of bipolar disorders and is associated with a higher incidence of reproductive adverse effects. Cyclooxygenase (COX)-2 and its derivative, prostaglandin E2 (PGE2), play regulatory roles in the human ovulatory process. Whether LiCl affects ovulation by regulating COX2 expression and PGE2 production in the human ovary is still largely unknown. The aim of this study was to investigate the effect of LiCl on the expression of COX-2 and production of PGE2 in human granulosa-lutein (hGL) cells, as well as the mechanisms underlying this effect. Both immortalized and primary hGL cells were used as research models. Using dual inhibition approaches, our results show that LiCl initiates the hGL cellular action by inhibiting the activity of glycogen synthase kinase-3β [GSK-3β (phosphorylation of GSK-3β)] and activation of extracellular signal-regulated kinase 1/2 (ERK1/2), but not by affecting protein kinase B or cAMP response element binding protein signaling. Additionally, the phosphorylation of GSK-3β, but not ERK1/2, resulted in the stabilization and nuclear localization of β-catenin. Furthermore, knockdown of either β-catenin or GSK-3β reversed the LiCl-induced upregulation of COX-2 expression. These results indicate that LiCl upregulates the expression of COX-2 and the subsequent production of PGE2 through the canonical GSK-3β/β-catenin signaling pathway in hGL cells. Copyright © 2017 Endocrine Society.

  6. Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage, Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients.

    Directory of Open Access Journals (Sweden)

    Serena Guidotti

    Full Text Available Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA, but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy.In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763 were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS production (2',7'-dichlorofluorescin diacetate staining. Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21, flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining.In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10.In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via

  7. Acquired radioresistance of cancer and the AKT/GSK3β/cyclin D1 overexpression cycle

    International Nuclear Information System (INIS)

    Shimura, Tsutomu

    2011-01-01

    Fractionated radiotherapy (RT) is widely used in cancer therapy for its advantages in the preservation of normal tissues. However, repopulation of surviving tumor cells during fractionated RT limits the efficacy of RT. In fact, repopulating tumors often acquire radioresistance and this is the major cause of failure of RT. We have recently demonstrated that human tumor cells acquire radioresistance when exposed to fractionated radiation (FR) of X-rays every 12 hours for 1 month. The acquired radioresistance was associated with overexpression of cyclin D1, a result of a series of molecular changes; constitutive activation of DNA-PK and AKT with concomitant down-regulation of glycogen synthase kinase-3β (GSK3β) which results in suppression of cyclin D1 proteolysis. Aberrant cyclin D1 overexpression in S-phase induced DNA double strand breaks which activated DNA-PK and established the vicious cycle of cycling D1 overexpression. This overexpression of cyclin D1 is responsible for the radioresistance phenotype of long-term FR cells, since this phenotype was completely abrogated by treatment of FR cells by the AKT/PKB signaling inhibitor (API-2), an AKT inhibitor or by a Cdk4 inhibitor. Thus, targeting the AKT/GSK3β/cyclin D1/Cdk4 pathway can be an efficient modality to suppress acquired radioresistance of tumor cells. In this article, I overview the newly discovered molecular mechanisms underlying acquired radioresistance of tumor cells induced by FR, and propose a strategy for eradication of tumors using fractionated RT by overcoming tumor radioresistance. (author)

  8. Quercetin Protects against Okadaic Acid-Induced Injury via MAPK and PI3K/Akt/GSK3β Signaling Pathways in HT22 Hippocampal Neurons.

    Directory of Open Access Journals (Sweden)

    Wei Jiang

    Full Text Available Increasing evidence shows that oxidative stress and the hyperphosphorylation of tau protein play essential roles in the progression of Alzheimer's disease (AD. Quercetin is a major flavonoid that has anti-oxidant, anti-cancer and anti-inflammatory properties. We investigated the neuroprotective effects of quercetin to HT22 cells (a cell line from mouse hippocampal neurons. We found that Okadaic acid (OA induced the hyperphosphorylation of tau protein at Ser199, Ser396, Thr205, and Thr231 and produced oxidative stress to the HT22 cells. The oxidative stress suppressed the cell viability and decreased the levels of lactate dehydrogenase (LDH, superoxide dismutase (SOD, mitochondria membrane potential (MMP and Glutathione peroxidase (GSH-Px. It up-regulated malondialdehyde (MDA production and intracellular reactive oxygen species (ROS. In addition, phosphoinositide 3 kinase/protein kinase B/Glycogen synthase kinase3β (PI3K/Akt/GSK3β and mitogen activated protein kinase (MAPK were also involved in this process. We found that pre-treatment with quercetin can inhibited OA-induced the hyperphosphorylation of tau protein and oxidative stress. Moreover, pre-treatment with quercetin not only inhibited OA-induced apoptosis via the reduction of Bax, and up-regulation of cleaved caspase 3, but also via the inhibition of PI3K/Akt/GSK3β, MAPKs and activation of NF-κB p65. Our findings suggest the therapeutic potential of quercetin to treat AD.

  9. The F-box Protein KIB1 Mediates Brassinosteroid-Induced Inactivation and Degradation of GSK3-like Kinases in Arabidopsis.

    Science.gov (United States)

    Zhu, Jia-Ying; Li, Yuyao; Cao, Dong-Mei; Yang, Hongjuan; Oh, Eunkyoo; Bi, Yang; Zhu, Shengwei; Wang, Zhi-Yong

    2017-06-01

    The glycogen synthase kinase-3 (GSK3) family kinases are central cellular regulators highly conserved in all eukaryotes. In Arabidopsis, the GSK3-like kinase BIN2 phosphorylates a range of proteins to control broad developmental processes, and BIN2 is degraded through unknown mechanism upon receptor kinase-mediated brassinosteroid (BR) signaling. Here we identify KIB1 as an F-box E3 ubiquitin ligase that promotes the degradation of BIN2 while blocking its substrate access. Loss-of-function mutations of KIB1 and its homologs abolished BR-induced BIN2 degradation and caused severe BR-insensitive phenotypes. KIB1 directly interacted with BIN2 in a BR-dependent manner and promoted BIN2 ubiquitination in vitro. Expression of an F-box-truncated KIB1 caused BIN2 accumulation but dephosphorylation of its substrate BZR1 and activation of BR responses because KIB1 blocked BIN2 binding to BZR1. Our study demonstrates that KIB1 plays an essential role in BR signaling by inhibiting BIN2 through dual mechanisms of blocking substrate access and promoting degradation. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. 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

    2017-01-01

    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. PMID:28067275

  11. 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.

  12. Silymarin Ameliorates Diabetes-Induced Proangiogenic Response in Brain Endothelial Cells through a GSK-3β Inhibition-Induced Reduction of VEGF Release

    Directory of Open Access Journals (Sweden)

    Ahmed Alhusban

    2017-01-01

    Full Text Available Diabetes mellitus (DM is a major risk factor for cardiovascular disease. Additionally, it was found to induce a dysfunctional angiogenic response in the brain that was attributed to oxidative stress. Milk thistle seed extract (silymarin has potent antioxidant properties, though its potential use in ameliorating diabetes-induced aberrant brain angiogenesis is unknown. Glycogen synthase kinase-3β is a regulator of angiogenesis that is upregulated by diabetes. Its involvement in diabetes-induced angiogenesis is unknown. To evaluate the potential of silymarin to ameliorate diabetes-induced aberrant angiogenesis, human brain endothelial cells (HBEC-5i were treated with 50 μg/mL advanced glycation end (AGE products in the presence or absence of silymarin (50, 100 μM. The angiogenic potential of HBEC-5i was evaluated in terms of migration and in vitro tube formation capacities. The involvement of GSK-3β was also evaluated. AGE significantly increased the migration and tube formation rates of HBEC-5i by about onefold (p=0.0001. Silymarin reduced AGE-induced migration in a dose-dependent manner where 50 μM reduced migration by about 50%, whereas the 100 μM completely inhibited AGE-induced migration. Similarly, silymarin 50 μg/mL blunted AGE-induced tube formation (p=0.001. This effect was mediated through a GSK-3β-dependent inhibition of VEGF release. In conclusion, silymarin inhibits AGE-induced aberrant angiogenesis in a GSK-3β-mediated inhibition of VEGF release.

  13. Acceleration of bone development and regeneration through the Wnt/β-catenin signaling pathway in mice heterozygously deficient for GSK-3β

    Energy Technology Data Exchange (ETDEWEB)

    Arioka, Masaki [Department of Clinical Pharmacology, Faculty of Medical Sciences, Kyushu University, Fukuoka (Japan); Department of Oral and Maxillofacial Surgery, Faculty of Dental Science, Kyushu University, Fukuoka (Japan); Takahashi-Yanaga, Fumi, E-mail: yanaga@clipharm.med.kyushu-u.ac.jp [Department of Clinical Pharmacology, Faculty of Medical Sciences, Kyushu University, Fukuoka (Japan); Global Medical Science Education Unit, Faculty of Medical Sciences, Kyushu University, Fukuoka (Japan); Sasaki, Masanori [Department of Oral and Maxillofacial Surgery, Faculty of Dental Science, Kyushu University, Fukuoka (Japan); Yoshihara, Tatsuya; Morimoto, Sachio [Department of Clinical Pharmacology, Faculty of Medical Sciences, Kyushu University, Fukuoka (Japan); Takashima, Akihiko [Department of Aging Neurobiology, National Center for Geriatrics and Gerontology, Oobu (Japan); Mori, Yoshihide [Department of Oral and Maxillofacial Surgery, Faculty of Dental Science, Kyushu University, Fukuoka (Japan); Sasaguri, Toshiyuki [Department of Clinical Pharmacology, Faculty of Medical Sciences, Kyushu University, Fukuoka (Japan)

    2013-11-01

    Highlights: •The Wnt/β-catenin signaling pathway was activated in GSK-3β{sup +/−} mice. •The cortical and trabecular bone volumes were increased in GSK-3β{sup +/−} mice. •Regeneration of a partial bone defect was accelerated in GSK-3β{sup +/−} mice. -- Abstract: Glycogen synthase kinase (GSK)-3β plays an important role in osteoblastogenesis by regulating the Wnt/β-catenin signaling pathway. Therefore, we investigated whether GSK-3β deficiency affects bone development and regeneration using mice heterozygously deficient for GSK-3β (GSK-3β{sup +/−}). The amounts of β-catenin, c-Myc, cyclin D1, and runt-related transcription factor-2 (Runx2) in the bone marrow cells of GSK-3β{sup +/−} mice were significantly increased compared with those of wild-type mice, indicating that Wnt/β-catenin signals were enhanced in GSK-3β{sup +/−} mice. Microcomputed tomography of the distal femoral metaphyses demonstrated that the volumes of both the cortical and trabecular bones were increased in GSK-3β{sup +/−} mice compared with those in wild-type mice. Subsequently, to investigate the effect of GSK-3β deficiency on bone regeneration, we established a partial bone defect in the femur and observed new bone at 14 days after surgery. The volume and mineral density of the new bone were significantly higher in GSK-3β{sup +/−} mice than those in wild-type mice. These results suggest that bone formation and regeneration in vivo are accelerated by inhibition of GSK-3β, probably through activation of the Wnt/β-catenin signaling pathway.

  14. Inositol synthesis regulates the activation of GSK-3α in neuronal cells.

    Science.gov (United States)

    Ye, Cunqi; Greenberg, Miriam L

    2015-04-01

    The synthesis of inositol provides precursors of inositol lipids and inositol phosphates that are pivotal for cell signaling. Mood stabilizers lithium and valproic acid, used for treating bipolar disorder, cause cellular inositol depletion, which has been proposed as a therapeutic mechanism of action of both drugs. Despite the importance of inositol, the requirement for inositol synthesis in neuronal cells is not well understood. Here, we examined inositol effects on proliferation of SK-N-SH neuroblastoma cells. The essential role of inositol synthesis in proliferation is underscored by the findings that exogenous inositol was dispensable for proliferation, and inhibition of inositol synthesis decreased proliferation. Interestingly, the inhibition of inositol synthesis by knocking down INO1, which encodes inositol-3-phosphate synthase, the rate-limiting enzyme of inositol synthesis, led to the inactivation of GSK-3α by increasing the inhibitory phosphorylation of this kinase. Similarly, the mood stabilizer valproic acid effected transient decreases in intracellular inositol, leading to inactivation of GSK-3α. As GSK-3 inhibition has been proposed as a likely therapeutic mechanism of action, the finding that inhibition of inositol synthesis results in the inactivation of GSK-3α suggests a unifying hypothesis for mechanism of mood-stabilizing drugs. Inositol is an essential metabolite that serves as a precursor for inositol lipids and inositol phosphates. We report that inhibition of the rate-limiting enzyme of inositol synthesis leads to the inactivation of glycogen synthase kinase (GSK) 3α by increasing inhibitory phosphorylation of this kinase. These findings have implications for the therapeutic mechanisms of mood stabilizers and suggest that inositol synthesis and GSK 3α activity are intrinsically related. © 2014 International Society for Neurochemistry.

  15. GSK-3β signaling determines autophagy activation in the breast tumor cell line MCF7 and inclusion formation in the non-tumor cell line MCF10A in response to proteasome inhibition.

    Science.gov (United States)

    Gavilán, E; Sánchez-Aguayo, I; Daza, P; Ruano, D

    2013-04-04

    The ubiquitin-proteasome system and the autophagy-lysosome pathway are the two main mechanisms for eukaryotic intracellular protein degradation. Proteasome inhibitors are used for the treatment of some types of cancer, whereas autophagy seems to have a dual role in tumor cell survival and death. However, the relationship between both pathways has not been extensively studied in tumor cells. We have investigated both proteolytic systems in the human epithelial breast non-tumor cell line MCF10A and in the human epithelial breast tumor cell line MCF7. In basal condition, tumor cells showed a lower proteasome function but a higher autophagy activity when compared with MCF10A cells. Importantly, proteasome inhibition (PI) leads to different responses in both cell types. Tumor cells showed a dose-dependent glycogen synthase kinase-3 (GSK-3)β inhibition, a huge increase in the expression of the transcription factor CHOP and an active processing of caspase-8. By contrast, MCF10A cells fully activated GSK-3β and showed a lower expression of both CHOP and processed caspase-8. These molecular differences were reflected in a dose-dependent autophagy activation and cell death in tumor cells, while non-tumor cells exhibited the formation of inclusion bodies and a decrease in the cell death rate. Importantly, the behavior of the MCF7 cells can be reproduced in MCF10A cells when GSK-3β and the proteasome were simultaneously inhibited. Under this situation, MCF10A cells strongly activated autophagy, showing minimal inclusion bodies, increased CHOP expression and cell death rate. These findings support GSK-3β signaling as a key mechanism in regulating autophagy activation or inclusion formation in human tumor or non-tumor breast cells, respectively, which may shed new light on breast cancer control.

  16. Ablation of periostin inhibits post-infarction myocardial regeneration in neonatal mice mediated by the phosphatidylinositol 3 kinase/glycogen synthase kinase 3β/cyclin D1 signalling pathway.

    Science.gov (United States)

    Chen, Zhenhuan; Xie, Jiahe; Hao, Huixin; Lin, Hairuo; Wang, Long; Zhang, Yingxue; Chen, Lin; Cao, Shiping; Huang, Xiaobo; Liao, Wangjun; Bin, Jianping; Liao, Yulin

    2017-05-01

    To resolve the controversy as to whether periostin plays a role in myocardial regeneration after myocardial infarction (MI), we created a neonatal mouse model of MI to investigate the influence of periostin ablation on myocardial regeneration and clarify the underlying mechanisms. Neonatal periostin-knockout mice and their wildtype littermates were subjected to MI or sham surgery. In the wildtype mice after MI, fibrosis was detectable at 3 days and fibrotic tissue was completely replaced by regenerated myocardium at 21 days. In contrast, in the knockout mice, significant fibrosis in the infarcted area was present at even 3 weeks after MI. Levels of phosphorylated-histone 3 and aurora B in the myocardium, detected by immunofluorescence and western blotting, were significantly lower in knockout than in wildtype mice at 7 days after MI. Similarly, angiogenesis was decreased in the knockout mice after MI. Expression of both the endothelial marker CD-31 and α-smooth muscle actin was markedly lower in the knockout than in wildtype mice at 7 days after MI. The knockout MI group had elevated levels of glycogen synthase kinase (GSK) 3β and decreased phosphatidylinositol 3-kinase (PI3K), phosphorylated serine/threonine protein kinase B (p-Akt), and cyclin D1, compared with the wildtype MI group. Similar effects were observed in experiments using cultured cardiomyocytes from neonatal wildtype or periostin knockout mice. Administration of SB216763, a GSK3β inhibitor, to knockout neonatal mice decreased myocardial fibrosis and increased angiogenesis in the infarcted area after MI. Ablation of periostin suppresses post-infarction myocardial regeneration by inhibiting the PI3K/GSK3β/cyclin D1 signalling pathway, indicating that periostin is essential for myocardial regeneration. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Cardiology

  17. P2X7R-dependent regulation of glycogen synthase kinase 3β and claudin-18 in alveolar epithelial type I cells of mice lung.

    Science.gov (United States)

    Barth, K; Bläsche, R; Neißer, A; Bramke, S; Frank, J A; Kasper, M

    2016-12-01

    The purinergic receptor P2X7 represents an ATP-gated ionotropic receptor with a selective localization in alveolar epithelial type I cells of the lung. Despite the involvement of the receptor in inflammatory processes of the lung, it is not established whether this receptor plays a specific role in the alveolar epithelial cell biology. There is evidence that P2X7 receptor influences Wnt/β-catenin signalling pathways in alveolar epithelial cells under conditions of injury. Here, we investigated the expression of GSK-3β, a potent protein kinase involved in alveolar epithelial barrier functions, and of tight junction molecules occludin, claudin-4 and claudin-18 in wild-type and P2X7 -/- mice. Western blot analysis, immunohistochemistry and quantitative real-time RT-PCR revealed a remarkable increase in claudin-18 mRNA and protein in lungs of P2X7 -/- mice animals. Furthermore, alveolar epithelial cells from P2X7 -/- animals showed decreased levels of GSK-3β protein and its inactive form GSK-3β (pS9). Conversely, claudin-18 knockout mice exhibited decreased P2X7 mRNA transcript abundance as measured by mRNA expression microarray and quantitative PCR. Our data are consistent with the hypothesis that P2X7R contributes to alveolar epithelial barrier function through effects on GSK-3β. Furthermore, these data suggest a potential reciprocal regulation of claudin-18 and P2X7R in the alveolar epithelium.

  18. Inositol synthesis regulates activation of GSK-3α in neuronal cells

    Science.gov (United States)

    Ye, Cunqi; Greenberg, Miriam L.

    2015-01-01

    The synthesis of inositol provides precursors of inositol lipids and inositol phosphates that are pivotal for cell signaling. Mood-stabilizers lithium and valproic acid (VPA), used for treating bipolar disorder, cause cellular inositol depletion, which has been proposed as a therapeutic mechanism of action of both drugs. Despite the importance of inositol, the requirement for inositol synthesis in neuronal cells is not well understood. Here, we examined inositol effects on proliferation of SK-N-SH neuroblastoma cells. The essential role of inositol synthesis in proliferation is underscored by the findings that exogenous inositol was dispensable for proliferation, and inhibition of inositol synthesis decreased proliferation. Interestingly, the inhibition of inositol synthesis by knocking down INO1, which encodes inositol-3-phosphate synthase, the rate-limiting enzyme of inositol synthesis, led to inactivation of GSK-3α by increasing the inhibitory phosphorylation of this kinase. Similarly, the mood-stabilizer VPA effected transient decreases in intracellular inositol, leading to inactivation of GSK-3α. As GSK-3 inhibition has been proposed as a likely therapeutic mechanism of action, the finding that inhibition of inositol synthesis results in inactivation of GSK-3α suggests a unifying hypothesis for mechanism of mood-stabilizing drugs. PMID:25345501

  19. Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes

    Directory of Open Access Journals (Sweden)

    Peraldi Pascal

    2008-02-01

    Full Text Available Abstract Background Multipotent stem cells exist within adipose tissue throughout life. An abnormal recruitment of these adipose precursor cells could participate to hyperplasia of adipose tissue observed in severe obesity or to hypoplasia of adipose tissue observed in lipodystrophy. Therefore, pharmacological molecules that control the pool of stem cells in adipose tissue are of great interest. Glycogen Synthase Kinase (GSK 3 has been previously described as involved in differentiation of preadipose cells and might be a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. However, the impact of GSK3 inhibition on human adipose-derived stem cells remained to be investigated. The aim of this study was to investigate GSK3 as a possible target for pharmacological inhibition of stem cell adipogenesis. To reach this goal, we studied the effects of pharmacological inhibitors of GSK3, i.e. lithium chloride (LiCl and BIO on proliferation and adipocyte differentiation of multipotent stem cells derived from human adipose tissue. Results Our results showed that GSK3 inhibitors inhibited proliferation and clonogenicity of human stem cells, strongly suggesting that GSK3 inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue. The impact of GSK3 inhibition on differentiation of hMADS cells was also investigated. Adipogenic and osteogenic differentiations were inhibited upon hMADS treatment with BIO. Whereas a chronic treatment was required to inhibit osteogenesis, a treatment that was strictly restricted to the early step of differentiation was sufficient to inhibit adipogenesis. Conclusion These results demonstrated the feasibility of a pharmacological approach to regulate adipose-derived stem cell function and that GSK3 could represent a potential target for controlling adipocyte precursor pool under conditions where fat tissue formation is impaired.

  20. 5-AIQ inhibits H{sub 2}O{sub 2}-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3β signaling pathway in H9c2 cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Park, Eun-Seok; Kang, Jun Chul; Kang, Do-Hyun; Jang, Yong Chang [Department of Applied Biochemistry, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of); Yi, Kyu Yang [Bio-Organic Science Division, Korea Research Institute of Chemical Technology, Daejeon, Chungnam, 305-600 (Korea, Republic of); Chung, Hun-Jong [Industrial Medicine Department, Chungju Hospital, Konkuk Medical School, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of); Park, Jong Seok [Department of Biomedical Laboratory Science, Taegu Health College, Taegu 702-722 (Korea, Republic of); Kim, Bokyung [Department of Physiology, Konkuk Medical School, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of); Feng, Zhong-Ping [Department of Physiology, College of Medicine, University of Toronto, Toronto, Ont., Canada M5S 1A8 (Canada); Shin, Hwa-Sup, E-mail: hsshin@kku.ac.kr [Department of Applied Biochemistry, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of)

    2013-04-01

    Poly(adenosine 5′-diphosphate ribose) polymerase (PARP) is a nuclear enzyme activated by DNA strand breaks and plays an important role in the tissue injury associated with ischemia and reperfusion. The aim of the present study was to investigate the protective effect of 5-aminoisoquinolinone (5-AIQ), a PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cardiomyocytes. 5-AIQ pretreatment significantly protected against H{sub 2}O{sub 2}-induced cell death, as determined by the XTT assay, cell counting, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and Western blot analysis of apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. Upregulation of antioxidant enzymes such as manganese superoxide dismutase and catalase accompanied the protective effect of 5-AIQ on H{sub 2}O{sub 2}-induced cell death. Our data also showed that 5-AIQ pretreatment protected H9c2 cells from H{sub 2}O{sub 2}-induced apoptosis by triggering activation of Akt and glycogen synthase kinase-3β (GSK-3β), and that the protective effect of 5-AIQ was diminished by the PI3K inhibitor LY294002 at a concentration that effectively abolished 5-AIQ-induced Akt and GSK-3β activation. In addition, inhibiting the Akt/GSK-3β pathway by LY294002 significantly attenuated the 5-AIQ-mediated decrease in cleaved caspase-3 and Bax activation and H9c2 cell apoptosis induction. Taken together, these results demonstrate that 5-AIQ prevents H{sub 2}O{sub 2}-induced apoptosis in H9c2 cells by reducing intracellular reactive oxygen species production, regulating apoptosis-related proteins, and activating the Akt/GSK-3β pathway. - Highlights: ► 5-AIQ, a PARP inhibitor, decreased H{sub 2}O{sub 2}-induced H9c2 cell death and apoptosis. ► 5-AIQ upregulated antioxidant Mn-SOD and catalase, while decreasing ROS production. ► 5-AIQ decreased H{sub 2}O{sub 2}-induced increase in cleaved caspase-3 and Bax and decrease in Bcl2. ► 5-AIQ activated Akt and GSK-3

  1. AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar [Department of Physiology, University of Tuebingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen (Germany)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Akt/SGK dependent phosphorylation of GSK3{alpha},{beta} regulates T lymphocytes. Black-Right-Pointing-Pointer T cells from mice expressing Akt/SGK insensitive GSK3{alpha},{beta} (gsk3{sup KI}) release less IL-2. Black-Right-Pointing-Pointer CD4{sup +} cells from gsk3{sup KI} mice express less CD62L. Black-Right-Pointing-Pointer CD8{sup +} cells from gsk3{sup KI} mice are relatively resistant to activation induced cell death. Black-Right-Pointing-Pointer Perforin expression is enhanced in gsk3{sup KI} T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3{alpha},{beta}. Lithium, a known unspecific GSK3 inhibitor protects against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3{alpha},{beta} inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3{sup KI}). T cells from gsk3{sup KI} mice were compared to T cells from corresponding wild type mice (gsk3{sup WT}). As a result, in gsk3{sup KI} CD4{sup +} cells surface CD62L (L-selectin) was significantly less abundant than in gsk3{sup WT} CD4{sup +} cells. Upon activation in vitro T cells from gsk3{sup KI} mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3{sup KI} T cells, suggesting that GSK3 induces effector function in CD8{sup +} T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3{alpha},{beta} is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.

  2. Detrimental ELAVL-1/HuR-dependent GSK3β mRNA stabilization impairs resolution in acute respiratory distress syndrome.

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    Olivia Hoffman

    Full Text Available A hallmark of acute respiratory distress syndrome (ARDS is accumulation of protein-rich edema in the distal airspaces and its removal is critical for patient survival. Previous studies have shown a detrimental role of Glycogen Synthase Kinase (GSK 3β during ARDS via inhibition of alveolar epithelial protein transport. We hypothesized that post-transcriptional regulation of GSK3β could play a functional role in ARDS resolution. To address this hypothesis, we performed an in silico analysis to identify regulatory genes whose expression correlation to GSK3β messenger RNA utilizing two lung cancer cell line array datasets. Among potential regulatory partners of GSK3β, these studies identified the RNA-binding protein ELAVL-1/HuR (Embryonic Lethal, Abnormal Vision, Drosophila-Like as a central component in a likely GSK3β signaling network. ELAVL-1/HuR is a RNA-binding protein that selectively binds to AU-rich elements of mRNA and enhances its stability thereby increasing target gene expression. Subsequent studies with siRNA suppression of ELAVL-1/HuR demonstrated deceased GSK3β mRNA and protein expression and improved clearance of FITC-albumin in A549 cells. Conversely, stabilization of ELAVL-1/HuR with the proteasome inhibitor MG-132 resulted in induction of GSK3β at mRNA and protein level and attenuated FITC-albumin clearance. Utilizing ventilator-induced lung injury or intra-tracheal installation of hydrochloric acid to induce ARDS in mice, we observed increased mRNA and protein expression of ELAVL-1/HuR and GSK3β. Together, our findings indicate a previously unknown interaction between GSK3β and ELAV-1 during ARDS, and suggest the inhibition of the ELAV-1- GSK3β pathways as a novel ARDS treatment approach.

  3. Activating the Wnt/β-Catenin Pathway for the Treatment of Melanoma--Application of LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3.

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    Jennifer M Atkinson

    Full Text Available It has previously been observed that a loss of β-catenin expression occurs with melanoma progression and that nuclear β-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/β-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3α and GSK3β isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized β-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10 nM after 72hr of treatment in contrast to other solid tumor cell lines (IC50 >10 uM as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that β-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by β-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma should be further explored.

  4. Hydrogen sulfide preconditioning protects rat liver against ischemia/reperfusion injury by activating Akt-GSK-3β signaling and inhibiting mitochondrial permeability transition.

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    Qingqing Zhang

    Full Text Available Hydrogen sulfide (H2S is the third most common endogenously produced gaseous signaling molecule, but its impact on hepatic ischemia/reperfusion (I/R injury, especially on mitochondrial function, remains unclear. In this study, rats were randomized into Sham, I/R, ischemia preconditioning (IPC or sodium hydrosulfide (NaHS, an H2S donor preconditioning groups. To establish a model of segmental (70% warm hepatic ischemia, the hepatic artery, left portal vein and median liver lobes were occluded for 60 min and then unclamped to allow reperfusion. Preconditioning with 12.5, 25 or 50 μmol/kg NaHS prior to the I/R insult significantly increased serum H2S levels, and, similar to IPC, NaHS preconditioning decreased alanine aminotransferase (ALT and aspartate aminotransferase (AST levels in the plasma and prevented hepatocytes from undergoing I/R-induced necrosis. Moreover, a sub-toxic dose of NaHS (25 μmol/kg did not disrupt the systemic hemodynamics but dramatically inhibited mitochondrial permeability transition pore (MPTP opening and thus prevented mitochondrial-related cell death and apoptosis. Mechanistic studies revealed that NaHS preconditioning markedly increased the expression of phosphorylated protein kinase B (p-Akt, phosphorylated glycogen synthase kinase-3 beta (p-GSK-3β and B-cell lymphoma-2 (Bcl-2 and decreased the release of mitochondrial cytochrome c and cleaved caspase-3/9 levels. Therefore, NaHS administration prior to hepatic I/R ameliorates mitochondrial and hepatocellular damage through the inhibition of MPTP opening and the activation of Akt-GSK-3β signaling. Furthermore, this study provides experimental evidence for the clinical use of H2S to reduce liver damage after perioperative I/R injury.

  5. GSK-3β inhibitor TWS119 attenuates rtPA-induced hemorrhagic transformation and activates the Wnt/β-catenin signaling pathway after acute ischemic stroke in rats.

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    Wang, Wei; Li, Mingchang; Wang, Yuefei; Li, Qian; Deng, Gang; Wan, Jieru; Yang, Qingwu; Chen, Qianxue; Wang, Jian

    2016-12-01

    Hemorrhagic transformation (HT) is a devastating complication for patients with acute ischemic stroke who are treated with tissue plasminogen activator (tPA). It is associated with high morbidity and mortality, but no effective treatments are currently available to reduce HT risk. Therefore, methods to prevent HT are urgently needed. In this study, we used TWS119, an inhibitor of glycogen synthase kinase 3β (GSK-3β), to evaluate the role of the Wnt/β-catenin signaling pathway in recombinant tPA (rtPA)-induced HT. Sprague-Dawley rats were subjected to a middle cerebral artery occlusion (MCAO) model of ischemic stroke and then were administered rtPA, rtPA combined with TWS119, or vehicle at 4 h. The animals were sacrificed 24 h after infarct induction. Rats treated with rtPA showed evident HT, had more severe neurologic deficit, brain edema, and blood-brain barrier breakdown, and had larger infarction volume than did the vehicle group. Rats treated with TWS119 had significantly improved outcomes compared with those of rats treated with rtPA alone. In addition, Western blot analysis showed that TWS119 increased the protein expression of β-catenin, claudin-3, and ZO-1 while suppressing the expression of GSK-3β. These results suggest that TWS119 reduces rtPA-induced HT and attenuates blood-brain barrier disruption, possibly through activation of the Wnt/β-catenin signaling pathway. This study provides a potential therapeutic strategy to prevent tPA-induced HT after acute ischemic stroke.

  6. Effects of prenatal chronic mild stress exposure on hippocampal cell proliferation, expression of GSK-3α, β and NR2B in adult offspring during fear extinction in rats.

    Science.gov (United States)

    Li, Min; Li, Xiaobai; Zhang, Xinxin; Ren, Jintao; Jiang, Han; Wang, Yan; Ma, Yuchao; Cheng, Wenwen

    2014-06-01

    Stress during pregnancy has been implicated as a risk factor for the development of many mental disorders; however, the influence of prenatal stress on the fear or anxiety-related behaviors, especially the fear extinction in adult offspring has been little investigated. In order to investigate how prenatal stress affects fear extinction, which is regarded as a form of new learning that counteracts the expression of Pavlovian's conditioned fear, a rat model of prenatal chronic mild stress (PNS) was used to evaluate the effects of PNS on fear extinction in adult offspring. The expression of hippocampal glycogen synthase kinase-3s (GSK-3α, β), N-methyl-d-aspartic acid receptors (NMDARs)-2B and the hippocampal cell proliferation in dentate gyrus in the adult offspring during fear extinction were studied. Our results showed that PNS significantly reduced body weight of pups, indicating PNS might induce growth retardation in offspring. Moreover, PNS significantly enhanced the freezing behavior of offspring at the phase of extinction, suggesting PNS impaired the abilities of fear extinction learning. In addition, PNS significantly increased the levels of GSK-3α, β and NR2B, but reduced hippocampal cell proliferation during fear extinction. Taken together, our findings suggest that maternal stress during pregnancy can impair the fear extinction of adult offspring, probably by affecting the neural plasticity of brain. Copyright © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

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

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    Parisi Federica

    2011-09-01

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

  8. Enhanced Chondrogenic Differentiation of Human Umbilical Cord Wharton's Jelly Derived Mesenchymal Stem Cells by GSK-3 Inhibitors.

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    Prapot Tanthaisong

    Full Text Available Articular cartilage is an avascular, alymphatic, and aneural system with very low regeneration potential because of its limited capacity for self-repair. Mesenchymal stem cells (MSCs are the preferred choice for cell-based therapies. Glycogen synthase kinase 3 (GSK-3 inhibitors are compounds that can induce the Wnt signaling pathway, which is involved in chondrogenesis and cartilage development. Here, we investigated the influence of lithium chloride (LiCl and SB216763 synergistically with TGF-β3 on chondrogenic differentiation in human mesenchymal stem cells derived from Wharton's jelly tissue (hWJ-MSCs. hWJ-MSCs were cultured and chondrogenic differentiation was induced in monolayer and pellet experiments using chondrogenic medium, chondrogenic medium supplemented with LiCl, or SB216763 for 4 weeks. After in vitro differentiation, cultured cells were examined for the expression of Sox9, ACAN, Col2a1, and β-catenin markers. Glycosaminoglycan (GAG accumulation was also examined by Alcian blue staining. The results indicated that SB216763 was more effective than LiCl as evidenced by a higher up-regulation of the expression of cartilage-specific markers, including Sox9, ACAN, Col2a1 as well as GAG accumulation. Moreover, collagen type II expression was strongly observed in cells cultured in the chondrogenic medium + SB216763 as evidenced by western blot analysis. Both treatments appeared to mediate the Wnt signaling pathway by up-regulating β-catenin gene expression. Further analyses showed that all treatments suppressed the progression of chondrocyte hypertrophy, determined by decreased expression of Col10a1 and Runx2. These results indicate that LiCl and SB216763 are potential candidates for further in vivo therapeutic trials and would be of great importance for cartilage regeneration.

  9. Bis(maltolato)-oxovanadium (IV)-induced phosphorylation of PKB, GSK-3 and FOXO1 contributes to its glucoregulatory responses (review).

    Science.gov (United States)

    Vardatsikos, George; Mehdi, Mohamad Z; Srivastava, Ashok K

    2009-09-01

    Over the last several decades, a large body of evidence has accumulated to suggest that organo-vanadium compounds (OVC) are more potent than inorganic vanadium salts in regulating hyperglycemia and insulin-resistance in rodent models of both type I and type II diabetes. Among these OVC, vanadium (IV) oxo bis(maltolato) (BMOV) was the first to be investigated for its higher potency over inorganic vanadium salts in eliciting insulin-like properties in both in vitro and in vivo systems. While the precise molecular mechanism by which BMOV exerts its insulin-mimetic effects remains poorly defined, studies have shown that BMOV is a potent activator of several key components of the insulin signaling pathways, such as phosphatidyl-inositol 3-kinase (PI3-K), and its downstream effector, protein kinase B (PKB). In addition, BMOV-induced phosphorylation of PKB has also been associated with the enhanced phosphorylation of glycogen synthase kinase-3 (GSK-3) and forkhead box protein 1 (FOXO1). Since PKB is instrumental in mediating the effects of insulin on glucose transport, glycogen synthesis and gluconeogenesis, it is reasonable to suggest that activation of this pathway by BMOV serves as a mechanism for its insulin-like effects.

  10. Rescue therapy with Tanshinone IIA hinders transition of acute kidney injury to chronic kidney disease via targeting GSK3β

    Science.gov (United States)

    Jiang, Chunming; Zhu, Wei; Yan, Xiang; Shao, Qiuyuan; Xu, Biao; Zhang, Miao; Gong, Rujun

    2016-01-01

    Acute kidney injury (AKI) remains challenging for clinical practice and poses a risk of developing progressive chronic kidney disease (CKD) with no definitive treatment available yet. Tanshinone IIA, an active ingredient of Chinese herbal Salvia miltiorrhiza, has been widely used in Asia for the remarkable organoprotective activities. Its effect on established AKI, however, remains unknown. In mice with folic acid-induced AKI, delayed treatment with Tanshinone IIA, commenced early or late after injury, diminished renal expression of kidney injury markers, reduced apoptosis and improved kidney dysfunction, concomitant with mitigated histologic signs of AKI to CKD transition, including interstitial fibrosis and tubular atrophy, and with an ameliorated inflammatory infiltration in tubulointerstitium and a favored M2-skewed macrophage polarization. Mechanistically, Tanshinone IIA blunted glycogen synthase kinase (GSK)3β overactivity and hyperactivation of its downstream mitogen-activated protein kinases that are centrally implicated in renal fibrogenesis and inflammation. Inhibition of GSK3β is likely a key mechanism mediating the therapeutic activity of Tanshinone IIA, because sodium nitroprusside, a GSK3β activator, largely offset its renoprotective effect. In confirmatory studies, rescue treatment with Tanshinone IIA likewise ameliorated ischemia/reperfusion-induced kidney destruction in mice. Our data suggest that Tanshinone IIA represents a valuable treatment that improves post-AKI kidney salvage via targeting GSK3β. PMID:27857162

  11. Activation of Nrf2/HO-1 Pathway by Glycogen Synthase Kinase-3β Inhibition Attenuates Renal Ischemia/Reperfusion Injury in Diabetic Rats

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    Xiaohua Shen

    2017-06-01

    Full Text Available Background/Aims: Diabetes mellitus can exacerbate renal ischemia-reperfusion (I/R injury (RI/RI. The aim of the present study was to evaluate the protective effect of GSK-3β inhibition (TDZD-8 on I/R-induced renal injury through the Nrf2/HO-1 pathway in a streptozocin (STZ-induced diabetic rat model. Methods: STZ-induced diabetic rats preconditioned with TDZD-8 and ZnPP were subjected to renal I/R. The extent of renal morphologic lesions. Renal function was assessed from blood urea nitrogen (BUN and serum creatinine (Scr, as determined utlizing commercial kits. Oxidative stress and inflammatory activity in the kidney tissue was estimated from levels of malondialdehyde (MDA, interleukin-10 (IL-10, tumor necrosis factor-α (TNF-α, and nitric oxide (NO, as well as the activities of superoxide dismutase (SOD and glutathione (GSH using qRT-PCR and ELISA. The expressions of Nrf2, HO-1, Bcl-2 and NF-κB in the renal tissue were measured by qRT-PCR and western blotting. Results: I/R-induced renal inflammation was reduced significantly by TDZD-8 pretreatment. Preconditioning with TDZD-8 suppressed NF-κB expression and enhanced Bcl-2 expression in the renal tissue. The upregulated level of malondialdehyde (MDA, and reduced activities of superoxide dismutase (SOD and glutathione (GSH in I/R-shocked rats were markedly restored by TDZD-8 pretreatment. Furthermore, pretreatment with TDZD-8 enhanced activation of the Nrf2/HO-1 pathway in the renal tissue of diabetic RI/RI rats. Conclusion: These findings suggest that preconditioning with TDZD-8 may protect the kidney from I/R-induced damage via the activation of the Nrf2/HO-1 pathway in STZ-induced diabetic rats. Further detailed studies are needed to further clarify the underlying mechanisms.

  12. AKT/GSK3 signaling pathways and schizophrenia

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    Effat eEmamian

    2012-03-01

    Full Text Available Schizophrenia is a prevalent complex trait disorder manifested by severe neurocognitive dysfunctions and lifelong disability. During the past few years several studies have provided direct evidence for the involvement of different signaling pathways in schizophrenia. In this review, we mainly focus on AKT/GSK3 signaling pathways in schizophrenia. The original study on the involvement of this pathway in schizophrenia was published by Emamian et al in 2004. This study reported convergent evidence for a decrease in AKT1 protein levels and levels of phosphorylation of GSK3β in the peripheral lymphocytes and brains of individuals with schizophrenia; a significant association between schizophrenia and an AKT1 haplotype; and a greater sensitivity to the sensorimotor gating−disruptive effect of amphetamine, conferred by AKT1 deficiency. It also showed that haloperidol can induce a stepwise increase in regulatory phosphorylation of AKT1 in the brains of treated mice that could compensate for the impaired function of this signaling pathway in schizophrenia. Following this study, several independent studies were published that not only confirmed the association of this signaling pathway with schizophrenia across different populations, but also shed light on the mechanisms by which AKT/GSK3 pathway may contribute to the development of this complex disorder. In this review, following an introduction on the role of AKT in human diseases and its functions in neuronal & non-neuronal cells, a review on the results of studies published on AKT/GSK3 signaling pathway in schizophrenia after the original 2004 paper will be provided. A brief review on other signaling pathways involved in schizophrenia and the possible connections with AKT/GSK3 signaling pathway will be discussed. Moreover, some possible molecular mechanisms acting through this pathway will be discussed besides the mechanisms by which they may contribute to the pathogenesis of schizophrenia. Finally

  13. Pharmacological postconditioning with atorvastatin calcium attenuates myocardial ischemia/reperfusion injury in diabetic rats by phosphorylating GSK3β.

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    Chen, Linyan; Cai, Ping; Cheng, Zhendong; Zhang, Zaibao; Fang, Jun

    2017-07-01

    Diabetes is an independent risk factor for myocardial ischemia, and many epidemiological data and laboratory studies have revealed that diabetes significantly exacerbated myocardial ischemia/reperfusion injury and ameliorated protective effects. The present study aimed to determine whether pharmacological postconditioning with atorvastatin calcium lessened diabetic myocardial ischemia/reperfusion injury, and investigated the role of glycogen synthase kinase (GSK3β) in this. A total of 72 streptozotocin-induced diabetic rats were randomly divided into six groups, and 24 age-matched male non-diabetic Sprague-Dawley rats were randomly divided into two groups. Rats all received 40 min myocardial ischemia followed by 180 min reperfusion, except sham-operated groups. Compared with the non-diabetic ischemia/reperfusion model group, the diabetic ischemia/reperfusion group had a comparable myocardial infarct size, but a higher level of serum cardiac troponin I (cTnI) and morphological alterations to their myocardial cells. Compared with the diabetic ischemia/reperfusion group, the group that received pharmacological postconditioning with atorvastatin calcium had smaller myocardial infarct sizes, lower levels of cTnI, reduced morphological alterations to myocardial cells, higher levels of p-GSK3β, heat shock factor (HSF)-1 and heat shock protein (HSP)70. The cardioprotective effect conferred by atorvastatin calcium did not attenuate myocardial ischemia/reperfusion injury following application of TDZD-8, which phosphorylates and inactivates GSK3β. Pharmacological postconditioning with atorvastatin calcium may attenuate diabetic heart ischemia/reperfusion injury in the current context. The phosphorylation of GSK3β serves a critical role during the cardioprotection in diabetic rats, and p-GSK3β may accelerate HSP70 production partially by activating HSF-1 during myocardial ischemic/reperfusion injury.

  14. Multifaceted Roles of GSK-3 in Cancer and Autophagy-Related Diseases

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    Romina Mancinelli

    2017-01-01

    Full Text Available GSK-3 is a ubiquitously expressed serine/threonine kinase existing as GSK-3α and GSK-3β isoforms, both active under basal conditions and inactivated upon phosphorylation by different upstream kinases. Initially discovered as a regulator of glycogen synthesis, GSK-3 is also involved in several signaling pathways controlling many different key functions. Here, we discuss recent advances regarding (i GSK-3 structure, function, regulation, and involvement in several cancers, including hepatocarcinoma, cholangiocarcinoma, breast cancer, prostate cancer, leukemia, and melanoma (active GSK-3 has been shown to induce apoptosis in some cases or inhibit apoptosis in other cases and to induce cancer progression or inhibit tumor cell proliferation, suggesting that different GSK-3 modulators may address different specific targets; (ii GSK-3 involvement in autophagy modulation, reviewing signaling pathways involved in neurodegenerative and liver diseases; (iii GSK-3 role in oxidative stress and autophagic cell death, focusing on liver injury; (iv GSK-3 as a possible therapeutic target of natural substances and synthetic inhibitors in many diseases; and (v GSK-3 role as modulator of mammalian aging, related to metabolic alterations characterizing senescent cells and age-related diseases. Studies summarized here underline the GSK-3 multifaceted role and indicate such kinase as a molecular target in different pathologies, including diseases associated with autophagy dysregulation.

  15. Synthesis of Novel Oxazolo[4,5-b]pyridine-2-one based 1,2,3-triazoles as Glycogen Synthase Kinase-3β Inhibitors with Anti-inflammatory Potential.

    Science.gov (United States)

    Tantray, Mushtaq A; Khan, Imran; Hamid, Hinna; Alam, Mohammad Sarwar; Umar, Sadiq; Ali, Yakub; Sharma, Kalicharan; Hussain, Firasat

    2016-06-01

    A novel series of oxazolo[4,5-b]pyridine-2-one based 1,2,3-triazoles has been synthesized by click chemistry approach and evaluated for in vitro GSK-3β inhibitory activity. Compound 4g showed maximum inhibition with IC50 value of 0.19 μm. Keeping in view the effect of GSK-3β inhibition on inflammation, compounds 4g, 4d, 4f, 4i, 4n and 4q exhibiting significant GSK-3β inhibition were examined for in vivo anti-inflammatory activity in rat paw edema model. The compounds 4g, 4d, 4f and 4i showed pronounced in vivo anti-inflammatory activity (76.36, 74.54, 72.72 and 70.90%, respectively, after 5h post-carrageenan administration) and were further found to inhibit the pro-inflammatory mediators, viz. NO, TNF-α, IL-1β, and IL-6 substantially in comparison with indomethacin, an anti-inflammatory drug as well as SB216763, a GSK-3β inhibitor, reported to exert a similar effect. Histopathology studies confirmed the tolerance of gastric mucosa to these compounds. © 2016 John Wiley & Sons A/S.

  16. An X11alpha/FSBP complex represses transcription of the GSK3beta gene promoter.

    LENUS (Irish Health Repository)

    Lau, Kwok-Fai

    2010-08-04

    X11alpha is a neuronal adaptor protein that interacts with the amyloid precursor protein (APP) through a centrally located phosphotyrosine binding domain to inhibit the production of Abeta peptide that is deposited in Alzheimer\\'s disease brains. X11alpha also contains two C-terminal postsynaptic density-95, large discs, zona occludens 1 (PDZ) domains, and we show here that through its PDZ domains, X11alpha interacts with a novel transcription factor, fibrinogen silencer binding protein. Moreover, we show that an X11alpha\\/fibrinogen silencer binding protein complex signals to the nucleus to repress glycogen synthase kinase-3beta promoter activity. Glycogen synthase kinase-3beta is a favoured candidate kinase for phosphorylating tau in Alzheimer\\'s disease. Our findings show a new function for X11alpha that may impact on Alzheimer\\'s disease pathogenesis.

  17. Atorvastatin Alleviates Experimental Diabetic Cardiomyopathy by Regulating the GSK-3β-PP2Ac-NF-κB Signaling Axis.

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    Xiao-Min Ren

    Full Text Available Recent studies reported that atorvastatin (ATOR alleviated progression of experimental diabetic cardiomyopathy (DCM, possibly by protecting against apoptosis. However, the underlying mechanisms of this protective effect remain unclear. Therefore, our study investigated the role of the glycogen synthase kinase (GSK-3β-protein phosphatase 2A(PP2A-NF-κB signaling pathway in the anti-apoptotic and cardioprotective effects of ATOR on cardiomyocytes cultured in high glucose (HG and in DCM. Our results showed that, in HG-cultured cardiomyocytes, phosphorylation of GSK-3β was decreased, while that of the PP2A catalytic subunit C (PP2Ac and IKK/IкBα was increased, followed by NF-кB nuclear translocation and apoptosis. IKK/IкBα phosphorylation and NF-кB nuclear translocation were also increased by treatment of cells with okadaic acid (OA, a selective PP2A inhibitor, or by silencing PP2Ac expression. The opposite results were obtained by silencing GSK-3β expression, which resulted in PP2Ac activation. Furthermore, IKK/IкBα phosphorylation and NF-кB nuclear translocation were markedly inhibited and apoptosis attenuated in cells treated with ATOR. These effects occurred through inactivation of GSK-3β and subsequent activation of PP2Ac. They were abolished by treatment of cells with OA or PP2Ac siRNA. In mice with type 1 diabetes mellitus, treatment with ATOR, at 10 mg-kg-1-d-1, significantly suppressed GSK-3β activation, IKK/IкBα phosphorylation, NF-кB nuclear translocation and caspase-3 activation, while also activating PP2Ac. Finally, improvements in histological abnormalities, fibrosis, apoptosis and cardiac dysfunction were observed in diabetic mice treated with ATOR. These findings demonstrated that ATOR protected against HG-induced apoptosis in cardiomyocytes and alleviated experimental DCM by regulating the GSK-3β-PP2A-NF-κB signaling pathway.

  18. Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation

    International Nuclear Information System (INIS)

    Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

    2014-01-01

    Highlights: • Lithium suppresses Akt activity by reducing PI3K-mediated Akt phosphorylation. • Lithium enhances GSK-3β activity by reducing Akt-mediated GSK-3β phosphorylation. • Lithium suppresses GSK-3β activity through its direct inhibition. - Abstract: Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li 2 CO 3 significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li 2 CO 3 did not affect PI3K-mediated PI(3,4,5)P 3 production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li 2 CO 3 on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li 2 CO 3 significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li 2 CO 3 significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity

  19. Morphine preconditioning confers cardioprotection in doxorubicin-induced failing rat hearts via ERK/GSK-3β pathway independent of PI3K/Akt

    International Nuclear Information System (INIS)

    He, Shu-Fang; Jin, Shi-Yun; Wu, Hao; Wang, Bin; Wu, Yun-Xiang; Zhang, Shu-Jie; Irwin, Michael G.; Wong, Tak-Ming; Zhang, Ye

    2015-01-01

    Preconditioning against myocardial ischemia–reperfusion (I/R) injury can be suppressed in some pathological conditions. This study was designed to investigate whether morphine preconditioning (MPC) exerts cardioprotection in doxorubicin (DOX)-induced heart failure in rats and the mechanisms involved. Phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt), extracellular signal-regulated kinase (ERK) and glycogen synthase kinase (GSK)-3β pathways were examined. Normal and DOX-induced failing rat hearts were subjected to I/R injury using a Langendorff perfusion system with or without MPC or ischemic preconditioning (IPC). The PI3K inhibitor (wortmannin) or ERK inhibitor (PD98059) was infused before MPC. In normal hearts, both MPC and IPC significantly reduced infarct size and the rise in lactate dehydrogenase (LDH) level caused by I/R injury. Pretreatment with wortmannin or PD98059 abrogated the protective effects of MPC and suppressed the phosphorylation of Akt, ERK and GSK-3β. In failing rat hearts, however, MPC retained its cardioprotection while IPC did not. This protective effect was abolished by PD98059 but not wortmannin. MPC increased the level of p-ERK rather than p-Akt. The phosphorylation of GSK-3β induced by MPC was reversed by PD98059 only. IPC did not elevate the expression of p-ERK, p-Akt and p-GSK-3β in failing rat hearts. We conclude that MPC is cardioprotective in rats with DOX-induced heart failure while IPC is not. The effect of MPC appears to be mediated via the ERK/GSK-3β pathway independent of PI3K/Akt. - Highlights: • Morphine and ischemic preconditioning are cardioprotective in normal rat hearts. • Ischemic preconditioning fails to confer cardioprotection in rats with heart failure. • Morphine retains cardioprotection in doxorubicin-induced heart failure. • Morphine exerts cardioprotection via the ERK/GSK-β pathway independent of PI3K/Akt.

  20. Cardioprotective effects of tanshinone IIA pretreatment via kinin B2 receptor-Akt-GSK-3β dependent pathway in experimental diabetic cardiomyopathy

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    Yuan Yuan

    2011-01-01

    Full Text Available Abstract Aims Diabetic cardiomyopathy, characterized by myocardial structural and functional changes, is a specific cardiomyopathy develops in patients with diabetes mellitus. The present study was to investigate the role of kinin B2 receptor-Akt-glycogen synthase kinase (GSK-3β signalling pathway in mediating the protective effects of tanshinone IIA (TSN on diabetic cardiomyopathy. Methods and results Streptozocin (STZ induced diabetic rats (n = 60 were randomized to receive TSN, TSN plus HOE140 (a kinin B2 receptor antagonist, or saline. Healthy Sprague-Dawley (SD rats (n = 20 were used as control. Left ventricular function, myocardial apoptosis, myocardial ultrastructure, Akt, GSK-3β and NF-κB phosphorylation, the expression of TNF-α, IL-6 and myeloperoxidase (MPO were examined. Cardiac function was well preserved as evidenced by increased left ventricular ejection fraction (LVEF and ± dp/dt (maximum speed of contraction/relaxation, along with decreased myocardial apoptotic death after TSN administration. TSN pretreatment alleviated mitochondria ultrastructure changes. TSN also enhanced Akt and GSK-3β phosphorylation and inhibited NF-κB phosphorylation, resulting in decreased TNF-α, IL-6 and MPO activities. Moreover, pretreatment with HOE140 abolished the beneficial effects of TSN: a decrease in LVEF and ± dp/dt, an inhibition of cardiomyocyte apoptosis, a destruction of cardiomyocyte mitochondria cristae, a reduction of Akt and GSK-3β phosphorylation, an enhancement of NF-κB phosphorylation and an increase of TNF-α, IL-6 and MPO production. Conclusion These data indicated that TSN is cardioprotective in the context of diabetic cardiomyopathy through kinin B2 receptor-Akt-GSK-3β dependent pathway.

  1. Cardioprotective effects of tanshinone IIA pretreatment via kinin B2 receptor-Akt-GSK-3β dependent pathway in experimental diabetic cardiomyopathy.

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    Sun, Dongdong; Shen, Min; Li, Jiayi; Li, Weijie; Zhang, Yingmei; Zhao, Li; Zhang, Zheng; Yuan, Yuan; Wang, Haichang; Cao, Feng

    2011-01-13

    Diabetic cardiomyopathy, characterized by myocardial structural and functional changes, is a specific cardiomyopathy develops in patients with diabetes mellitus. The present study was to investigate the role of kinin B2 receptor-Akt-glycogen synthase kinase (GSK)-3β signalling pathway in mediating the protective effects of tanshinone IIA (TSN) on diabetic cardiomyopathy. Streptozocin (STZ) induced diabetic rats (n = 60) were randomized to receive TSN, TSN plus HOE140 (a kinin B2 receptor antagonist), or saline. Healthy Sprague-Dawley (SD) rats (n = 20) were used as control. Left ventricular function, myocardial apoptosis, myocardial ultrastructure, Akt, GSK-3β and NF-κB phosphorylation, the expression of TNF-α, IL-6 and myeloperoxidase (MPO) were examined. Cardiac function was well preserved as evidenced by increased left ventricular ejection fraction (LVEF) and ± dp/dt (maximum speed of contraction/relaxation), along with decreased myocardial apoptotic death after TSN administration. TSN pretreatment alleviated mitochondria ultrastructure changes. TSN also enhanced Akt and GSK-3β phosphorylation and inhibited NF-κB phosphorylation, resulting in decreased TNF-α, IL-6 and MPO activities. Moreover, pretreatment with HOE140 abolished the beneficial effects of TSN: a decrease in LVEF and ± dp/dt, an inhibition of cardiomyocyte apoptosis, a destruction of cardiomyocyte mitochondria cristae, a reduction of Akt and GSK-3β phosphorylation, an enhancement of NF-κB phosphorylation and an increase of TNF-α, IL-6 and MPO production. These data indicated that TSN is cardioprotective in the context of diabetic cardiomyopathy through kinin B2 receptor-Akt-GSK-3β dependent pathway.

  2. Remote limb ischemic post conditioning during early reperfusion alleviates cerebral ischemic reperfusion injury via GSK-3β/CREB/ BDNF pathway.

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    Ramagiri, Sruthi; Taliyan, Rajeev

    2017-05-15

    Remote limb ischemic post conditioning (RIPOC) has been reported to attenuate cerebral ischemic reperfusion (I/R) injury, while the molecular mechanisms remain elusive. Various studies have highlighted the involvement of glycogen synthase kinase (GSK-3β) in cerebral I/R injury and cognitive disorders. Hence, the present study was designed to explore the role of GSK-3β and its downstream regulators in RIPOC mediated neuroprotection against cerebral I/R injury and associated cognitive impairment. Male Wistar rats are randomly assigned into four groups: Sham, bilateral common carotid arteries occlusion (BCCAO), RIPOC and BCCAO+RIPOC. BCCAO was achieved by transient occlusion of bilateral common carotid arteries for 20min, followed by reperfusion. Non-invasive RIPOC was induced by 3 cycles each of 10min occlusion and reperfusion of both femoral arteries by using tourniquets, during early reperfusion phase. A battery of behavioral and cognitive tests were performed. Biochemical estimation of oxidative markers, anti-oxidants and pro-inflammatory markers were estimated. Levels of GSK-3β, CREB and BDNF were estimated to confirm the molecular mechanism. Hippocampal structural abnormalities were confirmed by H and E staining. The neurobehavioral analysis revealed that neurological and cognitive deficits caused by BCCAO, were reduced by RIPOC intervention. Meanwhile, the results of biochemical tests suggested that RIPOC attenuates the BCCAO induced oxidative damage, neuroinflammation and hippocampal structural abnormalities. Further, RIPOC prevented the elevation of BCCAO induced GSK-3β. RIPOC exerts neuroprotective effect against I/R injury, putatively by attenuating GSK-3β expression and upregulating the levels of CREB and BDNF. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Mammalian diaphanous-related formin 1 regulates GSK3β-dependent microtubule dynamics required for T cell migratory polarization.

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    Baoxia Dong

    Full Text Available The mammalian diaphanous-related formin (mDia1, a Rho-regulated cytoskeletal modulator, has been shown to promote T lymphocyte chemotaxis and interaction with antigen presenting cells, but the mechanisms underpinning mDia1 roles in these processes have not been defined. Here we show that mDia1(-/- T cells exhibit impaired lymphocyte function-associated antigen 1 (LFA-1-mediated T cell adhesion, migration and in vivo trafficking. These defects are associated with impaired microtubule (MT polarization and stabilization, altered MT dynamics and reduced peripheral clustering of the MT plus-end-protein, adenomatous polyposis coli (APC in migrating T cells following LFA-1-engagement. Loss of mDia1 also leads to impaired inducible inactivation of the glycogen synthase kinase (GSK 3β as well as hyperphosphorylation and reduced levels of APC in migrating T cells. These findings identify essential roles for the mDia1 formin in modulating GSK3β-dependent MT contributions to induction of T-cell polarity, adhesion and motility.

  4. (R-(+-α-Lipoic acid protected NG108-15 cells against H2O2-induced cell death through PI3K-Akt/GSK-3β pathway and suppression of NF-κβ-cytokines

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    Kamarudin MNA

    2014-10-01

    Full Text Available Muhamad Noor Alfarizal Kamarudin, Nur Afiqah Mohd Raflee, Sharifah Salwa Syed Hussein, Jia Ye Lo, Hadi Supriady, Habsah Abdul KadirInstitute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, MalaysiaAbstract: Alpha-lipoic acid, a potent antioxidant with multifarious pharmacological benefits has been reported to be neuroprotective in several neuronal models and used to treat neurological disorders such as Alzheimer’s disease. Nonetheless, conclusive mechanisms of alpha-lipoic acid for its protective effects particularly in NG108-15 cells have never been investigated. In this study, the intricate neuroprotective molecular mechanisms by (R-(+-alpha-lipoic acid (R-LA against H2O2-induced cell death in an in vitro model of neurodegeneration were elucidated. Pretreatment with R-LA (2 hours significantly increased NG108-15 cell viability as compared to H2O2-treated cells and mitigated the induction of apoptosis as evidenced by Hoechst 33342/propidium iodide staining. R-LA (12.5–50 µM aggrandized the reduced glutathione over glutathione disulfide ratio followed by a reduction in the intracellular reactive oxygen species level and an increase in mitochondrial membrane potential following H2O2 exposure. Moreover, pretreatment with R-LA stimulated the activation of PI3K-Akt through mTORC1 and mTORC2 components (mTOR, rictor and raptor and production of antiinflammatory cytokine, IL-10 which led to the inactivation of glycogen synthase kinase-3β (GSK-3β and reduction of both Bax/Bcl2 and Bax/Bcl-xL ratios, accompanied by inhibition of the cleaved caspase-3. Additionally, this observation was preceded by the suppression of NF-κβ p65 translocation and production of proinflammatory cytokines (IL-6 and TNF-α. The current findings accentuate new mechanistic insight of R-LA against apoptogenic and brain inflammatory factors in a neuronal model. These results further advocate the therapeutic potential of R-LA for

  5. GSK-3beta is required for memory reconsolidation in adult brain.

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    Tetsuya Kimura

    Full Text Available Activation of GSK-3beta is presumed to be involved in various neurodegenerative diseases, including Alzheimer's disease (AD, which is characterized by memory disturbances during early stages of the disease. The normal function of GSK-3beta in adult brain is not well understood. Here, we analyzed the ability of heterozygote GSK-3beta knockout (GSK+/- mice to form memories. In the Morris water maze (MWM, learning and memory performance of GSK+/- mice was no different from that of wild-type (WT mice for the first 3 days of training. With continued learning on subsequent days, however, retrograde amnesia was induced in GSK+/- mice, suggesting that GSK+/- mice might be impaired in their ability to form long-term memories. In contextual fear conditioning (CFC, context memory was normally consolidated in GSK+/- mice, but once the original memory was reactivated, they showed reduced freezing, suggesting that GSK+/- mice had impaired memory reconsolidation. Biochemical analysis showed that GSK-3beta was activated after memory reactivation in WT mice. Intraperitoneal injection of a GSK-3 inhibitor before memory reactivation impaired memory reconsolidation in WT mice. These results suggest that memory reconsolidation requires activation of GSK-3beta in the adult brain.

  6. GSK3β is involved in the relief of mitochondria pausing in a Tau-dependent manner.

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    María Llorens-Martín

    Full Text Available Mitochondrial trafficking deficits have been implicated in the pathogenesis of several neurological diseases, including Alzheimer's disease (AD. The Ser/Thre kinase GSK3β is believed to play a fundamental role in AD pathogenesis. Given that GSK3β substrates include Tau protein, here we studied the impact of GSK3β on mitochondrial trafficking and its dependence on Tau protein. Overexpression of GSK3β in neurons resulted in an increase in motile mitochondria, whereas a decrease in the activity of this kinase produced an increase in mitochondria pausing. These effects were dependent on Tau proteins, as Tau (-/- neurons did not respond to distinct GSK3β levels. Furthermore, differences in GSK3β expression did not affect other parameters like mitochondria velocity or mitochondria run length. We conclude that GSK3B activity regulates mitochondrial axonal trafficking largely in a Tau-dependent manner.

  7. A SNP in steroid receptor coactivator-1 disrupts a GSK3β phosphorylation site and is associated with altered tamoxifen response in bone.

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    Hartmaier, R J; Richter, A S; Gillihan, R M; Sallit, J Z; McGuire, S E; Wang, J; Lee, A V; Osborne, C K; O'Malley, B W; Brown, P H; Xu, J; Skaar, T C; Philips, S; Rae, J M; Azzouz, F; Li, L; Hayden, J; Henry, N L; Nguyen, A T; Stearns, V; Hayes, D F; Flockhart, D A; Oesterreich, S

    2012-02-01

    The coregulator steroid receptor coactivator (SRC)-1 increases transcriptional activity of the estrogen receptor (ER) in a number of tissues including bone. Mice deficient in SRC-1 are osteopenic and display skeletal resistance to estrogen treatment. SRC-1 is also known to modulate effects of selective ER modulators like tamoxifen. We hypothesized that single nucleotide polymorphisms (SNP) in SRC-1 may impact estrogen and/or tamoxifen action. Because the only nonsynonymous SNP in SRC-1 (rs1804645; P1272S) is located in an activation domain, it was examined for effects on estrogen and tamoxifen action. SRC-1 P1272S showed a decreased ability to coactivate ER compared with wild-type SRC-1 in multiple cell lines. Paradoxically, SRC-1 P1272S had an increased protein half-life. The Pro to Ser change disrupts a putative glycogen synthase 3 (GSK3)β phosphorylation site that was confirmed by in vitro kinase assays. Finally, knockdown of GSK3β increased SRC-1 protein levels, mimicking the loss of phosphorylation at P1272S. These findings are similar to the GSK3β-mediated phospho-ubiquitin clock previously described for the related coregulator SRC-3. To assess the potential clinical significance of this SNP, we examined whether there was an association between SRC-1 P1272S and selective ER modulators response in bone. SRC-1 P1272S was associated with a decrease in hip and lumbar bone mineral density in women receiving tamoxifen treatment, supporting our in vitro findings for decreased ER coactivation. In summary, we have identified a functional genetic variant of SRC-1 with decreased activity, resulting, at least in part, from the loss of a GSK3β phosphorylation site, which was also associated with decreased bone mineral density in tamoxifen-treated women.

  8. Thr 163 phosphorylation causes Mcl-1 stabilization when degradation is independent of the adjacent GSK3-targeted phosphodegron, promoting drug resistance in cancer.

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    Shanna K Nifoussi

    Full Text Available The antiapoptotic Bcl-2 family member Mcl-1 is a PEST protein (containing sequences enriched in proline, glutamic acid, serine, and threonine and is subject to rapid degradation via multiple pathways. Impaired degradation leading to the maintenance of Mcl-1 expression is an important determinant of drug resistance in cancer. Phosphorylation at Thr 163 in the PEST region, stimulated by 12-O-tetradecanoylphorbol acetic acid (TPA-induced activation of extracellular signal-regulated kinase (ERK, is associated with Mcl-1 stabilization in BL41-3 Burkitt lymphoma cells. This contrasts with the observation that Thr 163 phosphorylation in normal fibroblasts primes glycogen synthase kinase (GSK3-induced phosphorylation at Ser 159, producing a phosphodegron that targets Mcl-1 for degradation. In the present follow-up studies in BL41-3 cells, Mcl-1 degradation was found to be independent of the GSK3-mediated pathway, providing a parallel to emerging findings showing that Mcl-1 degradation through this pathway is lost in many different types of cancer. Findings in Mcl-1-transfected CHO cells corroborated those in BL41-3 cells in that the GSK3-targeted phosphodegron did not play a major role in Mcl-1 degradation, and a phosphomimetic T163E mutation resulted in marked Mcl-1 stabilization. TPA-treated BL41-3 cells, in addition to exhibiting Thr 163 phosphorylation and Mcl-1 stabilization, exhibited an ∼10-fold increase in resistance to multiple chemotherapeutic agents, including Ara-C, etoposide, vinblastine, or cisplatin. In these cancer cells in which Mcl-1 degradation is not dependent on the GSK3/phosphodegron-targeted pathway, ERK activation and Thr 163 phosphorylation are associated with pronounced Mcl-1 stabilization and drug resistance - effects that can be suppressed by inhibition of ERK activation.

  9. Involvement of the PI3K/Akt/GSK3β pathway in photodynamic injury of neurons and glial cells

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    Komandirov, M. A.; Knyazeva, E. A.; Fedorenko, Y. P.; Rudkovskii, M. V.; Stetsurin, D. A.; Uzdensky, A. B.

    2011-03-01

    Photodynamic treatment causes intense oxidative stress and kills cells. It is currently used in neurooncology. However, along with tumor it damages surrounding healthy neuronal and glial cells. In order to study the possible role of the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β signaling pathway in photodynamic damage to normal neurons and glia, we used isolated crayfish stretch receptor that consists only of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens (100 nM). The laser diode (670nm, 0.4W/cm2) was used as a light source. Application of specific inhibitors of the enzymes involved in this pathway showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Protein kinase Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3β participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β pathway was not involved as a whole in photodynamic injury of crayfish neurons and glial cells but its components, protein kinase Akt and glycogen synthase kinase-3β, independently and cell-specifically regulated photoinduced death of neurons and glial cells. These data showed that in this system necrosis was not non-regulated and catastrophic mode of cell death. It was controlled by some signaling proteins. The obtained results may be used for search of pharmacological agents that selectively modulate injury of normal neurons and glial cells during photodynamic therapy of brain tumors.

  10. GSK3α/β: A Novel Therapeutic Target for Neuroendocrine Tumors?

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    Aristizabal Prada, Elke Tatjana; Weis, Carla; Orth, Michael; Lauseker, Michael; Spoettl, Gerald; Maurer, Julian; Grabowski, Patricia; Grossman, Ashley; Auernhammer, Christoph Josef; Nölting, Svenja

    2017-10-02

    Introduction: GSK3α/β is a serine/threonine-kinase that plays a critical role in cancer. In this study, we evaluated the effects of the specific GSK3α/β inhibitor AR-A014418 in vitro to gain novel insights into GSK3α/β signaling in NETs. Human NET cell lines (BON1, QGP1, H727 and GOT1) were treated with different concentrations of AR-A014418 alone and in combination with lovastatin, everolimus, 5-fluorouracil (5-FU) and γ-irradiation. AR-A014418 significantly dose- and time-dependently decreased cell viability in all four NET cell lines through inhibition of EGFR- and mTORC1/p70S6K signaling, as well as Cyclin D3 downregulation and induction of pChk1. In all cell lines tested, FACS analysis showed an AR-A014418-induced increase in the sub-G1 phase, reflecting cell death. However, apoptosis induction was only observed in H727 cells. Furthermore, significant anti-migratory effects upon GSK3α/β inhibition were found and were associated with β-catenin downregulation in all cell lines tested. Compensatory up-regulation of pAkt and pERK in response to GSK3α/β inhibition was prevented by combining AR-A014418 with the ERK- and Akt-inhibitor lovastatin. Accordingly, the lovastatin/AR-A014418 combination was synergistic in BON1 and QGP1 cells. Moreover, AR-A014418 displayed promising chemo-sensitizing effects to 5-FU in QGP1 and slight radio-sensitizing properties in BON1 and QGP1 cells. Our data provide new insights into the role of GSK3α/β in NETs and suggest that GSK3α/β-inhibition could be a novel therapeutic option in NETs, especially in combination with lovastatin or 5-FU, depending on tumor entity. ©2017S. Karger AG, Basel.

  11. Neuroprotective effects of honokiol against beta-amyloid-induced neurotoxicity via GSK-3β and β-catenin signaling pathway in PC12 cells.

    Science.gov (United States)

    Xian, Yan-Fang; Ip, Siu-Po; Mao, Qing-Qiu; Lin, Zhi-Xiu

    2016-07-01

    Beta-amyloid (Aβ) accumulation, one of the most important pathogenic traits of Alzheimer's disease (AD), has been reported to induce neurotoxicity in vitro as well as in vivo. Honokiol, isolated from the bark of Magnolia officinalis, has neuroprotective effects in different models of AD in vivo and in vitro. However, the exact mechanism for its neuroprotective effect is not well understood. The present study aimed to investigate the molecular mechanisms underlying the protective action of honokiol against Aβ1-42-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. The results revealed that honokiol protected PC12 cells from Aβ1-42 induced cytotoxicity with increases in cell viability, GSH production and Bcl-2 expression, but decreases in the release of lactate dehydrogenase and cytochrome c, the amount of DNA fragmentation and MDA level, as well as Bax expression. Mechanistic study showed that honokiol could inhibit the activation of glycogen synthase kinase (GSK)-3β, attenuate the nuclear accumulation of β-catenin and suppress the phosphorylation of β-catenin (Ser33/Ser37/Thr41 site) in the Aβ1-42-treated PC12 cells. These results indicate that the anti-oxidative and anti-apoptotic effects of honokiol in Aβ1-42-treated PC12 cells may be mediated, at least in part, by regulation the GSK-3β and β-catenin signaling pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Pharmacological targeting of GSK-3 and NRF2 provides neuroprotection in a preclinical model of tauopathy

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    Antonio Cuadrado

    2018-04-01

    Full Text Available Tauopathies are a group of neurodegenerative disorders where TAU protein is presented as aggregates or is abnormally phosphorylated, leading to alterations of axonal transport, neuronal death and neuroinflammation. Currently, there is no treatment to slow progression of these diseases. Here, we have investigated whether dimethyl fumarate (DMF, an inducer of the transcription factor NRF2, could mitigate tauopathy in a mouse model. The signaling pathways modulated by DMF were also studied in mouse embryonic fibroblast (MEFs from wild type or KEAP1-deficient mice. The effect of DMF on neurodegeneration, astrocyte and microglial activation was examined in Nrf2+/+ and Nrf2−/− mice stereotaxically injected in the right hippocampus with an adeno-associated vector expressing human TAUP301L and treated daily with DMF (100 mg/kg, i.g during three weeks. DMF induces the NRF2 transcriptional through a mechanism that involves KEAP1 but also PI3K/AKT/GSK-3-dependent pathways. DMF modulates GSK-3β activity in mouse hippocampi. Furthermore, DMF modulates TAU phosphorylation, neuronal impairment measured by calbindin-D28K and BDNF expression, and inflammatory processes involved in astrogliosis, microgliosis and pro-inflammatory cytokines production. This study reveals neuroprotective effects of DMF beyond disruption of the KEAP1/NRF2 axis by inhibiting GSK3 in a mouse model of tauopathy. Our results support repurposing of this drug for treatment of these diseases. Keywords: DMF, Inflammation, Neurodegeneration, NRF2, Oxidative stress, TAU/ GSK-3

  13. Dynamic telomerase gene suppression via network effects of GSK3 inhibition.

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    Alan E Bilsland

    2009-07-01

    Full Text Available Telomerase controls telomere homeostasis and cell immortality and is a promising anti-cancer target, but few small molecule telomerase inhibitors have been developed. Reactivated transcription of the catalytic subunit hTERT in cancer cells controls telomerase expression. Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression.In a focused promoter screen, several GSK3 inhibitors suppressed hTERT reporter activity. GSK3 inhibition using 6-bromoindirubin-3'-oxime suppressed hTERT expression, telomerase activity and telomere length in several cancer cell lines and growth and hTERT expression in ovarian cancer xenografts. Microarray analysis, network modelling and oligonucleotide binding assays suggested that multiple transcription factors were affected. Extensive remodelling involving Sp1, STAT3, c-Myc, NFkappaB, and p53 occurred at the endogenous hTERT promoter. RNAi screening of the hTERT promoter revealed multiple kinase genes which affect the hTERT promoter, potentially acting through these factors. Prolonged inhibitor treatments caused dynamic expression both of hTERT and of c-Jun, p53, STAT3, AR and c-Myc.Our results indicate that GSK3 activates hTERT expression in cancer cells and contributes to telomere length homeostasis. GSK3 inhibition is a clinical strategy for several chronic diseases. These results imply that it may also be useful in cancer therapy. However, the complex network effects we show here have implications for either setting.

  14. Sulfuretin Attenuates MPP+-Induced Neurotoxicity through Akt/GSK3β and ERK Signaling Pathways

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    Ramesh Pariyar

    2017-12-01

    Full Text Available Parkinson’s disease (PD is the second most common neurodegenerative disease. It is caused by the death of dopaminergic neurons in the substantia nigra pars compacta. Oxidative stress and mitochondrial dysfunction contribute to the loss of dopaminergic neurons in PD. Sulfuretin is a potent antioxidant that is reported to be beneficial in the treatment of neurodegenerative diseases. In this study, we examined the protective effect of sulfuretin against 1-methyl-4-phenyl pyridinium (MPP+-induced cell model of PD in SH-SY5Y cells and the underlying molecular mechanisms. Sulfuretin significantly decreased MPP+-induced apoptotic cell death, accompanied by a reduction in caspase 3 activity and polyADP-ribose polymerase (PARP cleavage. Furthermore, it attenuated MPP+-induced production of intracellular reactive oxygen species (ROS and disruption of mitochondrial membrane potential (MMP. Consistently, sulfuretin decreased p53 expression and the Bax/Bcl-2 ratio. Moreover, sulfuretin significantly increased the phosphorylation of Akt, GSK3β, and ERK. Pharmacological inhibitors of PI3K/Akt and ERK abolished the cytoprotective effects of sulfuretin against MPP+. An inhibitor of GSK3β mimicked sulfuretin-induced protection against MPP+. Taken together, these results suggest that sulfuretin significantly attenuates MPP+-induced neurotoxicity through Akt/GSK3β and ERK signaling pathways in SH-SY5Y cells. Our findings suggest that sulfuretin might be one of the potential candidates for the treatment of PD.

  15. Zinc ions regulate opening of tight junction favouring efflux of macromolecules via the GSK3β/snail-mediated pathway.

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    Xiao, Ruyue; Yuan, Lan; He, Weijiang; Yang, Xiaoda

    2018-01-24

    Zinc is an essential trace element presenting in particularly high concentration in the brain. In some regions, e.g. lateral amygdala, subiculum and hippocampus, rapidly-exchangeable zinc may transiently reach even up to 600 μM. To explore the possible roles of high-concentration Zn 2+ in regulating the blood-brain barrier (BBB), we investigated the effects of Zn 2+ on the functions and structures of the tight junction (TJ) with an in vitro model of a Madin-Darby canine kidney (MDCK) cell monolayer. The experimental results indicated that high concentrations (>200 μM) of Zn 2+ can affect the TJ integrity in a polarized manner. Basolateral addition of Zn 2+ led to reversible TJ opening with pore paths of r ∼ 2 nm or more depending on Zn 2+ concentration. The efflux/influx ratios of different sized probes were found to be ∼4.6 for FD4 (M W 4000) and ∼1.8 for Eu-DTPA (M W 560), suggesting that the Zn 2+ -induced paracelluar channels favour efflux especially for macromolecules. Further mechanistic studies revealed that the elevated intracellular Zn 2+ taken from the basolateral side can increase phosphorylation of glycogen synthase kinase (GSK) 3β, primarily due to the inhibition of calcineurin (CaN), thus resulting in the elevation of the snail transcriptional repressors. Subsequently, Zn 2+ can cause the down-regulation of claudin-1, breakage of occludin and ZO-1 rings, and collapse of basolateral F-actin structures. These overall factors result in the formation of a trumpet-like paracellular channel, which allows asymmetric solute permeation. The ERK1/2 and JNK1/2 pathways may also be involved in the Zn 2+ -induced TJ opening process, while the activation of matrix metalloproteinase was not observed. Our results may suggest a potential role of zinc in regulation of BBB permeability associated with brain clearance of metabolites through the glymphatic system.

  16. TAAR1 induces a disturbed GSK3β phosphorylation in recurrent miscarriages through the ODC.

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    Stavrou, Stavroula; Gratz, Michael; Tremmel, Eileen; Kuhn, Christina; Hofmann, Simone; Heidegger, Helene; Peryanova, Mina; Hermelink, Kerstin; Hutter, Stefan; Toth, Bettina; Mayr, Doris; Mahner, Sven; Jeschke, Udo; Vattai, Aurelia

    2018-02-01

    Thyroid hormones play an important role in the maintenance of pregnancy. Their derivates, endogenous amines, act via binding to the trace amine-associated receptor (TAAR1). The aim of our study was to analyse the regulation of TAAR1, serine/threonine kinase (pGSK3β) and ornithine decarboxylase (ODC) in placentas of healthy pregnancies, spontaneous (SM) and recurrent miscarriages (RM) and to investigate the influence of thyroid hormone derivates on TAAR1 expression in trophoblast model cells in vitro . Patients with SM ( n  = 15) and RM ( n  = 15) were compared with patients with healthy pregnancies ( n  = 15) (pregnancy weeks 7-13 each). Immunohistochemistry was applied to analyse placental TAAR1, pGSK3β and ODC expression. Protein expression of the receptors after stimulation with T 3 , T 1 AM and RO5203548 in BeWo trophoblast model cells was determined via Western blot. Double-immunofluorescence was used to determine placental expression of TAAR1 and ODC. Levels of TAAR1, pGSK3β and ODC were higher in placentas of RM in comparison to healthy controls. Stimulation of BeWo cells with T 3 , T 1 AM and RO5203548 significantly increased TAAR1 expression. ODC expression in BeWo cells was upregulated through T 3 . Via double-immunofluorescence, TAAR1 and ODC-positive EVT could be detected. Upregulation of placental TAAR1 may indicate an increased decarboxylation of thyroid hormones in miscarriages. Patients with RM may have a lack of T 3 through an enhanced transformation of T 3 into T 1 AM induced by the ODC. Future investigations could be carried out to analyse what role a prophylactic T 3 substitution plays for patients. © 2018 The authors.

  17. A GSK-3β Inhibitor Protects Against Radiation Necrosis in Mouse Brain

    International Nuclear Information System (INIS)

    Jiang, Xiaoyu; Perez-Torres, Carlos J.; Thotala, Dinesh; Engelbach, John A.; Yuan, Liya; Cates, Jeremy; Gao, Feng; Drzymala, Robert E.; Rich, Keith M.; Schmidt, Robert E.; Ackerman, Joseph J.H.; Hallahan, Dennis E.; Garbow, Joel R.

    2014-01-01

    Purpose: To quantify the effectiveness of SB415286, a specific inhibitor of GSK-3β, as a neuroprotectant against radiation-induced central nervous system (brain) necrosis in a mouse model. Methods and Materials: Cohorts of mice were treated with SB415286 or dimethyl sulfoxide (DMSO) prior to irradiation with a single 45-Gy fraction targeted to the left hemisphere (brain) using a gamma knife machine. The onset and progression of radiation necrosis (RN) were monitored longitudinally by noninvasive in vivo small-animal magnetic resonance imaging (MRI) beginning 13 weeks postirradiation. MRI-derived necrotic volumes for SB415286- and DMSO-treated mice were compared. MRI results were supported by correlative histology. Results: Mice treated with SB415286 showed significant protection from radiation-induced necrosis, as determined by in vivo MRI with histologic validation. MRI-derived necrotic volumes were significantly smaller at all postirradiation time points in SB415286-treated animals. Although the irradiated hemispheres of the DMSO-treated mice demonstrated many of the classic histologic features of RN, including fibrinoid vascular necrosis, vascular telangiectasia, hemorrhage, and tissue loss, the irradiated hemispheres of the SB415286-treated mice consistently showed only minimal tissue damage. These studies confirmed that treatment with a GSK-3β inhibitor dramatically reduced delayed time-to-onset necrosis in irradiated brain. Conclusions: The unilateral cerebral hemispheric stereotactic radiation surgery mouse model in concert with longitudinal MRI monitoring provided a powerful platform for studying the onset and progression of RN and for developing and testing new neuroprotectants. Effectiveness of SB415286 as a neuroprotectant against necrosis motivates potential clinical trials of it or other GSK-3β inhibitors

  18. Complex interactions between GSK3 and aPKC in Drosophila embryonic epithelial morphogenesis.

    Directory of Open Access Journals (Sweden)

    Nicole A Kaplan

    Full Text Available Generally, epithelial cells must organize in three dimensions to form functional tissue sheets. Here we investigate one such sheet, the Drosophila embryonic epidermis, and the morphogenetic processes organizing cells within it. We report that epidermal morphogenesis requires the proper distribution of the apical polarity determinant aPKC. Specifically, we find roles for the kinases GSK3 and aPKC in cellular alignment, asymmetric protein distribution, and adhesion during the development of this polarized tissue. Finally, we propose a model explaining how regulation of aPKC protein levels can reorganize both adhesion and the cytoskeleton.

  19. β-escin reverses multidrug resistance through inhibition of the GSK3β/β-catenin pathway in cholangiocarcinoma.

    Science.gov (United States)

    Huang, Gui-Li; Shen, Dong-Yan; Cai, Cheng-Fu; Zhang, Qiu-Yan; Ren, Hong-Yue; Chen, Qing-Xi

    2015-01-28

    To develop a safe and effective agent for cholangiocarcinoma (CCA) chemotherapy. A drug combination experiment was conducted to determine the effects of β-escin in combination with chemotherapy on CCA cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed to determine the effects of β-escin and common chemotherapeutics on the proliferation of human CCA cells (QBC939, Sk-ChA-1, and MZ-ChA-1). Immunocytochemistry was used to detect the expression of P-glycoprotein (P-gp) protein. Luciferase reporter assay was used to detect the activation of the Wnt/β-catenin pathway. The protein levels of P-gp, pS9-GSK3β, pT216-GSK3β, GSK3β, β-catenin, and p-β-catenin were further confirmed by western blotting. The drug sensitivity of QBC939 and QBC939/5-fluorouracil (5-FU) cells to 5-FU, vincristine sulfate (VCR), or mitomycin C was significantly enhanced by β-escin compared with either agent alone (Pescin (20 μmol/L) with 5-FU and VCR was synergic with a combination indexescin. Moreover, β-escin induced GSK3β phosphorylation at Tyr-216 and dephosphorylation at Ser-9, resulting in phosphorylation and degradation of β-catenin. Interestingly, activation of the GSK3β/β-catenin pathway induced by Wnt3a resulted in up-regulation of P-gp, which was effectively abolished by β-escin, indicating that β-escin down-regulated P-gp expression in a GSK3β-dependent manner. β-escin was a potent reverser of P-gp-dependent multidrug resistance, with said effect likely being achieved via inhibition of the GSK3β/β-catenin pathway and thus suggesting a promising strategy of developing combination drugs for CCA.

  20. Regulation of MDA-MB-231 cell proliferation by GSK-3β involves epigenetic modifications under high glucose conditions

    International Nuclear Information System (INIS)

    Gupta, Chanchal; Kaur, Jasmine; Tikoo, Kulbhushan

    2014-01-01

    Hyperglycemia is a critical risk factor for development and progression of breast cancer. We have recently reported that high glucose induces phosphorylation of histone H3 at Ser 10 as well as de-phosphorylation of GSK-3β at Ser 9 in MDA-MB-231 cells. Here, we elucidate the mechanism underlying hyperglycemia-induced proliferation in MDA-MB-231 breast cancer cells. We provide evidence that hyperglycemia led to increased DNA methylation and DNMT1 expression in MDA-MB-231 cells. High glucose condition led to significant increase in the expression of PCNA, cyclin D1 and decrease in the expression of PTPN 12, p21 and PTEN. It also induced hypermethylation of DNA at the promoter region of PTPN 12, whereas hypomethylation at Vimentin and Snail. Silencing of GSK-3β by siRNA prevented histone H3 phosphorylation and reduced DNMT1 expression. We show that chromatin obtained after immunoprecipitation with phospho-histone H3 was hypermethylated under high glucose condition, which indicates a cross-talk between DNA methylation and histone H3 phosphorylation. ChIP-qPCR analysis revealed up-regulation of DNMT1 and metastatic genes viz. Vimentin, Snail and MMP-7 by phospho-histone H3, which were down-regulated upon GSK-3β silencing. To the best of our knowledge, this is the first report which shows that interplay between GSK-3β activation, histone H3 phosphorylation and DNA methylation directs proliferation of breast cancer cells. - Highlights: • High glucose induces phosphorylation of histone H3 and dephosphorylation of GSK-3β. • Moreover, hyperglycemia also leads to increased DNA methylation in MDA-MB-231 cells. • Inhibition of GSK-3β prevented histone H3 phosphorylation and reduced DNMT1 levels. • Interplay exists between GSK-3β, histone H3 phosphorylation and DNA methylation

  1. Regulation of MDA-MB-231 cell proliferation by GSK-3β involves epigenetic modifications under high glucose conditions

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, Chanchal; Kaur, Jasmine; Tikoo, Kulbhushan, E-mail: tikoo.k@gmail.com

    2014-05-15

    Hyperglycemia is a critical risk factor for development and progression of breast cancer. We have recently reported that high glucose induces phosphorylation of histone H3 at Ser 10 as well as de-phosphorylation of GSK-3β at Ser 9 in MDA-MB-231 cells. Here, we elucidate the mechanism underlying hyperglycemia-induced proliferation in MDA-MB-231 breast cancer cells. We provide evidence that hyperglycemia led to increased DNA methylation and DNMT1 expression in MDA-MB-231 cells. High glucose condition led to significant increase in the expression of PCNA, cyclin D1 and decrease in the expression of PTPN 12, p21 and PTEN. It also induced hypermethylation of DNA at the promoter region of PTPN 12, whereas hypomethylation at Vimentin and Snail. Silencing of GSK-3β by siRNA prevented histone H3 phosphorylation and reduced DNMT1 expression. We show that chromatin obtained after immunoprecipitation with phospho-histone H3 was hypermethylated under high glucose condition, which indicates a cross-talk between DNA methylation and histone H3 phosphorylation. ChIP-qPCR analysis revealed up-regulation of DNMT1 and metastatic genes viz. Vimentin, Snail and MMP-7 by phospho-histone H3, which were down-regulated upon GSK-3β silencing. To the best of our knowledge, this is the first report which shows that interplay between GSK-3β activation, histone H3 phosphorylation and DNA methylation directs proliferation of breast cancer cells. - Highlights: • High glucose induces phosphorylation of histone H3 and dephosphorylation of GSK-3β. • Moreover, hyperglycemia also leads to increased DNA methylation in MDA-MB-231 cells. • Inhibition of GSK-3β prevented histone H3 phosphorylation and reduced DNMT1 levels. • Interplay exists between GSK-3β, histone H3 phosphorylation and DNA methylation.

  2. Small GSK-3 Inhibitor Shows Efficacy in a Motor Neuron Disease Murine Model Modulating Autophagy.

    Directory of Open Access Journals (Sweden)

    Estefanía de Munck

    Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive motor neuron degenerative disease that has no effective treatment up to date. Drug discovery tasks have been hampered due to the lack of knowledge in its molecular etiology together with the limited animal models for research. Recently, a motor neuron disease animal model has been developed using β-N-methylamino-L-alanine (L-BMAA, a neurotoxic amino acid related to the appearing of ALS. In the present work, the neuroprotective role of VP2.51, a small heterocyclic GSK-3 inhibitor, is analysed in this novel murine model together with the analysis of autophagy. VP2.51 daily administration for two weeks, starting the first day after L-BMAA treatment, leads to total recovery of neurological symptoms and prevents the activation of autophagic processes in rats. These results show that the L-BMAA murine model can be used to test the efficacy of new drugs. In addition, the results confirm the therapeutic potential of GSK-3 inhibitors, and specially VP2.51, for the disease-modifying future treatment of motor neuron disorders like ALS.

  3. Inhibition of GSK-3β activity can result in drug and hormonal resistance and alter sensitivity to targeted therapy in MCF-7 breast cancer cells

    Science.gov (United States)

    Sokolosky, Melissa; Chappell, William H; Stadelman, Kristin; Abrams, Stephen L; Davis, Nicole M; Steelman, Linda S; McCubrey, James A

    2014-01-01

    The PI3K/Akt/mTORC1 pathway plays prominent roles in malignant transformation, prevention of apoptosis, drug resistance, and metastasis. One molecule regulated by this pathway is GSK-3β. GSK-3β is phosphorylated by Akt on S9, which leads to its inactivation; however, GSK-3β also can regulate the activity of the PI3K/Akt/mTORC1 pathway by phosphorylating molecules such as PTEN, TSC2, p70S6K, and 4E-BP1. To further elucidate the roles of GSK-3β in chemotherapeutic drug and hormonal resistance of MCF-7 breast cancer cells, we transfected MCF-7 breast cancer cells with wild-type (WT), kinase-dead (KD), and constitutively activated (A9) forms of GSK-3β. MCF-7/GSK-3β(KD) cells were more resistant to doxorubicin and tamoxifen compared with either MCF-7/GSK-3β(WT) or MCF-7/GSK-3β(A9) cells. In the presence and absence of doxorubicin, the MCF-7/GSK-3β(KD) cells formed more colonies in soft agar compared with MCF-7/GSK-3β(WT) or MCF-7/GSK-3β(A9) cells. In contrast, MCF-7/GSK-3β(KD) cells displayed an elevated sensitivity to the mTORC1 blocker rapamycin compared with MCF-7/GSK-3β(WT) or MCF-7/GSK-3β(A9) cells, while no differences between the 3 cell types were observed upon treatment with a MEK inhibitor by itself. However, resistance to doxorubicin and tamoxifen were alleviated in MCF-7/GSK-3β(KD) cells upon co-treatment with an MEK inhibitor, indicating regulation of this resistance by the Raf/MEK/ERK pathway. Treatment of MCF-7 and MCF-7/GSK-3β(WT) cells with doxorubicin eliminated the detection of S9-phosphorylated GSK-3β, while total GSK-3β was still detected. In contrast, S9-phosphorylated GSK-3β was still detected in MCF-7/GSK-3β(KD) and MCF-7/GSK-3β(A9) cells, indicating that one of the effects of doxorubicin on MCF-7 cells was suppression of S9-phosphorylated GSK-3β, which could result in increased GSK-3β activity. Taken together, these results demonstrate that introduction of GSK-3β(KD) into MCF-7 breast cancer cells promotes resistance to

  4. Segregation of myoblast fusion and muscle-specific gene expression by distinct ligand-dependent inactivation of GSK-3β.

    Science.gov (United States)

    Pansters, N A M; van der Velden, J L J; Kelders, M C J M; Laeremans, H; Schols, A M W J; Langen, R C J

    2011-02-01

    Myogenic differentiation involves myoblast fusion and induction of muscle-specific gene expression, which are both stimulated by pharmacological (LiCl), genetic, or IGF-I-mediated GSK-3β inactivation. To assess whether stimulation of myogenic differentiation is common to ligand-mediated GSK-3β inactivation, myoblast fusion and muscle-specific gene expression were investigated in response to Wnt-3a. Moreover, crosstalk between IGF-I/GSK-3β/NFATc3 and Wnt/GSK-3β/β-catenin signaling was assessed. While both Wnt-3a and LiCl promoted myoblast fusion, muscle-specific gene expression was increased by LiCl, but not by Wnt-3a or β-catenin over-expression. Furthermore, LiCl and IGF-I, but not Wnt-3a, increased NFATc3 transcriptional activity. In contrast, β-catenin-dependent transcriptional activity was increased by Wnt-3a and LiCl, but not IGF-I. These results for the first time reveal a segregated regulation of myoblast fusion and muscle-specific gene expression following stimulation of myogenic differentiation in response to distinct ligand-specific signaling routes of GSK-3β inactivation.

  5. Research on the mechanism of HP mediated PI3K/AKT/GSK3β pathways in gastric cancer.

    Science.gov (United States)

    Geng, W; Zhang, H-Y

    2017-07-01

    This study aimed to investigate the mechanism of Helicobacter pylori (HP) mediated PI3K/AKT/GSK3β signal pathways in the occurrence of gastric cancer. For this purpose, a total of 90 patients were selected; 30 were suffering from gastric cancer and gastric ulcer (observation group I), 30 were suffering from pure gastric ulcer (observation group II) and 30 were with chronic gastritis (control group), and the lesion tissues were extracted by using endoscopy. Then, rapid urease test, RT-PCR and Western blot tests were conducted to detect the positivity rate of HP infection, the expression levels of PI3K/AKT/GSK3β mRNA and protein respectively. The results showed that the positivity rate of HP infection, expression level of the PI3K/AKT/GSK3β mRNA and protein from the samples of the observation group I were significantly higher than other two groups (p-value HP may mediate the PI3K/AKT/GSK3β signal pathways in the occurrence of gastric cancer.

  6. A combined study of GSK3β polymorphisms and brain network topological metrics in major depressive disorder.

    Science.gov (United States)

    Liu, Zhifen; Guo, Hao; Cao, Xiaohua; Cheng, Chen; Yang, Chunxia; Xu, Cheng; Zhang, Aixia; Sun, Ning; Li, Xinrong; Zhang, Kerang

    2014-09-30

    GSK3β genotypes may interact with major depressive disorder (MDD) and may have a role in determining regional gray matter volume differences from healthy comparison subjects. However, any associations of GSK3β genotypes with MDD related to abnormal functional brain activity have yet to be elucidated. In the present study, resting state functional brain networks were constructed by thresholding partial correlation matrices of 90 regions. Differences in the network features of GSK3β-rs6438552 genotypes were tested, and a 2×2 analysis of variance was performed to identify the main effects of genotypes, disease status, and their interactions in MDD. Compared with CC carriers, T+ carriers with MDD showed increased nodal centralities in many brain regions-mainly the limbic system, thalamus and parts of the parietal, temporal, occipital, and frontal regions. Decreased nodal centralities predominantly occurred in the sensorimotor area and parts of the frontal, occipital, and temporal lobes. Significant interactions between genotypes and disease status were found in the left thalamus, left superior occipital gyrus, and left inferior parietal lobe. Only altered nodal centrality in the left angular gyrus was negatively correlated with scores on the Hamilton Depression Rating Scale. Our results suggest the GSK3β genotypic effect of rs6438552 and its interaction with disease status may contribute to the altered topological organization of resting state functional brain networks in MDD patients. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  7. Expression analysis of E-cadherin, Slug and GSK3β in invasive ductal carcinoma of breast

    International Nuclear Information System (INIS)

    Prasad, Chandra P; Rath, Gayatri; Mathur, Sandeep; Bhatnagar, Dinesh; Parshad, Rajinder; Ralhan, Ranju

    2009-01-01

    Cancer progression is linked to a partially dedifferentiated epithelial cell phenotype. The signaling pathways Wnt, Hedgehog, TGF-β and Notch have been implicated in experimental and developmental epithelial mesenchymal transition (EMT). Recent findings from our laboratory confirm that active Wnt/β-catenin signaling is critically involved in invasive ductal carcinomas (IDCs) of breast. In the current study, we analyzed the expression patterns and relationships between the key Wnt/β-catenin signaling components- E-cadherin, Slug and GSK3β in IDCs of breast. Of the 98 IDCs analyzed, 53 (54%) showed loss/or reduced membranous staining of E-cadherin in tumor cells. Nuclear accumulation of Slug was observed in 33 (34%) IDCs examined. Loss or reduced level of cytoplasmic GSK3β expression was observed in 52/98 (53%) cases; while 34/98 (35%) tumors showed nuclear accumulation of GSK3β. Statistical analysis revealed associations of nuclear Slug expression with loss of membranous E-cadherin (p = 0.001); nuclear β-catenin (p = 0.001), and cytoplasmic β-catenin (p = 0.005), suggesting Slug mediated E-cadherin suppression via the activation of Wnt/β-catenin signaling pathway in IDCs. Our study also demonstrated significant correlation between GSK3β nuclear localization and tumor grade (p = 0.02), suggesting its association with tumor progression. The present study for the first time provided the clinical evidence in support of Wnt/β-catenin signaling upregulation in IDCs and key components of this pathway - E-cadherin, Slug and GSK3β with β-catenin in implementing EMT in these cells

  8. Evaluation and comparison of 3D-QSAR CoMSIA models for CDK1, CDK5, and GSK-3 inhibition by paullones

    DEFF Research Database (Denmark)

    Kunick, Conrad; Lauenroth, Kathrin; Wieking, Karen

    2004-01-01

    With a view to the rational design of selective GSK-3beta inhibitors, 3D-QSAR CoMSIA models were developed for the inhibition of the three serine/threonine kinases CDK1/cyclin B, CDK5/p25, and GSK-3beta by compounds from the paullone inhibitor family. The models are based on the kinase inhibition...

  9. Lithium protects against methamphetamine-induced neurotoxicity in PC12 cells via Akt/GSK3β/mTOR pathway

    International Nuclear Information System (INIS)

    Wu, Jintao; Zhu, Dexiao; Zhang, Jing; Li, Guibao; Liu, Zengxun; Sun, Jinhao

    2015-01-01

    Methamphetamine (MA) is neurotoxic, especially in dopaminergic neurons. Long-lasting exposure to MA causes psychosis and increases the risk of Parkinson's disease. Lithium (Li) is a known mood stabilizer and has neuroprotective effects. Previous studies suggest that MA exposure decreases the phosphorylation of Akt/GSK3β pathway in vivo, whereas Li facilitates the phosphorylation of Akt/GSK3β pathway. Moreover, GSK3β and mTOR are implicated in the locomotor sensitization induced by psychostimulants and mTOR plays a critical role in MA induced toxicity. However, the effect of MA on Akt/GSK3β/mTOR pathway has not been fully investigated in vitro. Here, we found that MA exposure significantly dephosphorylated Akt/GSK3β/mTOR pathway in PC12 cells. In addition, Li remarkably attenuated the dephosphorylation effect of MA exposure on Akt/GSK3β/mTOR pathway. Furthermore, Li showed obvious protective effects against MA toxicity and LY294002 (Akt inhibitor) suppressed the protective effects of Li. Together, MA exposure dephosphorylates Akt/GSK3β/mTOR pathway in vitro, while lithium protects against MA-induced neurotoxicity via phosphorylation of Akt/GSK3β/mTOR pathway. - Highlights: • Lithium protects against methamphetamine-induced neurotoxicity in vitro. • Methamphetamine exposure dephosphorylates Akt/GSK3β/mTOR pathway. • Lithium attenuates methamphetamine-induced toxicity via phosphorylating Akt/GSK3β/mTOR pathway

  10. Lithium protects against methamphetamine-induced neurotoxicity in PC12 cells via Akt/GSK3β/mTOR pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jintao; Zhu, Dexiao; Zhang, Jing; Li, Guibao [Department of Anatomy, School of Medicine, Shandong University, Jinan, Shandong, 250012 (China); Liu, Zengxun [Department of Psychiatry, School of Medicine, Shandong University, Jinan, Shandong, 250012 China (China); Sun, Jinhao, E-mail: sunjinhao@gmail.com [Department of Anatomy, School of Medicine, Shandong University, Jinan, Shandong, 250012 (China)

    2015-09-25

    Methamphetamine (MA) is neurotoxic, especially in dopaminergic neurons. Long-lasting exposure to MA causes psychosis and increases the risk of Parkinson's disease. Lithium (Li) is a known mood stabilizer and has neuroprotective effects. Previous studies suggest that MA exposure decreases the phosphorylation of Akt/GSK3β pathway in vivo, whereas Li facilitates the phosphorylation of Akt/GSK3β pathway. Moreover, GSK3β and mTOR are implicated in the locomotor sensitization induced by psychostimulants and mTOR plays a critical role in MA induced toxicity. However, the effect of MA on Akt/GSK3β/mTOR pathway has not been fully investigated in vitro. Here, we found that MA exposure significantly dephosphorylated Akt/GSK3β/mTOR pathway in PC12 cells. In addition, Li remarkably attenuated the dephosphorylation effect of MA exposure on Akt/GSK3β/mTOR pathway. Furthermore, Li showed obvious protective effects against MA toxicity and LY294002 (Akt inhibitor) suppressed the protective effects of Li. Together, MA exposure dephosphorylates Akt/GSK3β/mTOR pathway in vitro, while lithium protects against MA-induced neurotoxicity via phosphorylation of Akt/GSK3β/mTOR pathway. - Highlights: • Lithium protects against methamphetamine-induced neurotoxicity in vitro. • Methamphetamine exposure dephosphorylates Akt/GSK3β/mTOR pathway. • Lithium attenuates methamphetamine-induced toxicity via phosphorylating Akt/GSK3β/mTOR pathway.

  11. TORC1 Inhibits GSK3-Mediated Elo2 Phosphorylation to Regulate Very Long Chain Fatty Acid Synthesis and Autophagy

    DEFF Research Database (Denmark)

    Zimmermann, Christine; Santos, Aline; Gable, Kenneth

    2013-01-01

    Very long chain fatty acids (VLCFAs) are essential fatty acids with multiple functions, including ceramide synthesis. Although the components of the VLCFA biosynthetic machinery have been elucidated, how their activity is regulated to meet the cell's metabolic demand remains unknown. The goal...... of autophagy. Together, our data reveal a function for TORC1 and GSK3 in the regulation of VLCFA synthesis that has important implications for autophagy and cell homeostasis....... of this study was to identify mechanisms that regulate the rate of VLCFA synthesis, and we discovered that the fatty acid elongase Elo2 is regulated by phosphorylation. Elo2 phosphorylation is induced upon inhibition of TORC1 and requires GSK3. Expression of nonphosphorylatable Elo2 profoundly alters...

  12. GSK3β-dependent inhibition of AMPK potentiates activation of neutrophils and macrophages and enhances severity of acute lung injury

    Science.gov (United States)

    Park, Dae Won; Jiang, Shaoning; Liu, Yanping; Siegal, Gene P.; Inoki, Ken; Abraham, Edward

    2014-01-01

    Although AMP-activated protein kinase (AMPK) is involved in regulating carbohydrate and lipid metabolism, activated AMPK also plays an anti-inflammatory role in many cell populations. However, despite the ability of AMPK activation to diminish the severity of inflammatory responses, previous studies have found that AMPK activity is diminished in LPS-treated neutrophils and also in lungs of mice with LPS-induced acute lung injury (ALI). Since GSK3β participates in regulating AMPK activity, we examined potential roles for GSK3β in modulating LPS-induced activation of neutrophils and macrophages and in influencing severity of ALI. We found that GSK3β-dependent phosphorylation of T479-AMPK was associated with pT172 dephosphorylation and inactivation of AMPK following TLR4 engagement. GSK3β inhibitors BIO (6-bromoindirubin-3′-oxime), SB216763, or siRNA knockdown of GSK3β, but not the PI3K/AKT inhibitor LY294002, prevented Thr172-AMPK dephosphorylation. Exposure to LPS resulted in rapid binding between IKKβ and AMPKα, and phosphorylation of S485-AMPK by IKKβ. These results suggest that IKKβ-dependent phosphorylation of S485-AMPK was an essential step in subsequent phosphorylation and inactivation AMPK by GSK3β. Inhibition of GSK3β activity delayed IκBα degradation and diminished expression of the proinflammatory TNF-α in LPS-stimulated neutrophils and macrophages. In vivo, inhibition of GSK3β decreased the severity of LPS-induced lung injury as assessed by development of pulmonary edema, production of TNF-α and MIP-2, and release of the alarmins HMGB1 and histone 3 in the lungs. These results show that inhibition of AMPK by GSK3β plays an important contributory role in enhancing LPS-induced inflammatory responses, including worsening the severity of ALI. PMID:25239914

  13. Selective dicer suppression in the kidney alters GSK3β/β-catenin pathways promoting a glomerulocystic disease.

    Directory of Open Access Journals (Sweden)

    Anna Iervolino

    Full Text Available Dicer is a crucial enzyme for the maturation of miRNAs. Mutations in the Dicer gene are highly associated with Pleuro Pulmonary Blastoma-Family Dysplasia Syndrome (PPB-FDS, OMIM 601200, recently proposed to be renamed Dicer syndrome. Aside from the pulmonary phenotype (blastoma, renal nephroma and thyroid goiter are frequently part of Dicer syndrome. To investigate the renal phenotype, conditional knockout (cKO mice for Dicer in Pax8 expressing cells were generated. Dicer cKO mice progressively develop a glomerulocystic phenotype coupled with urinary concentration impairment, proteinuria and severe renal failure. Higher cellular turnover of the parietal cells of Bowman's capsule precedes the development of the cysts and the primary cilium progressively disappears with cyst-enlargement. Upregulation of GSK3β precedes the development of the glomerulocystic phenotype. Downregulation of β-catenin in the renal cortex and its cytosolic removal in the cells lining the cysts may be associated with observed accumulation of GSK3β. Alterations of β-catenin regulating pathways could promote cystic degeneration as in other models. Thus, miRNAs are fundamental in preserving renal morphology and function. Alteration of the GSK3β/β-catenin pathway could be a crucial mechanism linking miRNA dysregulation and the development of a glomerulocystic disease.

  14. PTEN silencing enhances neuronal proliferation and differentiation by activating PI3K/Akt/GSK3β pathway in vitro.

    Science.gov (United States)

    Song, Zhiwen; Han, Xiu; Shen, Liming; Zou, Hongjun; Zhang, Bin; Liu, Jinbo; Gong, Aihua

    2018-02-15

    The failure of neuronal proliferation and differentiation is a major obstacle for neural repair and regeneration after traumatic central nervous system (CNS) injury. PTEN acts as an intrinsic brake on the neuronal cells, but its roles and mechanism still remain to be clarified. Herein, for the first time we confirmed that PTEN had a dual effect on the neuronal cells in vitro. Firstly, we found that PTEN knockdown significantly promoted cell proliferation and differentiation. Then, PTEN knockdown activated PI3K/Akt and Wnt/β-catenin pathways in vitro. Further evidence revealed that GSK3β as a key node involved in PTEN controlling cell proliferation and differentiation in PC12 cells. In addition, we identified that PTEN-GSK3β pathway modulated neuronal proliferation via β-catenin. Taken together, these results suggest that PTEN silencing enhances neuronal proliferation and differentiation by activating PI3K/Akt/GSK3β pathway that it may be a promising therapeutic approach for CNS injury. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Roles of PI3K/AKT/GSK3/mTOR Pathway in Cell Signaling of Mental Illnesses

    Directory of Open Access Journals (Sweden)

    Yasuko Kitagishi

    2012-01-01

    Full Text Available Several pharmacological agents acting on monoamine neurotransmission are used for the management of mental illnesses. Regulation of PI3K/AKT and GSK3 pathways may constitute an important signaling center in the subcellular integration of the synaptic neurotransmission. The pathways also modulate neuronal cell proliferation, migration, and plasticity. There are evidences to suggest that inflammation of neuron contributes to the pathology of depression. Inflammatory activation of neuron contributes to the loss of glial elements, which are consistent with pathological findings characterizing the depression. A mechanism of anti-inflammatory reactions from antidepressant medications has been found to be associated with an enhancement of heme oxygenase-1 expression. This induction in brain is also important in neuroprotection and neuroplasticity. As enzymes involved in cell survival and neuroplasticity are relevant to neurotrophic factor dysregulation, the PI3K/AKT/GSK3 may provide an important signaling for the neuroprotection in depression. In this paper, we summarize advances on the involvement of the PI3K/AKT/GSK3 pathways in cell signaling of neuronal cells in mental illnesses.

  16. RAGE modulates hypoxia/reoxygenation injury in adult murine cardiomyocytes via JNK and GSK-3beta signaling pathways.

    Directory of Open Access Journals (Sweden)

    Linshan Shang

    2010-04-01

    Full Text Available Advanced glycation end-products (AGEs have been implicated in diverse pathological settings including diabetes, inflammation and acute ischemia/reperfusion injury in the heart. AGEs interact with the receptor for AGEs (RAGE and transduce signals through activation of MAPKs and proapoptotic pathways. In the current study, adult cardiomyocytes were studied in an in vitro ischemia/reperfusion (I/R injury model to delineate the molecular mechanisms underlying RAGE-mediated injury due to hypoxia/reoxygenation (H/R.Cardiomyocytes isolated from adult wild-type (WT, homozygous RAGE-null (RKO, and WT mice treated with soluble RAGE (sRAGE were subjected to hypoxia for 30 minutes alone or followed by reoxygenation for 1 hour. In specific experiments, RAGE ligand carboxymethyllysine (CML-AGE (termed "CML" in this manuscript was evaluated in vitro. LDH, a marker of cellular injury, was assayed in the supernatant in the presence or absence of signaling inhibitor-treated cardiomyocytes. Cardiomyocyte levels of heterogeneous AGEs were measured using ELISA. A pronounced increase in RAGE expression along with AGEs was observed in H/R vs. normoxia in WT cardiomyocytes. WT cardiomyocytes after H/R displayed increased LDH release compared to RKO or sRAGE-treated cardiomyocytes. Our results revealed significant increases in phospho-JNK in WT cardiomyocytes after H/R. In contrast, neither RKO nor sRAGE-treated cardiomyocytes exhibited increased phosphorylation of JNK after H/R stress. The impact of RAGE deletion on GSK-3beta phosphorylation in the cardiomyocytes subjected to H/R revealed significantly higher levels of phospho-GSK-3beta/total GSK-3beta in RKO, as well as in sRAGE-treated cardiomyocytes versus WT cardiomyocytes after H/R. Further investigation established a key role for Akt, which functions upstream of GSK-3beta, in modulating H/R injury in adult cardiomyocytes.These data illustrate key roles for RAGE-ligand interaction in the pathogenesis of

  17. GSKIP- and GSK3-mediated anchoring strengthens cAMP/PKA/Drp1 axis signaling in the regulation of mitochondrial elongation.

    Science.gov (United States)

    Loh, Joon-Khim; Lin, Ching-Chih; Yang, Ming-Chang; Chou, Chia-Hua; Chen, Wan-Shia; Hong, Ming-Chang; Cho, Chung-Lung; Hsu, Ching-Mei; Cheng, Jiin-Tsuey; Chou, An-Kuo; Chang, Chung-Hsing; Tseng, Chao-Neng; Wang, Chi-Huei; Lieu, Ann-Shung; Howng, Shen-Long; Hong, Yi-Ren

    2015-08-01

    GSK3β binding of GSKIP affects neurite outgrowth, but the physiological significance of PKA binding to GSKIP remains to be determined. We hypothesized that GSKIP and GSK3β mediate cAMP/PKA/Drp1 axis signaling and modulate mitochondrial morphology by forming a working complex comprising PKA/GSKIP/GSK3β/Drp1. We demonstrated that GSKIP wild-type overexpression increased phosphorylation of Drp1 S637 by 7-8-fold compared to PKA kinase-inactive mutants (V41/L45) and a GSK3β binding-defective mutant (L130) under H2O2 and forskolin challenge in HEK293 cells, indicating that not only V41/L45, but also L130 may be involved in Drp1-associated protection of GSKIP. Interestingly, silencing either GSKIP or GSK3β but not GSK3α resulted in a dramatic decrease in Drp1 S637 phosphorylation, revealing that both GSKIP and GSK3β are required in this novel PKA/GSKIP/GSK3β/Drp1 complex. Moreover, overexpressed kinase-dead GSK3β-K85R, which retains the capacity to bind GSKIP, but not K85M which shows total loss of GSKIP-binding, has a higher Drp1 S637 phosphorylation similar to the GSKIP wt overexpression group, indicating that GSK3β recruits Drp1 by anchoring rather than in a kinase role. With further overexpression of either V41/L45P or the L130P GSKIP mutant, the elongated mitochondrial phenotype was lost; however, ectopically expressed Drp1 S637D, a phosphomimetic mutant, but not S637A, a non-phosphorylated mutant, restored the elongated mitochondrial morphology, indicating that Drp1 is a downstream effector of direct PKA signaling and possibly has an indirect GSKIP function involved in the cAMP/PKA/Drp1 signaling axis. Collectively, our data revealed that both GSKIP and GSK3β function as anchoring proteins in the cAMP/PKA/Drp1 signaling axis modulating Drp1 phosphorylation. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Differential modulatory effects of GSK-3β and HDM2 on sorafenib-induced AIF nuclear translocation (programmed necrosis in melanoma

    Directory of Open Access Journals (Sweden)

    Mier James W

    2011-09-01

    Full Text Available Abstract Background GSK-3β phosphorylates numerous substrates that govern cell survival. It phosphorylates p53, for example, and induces its nuclear export, HDM2-dependent ubiquitination, and proteasomal degradation. GSK-3β can either enhance or inhibit programmed cell death, depending on the nature of the pro-apoptotic stimulus. We previously showed that the multikinase inhibitor sorafenib activated GSK-3β and that this activation attenuated the cytotoxic effects of the drug in various BRAF-mutant melanoma cell lines. In this report, we describe the results of studies exploring the effects of GSK-3β on the cytotoxicity and antitumor activity of sorafenib combined with the HDM2 antagonist MI-319. Results MI-319 alone increased p53 levels and p53-dependent gene expression in melanoma cells but did not induce programmed cell death. Its cytotoxicity, however, was augmented in some melanoma cell lines by the addition of sorafenib. In responsive cell lines, the MI-319/sorafenib combination induced the disappearance of p53 from the nucleus, the down modulation of Bcl-2 and Bcl-xL, the translocation of p53 to the mitochondria and that of AIF to the nuclei. These events were all GSK-3β-dependent in that they were blocked with a GSK-3β shRNA and facilitated in otherwise unresponsive melanoma cell lines by the introduction of a constitutively active form of the kinase (GSK-3β-S9A. These modulatory effects of GSK-3β on the activities of the sorafenib/MI-319 combination were the exact reverse of its effects on the activities of sorafenib alone, which induced the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF only in cells in which GSK-3β activity was either down modulated or constitutively low. In A375 xenografts, the antitumor effects of sorafenib and MI-319 were additive and associated with the down modulation of Bcl-2 and Bcl-xL, the nuclear translocation of AIF, and increased suppression of tumor angiogenesis

  19. Molecular cloning of a novel GSK3/shaggy-like gene from Triticum ...

    African Journals Online (AJOL)

    The deduced amino acid sequence showed a high homology with shaggy-like kinases from Triticum aestivum, Zea mays, Trifolium repens, Nicotine tabacum, Medicago sativa and Arabidopsis thaliana; therefore, the gene was named TmGSK1 (Triticum monococcum Glycogen Synthase Kinase 1,GenBank Accession No.

  20. The Role of Insulin/IGF-1/PI3K/Akt/GSK3β Signaling in Parkinson's Disease Dementia

    Directory of Open Access Journals (Sweden)

    Liying Yang

    2018-02-01

    Full Text Available Dementia, a condition that frequently afflicts patients in advanced stages of Parkinson's disease (PD, results in decreased quality of life and survival time. Nevertheless, the pathological mechanisms underlying Parkinson's disease dementia (PDD are not completely understood. The symptoms characteristic of PDD may be the result of functional and structural deficiencies. The present study implicates the accumulation of Lewy bodies in the cortex and limbic system as a potent trigger in the development of PDD. In addition, significant Alzheimer-type pathologies, including amyloid-β (Aβ plaques and NFTs, are observed in almost half of PDD patients. Interestingly, links between PDD pathogenesis and the mechanisms underlying the development of insulin resistance have begun to emerge. Furthermore, previous studies have demonstrated that insulin treatment reduces amyloid plaques in Alzheimer's disease (AD, and normalizes the production and functionality of dopamine and ameliorates motor impairments in 6-OHDA-induced rat PD models. GSK3β, a downstream substrate of PI3K/Akt signaling following induction by insulin and IGF-1, exerts an influence on AD and PD physiopathology. The genetic overexpression of GSK3β in cortex and hippocampus results in signs of neurodegeneration and spatial learning deficits in in vivo models (Lucas et al., 2001, whereas its inhibition results in improvements in cognitive impairment in these rodents, including AD and PD. Accordingly, insulin- or IGF-1-activated PI3K/Akt/GSK3β signaling may be involved in PDD pathogenesis, at least in the pathology of PD-type + AD-type.

  1. Cerebrolysin protects PC12 cells from CoCl2-induced hypoxia employing GSK3β signaling.

    Science.gov (United States)

    Hartwig, Kerstin; Fackler, Viktoria; Jaksch-Bogensperger, Heidi; Winter, Stefan; Furtner, Tanja; Couillard-Despres, Sebastien; Meier, Dieter; Moessler, Herbert; Aigner, Ludwig

    2014-11-01

    Cerebrolysin (EVER Neuro Pharma GmbH, Austria) is a peptidergic drug indicated for clinical use in stroke, traumatic brain injury and dementia. The therapeutic effect of Cerebrolysin is thought to ensure from its neurotrophic activity, which shares some properties with naturally occurring neurotrophic factors. However, the exact mechanism of action of Cerebrolysin is yet to be fully deciphered. This study aimed to investigate the neuroprotective effect of Cerebrolysin in a widely used in vitro model of hypoxia-induced neuronal cytotoxicity, namely cobalt chloride (CoCl2)-treatment of PC12 cells. CoCl2-cytotoxicity was indicated by a reduced cell-diameter, cell shrinkage, increased pro-apoptotic Caspase-activities and a decreased metabolic activity. Cerebrolysin maintained the cell-diameter of CoCl2-treated naïve PC12 cells, decreased the activation of Caspase 3/7 in CoCl2-stressed naïve PC12 cells and restored the cells' metabolic activity in CoCl2-impaired naïve and differentiated PC12 cells. Cerebrolysin treatment also decreased the levels of superoxide observed after exposure to CoCl2. Investigating the mechanism of action, we could demonstrate that Cerebrolysin application to CoCl2-stressed PC12 cells increased the phosphorylation of GSK3β, resulting in the inhibition of GSK3β. This might become clinically relevant for Alzheimer's disease, since GSK3β activity has been linked to the production of amyloid beta. Taken together, Cerebrolysin was found to have neuroprotective effects in CoCl2-induced cytotoxicity in PC12 cells. Copyright © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

  2. ARTD10 substrate identification on protein microarrays: regulation of GSK3β by mono-ADP-ribosylation.

    Science.gov (United States)

    Feijs, Karla Lh; Kleine, Henning; Braczynski, Anne; Forst, Alexandra H; Herzog, Nicolas; Verheugd, Patricia; Linzen, Ulrike; Kremmer, Elisabeth; Lüscher, Bernhard

    2013-01-19

    Although ADP-ribosylation has been described five decades ago, only recently a distinction has been made between eukaryotic intracellular poly- and mono-ADP-ribosylating enzymes. Poly-ADP-ribosylation by ARTD1 (formerly PARP1) is best known for its role in DNA damage repair. Other polymer forming enzymes are ARTD2 (formerly PARP2), ARTD3 (formerly PARP3) and ARTD5/6 (formerly Tankyrase 1/2), the latter being involved in Wnt signaling and regulation of 3BP2. Thus several different functions of poly-ADP-ribosylation have been well described whereas intracellular mono-ADP-ribosylation is currently largely undefined. It is for example not known which proteins function as substrate for the different mono-ARTDs. This is partially due to lack of suitable reagents to study mono-ADP-ribosylation, which limits the current understanding of this post-translational modification. We have optimized a novel screening method employing protein microarrays, ProtoArrays®, applied here for the identification of substrates of ARTD10 (formerly PARP10) and ARTD8 (formerly PARP14). The results of this substrate screen were validated using in vitro ADP-ribosylation assays with recombinant proteins. Further analysis of the novel ARTD10 substrate GSK3β revealed mono-ADP-ribosylation as a regulatory mechanism of kinase activity by non-competitive inhibition in vitro. Additionally, manipulation of the ARTD10 levels in cells accordingly influenced GSK3β activity. Together these data provide the first evidence for a role of endogenous mono-ADP-ribosylation in intracellular signaling. Our findings indicate that substrates of ADP-ribosyltransferases can be identified using protein microarrays. The discovered substrates of ARTD10 and ARTD8 provide the first sets of proteins that are modified by mono-ADP-ribosyltransferases in vitro. By studying one of the ARTD10 substrates more closely, the kinase GSK3β, we identified mono-ADP-ribosylation as a negative regulator of kinase activity.

  3. STK17B promotes carcinogenesis and metastasis via AKT/GSK-3β/Snail signaling in hepatocellular carcinoma.

    Science.gov (United States)

    Lan, Yaliang; Han, Jihua; Wang, Yan; Wang, Jiabei; Yang, Guangchao; Li, Keyu; Song, Ruipeng; Zheng, Tongsen; Liang, Yingjian; Pan, Shangha; Liu, Xirui; Zhu, Mingxi; Liu, Yao; Meng, Fanzheng; Mohsin, Manzoor; Cui, Yifeng; Zhang, Bo; Subash, Sharma; Liu, Lianxin

    2018-02-14

    Hepatocellular carcinoma (HCC) is a lethal malignancy worldwide with frequent intrahepatic and distant metastasis. Elucidating the underlying molecular mechanism that modulates HCC progression is critical for exploring novel therapeutic strategies. Serine/Threonine Kinase 17B (STK17B) is upregulated in HCC tissues, but its role in HCC progression remains elusive. In the present studies, we reported that STK17B had a critical role in HCC progression. STK17B was significantly upregulated in HCC cell lines and specimens, and patients with ectopic STK17B expression characterized with poor clinicopathological features. In vitro and in vivo assay demonstrated that inhibition of STK17B markedly inhibits HCC tumorigenesis and metastasis, while STK17B overexpression promoted these processes. Furthermore, we found that STK17B promoted EMT process via activating AKT/GSK-3β/Snail signal pathway, and miR-455-3p was identified as the upstream regulator of STK17B. Combination of high level of STK17B and low level of miR-455-3p predicted poor prognosis with higher accuracy for HCC patients. In conclusion, our research demonstrated that STK17B promotes HCC progression, induces EMT process via activating AKT/GSK-3β/Snail signal and predicts poor prognosis in HCC.

  4. TDCPP protects cardiomyocytes from hypoxia-reoxygenation injury induced apoptosis through mitigating calcium overload and promotion GSK-3β phosphorylation.

    Science.gov (United States)

    He, Xiju; Li, Shoutian; Fang, Xiaoxia; Liao, Yanhong

    2018-02-01

    TDCPP, Tris (1, 3-dichloro-2-propyl) phosphate belongs to a group of chemicals known as triester organophosphate flame retardants, It can alter calcium homeostasis at much lower concentrations in normal conditions, but the mechanism is unclear till now. Calcium overload is a leading cause of apoptosis in myocardial ischemia/reperfusion (I/R) injury, thus how to mitigate Ca 2+ -overload is deserved to be investigated. We therefore hypothesized that TDCPP could attenuate cardiomyocytes apoptosis in I/R injury. H/R (hypoxia/reoxygenation) experiments in vitro were used to simulate in vivo I/R injury. The present study aimed to explore the potential effect of TDCPP in cardiomyocytes after H/R injury, Ca 2+ imaging technique was used to explore SOCE(store-operated calcium entry) and Ca 2+ -overload levels, western blot technique was used to explore the potential target, the cell morphology, cell viability and mitochondrial membrane potential were also detected. The results have shown that: TDCPP could decrease SOCE, restore H9c2 cell viability, mitigate Ca 2+ -overload in H/R injury and reduce the mitochondrial membrane potential. Furthermore, TDCPP decreased STIM1 expression and promoted GSK3β phosphorylation. Collectively, for the first time, this study suggest the antiapoptosis roles of TDCPP in H/R injury are via mitigation Ca 2+ -overload and promoting GSK-3β phosphorylation. Copyright © 2017. Published by Elsevier Inc.

  5. Emodin Protects against Diabetic Cardiomyopathy by Regulating the AKT/GSK-3β Signaling Pathway in the Rat Model

    Directory of Open Access Journals (Sweden)

    Zhiqin Wu

    2014-09-01

    Full Text Available Diabetes mellitus (DM has been recognized as a major health problem. Emodin (Emo has been reported to exhibit protective effects against diabetic nephropathy. However, little has been known about the effect of Emo on diabetic cardiomyopathy (DCM. A type 2 DM model was induced in rats by low dose streptozotocin (STZ combined with high energy intake. We found that Emo-treated groups displayed significantly higher body weight (BW and lower heart weight (HW/BW. Furthermore, Emo could significantly decrease blood glucose, total cholesterol (TG levels, and triglyceride (TC levels in diabetic rats. Moreover, the Emo-treated group showed a marked increase in heart rate (HR and showed lower left ventricular end-diastolic diameter (LVEDD, left ventricular end-systolic diameter (LVESD, left ventricular posterior wall thickness (LWPWT, and interventricular septal diastolic wall thickness (IVSD. Emo induced a significant increase in phosphorylation of Akt and GSK-3β in myocardium. These results suggest that Emo may have great therapeutic potential in the treatment of DCM by Akt/GSK-3β signaling pathway.

  6. NONO ubiquitination is mediated by FBW7 and GSK3 β via a degron lost upon chromosomal rearrangement in cancer.

    Science.gov (United States)

    Alfano, Luigi; Caporaso, Antonella; Altieri, Angela; Costa, Caterina; Forte, Iris M; Iannuzzi, Carmelina A; Barone, Daniela; Esposito, Luca; Giordano, Antonio; Pentimalli, Francesca

    2018-05-01

    NONO is an RNA-binding protein involved in transcription, mRNA splicing, DNA repair, and checkpoint activation in response to UV radiation. NONO expression has been found altered in several tumor types, including prostate, colon, breast, melanoma, and in papillary renal carcinoma, in which an X chromosome inversion generates a NONO-TFE3 fusion protein. Upon such rearrangement, NONO loses its C-terminal domain. Through bioinformatics analysis, we identified a putative degron motif, known to be recognized by the Skp1-Cul1-F-box-protein (SCF) complex. Here, we evaluated how this domain could affect NONO protein biology. We showed that NONO interacts with the nuclear FBW7α isoform and its ubiquitination is regulated following modulation of the GSK3β kinase. Mutation of T428A/T432A within the degron impaired polyubiquitination upon FBW7α and GSK3β overexpression. Overall, our data suggest that NONO is likely subjected to proteasome-mediated degradation and add NONO to the list of proteins targeted by FBW7, which is itself often deregulated in cancer. © 2017 Wiley Periodicals, Inc.

  7. New organometallic imines of rhenium(i) as potential ligands of GSK-3β: synthesis, characterization and biological studies.

    Science.gov (United States)

    Muñoz-Osses, Michelle; Godoy, Fernando; Fierro, Angélica; Gómez, Alejandra; Metzler-Nolte, Nils

    2018-01-23

    Substituted amino-piperazine derivatives were synthesized and used as precursors for the preparation of a series of new organometallic Re(i) imine complexes with the general formula [(η 5 -C 5 H 4 CH[double bond, length as m-dash]N-(CH 2 ) 5 -Pz-R)Re(CO) 3 ] (Pz-R: -alkyl or aryl piperazine). The piperazine-based ligands were designed to be potential inhibitors of GSK-3β kinase. All the ligands and complexes were fully characterized and evaluated against the HT-29 and PT-45 cancer cell lines, in which GSK-3β plays a crucial role. In this context, we carried out biological evaluation using the MTT colorimetric assay. In terms of structure activity relationship, our findings indicated improved biological activity when aromaticity increased in the organic ligands (3d). In addition, the presence of the rhenium fragment in the imines (5a-d) leads to better activity with IC 50 values in the range of 25-100 μM. In addition, our experimental studies were complemented by computational studies, where the volume and electrostatic surface of the organic ligands and organometallic compounds as well as their binding to the kinase protein are calculated.

  8. Arctigenin Attenuates Learning and Memory Deficits through PI3k/Akt/GSK-3β Pathway Reducing Tau Hyperphosphorylation in Aβ-Induced AD Mice.

    Science.gov (United States)

    Qi, Yue; Dou, De-Qiang; Jiang, Hong; Zhang, Bing-Bing; Qin, Wen-Yan; Kang, Kai; Zhang, Na; Jia, Dong

    2017-01-01

    Arctigenin is a phenylpropanoid dibenzylbutyrolactone lignan compound possessing antitumor, anti-inflammatory, anti-influenza, antioxidant, antibacterial, and hypoglycaemic activities. Our previous study demonstrated that arctigenin exerts neuroprotective effects both in vitro and in vivo in a Parkinson's disease model. However, the exact mechanism through which arctigenin improves amyloid beta-induced memory impairment by inhibiting the production of the hyperphosphorylated tau protein is unknown. Amyloid β 1-42 was slowly administered via the intracerebroventricular route in a volume of 3 µL (≈ 410 pmmol/mouse) to mice. The mice were administered arctigenin (10, 40, or 150 mg/kg) or vehicle starting from the second day after amyloid β 1-42 injection to the end of the experiment. Behavioural tests were performed from days 9 to 15. On day 16 after the intracerebroventricular administration of amyloid β 1-42 , the mice were sacrificed for biochemical analysis. Arctigenin (10-150 mg/kg) significantly attenuated the impairment of spontaneous alternation behaviours in the Y-maze task, decreased the escape latency in the Morris water maze test, and increased the swimming times and swimming distances to the platform located in the probe test. Arctigenin attenuated the level of phosphorylated tau at the Thr-181, Thr-231, and Ser-404 sites in the hippocampus, and increased the phosphorylation levels of phosphatidylinositol-3-kinase, threonine/serine protein kinase B, and glycogen synthase kinase-3 β . Arctigenin effectively provides protection against learning and memory deficits and in inhibits hyperphosphorylated tau protein expression in the hippocampus. The possible mechanism may occur via the phosphatidylinositol-3-kinase/protein kinase B-dependent glycogen synthase kinase-3 β signalling pathway. Georg Thieme Verlag KG Stuttgart · New York.

  9. Prenatal alcohol exposure alters p35, CDK5 and GSK3β in the medial frontal cortex and hippocampus of adolescent mice

    Directory of Open Access Journals (Sweden)

    Samantha L. Goggin

    2014-01-01

    Full Text Available Fetal alcohol spectrum disorders (FASDs are the number one cause of preventable mental retardation. An estimated 2–5% of children are diagnosed as having a FASD. While it is known that children prenatally exposed to alcohol experience cognitive deficits and a higher incidence of psychiatric illness later in life, the pathways underlying these abnormalities remain uncertain. GSK3β and CDK5 are protein kinases that are converging points for a vast number of signaling cascades, including those controlling cellular processes critical to learning and memory. We investigated whether levels of GSK3β and CDK5 are affected by moderate prenatal alcohol exposure (PAE, specifically in the hippocampus and medial frontal cortex of the adolescent mouse. In the present work we utilized immunoblotting techniques to demonstrate that moderate PAE increased hippocampal p35 and β-catenin, and decreased total levels of GSK3β, while increasing GSK3β Ser9 and Tyr216 phosphorylation. Interestingly, different alterations were seen in the medial frontal cortex where p35 and CDK5 were decreased and increased total GSK3β was accompanied by reduced Tyr216 of the enzyme. These results suggest that kinase dysregulation during adolescence might be an important contributing factor to the effects of PAE on hippocampal and medial frontal cortical functioning; and by extension, that global modulation of these kinases may produce differing effects depending on brain region.

  10. Bi-phasic regulation of glycogen content in astrocytes via Cav-1/PTEN/PI3K/AKT/GSK-3β pathway by fluoxetine.

    Science.gov (United States)

    Bai, Qiufang; Song, Dan; Gu, Li; Verkhratsky, Alexei; Peng, Liang

    2017-04-01

    Here, we present the data indicating that chronic treatment with fluoxetine regulates Cav-1/PTEN/PI3K/AKT/GSK-3β signalling pathway and glycogen content in primary cultures of astrocytes with bi-phasic concentration dependence. At lower concentrations, fluoxetine downregulates gene expression of Cav-1, decreases membrane content of PTEN, increases activity of PI3K/AKT, and elevates GSK-3β phosphorylation thus suppressing its activity. At higher concentrations, fluoxetine acts in an inverse fashion. As expected, fluoxetine at lower concentrations increased while at higher concentrations decreased glycogen content in astrocytes. Our findings indicate that bi-phasic regulation of glycogen content via Cav-1/PTEN/PI3K/AKT/GSK-3β pathway by fluoxetine may be responsible for both therapeutic and side effects of the drug.

  11. miR-19 targeting of GSK3β mediates sulforaphane suppression of lung cancer stem cells.

    Science.gov (United States)

    Zhu, Jianyun; Wang, Shijia; Chen, Yue; Li, Xiaoting; Jiang, Ye; Yang, Xue; Li, Yuan; Wang, Xiaoqian; Meng, Yu; Zhu, Mingming; Ma, Xiao; Huang, Cong; Wu, Rui; Xie, Chunfeng; Geng, Shanshan; Wu, Jieshu; Zhong, Caiyun; Han, Hongyu

    2017-06-01

    Cancer stem cells (CSCs) play a central role in the development of cancer. The canonical Wnt/β-catenin pathway is critical for maintaining stemness of CSCs. Phytochemicals from dietary compounds possess anti-CSCs properties and have been characterized as promising therapeutic agents for the prevention and treatment of many cancers. To date, the involvement and function of miR-19, a key oncogenic miRNA, in regulating Wnt/β-catenin pathway and lung CSCs has not been defined. Meanwhile, the effect of sulforaphane (SFN) on lung CSCs also remains to be elucidated. Here, we reported that lung CSCs up-regulated miR-19a and miR-19b expression. Overexpression of miR-19a/19b enhanced the ability of tumorsphere formation, up-regulated the expression of lung CSCs markers, increased Wnt/β-catenin pathway activation and β-catenin/TCF transcriptional activity in lung CSCs. In contrary, down-regulation of miR-19 suppressed lung CSCs activity and Wnt/β-catenin activation. We further revealed that miR-19 activated Wnt/β-catenin pathway by directly targeting GSK3β, the key negative modulator of this pathway. Moreover, we showed that SFN exhibited inhibitory effect on lung CSCs through suppressing miR-19 and Wnt/β-catenin pathway. Taken together, these data illustrate the role of miR-19 in regulating lung CSCs traits and miR-19/GSK3β/β-catenin axis in SFN intervention of lung CSCs. Findings from this study could provide important new insights into the molecular mechanisms of lung CSCs regulation as well as its target intervention. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Novel Therapeutic Strategy for the Prevention of Bone Fractures

    Science.gov (United States)

    2014-08-01

    treated mice using behavioral outcome measures in Small Animal Behavior Core (SABC) at Georgia Regents University. Rotarod tests, measures of free ...GDF-8 growth and differentiation factor-8, or myostatin GDF-11 growth and differentiation factor-11 Gsk3a gene encoding glycogen synthase...phosphorylation of glycogen synthase kinase-3 (GSK-3) in response to lithium. Evidence for autoregulation of GSK-3. J. Biol. Chem. 278:33067-77. Zhong,N. and R

  13. Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

    International Nuclear Information System (INIS)

    Varma, Shailly; Shrivastav, Anuraag; Changela, Sheena; Khandelwal, Ramji L.

    2008-01-01

    Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3β (GSK-3β) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-α (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity

  14. Escitalopram attenuates β-amyloid-induced tau hyperphosphorylation in primary hippocampal neurons through the 5-HT1A receptor mediated Akt/GSK-3β pathway.

    Science.gov (United States)

    Wang, Yan-Juan; Ren, Qing-Guo; Gong, Wei-Gang; Wu, Di; Tang, Xiang; Li, Xiao-Li; Wu, Fang-Fang; Bai, Feng; Xu, Lin; Zhang, Zhi-Jun

    2016-03-22

    Tau hyperphosphorylation is an important pathological feature of Alzheimer's disease (AD). To investigate whether escitalopram could inhibit amyloid-β (Aβ)-induced tau hyperphosphorylation and the underlying mechanisms, we treated the rat primary hippocampal neurons with Aβ1-42 and examined the effect of escitalopram on tau hyperphosphorylation. Results showed that escitalopram decreased Aβ1-42-induced tau hyperphosphorylation. In addition, escitalopram activated the Akt/GSK-3β pathway, and the PI3K inhibitor LY294002 blocked the attenuation of tau hyperphosphorylation induced by escitalopram. Moreover, the 5-HT1A receptor agonist 8-OH-DPAT also activated the Akt/GSK-3β pathway and decreased Aβ1-42-induced tau hyperphosphorylation. Furthermore, the 5-HT1A receptor antagonist WAY-100635 blocked the activation of Akt/GSK-3β pathway and the attenuation of tau hyperphosphorylation induced by escitalopram. Finally, escitalopram improved Aβ1-42 induced impairment of neurite outgrowth and spine density, and reversed Aβ1-42 induced reduction of synaptic proteins. Our results demonstrated that escitalopram attenuated Aβ1-42-induced tau hyperphosphorylation in primary hippocampal neurons through the 5-HT1A receptor mediated Akt/GSK-3β pathway.

  15. The JNK- and AKT/GSK3β- Signaling Pathways Converge to Regulate Puma Induction and Neuronal Apoptosis Induced by Trophic Factor Deprivation

    Science.gov (United States)

    Karajgikar, Meera; Hamilton, Alison; Ferguson, Stephen S.; Cregan, Sean P.

    2012-01-01

    The AKT, GSK3 and JNK family kinases have been implicated in neuronal apoptosis associated with neuronal development and several neurodegenerative conditions. However, the mechanisms by which these kinase pathways regulate apoptosis remain unclear. In this study we have investigated the role of these kinases in neuronal cell death using an established model of trophic factor deprivation induced apoptosis in cerebellar granule neurons. BCL-2 family proteins are known to be central regulators of apoptosis and we have determined that the pro-apoptotic family member Puma is transcriptionally up-regulated in trophic factor deprived neurons and that Puma induction is required for apoptosis in vitro and in vivo. Importantly, we demonstrate that Puma induction is dependent on both JNK activation and AKT inactivation. AKT is known to regulate a number of downstream pathways, however we have determined that PI3K-AKT inactivation induces Puma expression through a GSK3β-dependent mechanism. Finally we demonstrate that the JNK and AKT/GSK3β pathways converge to regulate FoxO3a-mediated transcriptional activation of Puma. In summary we have identified a novel and critical link between the AKT, GSK3β and JNK kinases and the regulation of Puma induction and suggest that this may be pivotal to the regulation of neuronal apoptosis in neurodegenerative conditions. PMID:23056511

  16. Sulforaphane prevents angiotensin II-induced cardiomyopathy by activation of Nrf2 via stimulating the Akt/GSK-3ß/Fyn pathway

    Directory of Open Access Journals (Sweden)

    Ying Xin

    2018-05-01

    Conclusion: These results suggest that Nrf2 plays a central role in the prevention of Ang II-induced cardiomyopathy, and SFN prevents Ang II-induced cardiomyopathy partially via the Akt/GSK-3β/Fyn-mediated Nrf2 activation.

  17. Toosendanin inhibits growth and induces apoptosis in colorectal cancer cells through suppression of AKT/GSK-3β/β-catenin pathway.

    Science.gov (United States)

    Wang, Ge; Feng, Cheng-Cheng; Chu, Shao-Jun; Zhang, Rui; Lu, Yun-Min; Zhu, Jin-Shui; Zhang, Jing

    2015-11-01

    AKT/GSK-3β/β-catenin signaling pathway plays an important role in the progression of colorectal cancer (CRC). Toosendanin (TSN) is a triterpenoid extracted from the bark or fruits of Melia toosendan Sieb et Zucc and possesses antitumour effects on various human cancer cells. However, its effect on CRC remains poorly understood. The present study investigated the effect of TSN on CRC SW480 cells and the AKT/GSK-3β/β-catenin signaling. Proliferation assay, flow cytometry and Hoechst 33342 nuclear staining demonstrated TSN dose-dependently inhibited cell viability and induced cell apoptosis as well as cell cycle arrest in S phase. Confocal laser scanning microscope showed β-catenin transferred to the outside of the nucleus in TSN-treated cells. Quantitative real-time PCR and western blot analysis found that TSN effectively modulated molecules related to apoptosis and AKT/GSK-3β/β-catenin signaling. Moreover, TSN administration significantly inhibited CRC growth in a mouse tumor xenograft model. In conclusion, our findings indicate that TSN inhibits growth and induces apoptosis in CRC cells through suppression of AKT/GSK-3β/β-catenin pathway, suggesting that TSN may have potential for use in CRC treatment.

  18. GSK-3β promotes PA-induced apoptosis through changing β-arrestin 2 nucleus location in H9c2 cardiomyocytes.

    Science.gov (United States)

    Chang, Fen; Liu, Jing; Fu, Hui; Wang, Jinlan; Li, Fang; Yue, Hongwei; Li, Wenjing; Zhao, Jing; Yin, Deling

    2016-09-01

    Palmitic acid (PA), a type of saturated fatty acids, induces cardiovascular diseases by causing cardiomyocyte apoptosis with unclear mechanisms. Akt participates in PA-induced cardiomyocyte apoptosis. GSK-3β is a substrate of Akt, we investigated its role in PA-induced apoptosis. We reveal that PA inhibits GSK-3β phosphorylation accompanied by inactivation of Akt in H9c2 cardiomyocytes. We also reveal that inhibition the activity of GSK-3β by its inhibitor LiCl or knockdown by siRNA significantly attenuates PA-induced cardiomyocyte apoptosis, this suggesting that GSK-3β plays a pro-apoptotic role. To detect its downstream factors, we analyzed the roles of JNK, p38 MAPK and β-arrestin 2 (β-Arr2). Here, we report that GSK-3β regulate PA-induced cardiomyocyte apoptosis by affecting the distribution of β-Arr2. PA diminishes the protein level of β-Arr2 and changes its distribution from nucleus to cytoplasm. Either inhibition of β-Arr2 by its siRNA or overexpression of its protein level by transfection of β-Arr2 full-length plasmid promotes PA-induced cardiomyocyte apoptosis, which remind us to focus on the changes of its location. β-Arr2 siRNA decreased the background level of β-Arr2 in nucleus in normal H9c2 cells. Overexpression of β-Arr2 increased cytoplasm level of β-Arr2 as PA did. While LiCl, the inhibitor of GSK-3β decreased PA-induced apoptosis, accompany with increased nucleus level of β-Arr2. Then we concluded that GSK-3β is closely associated with cardiomyocyte apoptosis induced by PA, it performs its pro-apoptotic function by affecting the location of β-Arr2. LiCl inhibits PA-induced cardiomyocyte apoptosis, which might provide novel therapeutic for cardiovascular diseases induced by metabolic syndrome.

  19. Curcumin activates Wnt/β-catenin signaling pathway through inhibiting the activity of GSK-3β in APPswe transfected SY5Y cells.

    Science.gov (United States)

    Zhang, Xiong; Yin, Wen-ke; Shi, Xiao-dong; Li, Yu

    2011-04-18

    Wnt/β-catenin signaling pathway plays an important role in the genesis and development of Alzheimer's disease. The study aims to investigate the effect of Curcumin on the expression of GSK-3β, β-catenin and CyclinD1 in vitro, which are tightly correlated with Wnt/β-catenin signaling pathway, and also to explore the mechanisms, which will provide a novel therapeutic intervention for treatment of Alzheimer's disease. Plasmid APPswe and BACE1-mychis were transiently co-transfected into SHSY5Y cells by Liposfectamin™2000. The cells were treated with Curcumin at 0, 1.25, 5.0, 20.0 μmol/L for 24 h, or with Curcumin at 5.0 μmol/L for 0, and 12, 24 and 48 h for time course assay. Cell lysates were collected for RT-PCR, Western blot assay and immunofluorescent staining were carried out for detecting the effect of Curcumin on the expression of GSK-3β, β-catenin and CyclinD1. RT-PCR and Western blot results showed that the expression of GSK-3β mRNA and protein significantly decreased in the transfected cells treated with Curcumin, and that the changes were in a dose and time-dependent manner (PCurcumin. Therefore, GSK-3β is a potential target for treatment of AD. Curcumin could activate the Wnt/β-catenin signaling pathway through inhibiting the expression of GSK-3β and inducing the expression of β-catenin and CyclinD1, which will provide a new theory for treatment of neurodegenerative diseases by Curcumin. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Moringin activates Wnt canonical pathway by inhibiting GSK3β in a mouse model of experimental autoimmune encephalomyelitis

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    Giacoppo S

    2016-10-01

    Full Text Available Sabrina Giacoppo,1 Thangavelu Soundara Rajan,1 Gina Rosalinda De Nicola,2 Renato Iori,2 Placido Bramanti,1 Emanuela Mazzon1 1IRCCS Centre Neurolesi “Bonino-Pulejo”, Messina, Italy; 2Council for Agricultural Research and Economics, Research Centre for Industrial Crops (CREA-CIN, Bologna, Italy Abstract: Aberrant canonical Wnt–β-catenin signaling has been reported in multiple sclerosis (MS, although the results are controversial. The present study aimed to examine the role of the Wnt–β-catenin pathway in experimental MS and also to test moringin (4-[α-L-rhamnopyranosyloxy]-benzyl isothiocyanate, resulting from exogenous myrosinase hydrolysis of the natural phytochemical glucomoringin 4(α-L-rhamnosyloxy-benzyl glucosinolate as a modulator of neuroinflammation via the β-catenin–PPARγ axis. Experimental autoimmune encephalomyelitis (EAE, the most common model of MS, was induced in C57BL/6 mice by immunization with MOG35–55. Released moringin (10 mg/kg glucomoringin +5 µL myrosinase/mouse was administered daily for 1 week before EAE induction and continued until mice were killed on day 28 after EAE induction. Our results clearly showed that the Wnt–β-catenin pathway was downregulated in the EAE model, whereas moringin pretreatment was able to avert this. Moringin pretreatment normalizes the aberrant Wnt–β-catenin pathway, resulting in GSK3β inhibition and β-catenin upregulation, which regulates T-cell activation (CD4 and FoxP3, suppresses the main inflammatory mediators (IL-1β, IL-6, and COX2, through activation of PPARγ. In addition, moringin attenuates apoptosis by reducing the expression of the Fas ligand and cleaved caspase 9, and in parallel increases antioxidant Nrf2 expression in EAE mice. Taken together, our results provide an interesting discovery in identifying moringin as a modulator of the Wnt–β-catenin signaling cascade and as a new potential therapeutic target for MS treatment. Keywords: Wnt

  1. SPSB3 targets SNAIL for degradation in GSK-3β phosphorylation-dependent manner and regulates metastasis.

    Science.gov (United States)

    Liu, Y; Zhou, H; Zhu, R; Ding, F; Li, Y; Cao, X; Liu, Z

    2018-02-08

    Epithelial-mesenchymal transition (EMT) is a process during which normal epithelial cells acquire mesenchymal characteristics. EMT has a critical role in various human diseases especially in cancer. EMT facilitates tumor initiation and progression by mediating cancer cell stemness and motility. Zinc finger transcription factor SNAIL is one of the most important initiators of EMT. Therefore, it is of great significance to understand the regulating mechanism of SNAIL. In this study, we carried out a luciferase-based genome-wide screening using small interfering RNA library against ~200 of E3 ligases and ubiquitin-related genes and identified SOCS box protein SPSB3 as a novel E3 ligase component that targets SNAIL into polyubiquitination and degradation in response to GSK-3β phosphorylation of SNAIL. Functionally, we observed that SPSB3 overexpression greatly inhibits tumor metastasis by regulating SNAIL degradation both in vitro and in vivo. The expression of SPSB3 and SNAIL are negatively correlated in human esophageal squamous cell carcinoma tissues, and low SPSB3 expression indicates lymph node metastasis. Moreover, high SPSB3 expression indicates good survivals in various kinds of cancer. Collectively, these findings suggest that SPSB3-mediated SNAIL degradation has a vital role in regulating EMT and cancer progression.

  2. Deletion of Irs2 causes reduced kidney size in mice: role for inhibition of GSK3beta?

    LENUS (Irish Health Repository)

    Carew, Rosemarie M.

    2010-07-06

    Abstract Background Male Irs2-\\/- mice develop fatal type 2 diabetes at 13-14 weeks. Defects in neuronal proliferation, pituitary development and photoreceptor cell survival manifest in Irs2-\\/- mice. We identify retarded renal growth in male and female Irs2-\\/- mice, independent of diabetes. Results Kidney size and kidney:body weight ratio were reduced by approximately 20% in Irs2-\\/- mice at postnatal day 5 and was maintained in maturity. Reduced glomerular number but similar glomerular density was detected in Irs2-\\/- kidney compared to wild-type, suggesting intact global kidney structure. Analysis of insulin signalling revealed renal-specific upregulation of PKBβ\\/Akt2, hyperphosphorylation of GSK3β and concomitant accumulation of β-catenin in Irs2-\\/- kidney. Despite this, no significant upregulation of β-catenin targets was detected. Kidney-specific increases in Yes-associated protein (YAP), a key driver of organ size were also detected in the absence of Irs2. YAP phosphorylation on its inhibitory site Ser127 was also increased, with no change in the levels of YAP-regulated genes, suggesting that overall YAP activity was not increased in Irs2-\\/- kidney. Conclusions In summary, deletion of Irs2 causes reduced kidney size early in mouse development. Compensatory mechanisms such as increased β-catenin and YAP levels failed to overcome this developmental defect. These data point to Irs2 as an important novel mediator of kidney size.

  3. Role of GSK-3β in Regulation of Canonical Wnt/β-catenin Signaling and PI3-K/Akt Oncogenic Pathway in Colon Cancer.

    Science.gov (United States)

    Jain, Shelly; Ghanghas, Preety; Rana, Chandan; Sanyal, S N

    2017-08-09

    Non-steroidal anti-inflammatory drugs (NSAIDs) are emerging as novel chemopreventive agents because of their ability in blocking cellular proliferation, and thereby tumor development, and also by promoting apoptosis. GSK-3β, a serine threonine kinase and a negative regulator of the oncogenic Wnt/β-catenin signaling pathway, plays a critical role in the regulation of oncogenesis. Celecoxib and etoricoxib, the two cyclooxygenase-2 (COX-2) selective NSAIDs, and Diclofenac, a preferential COX-2 inhibitory NSAID, had shown uniformly the chemopreventive and anti-neoplastic effects in the early stage of colon cancer by promoting apoptosis as well as an over-expression of GSK-3β while down-regulating the PI3-K/Akt oncogenic pathway.

  4. Escitalopram attenuates ?-amyloid-induced tau hyperphosphorylation in primary hippocampal neurons through the 5-HT1A receptor mediated Akt/GSK-3? pathway

    OpenAIRE

    Wang, Yan-Juan; Ren, Qing-Guo; Gong, Wei-Gang; Wu, Di; Tang, Xiang; Li, Xiao-Li; Wu, Fang-Fang; Bai, Feng; Xu, Lin; Zhang, Zhi-Jun

    2016-01-01

    Tau hyperphosphorylation is an important pathological feature of Alzheimer's disease (AD). To investigate whether escitalopram could inhibit amyloid-? (A?)-induced tau hyperphosphorylation and the underlying mechanisms, we treated the rat primary hippocampal neurons with A?1-42 and examined the effect of escitalopram on tau hyperphosphorylation. Results showed that escitalopram decreased A?1?42-induced tau hyperphosphorylation. In addition, escitalopram activated the Akt/GSK-3? pathway, and t...

  5. Galectin-3 silencing inhibits epirubicin-induced ATP binding cassette transporters and activates the mitochondrial apoptosis pathway via β-catenin/GSK-3β modulation in colorectal carcinoma.

    Directory of Open Access Journals (Sweden)

    Yung-Kuo Lee

    Full Text Available Multidrug resistance (MDR, an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp, MDR-associated protein (MRP 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.

  6. Chronic administration of aripiprazole activates GSK3?-dependent signalling pathways, and up-regulates GABAA receptor expression and CREB1 activity in rats

    OpenAIRE

    Pan, Bo; Huang, Xu-Feng; Deng, Chao

    2016-01-01

    Aripiprazole is a D2-like receptor (D2R) partial agonist with a favourable clinical profile. Previous investigations indicated that acute and short-term administration of aripiprazole had effects on PKA activity, GSK3?-dependent pathways, GABAA receptors, NMDA receptor and CREB1 in the brain. Since antipsychotics are used chronically in clinics, the present study investigated the long-term effects of chronic oral aripiprazole treatment on these cellular signalling pathways, in comparison with...

  7. NCYM, a Cis-antisense gene of MYCN, encodes a de novo evolved protein that inhibits GSK3β resulting in the stabilization of MYCN in human neuroblastomas.

    Directory of Open Access Journals (Sweden)

    Yusuke Suenaga

    2014-01-01

    Full Text Available The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.

  8. Amiodarone Induces Overexpression of Similar to Versican b to Repress the EGFR/Gsk3b/Snail Signaling Axis during Cardiac Valve Formation of Zebrafish Embryos.

    Directory of Open Access Journals (Sweden)

    Hung-Chieh Lee

    Full Text Available Although Amiodarone, a class III antiarrhythmic drug, inhibits zebrafish cardiac valve formation, the detailed molecular pathway is still unclear. Here, we proved that Amiodarone acts as an upstream regulator, stimulating similar to versican b (s-vcanb overexpression at zebrafish embryonic heart and promoting cdh-5 overexpression by inhibiting snail1b at atrioventricular canal (AVC, thus blocking invagination of endocardial cells and, as a result, preventing the formation of cardiac valves. A closer investigation showed that an intricate set of signaling events ultimately caused the up-regulation of cdh5. In particular, we investigated the role of EGFR signaling and the activity of Gsk3b. It was found that knockdown of EGFR signaling resulted in phenotypes similar to those of Amiodarone-treated embryos. Since the reduced phosphorylation of EGFR was rescued by knockdown of s-vcanb, it was concluded that the inhibition of EGFR activity by Amiodarone is s-vcanb-dependent. Moreover, the activity of Gsk3b, a downstream effector of EGFR, was greatly increased in both Amiodarone-treated embryos and EGFR-inhibited embryos. Therefore, it was concluded that reduced EGFR signaling induced by Amiodarone treatment results in the inhibition of Snail functions through increased Gsk3b activity, which, in turn, reduces snail1b expression, leading to the up-regulation the cdh5 at the AVC, finally resulting in defective formation of valves. This signaling cascade implicates the EGFR/Gsk3b/Snail axis as the molecular basis for the inhibition of cardiac valve formation by Amiodarone.

  9. KPNA2 promotes migration and invasion in epithelial ovarian cancer cells by inducing epithelial-mesenchymal transition via Akt/GSK-3β/Snail activation.

    Science.gov (United States)

    Huang, Long; Zhou, Yun; Cao, Xin-Ping; Lin, Jia-Xin; Zhang, Lan; Huang, Shu-Ting; Zheng, Min

    2018-01-01

    Background : Increased karyopherin alpha 2 (KPNA2) expression has been demonstrated in epithelial ovarian carcinoma (EOC) tissue. However, its role in the disease is not clear. Here, we investigate the mechanism of involvement of KPNA2 in EOC. Methods : Stable cell lines expressing KPNA2, or KPNA2 shRNAs, were constructed. The effects of KPNA2 overexpression and knockdown on EOC cell migration, invasion, and epithelial-to-mesenchymal transition (EMT) were evaluated using relevant assays and western blot analysis. Key components of the Akt/GSK-3β/Snail signaling pathway were detected using western blotting and immunofluorescence. Results: KPNA2 overexpression increased the migration and invasion of EOC cells (EFO-21 and SK-OV3); these cells also exhibited characteristics of EMT. Key proteins in the Akt/GSK-3β/Snail signaling pathway were also upregulated in cells overexpressing KPNA2. In contrast, knockdown of KPNA2 effectively suppressed migration and invasion of these EOC cells. Conclusions: KPNA2 may reduce the migration and invasion of EOC by inhibiting the Akt/GSK-3β/Snail signaling pathway and suppressing EMT.

  10. CCL21 Facilitates Chemoresistance and Cancer Stem Cell-Like Properties of Colorectal Cancer Cells through AKT/GSK-3β/Snail Signals

    Directory of Open Access Journals (Sweden)

    Lin-Lin Lu

    2016-01-01

    Full Text Available Some evidence indicated that chemoresistance associates with the acquisition of cancer stem-like properties. Recent studies suggested that chemokines can promote the chemoresistance and stem cell properties in various cancer cells, while the underling mechanism is still not completely illustrated. In our study, we found that CCL21 can upregulate the expression of P-glycoprotein (P-gp and stem cell property markers such as Bmi-1, Nanog, and OCT-4 in colorectal cancer (CRC HCT116 cells and then improve the cell survival rate and mammosphere formation. Our results suggested that Snail was crucial for CCL21-mediated chemoresistance and cancer stem cell property in CRC cells. Further, we observed that CCL21 treatment increased the protein but not mRNA levels of Snail, which suggested that CCL21 upregulates Snail via posttranscriptional ways. The downstream signals AKT/GSK-3β mediated CCL21 induced the upregulation of Snail due to the fact that CCL21 treatment can obviously phosphorylate both AKT and GSK-3β. The inhibitor of PI3K/Akt, LY294002 significantly abolished CCL21 induced chemoresistance and mammosphere formation of HCT116 cells. Collectively, our results in the present study revealed that CCL21 can facilitate chemoresistance and stem cell property of CRC cells via the upregulation of P-gp, Bmi-1, Nanog, and OCT-4 through AKT/GSK-3β/Snail signals, which suggested a potential therapeutic approach to CRC patients.

  11. PKA-GSK3β and β-Catenin Signaling Play a Critical Role in Trans-Resveratrol Mediated Neuronal Differentiation in Human Cord Blood Stem Cells.

    Science.gov (United States)

    Jahan, S; Singh, S; Srivastava, A; Kumar, V; Kumar, D; Pandey, A; Rajpurohit, C S; Purohit, A R; Khanna, V K; Pant, A B

    2018-04-01

    The role of resveratrol (RV), a natural polyphenol, is well documented, although its role on neurogenesis is still controversial and poorly understood. Therefore, to decipher the cellular insights of RV on neurogenesis, we investigated the potential effects of the compound on the survival, proliferation, and neuronal differentiation of human cord blood-derived mesenchymal stem cells (hCBMSCs). For neuronal differentiation, purified and characterized hCBMSCs were exposed to biological safe doses of RV (10 μM) alone and in combination with nerve growth factor (NGF-50 ng). The cells exposed only to NGF (50 ng/mL) served as positive control for neuronal differentiation. The genes showing significant involvement in the process of neuronal differentiation were further funneled down at transcriptional and translational level. It was observed that RV promotes PKA-mediated neuronal differentiation in hCBMSCs by inducing canonical pathway. The studies with pharmacological inhibitors also confirmed that PKA significantly induces β-catenin expression via GSK3β induction and stimulates CREB phosphorylation and pERK1/2 induction. Besides that, the studies also revealed that RV additionally possesses the binding sites for molecules other than PKA and GSK3β, with which it interacts. The present study therefore highlights the positive impact of RV over the survival, proliferation, and neuronal differentiation in hCBMSCs via PKA-mediated induction of GSK3β, β catenin, CREB, and ERK1/2.

  12. Selectivity criterion for pyrazolo[3,4-b]pyrid[az]ine derivatives as GSK-3 inhibitors: CoMFA and molecular docking studies.

    Science.gov (United States)

    Patel, Dhilon S; Bharatam, Prasad V

    2008-05-01

    In the development of drugs targeted for GSK-3, its selective inhibition is an important requirement owing to the possibility of side effects arising from other kinases for the treatment of diabetes mellitus. A three-dimensional quantitative structure-activity relationship study (3D-QSAR) has been carried out on a set of pyrazolo[3,4-b]pyrid[az]ine derivatives, which includes non-selective and selective GSK-3 inhibitors. The CoMFA models were derived from a training set of 59 molecules. A test set containing 14 molecules (not used in model generation) was used to validate the CoMFA models. The best CoMFA model generated by applying leave-one-out (LOO) cross-validation study gave cross-validation r(cv)(2) and conventional r(conv)(2) values of 0.60 and 0.97, respectively, and r(pred)(2) value of 0.55, which provide the predictive ability of model. The developed models well explain (i) the observed variance in the activity and (ii) structural difference between the selective and non-selective GSK-3 inhibitors. Validation based on the molecular docking has also been carried out to explain the structural differences between the selective and non-selective molecules in the given series of molecules.

  13. Whey Protein Concentrate Renders MDA-MB-231 Cells Sensitive to Rapamycin by Altering Cellular Redox State and Activating GSK3β/mTOR Signaling.

    Science.gov (United States)

    Cheng, Shih-Hsuan; Tseng, Yang-Ming; Wu, Szu-Hsien; Tsai, Shih-Meng; Tsai, Li-Yu

    2017-11-21

    Whey protein concentrate (WPC) is an amino acid-rich supplement that has been shown to increase cellular antioxidant capacity. Mammalian target of rapamycin (mTOR) is a crucial regulator of signaling in mammalian cells, and serves as a therapeutic target for triple-negative breast cancer (TNBC). This study was designed to investigate the effect of combining WPC with rapamycin on MDA-MB-231 human breast cancer cells. These cells were found to be insensitive to rapamycin and exhibited higher glutathione (GSH) and reactive oxygen species levels than non-tumorigenic MCF-10A cells. However, for MDA-MB-231 cells, the half maximal inhibitory concentration of rapamycin was lower when this drug was administered in combination with WPC than when used alone. Furthermore, combining WPC with rapamycin depleted GSH levels and reduced Nrf2 nuclear accumulation. In addition, WPC activated GSK3β/mTOR signaling, and GSK3β appeared to be involved in the WPC-mediated Nrf2 reduction and mTOR activation. In conclusion, WPC induced rapamycin sensitivity in MDA-MB-231 cells by altering their redox state and activating GSK3β/mTOR signaling. These results not only suggest a novel therapeutic approach for breast cancer treatment, but also provide insight into the critical pathways affecting the resistance to mTOR inhibition observed in a subgroup of TNBC patients.

  14. TaSK5, an abiotic stress-inducible GSK3/shaggy-like kinase from wheat, confers salt and drought tolerance in transgenic Arabidopsis.

    Science.gov (United States)

    Christov, Nikolai Kirilov; Christova, Petya Koeva; Kato, Hideki; Liu, Yuelin; Sasaki, Kentaro; Imai, Ryozo

    2014-11-01

    A novel cold-inducible GSK3/shaggy-like kinase, TaSK5, was isolated from winter wheat using a macroarray-based differential screening approach. TaSK5 showed high similarity to Arabidopsis subgroup I GSK3/shaggy-like kinases ASK-alpha, AtSK-gamma and ASK-epsilon. RNA gel blot analyses revealed TaSK5 induction by cold and NaCl treatments and to a lesser extent by drought treatment. TaSK5 functionally complemented the cold- and salt-sensitive phenotypes of a yeast GSK3/shaggy-like kinase mutant, △mck1. Transgenic Arabidopsis plants overexpressing TaSK5 cDNA showed enhanced tolerance to salt and drought stresses. By contrast, the tolerance of the transgenic plants to freezing stress was not altered. Microarray analysis revealed that a number of abiotic stress-inducible genes were constitutively induced in the transgenic Arabidopsis plants, suggesting that TaSK5 may function in a novel signal transduction pathway that appears to be unrelated to DREB1/CBF regulon and may involve crosstalk between abiotic and hormonal signals. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  15. GSK3 beta forms a tetrameric complex with endogenous PS1-CTF/NTF and beta-catenin. Effects of the D257/D385A and FAD-linked mutations.

    Science.gov (United States)

    Tesco, G; Tanzi, R E

    2000-01-01

    We have previously shown that the endogenous C-terminal fragment of presenilin 1 co-immunoprecipitates with endogenous beta-catenin. Since PS1 has been suggested to be involved in beta-catenin stabilization, we further investigated whether GSK3 beta, responsible for beta-catenin phosphorylation and degradation, is part of the PS1/beta-catenin complex. In naïve H4 and CHO cells, PS1 co-immunoprecipitated with both endogenous beta-catenin and GSK3 beta. In addition, GSK3 beta endogenously binds to the PS1-CTF/NTF complex and beta-catenin in naïve CHO cells. GSK3 beta also co-immunoprecipitated with PS1 full length in CHO cell lines overexpressing PS1 wild type. Given that it has been recently shown that PS1 mutations of aspartate 257 or 385 result in prevention of PS1 endoproteolysis and inhibition of gamma-secretase activity, we also tested whether PS1 endoproteolysis is required for beta-catenin/GSK3 beta/PS1 binding and whether PS1 FAD-linked mutations affect GSK3 beta recruitment in the PS1/beta-catenin complex. GSK3 beta was detected in PS1 immunoprecipitates from H4 cell lines overexpressing PS1 wild type, delta E10, A286E, L246V and in CHO cell lines overexpressing aspartate or M146L mutations. The latter data show that the absence of PS1 endoproteolysis (D257A/D385A and delta E10) or the presence of PS1-FAD mutations does not interfere with beta-catenin/GSK3 beta/PS1 complex formation.

  16. GSK3β and ERK regulate the expression of 78 kDa SG2NA and ectopic modulation of its level affects phases of cell cycle.

    Science.gov (United States)

    Pandey, Shweta; Talukdar, Indrani; Jain, Buddhi P; Goswami, Shyamal K

    2017-08-08

    Striatin and SG2NA are essential constituents of the multi-protein STRIPAK assembly harbouring protein phosphatase PP2A and several kinases. SG2NA has several isoforms generated by mRNA splicing and editing. While the expression of striatin is largely restricted to the striatum in brain, that of SG2NAs is ubiquitous. In NIH3T3 cells, only the 78 kDa isoform is expressed. When cells enter into the S phase, the level of SG2NA increases; reaches maximum at the G2/M phase and declines thereafter. Downregulation of SG2NA extends G1 phase and its overexpression extends G2. Ectopic expression of the 35 kDa has no effects on the cell cycle. Relative abundance of phospho-SG2NA is high in the microsome and cytosol and the nucleus but low in the mitochondria. Okadoic acid, an inhibitor of PP2A, increases the level of SG2NA which is further enhanced upon inhibition of proteasomal activity. Phospho-SG2NA is thus more stable than the dephosphorylated form. Inhibition of GSK3β by LiCl reduces its level, but the inhibition of ERK by PD98059 increases it. Thus, ERK decreases the level of phospho-SG2NA by inhibiting GSK3β. In cells depleted from SG2NA by shRNA, the levels of pGSK3β and pERK are reduced, suggesting that these kinases and SG2NA regulate each other's expression.

  17. The balance of TCF7L2 variants with differential activities in Wnt-signaling is regulated by lithium in a GSK3β-independent manner

    International Nuclear Information System (INIS)

    Struewing, Ian; Boyechko, Tania; Barnett, Corey; Beildeck, Marcy; Byers, Stephen W.; Mao, Catherine D.

    2010-01-01

    Research highlights: → Identification of a novel effect of lithium on the expression of TCF7L2 RNA isoforms and protein variants. → The extent of lithium-induced TCF7L2 form switch mirrors cell responsiveness to Wnt/β-catenin signaling. → Demonstration that lithium has dual GSK3β-dependent and -independent effects on TCF7L2 expression. → Demonstration that TCF7L2 expression is repressed by the transcriptionally active TCF7L2E form. → Evidence for a lithium-induced de-repression mechanism of TCF7L2 expression via TCF7L2 variant switch. -- Abstract: TCF7L2 transcription factor is a downstream effector of the canonical Wnt/β-catenin signaling, which controls cell fate and homeostasis. However, the complexity of TCF7L2 expression with numerous mRNA isoforms coding for proteins with distinct N- and C-termini allows variability in TCF7L2 functions and regulations. Here, we show that although TCF7L2 mRNA isoforms distinguish fetal, immortalized and adult differentiated endothelial cells (EC), they cannot explain the lack of significant β-catenin/TCF7 activities in ECs. Lithium, a Wnt-signaling activator, increases TCF7L2 mRNA levels and induces an RNA isoform switch favoring the expression of TCF7L2-short forms lacking the C-termini domains. Although the latter occurs in different cell types, its extent depends on the overall increase of TCF7L2 transcription, which correlates with cell responsiveness to Wnt/β-catenin signaling. While GSK3β down-regulation increases TCF7L2 expression, there is no concomitant change in TCF7L2 mRNA isoforms, which demonstrate the dual effects of lithium on TCF7L2 expression via a GSK3β-dependent up-regulation and a GSK3β-independent modulation of RNA splicing. TCF7L2E-long forms display a repressor activity on TCF7L2-promoter reporters and lithium induces a decrease of the endogenous TCF7L2 forms bound to native TCF7L2-promoter chromatin at two novel distal TCF7-binding sites. Altogether our data reveal a lithium

  18. Curcumol induces cell cycle arrest in colon cancer cells via reactive oxygen species and Akt/ GSK3β/cyclin D1 pathway.

    Science.gov (United States)

    Wang, Juan; Li, Xu-Mei; Bai, Zhun; Chi, Bi-Xia; Wei, Yan; Chen, Xu

    2018-01-10

    Curcuma kwangsiensis S. G. Lee & C. F. Liang (Guangxi ezhu, in Chinese) belongs to the Zingiberaceae family, has been used as a traditionally Chinese medicine nearly 2000 year. Curcumol is one of the guaiane-type sesquiterpenoid hemiketal isolated from medicine plant Curcuma kwangsiensis S. G. Lee & C. F. Liang, which has been reported possesses anti-cancer effects. Our previous study found that the most contribution to inhibit nasopharyngeal carcinoma cell growth was curcumol. To assess the effect of curcumol on cell cycle arrest against human colon cancer cells (CRC) cells (LoVo and SW480) and explore its mechanism in vitro and in vivo. Curcumol was dissolved in absolute ethyl alcohol. The concentration of absolute ethyl alcohol in the control group or in experimental samples was always 1/500 (v/v) of the final medium volume. LoVo and SW480 cells were treated with different concentrations of curcumol (0, 53, 106, 212 and 424μM). And then the cell cycle of each group was examined by flow cytometry. The protein levels of PI3K, p-Akt, cyclin D1, cyclin E, CDK2, CDK4 and GSK3β were determined by Western blot. The mRNA expression of PI3K, Akt, cyclin D1, CDK4, P27, p21, and P16 in the treated cells were analyzed by real-time RT-PCR. In addition, the antitumor activity of curcumol was evaluated in nude mice bearing orthotopic tumor implants. Curcumol induced cell cycle arrest in G1/S phase. RT-qPCR and Western blot data showed that curcumol enhanced the expression of GSK3β, P27, p21 and P16, and decreased the levels of PI3K, phosphorylated Akt (p-Akt), cyclin D1, CDK4, cyclin E and CDK2. Furthermore, curcumol induced reactive oxygen species (ROS) generation in LoVo cells, and ROS scavenger N-acetylcysteine (NAC) significantly reversed curcumol-induced cell growth inhibition. Besides, curcumol also prevented the growth of human colon cancer cells xenografts in nude mouse, accompanied by the reduction of PI3K, Akt, cyclin D1, CDK4, cycln E and significant increase of

  19. Reactive oxygen species mediate Cr(VI)-induced carcinogenesis through PI3K/AKT-dependent activation of GSK-3β/β-catenin signaling

    Energy Technology Data Exchange (ETDEWEB)

    Son, Young-Ok; Pratheeshkumar, Poyil; Wang, Lei; Wang, Xin; Fan, Jia; Kim, Dong-Hern; Lee, Ju-Yeon; Zhang, Zhuo [Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Lee, Jeong-Chae [Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); School of Dentistry and Institute of Oral Biosciences, Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Shi, Xianglin, E-mail: xshi5@email.uky.edu [Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States)

    2013-09-01

    Cr(VI) compounds are known human carcinogens that primarily target the lungs. Cr(VI) produces reactive oxygen species (ROS), but the exact effects of ROS on the signaling molecules involved in Cr(VI)-induced carcinogenesis have not been extensively studied. Chronic exposure of human bronchial epithelial cells to Cr(VI) at nanomolar concentrations (10–100 nM) for 3 months not only induced cell transformation, but also increased the potential of these cells to invade and migrate. Injection of Cr(VI)-stimulated cells into nude mice resulted in the formation of tumors. Chronic exposure to Cr(VI) increased levels of intracellular ROS and antiapoptotic proteins. Transfection with catalase or superoxide dismutase (SOD) prevented Cr(VI)-mediated increases in colony formation, cell invasion, migration, and xenograft tumors. While chronic Cr(VI) exposure led to activation of signaling cascades involving PI3K/AKT/GSK-3β/β-catenin and PI3K/AKT/mTOR, transfection with catalase or SOD markedly inhibited Cr(VI)-mediated activation of these signaling proteins. Inhibitors specific for AKT or β-catenin almost completely suppressed the Cr(VI)-mediated increase in total and active β-catenin proteins and colony formation. In particular, Cr(VI) suppressed autophagy of epithelial cells under nutrition deprivation. Furthermore, there was a marked induction of AKT, GSK-3β, β-catenin, mTOR, and carcinogenic markers in tumor tissues formed in mice after injection with Cr(VI)-stimulated cells. Collectively, our findings suggest that ROS is a key mediator of Cr(VI)-induced carcinogenesis through the activation of PI3K/AKT-dependent GSK-3β/β-catenin signaling and the promotion of cell survival mechanisms via the inhibition of apoptosis and autophagy. - Highlights: • Chronic exposure to Cr(VI) induces carcinogenic properties in BEAS-2B cells. • ROS play an important role in Cr(VI)-induced tumorigenicity of BEAS-2B cells. • PI3K/AKT/GSK-3β/β-catenin signaling involved in Cr

  20. Reactive oxygen species mediate Cr(VI)-induced carcinogenesis through PI3K/AKT-dependent activation of GSK-3β/β-catenin signaling

    International Nuclear Information System (INIS)

    Son, Young-Ok; Pratheeshkumar, Poyil; Wang, Lei; Wang, Xin; Fan, Jia; Kim, Dong-Hern; Lee, Ju-Yeon; Zhang, Zhuo; Lee, Jeong-Chae; Shi, Xianglin

    2013-01-01

    Cr(VI) compounds are known human carcinogens that primarily target the lungs. Cr(VI) produces reactive oxygen species (ROS), but the exact effects of ROS on the signaling molecules involved in Cr(VI)-induced carcinogenesis have not been extensively studied. Chronic exposure of human bronchial epithelial cells to Cr(VI) at nanomolar concentrations (10–100 nM) for 3 months not only induced cell transformation, but also increased the potential of these cells to invade and migrate. Injection of Cr(VI)-stimulated cells into nude mice resulted in the formation of tumors. Chronic exposure to Cr(VI) increased levels of intracellular ROS and antiapoptotic proteins. Transfection with catalase or superoxide dismutase (SOD) prevented Cr(VI)-mediated increases in colony formation, cell invasion, migration, and xenograft tumors. While chronic Cr(VI) exposure led to activation of signaling cascades involving PI3K/AKT/GSK-3β/β-catenin and PI3K/AKT/mTOR, transfection with catalase or SOD markedly inhibited Cr(VI)-mediated activation of these signaling proteins. Inhibitors specific for AKT or β-catenin almost completely suppressed the Cr(VI)-mediated increase in total and active β-catenin proteins and colony formation. In particular, Cr(VI) suppressed autophagy of epithelial cells under nutrition deprivation. Furthermore, there was a marked induction of AKT, GSK-3β, β-catenin, mTOR, and carcinogenic markers in tumor tissues formed in mice after injection with Cr(VI)-stimulated cells. Collectively, our findings suggest that ROS is a key mediator of Cr(VI)-induced carcinogenesis through the activation of PI3K/AKT-dependent GSK-3β/β-catenin signaling and the promotion of cell survival mechanisms via the inhibition of apoptosis and autophagy. - Highlights: • Chronic exposure to Cr(VI) induces carcinogenic properties in BEAS-2B cells. • ROS play an important role in Cr(VI)-induced tumorigenicity of BEAS-2B cells. • PI3K/AKT/GSK-3β/β-catenin signaling involved in Cr

  1. The balance of TCF7L2 variants with differential activities in Wnt-signaling is regulated by lithium in a GSK3{beta}-independent manner

    Energy Technology Data Exchange (ETDEWEB)

    Struewing, Ian; Boyechko, Tania; Barnett, Corey [Department of Clinical Sciences, University of Kentucky, Lexington, KY (United States); Beildeck, Marcy; Byers, Stephen W. [Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University School of Medicine, Washington, DC (United States); Mao, Catherine D., E-mail: cdmao2@uky.edu [Department of Clinical Sciences, University of Kentucky, Lexington, KY (United States); Markey Cancer Center, University of Kentucky, Lexington, KY (United States)

    2010-08-20

    Research highlights: {yields} Identification of a novel effect of lithium on the expression of TCF7L2 RNA isoforms and protein variants. {yields} The extent of lithium-induced TCF7L2 form switch mirrors cell responsiveness to Wnt/{beta}-catenin signaling. {yields} Demonstration that lithium has dual GSK3{beta}-dependent and -independent effects on TCF7L2 expression. {yields} Demonstration that TCF7L2 expression is repressed by the transcriptionally active TCF7L2E form. {yields} Evidence for a lithium-induced de-repression mechanism of TCF7L2 expression via TCF7L2 variant switch. -- Abstract: TCF7L2 transcription factor is a downstream effector of the canonical Wnt/{beta}-catenin signaling, which controls cell fate and homeostasis. However, the complexity of TCF7L2 expression with numerous mRNA isoforms coding for proteins with distinct N- and C-termini allows variability in TCF7L2 functions and regulations. Here, we show that although TCF7L2 mRNA isoforms distinguish fetal, immortalized and adult differentiated endothelial cells (EC), they cannot explain the lack of significant {beta}-catenin/TCF7 activities in ECs. Lithium, a Wnt-signaling activator, increases TCF7L2 mRNA levels and induces an RNA isoform switch favoring the expression of TCF7L2-short forms lacking the C-termini domains. Although the latter occurs in different cell types, its extent depends on the overall increase of TCF7L2 transcription, which correlates with cell responsiveness to Wnt/{beta}-catenin signaling. While GSK3{beta} down-regulation increases TCF7L2 expression, there is no concomitant change in TCF7L2 mRNA isoforms, which demonstrate the dual effects of lithium on TCF7L2 expression via a GSK3{beta}-dependent up-regulation and a GSK3{beta}-independent modulation of RNA splicing. TCF7L2E-long forms display a repressor activity on TCF7L2-promoter reporters and lithium induces a decrease of the endogenous TCF7L2 forms bound to native TCF7L2-promoter chromatin at two novel distal TCF7

  2. N-Cadherin-Mediated Activation of PI3K/Akt-GSK-3β Signaling Attenuates Nucleus Pulposus Cell Apoptosis Under High-Magnitude Compression

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    Pei Li

    2017-11-01

    Full Text Available Background/Aims: Mechanical overloading-induced nucleus pulposus (NP apoptosis plays an important role in the pathogenesis of intervertebral disc degeneration. N-cadherin (N-CDH-mediated signaling preserves normal NP cell phenotype. This study aims to investigate the effects of N-CDH on NP cell apoptosis under high-magnitude compression and the underlying mechanism behind this process. Methods: Rat NP cells seeded on scaffold were perfusion-cultured using a self-developed perfusion bioreactor for 5 days and experienced different magnitudes (2% and 20% compressive deformation, respectively of compression at a frequency of 1.0 Hz for 4 hours once per day. The un-loaded NP cells were used as controls. Lentivirus-mediated N-CDH overexpression and inhibitor LY294002 were used to further investigate the role of N-CDH and PI3K/Akt pathway under high-magnitude compression, respectively. NP cell apoptosis was evaluated by caspase-3 activity measured using a commercial kit, flow cytometry, and expression of apoptosis-related molecules analyzed by real-time PCR and western blotting assays. Results: High-magnitude compression significantly increased apoptotic NP cells, caspase-3 activity and expression of pro-apoptotic molecules (Bax and caspase-3/cleaved caspase-3, but decreased expression of anti-apoptotic molecule (Bcl-2. High-magnitude compression decreased expression of N-CDH, p-Akt and p-GSK-3β. However, N-CDH overexpression attenuated NP cell apoptosis and increased expression of p-Akt and p-GSK-3β under high-magnitude compression. Further analysis showed that inhibition of the PI3K/Akt pathway suppressed NP cell apoptosis and decreased expression of p-GSK-3β, but had no significant effects on N-CDH expression under high-magnitude compression. Conclusion: N-CDH can attenuate NP cell apoptosis through activating the PI3K/Akt-GSK-3β signaling under high-magnitude compression.

  3. Cadmium induces carcinogenesis in BEAS-2B cells through ROS-dependent activation of PI3K/AKT/GSK-3β/β-catenin signaling

    International Nuclear Information System (INIS)

    Son, Young-Ok; Wang, Lei; Poyil, Pratheeshkumar; Budhraja, Amit; Hitron, J. Andrew; Zhang, Zhuo; Lee, Jeong-Chae; Shi, Xianglin

    2012-01-01

    Cadmium has been widely used in industry and is known to be carcinogenic to humans. Although it is widely accepted that chronic exposure to cadmium increases the incidence of cancer, the mechanisms underlying cadmium-induced carcinogenesis are unclear. The main aim of this study was to investigate the role of reactive oxygen species (ROS) in cadmium-induced carcinogenesis and the signal transduction pathways involved. Chronic exposure of human bronchial epithelial BEAS-2B cells to cadmium induced cell transformation, as evidenced by anchorage-independent growth in soft agar and clonogenic assays. Chronic cadmium treatment also increased the potential of these cells to invade and migrate. Injection of cadmium-stimulated cells into nude mice resulted in the formation of tumors. In contrast, the cadmium-mediated increases in colony formation, cell invasion and migration were prevented by transfection with catalase, superoxide dismutase-1 (SOD1), or SOD2. In particular, chronic cadmium exposure led to activation of signaling cascades involving PI3K, AKT, GSK-3β, and β-catenin and transfection with each of the above antioxidant enzymes markedly inhibited cadmium-mediated activation of these signaling proteins. Inhibitors specific for AKT or β-catenin almost completely suppressed the cadmium-mediated increase in total and active β-catenin proteins and colony formation. Moreover, there was a marked induction of AKT, GSK-3β, β-catenin, and carcinogenic markers in tumor tissues formed in mice after injection with cadmium-stimulated cells. Collectively, our findings suggest a direct involvement of ROS in cadmium-induced carcinogenesis and implicate a role of AKT/GSK-3β/β-catenin signaling in this process. -- Highlights: ► Chronic exposure to cadmium induces carcinogenic properties in BEAS-2B cells. ► ROS involved in cadmium-induced tumorigenicity of BEAS-2B cells. ► Cadmium activates ROS-dependent AKT/GSK-3β/β-catenin-mediated signaling. ► ROS

  4. Investigation of naphthofuran moiety as potential dual inhibitor against BACE-1 and GSK-3β: molecular dynamics simulations, binding energy, and network analysis to identify first-in-class dual inhibitors against Alzheimer's disease.

    Science.gov (United States)

    Kumar, Akhil; Srivastava, Gaurava; Srivastava, Swati; Verma, Seema; Negi, Arvind S; Sharma, Ashok

    2017-08-01

    BACE-1 and GSK-3β are potential therapeutic drug targets for Alzheimer's disease. Recently, both the targets received attention for designing dual inhibitors for Alzheimer's disease. Until now, only two-scaffold triazinone and curcumin have been reported as BACE-1 and GSK-3β dual inhibitors. Docking, molecular dynamics, clustering, binding energy, and network analysis of triazinone derivatives with BACE-1 and GSK-3β was performed to get molecular insight into the first reported dual inhibitor. Further, we designed and evaluated a naphthofuran series for its ability to inhibit BACE-1 and GSK-3β with the computational approaches. Docking study of naphthofuran series showed a good binding affinity towards both the targets. Molecular dynamics, binding energy, and network analysis were performed to compare their binding with the targets and amino acids responsible for binding. Naphthofuran series derivatives showed good interaction within the active site residues of both of the targets. Hydrogen bond occupancy and binding energy suggested strong binding with the targets. Dual-inhibitor binding was mostly governed by the hydrophobic interactions for both of the targets. Per residue energy decomposition and network analysis identified the key residues involved in the binding and inhibiting BACE-1 and GSK-3β. The results indicated that naphthofuran series derivative 11 may be a promising first-in-class dual inhibitor against BACE-1 and GSK-3β. This naphthofuran series may be further explored to design better dual inhibitors. Graphical abstract Naphthofuran derivative as a dual inhibitor for BACE-1 and GSK-3β.

  5. Fatty acid binding protein 4 promotes epithelial-mesenchymal transition in cervical squamous cell carcinoma through AKT/GSK3β/Snail signaling pathway.

    Science.gov (United States)

    Jin, Jiangbo; Zhang, Ziyu; Zhang, Song; Chen, Xinyu; Chen, Zhen; Hu, Ping; Wang, Jianbin; Xie, Caifeng

    2018-02-05

    Fatty acid binding protein 4 (FABP4) is a member of the fatty acid binding protein family which involved in a variety of biological cellular processes, including tumorigenesis. However, the role of this key adipokine in cervical cancer is still unclear. In this study, we explored the function of FABP4 in cervical cancer and the underlying molecular mechanisms. FABP4 was specifically elevated in tissue samples from patients with cervical squamous cell carcinoma (CSCC) but not with cervical adenocarcinoma, and the level of FABP4 was correlated with E-cadherin and Vimentin expression. In vitro, exogenous FABP4 promoted the migration and invasion of CSCC cells in a dose-dependent manner, and reorganized the actin cytoskeletons in F-Actin staining and TGF-β induced EMT assays. Importantly, the AKT/GSK3β/Snail pathway appears to be involved in FABP4-induced EMT in CSCC cells. In conclusion, our research demonstrated elevated FABP4 promoted EMT via the activation of AKT/GSK3β/Snail pathway in CSCC. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Apolipoprotein E-Mimetic Peptide COG1410 Promotes Autophagy by Phosphorylating GSK-3β in Early Brain Injury Following Experimental Subarachnoid Hemorrhage

    Directory of Open Access Journals (Sweden)

    Xinshen Li

    2018-03-01

    Full Text Available COG1410, a mimetic peptide derived from the apolipoprotein E (apoE receptor binding region, exerts positive effect on neurological deficits in early brain injury (EBI after experimental subarachnoid hemorrhage (SAH. Currently the neuroprotective effect of COG1410 includes inhibiting BBB disruption, reducing neuronal apoptosis, and neuroinflammation. However, the effect and mechanism of COG1410 to subcellular organelles disorder have not been fully investigated. As the main pathway for recycling long-lived proteins and damaged organelles, neuronal autophagy is activated in SAH and exhibits neuroprotective effects by reducing the insults of EBI. Pharmacologically elevated autophagy usually contributes to alleviated brain injury, while few of the agents achieved clinical transformation. In this study, we explored the activation of autophagy during EBI by measuring the Beclin-1 and LC3B-II protein levels. Administration of COG1410 notably elevated the autophagic markers expression in neurons, simultaneously reversed the neurological deficits. Furthermore, the up-regulated autophagy by COG1410 was further promoted by p-GSK-3β agonist, whereas decreased by p-GSK-3β inhibitor. Taken together, these data suggest that the COG1410 might be a promising therapeutic strategy for EBI via promoting autophagy in SAH.

  7. Preferential Selectivity of Inhibitors with Human Tau Protein Kinase Gsk3 Elucidates Their Potential Roles for Off-Target Alzheimer’s Therapy

    Directory of Open Access Journals (Sweden)

    Jagadeesh Kumar Dasappa

    2013-01-01

    Full Text Available Alzheimer’s disease (AD is a neurodegenerative disorder characterized by the accumulation of amyloid beta peptides (A and neurofibrillary tangles (NFTs. The abnormal phosphorylation of tau leads to the formation of NFTs produced by the action of tau kinases, resulting in the loss of neurons and synapse, leading to dementia. Hence, tau kinases have become potential drug target candidates for small molecule inhibitors. With an aim to explore the identification of a common inhibitor, this investigation was undertaken towards analyzing all 10 tau kinases which are implicated in phosphorylation of AD. A set of 7 inhibitors with varied scaffolds were collected from the Protein Data Bank (PDB. The analysis, involving multiple sequence alignment, 3D structural alignment, catalytic active site overlap, and docking studies, has enabled elucidation of the pharmacophoric patterns for the class of 7 inhibitors. Our results divulge that tau protein kinases share a specific set of conserved structural elements for the binding of inhibitors and ATP, respectively. The scaffold of 3-aminopyrrolidine (inhibitor 6 exhibits high preferential affinity with GSK3. Surprisingly, the PDB does not contain the structural details of GSK3 with this specific inhibitor. Thus, our investigations provide vital clues towards design of novel off-target drugs for Alzheimer’s.

  8. Silica nanoparticles induce multinucleation through activation of PI3K/Akt/GSK-3β pathway and downregulation of chromosomal passenger proteins in L-02 cells

    Energy Technology Data Exchange (ETDEWEB)

    Geng, Weijia; Li, Yang; Yu, Yongbo; Yu, Yang; Duan, Junchao; Jiang, Lizhen; Li, Qiuling; Sun, Zhiwei, E-mail: zwsun@ccmu.edu.cn, E-mail: zwsun@hotmail.com [Capital Medical University, School of Public Health (China)

    2016-04-15

    Silica nanoparticles (SNPs) are applicable in various fields due to their unique physicochemical characteristics. However, concerns over their potential adverse effects have been raised. In our previous studies, we reported that SNPs could induce abnormal high incidence of multinucleation. The aim of this study is to further investigate the mechanisms of multinucleation induced by SNPs (68 nm) in human normal liver L-02 cells (L-02 cells). In order to determine the cytotoxicity of SNPs, MTT assay was performed, and the cell viability was decreased in a dose-dependent manner. The intracellular reactive oxygen species (ROS) detected by flow cytometry and multinucleation observed by Giemsa stain showed that ROS generation and rate of multinucleated cells increased after SNPs exposure. N-acetyl-cysteine (NAC), a glutathione precursor against SNP-induced toxicity, was used as a ROS inhibitor to elucidate the relationship between ROS and multinucleation. The presence of NAC resulted in inhibition of both ROS generation and rate of multinucleation. Moreover, Western blot analysis showed that the protein levels of Cdc20, Aurora B, and Survivin were down-regulated, and the PI3K/Akt/GSK-3β pathway was activated by SNPs. In conclusion, our findings strongly suggested that multinucleation induced by SNPs was related to PI3K/Akt/GSK-3β signal pathway activation and downregulation of G2/M phase-related protein and chromosomal passenger proteins.

  9. Nebula/DSCR1 upregulation delays neurodegeneration and protects against APP-induced axonal transport defects by restoring calcineurin and GSK-3β signaling.

    Directory of Open Access Journals (Sweden)

    Jillian L Shaw

    Full Text Available Post-mortem brains from Down syndrome (DS and Alzheimer's disease (AD patients show an upregulation of the Down syndrome critical region 1 protein (DSCR1, but its contribution to AD is not known. To gain insights into the role of DSCR1 in AD, we explored the functional interaction between DSCR1 and the amyloid precursor protein (APP, which is known to cause AD when duplicated or upregulated in DS. We find that the Drosophila homolog of DSCR1, Nebula, delays neurodegeneration and ameliorates axonal transport defects caused by APP overexpression. Live-imaging reveals that Nebula facilitates the transport of synaptic proteins and mitochondria affected by APP upregulation. Furthermore, we show that Nebula upregulation protects against axonal transport defects by restoring calcineurin and GSK-3β signaling altered by APP overexpression, thereby preserving cargo-motor interactions. As impaired transport of essential organelles caused by APP perturbation is thought to be an underlying cause of synaptic failure and neurodegeneration in AD, our findings imply that correcting calcineurin and GSK-3β signaling can prevent APP-induced pathologies. Our data further suggest that upregulation of Nebula/DSCR1 is neuroprotective in the presence of APP upregulation and provides evidence for calcineurin inhibition as a novel target for therapeutic intervention in preventing axonal transport impairments associated with AD.

  10. Melatonin Modulates Endoplasmic Reticulum Stress and Akt/GSK3-Beta Signaling Pathway in a Rat Model of Renal Warm Ischemia Reperfusion

    Directory of Open Access Journals (Sweden)

    Kaouther Hadj Ayed Tka

    2015-01-01

    Full Text Available Melatonin (Mel is widely used to attenuate ischemia/reperfusion (I/R injury in several organs. Nevertheless, the underlying mechanisms remain unclear. This study was conducted to explore the effect of Mel on endoplasmic reticulum (ER stress, Akt and MAPK cascades after renal warm I/R. Eighteen Wistar rats were randomized into three groups: Sham, I/R, and Mel + I/R. The ischemia period was 60 min followed by 120 min of reperfusion. Mel (10 mg/kg was administrated 30 min prior to ischemia. The creatinine clearance, MDA, LDH levels, and histopathological changes were evaluated. In addition, Western blot was performed to study ER stress and its downstream apoptosis as well as phosphorylation of Akt, GSK-3β, VDAC, ERK, and P38. Mel decreased cytolysis and lipid peroxidation and improved renal function and morphology compared to I/R group. Parallely, it significantly reduced the ER stress parameters including GRP 78, p-PERK, XBP 1, ATF 6, CHOP, and JNK. Simultaneously, p-Akt level was significantly enhanced and its target molecules GSK-3β and VDAC were inhibited. Furthermore, the ERK and P38 phosphorylation were evidently augmented after Mel administration in comparison to I/R group. In conclusion, Mel improves the recovery of renal function by decreasing ER stress and stimulating Akt pathway after renal I/R injury.

  11. Phosphorylation of PPP(S/T)P motif of the free LRP6 intracellular domain is not required to activate the Wnt/beta-catenin pathway and attenuate GSK3beta activity.

    Science.gov (United States)

    Beagle, Brandon; Mi, Kaihong; Johnson, Gail V W

    2009-11-01

    The canonical Wnt/beta-catenin signaling pathway plays a critical role in numerous physiological and pathological processes. LRP6 is an essential co-receptor for Wnt/beta-catenin signaling; as transduction of the Wnt signal is strongly dependent upon GSK3beta-mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane-anchored LRP6 intracellular domain. Previously, we showed that the free LRP6 intracellular domain (LRP6-ICD) can activate the Wnt/beta-catenin pathway in a beta-catenin and TCF/LEF-1 dependent manner, as well as interact with and attenuate GSK3beta activity. However, it is unknown if the ability of LRP6-ICD to attenuate GSK3beta activity and modulate activation of the Wnt/beta-catenin pathway requires phosphorylation of the LRP6-ICD PPP(S/T)P motifs, in a manner similar to the membrane-anchored LRP6 intracellular domain. Here we provide evidence that the LRP6-ICD does not have to be phosphorylated at its PPP(S/T)P motif by GSK3beta to stabilize endogenous cytosolic beta-catenin resulting in activation of TCF/LEF-1 and the Wnt/beta-catenin pathway. LRP6-ICD and a mutant in which all 5 PPP(S/T)P motifs were changed to PPP(A)P motifs equivalently interacted with and attenuated GSK3beta activity in vitro, and both constructs inhibited the in situ GSK3beta-mediated phosphorylation of beta-catenin and tau to the same extent. These data indicate that the LRP6-ICD attenuates GSK3beta activity similar to other GSK3beta binding proteins, and is not a result of it being a GSK3beta substrate. Our findings suggest the functional and regulatory mechanisms governing the free LRP6-ICD may be distinct from membrane-anchored LRP6, and that release of the LRP6-ICD may provide a complimentary signaling cascade capable of modulating Wnt-dependent gene expression. (c) 2009 Wiley-Liss, Inc.

  12. Obg-like ATPase 1 regulates global protein serine/threonine phosphorylation in cancer cells by suppressing the GSK3β-inhibitor 2-PP1 positive feedback loop.

    Science.gov (United States)

    Xu, Dong; Song, Renduo; Wang, Guohui; Jeyabal, Prince V S; Weiskoff, Amanda M; Ding, Kefeng; Shi, Zheng-Zheng

    2016-01-19

    OLA1 is an Obg family P-loop NTPase that possesses both GTP- and ATP-hydrolyzing activities. Here we report that OLA1 is a GSK3β interacting protein, and through its ATPase activity, inhibits the GSK3β-mediated activation of protein serine/threonine phosphatase 1 (PP1). It is hypothesized that GSK3β phosphorylates inhibitor 2 (I-2) of PP1 at Thr-72 and activates the PP1 · I-2 complex, which in turn dephosphorylates and stimulates GSK3β, thus forming a positive feedback loop. We revealed that the positive feedback loop is normally suppressed by OLA1, and becomes over-activated under OLA1 deficiency, resulting in increased cellular PP1 activity and dephosphorylation of multiple Ser/Thr phosphoproteins, and more strikingly, decreased global protein threonine phosphorylation. Furthermore, using xenograft models of colon cancer (H116) and ovarian cancer (SKOV3), we established a correlation among downregulation of OLA1, over-activation of the positive feedback loop as indicated by under-phosphorylation of I-2, and more aggressive tumor growth. This study provides the first evidence for the existence of a GSK3β-I-2-PP1 positive feedback loop in human cancer cells, and identifies OLA1 as an endogenous suppressor of this signaling motif.

  13. Targeting the AKT/GSK3β/cyclin D1/Cdk4 survival signaling pathway for eradication of tumor radioresistance acquired by fractionated radiotherapy.

    Science.gov (United States)

    Shimura, Tsutomu; Kakuda, Satoshi; Ochiai, Yasushi; Kuwahara, Yoshikazu; Takai, Yoshihiro; Fukumoto, Manabu

    2011-06-01

    Radioresistance is a major cause of treatment failure of radiotherapy (RT) in human cancer. We have recently revealed that acquired radioresistance of tumor cells induced by fractionated radiation is attributable to cyclin D1 overexpression as a consequence of the downregulation of GSK3β-dependent cyclin D1 proteolysis mediated by a constitutively activated serine-threonine kinase, AKT. This prompted us to hypothesize that targeting the AKT/GSK3β/cyclin D1 pathway may improve fractionated RT by suppressing acquired radioresistance of tumor cells. Two human tumor cell lines with acquired radioresistance were exposed to X-rays after incubation with either an AKT inhibitor, AKT/PKB signaling inhibitor-2 (API-2), or a Cdk4 inhibitor (Cdk4-I). Cells were then subjected to immunoblotting, clonogenic survival assay, cell growth analysis, and cell death analysis with TUNEL and annexin V staining. In vivo radiosensitivity was assessed by growth of human tumors xenografted into nude mice. Treatment with API-2 resulted in downregulation of cyclin D1 expression in cells with acquired radioresistance. Cellular radioresistance disappeared completely both in vitro and in vivo with accompanying apoptosis when treated with API-2. Furthermore, inhibition of cyclin D1/Cdk4 by Cdk4-I was sufficient for abolishing radioresistance. Treatment with either API-2 or Cdk4-I was also effective in suppressing resistance to cis-platinum (II)-diamine-dichloride in the cells with acquired radioresistance. Interestingly, the radiosensitizing effect of API-2 was canceled by overexpression of cyclin D1 whereas Cdk4-I was still able to sensitize cells with cyclin D1 overexpression. Cyclin D1/Cdk4 is a critical target of the AKT survival signaling pathway responsible for tumor radioresistance. Targeting the AKT/GSK3β/cyclin D1/Cdk4 pathway would provide a novel approach to improve fractionated RT and would have an impact on tumor eradication in combination with chemotherapy. Copyright © 2011

  14. Rho iso-alpha acids from hops inhibit the GSK-3/NF-κB pathway and reduce inflammatory markers associated with bone and cartilage degradation

    Directory of Open Access Journals (Sweden)

    Bland Jeffrey S

    2009-08-01

    Full Text Available Abstract Background Rho iso-alpha acids (RIAA from hops have been shown to have anti-inflammatory properties. To understand the mechanisms, we evaluated the effect of RIAA in cell signaling pathways and inflammatory markers using various in vitro models. We also investigated their therapeutic effect in mice with collagen-induced arthritis. Methods The LPS-stimulated RAW 264.7 macrophages were used to evaluate the effect of RIAA on the NF-κB and MAPK signaling pathways; phosphorylation of ERK1/2, p38 and JNK was assessed by western blotting and NF-κB binding by electrophoretic mobility shift assays. Effect on the NF-κB activity was evaluated by the luciferase reporter assays in LPS-stimulated RAW 264.7 cells. GSK-3α/β kinase activity was measured in cell-free assays. The inhibitory effect of RIAA on inflammatory markers was assessed by measuring nitric oxide in LPS-stimulated RAW 264.7 cells, RANKL-mediated TRAP activity in transformed osteoclasts, and TNF-α/IL-1β-mediated MMP-13 expression in SW1353 cells. Mice with collagen-induced arthritis were fed with RIAA for 2 weeks. Symptoms of joint swelling, arthritic index and joint damage were assessed. Results RIAA selectively inhibited the NF-κB pathway while having no effect on ERK1/2, p38 and JNK phosphorylation in LPS-stimulated RAW 264.7 cells. RIAA also inhibited GSK-3α/β kinase activity and GSK-3β dependent phosphorylation of β-catenin in RAW 264.7 cells. In addition, RIAA inhibited NF-κB-mediated inflammatory markers in various cell models, including nitric oxide in LPS-stimulated RAW 264.7 cells, RANKL-mediated TRAP activity in transformed osteoclasts, and TNF-α/IL-1β-mediated MMP-13 expression in SW1353 human chondrosarcoma cells. Finally, in a mouse model of collagen-induced arthritis, RIAA ameliorated joint damage as evidenced by significant reduction of the arthritis index and histology score; at 250 mg/kg-body weight, RIAA had efficacy similar to that of 20 mg

  15. An Isoxazole Chalcone Derivative Enhances Melanogenesis in B16 Melanoma Cells via the Akt/GSK3β/β-Catenin Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Li Yin

    2017-11-01

    Full Text Available Plants or plant-derived products have been routinely used in several traditional medicine systems for vitiligo treatment. It is well-known that melanogenesis can be promoted by certain flavonoid compounds isolated from the traditional Uyghur medicinal plant, Kaliziri. Therefore, Chalcones, one class of flavonoid compounds, has become an interesting target for the development of anti-vitiligo agents. A series of novel isoxazole chalcone derivatives have been designed, synthesized, and evaluated for biological activities by our group. Among them, derivative 1-(4-((3-phenylisoxazol-5-ylmethoxyphenyl-3-phenylprop-2-en-1-one (PMPP was identified as a potent tyrosinase activator with better activity and lower toxicity than the positive control 8-methoxypsoralen (8-MOP in this study. Further investigations revealed that Akt and GSK3β were the signaling pathways involved in the hyperpigmentation of PMPP. Overall, these studies may provide a convenient and novel approach for the further development of anti-vitiligo agents.

  16. Enriched environment prevents hypobaric hypoxia induced memory impairment and neurodegeneration: role of BDNF/PI3K/GSK3β pathway coupled with CREB activation.

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    Vishal Jain

    Full Text Available Adverse environmental conditions such as hypobaric hypoxia (HH cause memory impairment by affecting cellular machinery leading to neurodegeneration. Providing enriched environment (EE is found to be beneficial for curing several neurodegenerative disorders. The protective role of EE in preventing HH induced neuronal death has been reported previously but the involved mechanism is still not clearly understood. The present study is an attempt to verify the impact of EE on spatial memory during HH and also to explore the possible role of neurotrophin in EE mediated neuroprotection. Signaling mechanism involved in neuroprotection was also explored. Male Sprague Dawley rats were simulated to HH condition in an Animal Decompression Chamber at an altitude of 25000 feet in standard and enriched cages for 7 days. Spatial memory was assessed through Morris Water Maze. Role of different neurotrophins was explored by gene silencing and inhibitors for their respective receptors. Further, using different blockers signaling pathway was also explored. Finding of the present study suggested that EE prevents HH mediated memory impairment and neurodegeneration. Also brain-derived neurotrophic factor (BDNF plays a major role in EE mediated neuroprotection and it effectively prevented neurodegeneration by activating PI3K/AKT pathway resulting in GSK3β inactivation which further inhibits apoptosis. Moreover GSK3β phosphorylation and hence its inactivation upregulates CREB phosphorylation which may also accounts for activation of survival machinery in cells and provides neuroprotection. From these observations it can be postulated that EE has a therapeutic potential in amelioration of HH induced memory impairment and neurodegeneration. Hence it may be used as a non invasive and non pharmacological intervention against various neurological disorders.

  17. Enriched environment prevents hypobaric hypoxia induced memory impairment and neurodegeneration: role of BDNF/PI3K/GSK3β pathway coupled with CREB activation.

    Science.gov (United States)

    Jain, Vishal; Baitharu, Iswar; Prasad, Dipti; Ilavazhagan, Govindasamy

    2013-01-01

    Adverse environmental conditions such as hypobaric hypoxia (HH) cause memory impairment by affecting cellular machinery leading to neurodegeneration. Providing enriched environment (EE) is found to be beneficial for curing several neurodegenerative disorders. The protective role of EE in preventing HH induced neuronal death has been reported previously but the involved mechanism is still not clearly understood. The present study is an attempt to verify the impact of EE on spatial memory during HH and also to explore the possible role of neurotrophin in EE mediated neuroprotection. Signaling mechanism involved in neuroprotection was also explored. Male Sprague Dawley rats were simulated to HH condition in an Animal Decompression Chamber at an altitude of 25000 feet in standard and enriched cages for 7 days. Spatial memory was assessed through Morris Water Maze. Role of different neurotrophins was explored by gene silencing and inhibitors for their respective receptors. Further, using different blockers signaling pathway was also explored. Finding of the present study suggested that EE prevents HH mediated memory impairment and neurodegeneration. Also brain-derived neurotrophic factor (BDNF) plays a major role in EE mediated neuroprotection and it effectively prevented neurodegeneration by activating PI3K/AKT pathway resulting in GSK3β inactivation which further inhibits apoptosis. Moreover GSK3β phosphorylation and hence its inactivation upregulates CREB phosphorylation which may also accounts for activation of survival machinery in cells and provides neuroprotection. From these observations it can be postulated that EE has a therapeutic potential in amelioration of HH induced memory impairment and neurodegeneration. Hence it may be used as a non invasive and non pharmacological intervention against various neurological disorders.

  18. Insulin and GSK3β-inhibition abrogates the infarct sparing-effect of ischemic postconditioning in ex vivo rat hearts.

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    Helgeland, Erik; Wergeland, Anita; Sandøy, Rune M; Askeland, Maren; Aspevik, Anne; Breivik, Lars; Jonassen, Anne K

    2017-06-01

    Pharmacological treatment of reperfusion injury using insulin and GSK3β inhibition has been shown to be cardioprotective, however, their interaction with the endogenous cardioprotective strategy, ischemic postconditioning, is not known. Langendorff perfused ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. For the first 15 min of reperfusion hearts received either vehicle (Ctr), insulin (Ins) or a GSK3β inhibitor (SB415286; SB41), with or without interruption of ischemic postconditioning (IPost; 3 × 30 s of global ischemia). In addition, the combination of insulin and SB41 for 15 min was assessed. Insulin, SB41 or IPost significantly reduced infarct size versus vehicle treated controls (IPost 33.5 ± 3.3%, Ins 33.5 ± 3.4%, SB41 30.5 ± 3.0% vs. Ctr 54.7 ± 6.8%, p insulin and SB415286 did not confer additional cardioprotection compared to the treatments given alone (SB41   + Ins 26.7 ± 3.5%, ns). Conversely, combining either of the pharmacological reperfusion treatments with IPost completely abrogated the cardioprotection afforded by the treatments separately (Ins + IPost 59.5 ± 3.4% vs. Ins 33.5 ± 3.4% and SB41 + IPost 50.2 ± 6.6% vs. SB41 30.5 ± 3.0%, both p insulin and SB41 interferes with the cardioprotection afforded by ischemic postconditioning.

  19. Activation of Akt/GSK3β and Akt/Bcl-2 signaling pathways in nickel-transformed BEAS-2B cells.

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    Pan, Jing-Ju; Chang, Qing-Shan; Wang, Xin; Son, Young-Ok; Liu, Jiankang; Zhang, Zhuo; Bi, Yong-Yi; Shi, Xianglin

    2011-11-01

    The Akt signaling pathway has been implicated in a wide range of cellular functions involving cell survival and proliferation, angiogenesis, metabolism and cell migration. Accumulating evidence suggests that Akt perturbations play an important role in human malignancy. Here, we investigated Akt perturbation in nickel-transformed cells. Chronic treatment of human bronchial epithelial BEAS-2B cells with low doses of nickel chloride resulted in cell transformation demonstrated by anchorage-independent (AI) growth, increased cell growth and alterations of cell growth pattern. Western blot assays show that phosphorylation of Akt at Ser473, but not that of p38, JNK and ERK, was increased in nickel-transformed cells compared with controls. Inhibition of Akt or PI3K by pharmacological or biochemical interference suppressed nickel AI growth and cell growth of transformed cells. Activation of Akt led to inhibition of GSK3β by phosphorylation at Ser9 in nickel-transformed cells. In addition, two major anti-apoptotic proteins of the Bcl family, Bcl-2 and Bcl-XL, were increased in nickel-transformed cells. By employing the small interfering RNA technique (siRNA), our results showed that siRNA Akt attenuated the expression of Bcl-2 and Bcl-XL in nickel-transformed cells, indicating that induction of Bcl-2 and Bcl-XL was likely mediated through Akt. ROS generation was decreased in nickel-transformed cells compared with controls. Moreover, down-regulation of retinoblastoma protein (Rb) was observed in nickel-transformed cells. Taken together, these findings demonstrate that activation of Akt, followed by GSK3β inhibition and Bcl-2, Bcl-XL up-regulation and decrease of ROS generation, along with a synergistic effect of Rb down-regulation may cause apoptosis resistance, contributing to the overall mechanism of nickel carcinogenesis.

  20. Xanthohumol ameliorates lipopolysaccharide (LPS-induced acute lung injury via induction of AMPK/GSK3β-Nrf2 signal axis

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    Hongming Lv

    2017-08-01

    Full Text Available Abundant natural flavonoids can induce nuclear factor-erythroid 2 related factor 2 (Nrf2 and/or AMP-activated protein kinase (AMPK activation, which play crucial roles in the amelioration of various inflammation- and oxidative stress-induced diseases, including acute lung injury (ALI. Xanthohumol (Xn, a principal prenylflavonoid, possesses anti-inflammation and anti-oxidant activities. However, whether Xn could protect from LPS-induced ALI through inducing AMPK/Nrf2 activation and its downstream signals, are still poorly elucidated. Accordingly, we focused on exploring the protective effect of Xn in the context of ALI and the involvement of underlying molecular mechanisms. Our findings indicated that Xn effectively alleviated lung injury by reduction of lung W/D ratio and protein levels, neutrophil infiltration, MDA and MPO formation, and SOD and GSH depletion. Meanwhile, Xn significantly lessened histopathological changes, reactive oxygen species (ROS generation, several cytokines secretion, and iNOS and HMGB1 expression, and inhibited Txnip/NLRP3 inflammasome and NF-κB signaling pathway activation. Additionally, Xn evidently decreased t-BHP-stimulated cell apoptosis, ROS generation and GSH depletion but increased various anti-oxidative enzymes expression regulated by Keap1-Nrf2/ARE activation, which may be associated with AMPK and GSK3β phosphorylation. However, Xn-mediated inflammatory cytokines and ROS production, histopathological changes, Txnip/NLRP3 inflammasome and NF-κB signaling pathway in WT mice were remarkably abrogated in Nrf2-/- mice. Our experimental results firstly provided a support that Xn effectively protected LPS-induced ALI against oxidative stress and inflammation damage which are largely dependent upon upregulation of the Nrf2 pathway via activation of AMPK/GSK3β, thereby suppressing LPS-activated Txnip/NLRP3 inflammasome and NF-κB signaling pathway.

  1. DIF-1 inhibits tumor growth in vivo reducing phosphorylation of GSK-3β and expressions of cyclin D1 and TCF7L2 in cancer model mice.

    Science.gov (United States)

    Takahashi-Yanaga, Fumi; Yoshihara, Tatsuya; Jingushi, Kentaro; Igawa, Kazuhiro; Tomooka, Katsuhiko; Watanabe, Yutaka; Morimoto, Sachio; Nakatsu, Yoshimichi; Tsuzuki, Teruhisa; Nakabeppu, Yusaku; Sasaguri, Toshiyuki

    2014-06-01

    We reported that differentiation-inducing factor-1 (DIF-1), synthesized by Dictyostelium discoideum, inhibited proliferation of various tumor cell lines in vitro by suppressing the Wnt/β-catenin signaling pathway. However, it remained unexplored whether DIF-1 also inhibits tumor growth in vivo. In the present study, therefore, we examined in-vivo effects of DIF-1 using three cancer models: Mutyh-deficient mice with oxidative stress-induced intestinal tumors and nude mice xenografted with the human colon cancer cell line HCT-116 and cervical cancer cell line HeLa. In exploration for an appropriate route of administration, we found that orally administered DIF-1 was absorbed through the digestive tract to elevate its blood concentration to levels enough to suppress tumor cell proliferation. Repeated oral administration of DIF-1 markedly reduced the number and size of intestinal tumors that developed in Mutyh-deficient mice, reducing the phosphorylation level of GSK-3β Ser(9) and the expression levels of early growth response-1 (Egr-1), transcription factor 7-like 2 (TCF7L2) and cyclin D1. DIF-1 also inhibited the growth of HCT-116- and HeLa-xenograft tumors together with decreasing phosphorylation level of GSK-3β Ser(9), although it was not statistically significant in HeLa-xenograft tumors. DIF-1 also suppressed the expressions of Egr-1, TCF7L2 and cyclin D1 in HCT-116-xenograft tumors and those of β-catenin, TCF7L2 and cyclin D1 in HeLa-xenograft tumors. This is the first report to show that DIF-1 inhibits tumor growth in vivo, consistent with its in-vitro action, suggesting that this compound may have potential as a novel anti-tumor agent. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. PTEN Gene Induces Cell Invasion and Migration via Regulating AKT/GSK-3β/β-Catenin Signaling Pathway in Human Gastric Cancer.

    Science.gov (United States)

    Ma, Jingjing; Guo, Xufeng; Zhang, Jixiang; Wu, Dandan; Hu, Xue; Li, Jiao; Lan, Qingzhi; Liu, Ya; Dong, Weiguo

    2017-12-01

    Abnormality of PTEN gene and Wnt/β-catenin signaling have been strongly implicated in various malignant cancers. Recently, it has been noted that a functional interaction/cross-talk was found between the PTEN/PI3K/AKT and Wnt/β-catenin, which plays a key role in the development of cancers. However, few related studies on gastric cancer are available. We examined the expression of PTEN and β-catenin in gastric cancer tissues and detected whether down-regulation of PTEN promotes the migration and invasion in gastric cancer cells along with its underlying mechanism. Immunocytochemistry, a wound healing assay, a Matrigel invasion assay, an immunofluorescence staining were performed to detect expression of PTEN and β-catenin in gastric cancer and adjacent normal tissues, cell migration, cell invasion, and the effects of PTEN knockdown on β-catenin in cells, respectively. Further, MMP-2 and MMP-9 activities were analyzed by zymography assay. The changes in related proteins were further quantified by western blotting. Low expression of PTEN was found in majority of gastric cancer tissues, which showed significant associations with differentiation grade in gastric cancer patients. Further, a negative correlation was revealed between PTEN and β-catenin protein expression in gastric cancer tissues (r = - 0.546, P PTEN knockdown promoted the migration and invasion of cells and caused an obvious increase in p-AKT, p-GSK-3β, β-catenin, E-cadherin, MMP-7, MMP-2, and MMP-9 in gastric cancer cells. Our results indicated PTEN gene might induce cell invasion and migration via regulating AKT/GSK-3β/β-catenin signaling pathway, playing a vital role in the progression of gastric cancer.

  3. Anti-cancer activity ofBacillus amyloliquefaciensAK-0 through cyclin D1 proteasomal degradationviaGSK3β-dependent phosphorylation of threonine-286.

    Science.gov (United States)

    Park, Gwang Hun; Song, Hun Min; Kim, Young Soo; Jeon, Yongho; Koo, Jin Suk; Jeong, Hyung Jin; Jeong, Jin Boo

    2017-06-01

    Microorganisms have been regarded as important sources of novel bioactive natural products. In this study, we evaluated the anti-cancer activity and the potential mechanism of Bacillus amyloliquefaciens AK-0 newly isolated from the rhizosphere soil of Korean ginseng. The ethyl acetate fraction from the culture medium of B. amyloliquefaciens AK-0 (EA-AK0) inhibited markedly the proliferation of human colorectal cancer cells such as HCT116, SW480, LoVo and HT-29. EA-AK0 effectively decreased cyclin D1 protein level in human colorectal cancer cells, while cyclin D1 mRNA level was not changed by EA-AK0 treatment. Inhibition of proteasomal degradation by MG132 blocked EA-AK0-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with MRB. In addition, EA-AK0 increased threonine-286 (T286) phosphorylation of cyclin D1, and a point mutation of T286 to alanine attenuated cyclin D1 degradation by EA-AK0. Inhibition of GSK3β by LiCl suppressed cyclin D1 phosphorylation and downregulation by EA-AKO. From these results, EA-AK0 may suppress the proliferation of human colorectal cancer cells by inducing cyclin D1 proteasomal degradation through GSK3β-dependent T286 phosphorylation. These results indicate that EA-AK0 could be used for treating colorectal cancer and serve as a potential candidate for anticancer drug development. In addition, these findings will be helpful for expanding the knowledge on the molecular anti-cancer mechanisms of EA-AK0.

  4. Sulforaphane suppresses EMT and metastasis in human lung cancer through miR-616-5p-mediated GSK3β/β-catenin signaling pathways.

    Science.gov (United States)

    Wang, Da-Xuan; Zou, Yu-Jiao; Zhuang, Xi-Bin; Chen, Shu-Xing; Lin, Yong; Li, Wen-Lan; Lin, Jun-Jin; Lin, Zhi-Qiang

    2017-02-01

    Sulforaphane is a common antioxidant selectively abundant in cruciferous plants, which exhibits effective anti-cancer actions in control of tumorigenesis or progression of various cancers. A recent study has shown that sulforaphane attenuates the EGFR signaling pathway in non-small cell lung cancer (NSCLC), suggesting its potential anti-metastatic effects. In this study we assessed the involvement of sulforaphane and miR-616-5p in epithelial-mesenchymal transition (EMT) and NSCLC metastasis. Sulforaphane suppressed the cell proliferation in human NSCLC cell lines H1299, 95C and 95D with IC 50 values of 9.52±1.23, 9.04±1.90 and 17.35±2.03 μmol/L, respectively. At low concentrations (1-5 μmol/L), sulforaphane dose-dependently inhibited the migration and invasion of 95D and H1299 cells with relatively high metastatic potential. The anti-metastatic action of sulforaphane was confirmed in 95D and H1299 cell xenografts in vivo. In fresh NSCLC tissue samples from 179 patients, miR-616-5p levels were upregulated in late-stage NSCLCs, and strongly correlated with risk of NSCLC recurrence and metastasis. Consistent with the clinic observation, miR-616-5p levels in the 3 NSCLC cell lines were correlated with their metastatic ability, and were decreased by sulforaphane treatment. Silencing miR-616-5p markedly suppressed the migration and invasion of 95D cells in vitro and NSCLC metastasis in vivo. Further studies revealed that miR-616-5p directly targeted GSK3β and decreased its expression, whereas sulforaphane decreased miR-616-5p levels by histone modification, and followed by inactivation of the GSK3β/β-catenin signaling pathway and inhibition of EMT, which was characterized by loss of epithelial markers and acquisition of a mesenchymal phenotype in NSCLC cells. Our findings suggest that sulforaphane is a potential adjuvant chemotherapeutic agent for the prevention of NSCLC recurrence and metastasis, and miR-616-5p can be clinically utilized as a biomarker or

  5. Tannoid principles of Emblica officinalis attenuated aluminum chloride induced apoptosis by suppressing oxidative stress and tau pathology via Akt/GSK-3βsignaling pathway.

    Science.gov (United States)

    Justin Thenmozhi, Arokiasamy; Dhivyabharathi, Mathiyazahan; Manivasagam, Thamilarasan; Essa, Musthafa Mohamed

    2016-12-24

    Fruits of Phyllanthus emblica Linn. or Emblica officinalis Gaertn. (Phyllanthaceae) are used in Ayurveda, Siddha, Unani, Arabic, Tibetan and various other folk medicinal systems to promote intelligence and memory. Recent study from our lab indicated the neuroprotective effect of tannoids principles of Emblica officinalis (EoT) against memory loss caused by aluminum chloride (AlCl 3 ) intoxication through attenuating acetylcholine esterase activity and the expression of amyloid β protein biosynthesis related markers. However the molecular mechanism of EoT has not yet been fully elucidated. The aim of the present study was to further investigate the neuroprotective mechanisms of EoT against AlCl 3 -induced cognitive deficits, tau hyperphosphorylation, oxidative stress and apoptosis. Rats were treated with AlCl 3 for 60 days to induce biochemical and physiological abnormalities similar to AD patients. AD rats were treated with EoT (100mg/kg., bw. oral) for 60 days. For the examination of neuroprotective effect of EoT, behavior analysis, biochemical estimations and western blot were performed in the hippocampus and cortex of control, EoT treated and untreated AD rats. Intraperitoneal injections of AlCl 3 (100mg/kg., b.w.) for 60 days enhanced the learning and memory deficits, levels of TBARS and diminished the levels of reduced glutathione and activities of enzymatic antioxidants as compared to control group. Moreover toxicity of AlCl 3 is accompanied by the enhanced expressions of Bax, caspases-3,-9, cytosolic cytochrome c (cyto c), and pTau along with diminished expressions of Bcl-2, mitochondrial cyto c, pGSK-3β and pAkt. Coadministration of EoT nullified the cognitive deficits, biochemical abnormalities and apoptosis induced by AlCl 3 treatment. Moreover EoT prevents tau hyperphosphorylation by targeting the GSK-3β/Akt signaling pathway. This study confirms that EoT would be used as a potential drug candidate for AD and other tau pathology-related neuronal

  6. DNA damage stress and inhibition of Jak2-V617F cause its degradation and synergistically induce apoptosis through activation of GSK3β.

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    Toshikage Nagao

    Full Text Available The cytoplasmic tyrosine kinase Jak2 plays a crucial role in cytokine receptor signaling in hematopoietic cells. The activated Jak2-V617F mutant is present in most cases of BCR/ABL-negative myeloproliferative neoplasms and constitutively activates downstream signals from homodimeric cytokine receptors, such as the erythropoietin receptor (EpoR. Here we examine the effects of DNA damage stress on Jak2 or Jak2-V617F and on induction of apoptosis in hematopoietic cells. Etoposide or doxorubicin dose-dependently decreased the expression level of Jak2 in UT7 or 32D cells expressing EpoR in the absence of Epo and that of exogenously expressed Jak2-V617F in UT7 cells when cotreated with the Jak2 inhibitor JakI-1 or AG490. Studies with pharmacological inhibitors and genetic manipulations further showed that downregulation of the PI3K/Akt pathway leading to the activation of GSK3β may be involved in downregulation of Jak2 or Jak2-V617F as well as in synergistic induction of Bax activation and apoptosis. The downregulation of Jak2 was inhibited by the proteasome inhibitor MG132 or by expression of both of loss-of-function mutants of c-Cbl and Cbl-b, E3 ubiquitin ligases which facilitated ubiquitination of Jak2-V617F when co-expressed in 293T cells. The pan-caspase inhibitor Boc-d-fmk also inhibited the Jak2 downregulation as well as appearance of a 100-kDa fragment that contained the N-terminal portion of Jak2 in response to DNA damage. Together, these data suggest that DNA damage stress with simultaneous inhibition of the kinase activity causes degradation of Jak2 or Jak2-V617F by caspase cleavage and proteasomal degradation through GSK3β activation, which is closely involved in synergistic induction of apoptosis in hematopoietic cells.

  7. An anti-cancer WxxxE-containing azurin polypeptide inhibits Rac1-dependent STAT3 and ERK/GSK-3β signaling in breast cancer cells.

    Science.gov (United States)

    Zhang, Zhe; Luo, Zhiyong; Min, Wenpu; Zhang, Lin; Wu, Yaqun; Hu, Xiaopeng

    2017-06-27

    In our previous study, we characterized a mycoplasmal small GTPase-like polypeptide of 240 amino acids that possesses an N-terminal WVLGE sequence. The N-terminal WVLGE sequence promotes activation of Rac1 and subsequent host cancer cell proliferation. To investigate the function of the WxxxE motif in the interaction with Rac1 and host tumor progression, we synthesized a 35-amino acid WVLGE-containing polypeptide derived from a cell-penetrating peptide derived from the azurin protein. We verified that the WVLGE-containing polypeptide targeted MCF-7 cells rather than MCF-10A cells. However, the WVLGE-containing polypeptide inhibited activation of Rac1 and induced cellular phenotypes that resulted from inhibition of Rac1. In addition, the WVLGE-containing polypeptide down-regulated phosphorylation of the STAT3 and ERK/GSK-3β signaling pathways, and this effect was abolished by either stimulation or inhibition of Rac1 activity. We also found that the WVLGE-containing polypeptide has a Rac1-dependent potential to suppress breast cancer growth in vitro and in vivo. We suggest that by acting as a Rac1 inhibitor, this novel polypeptide may be useful for the treatment of breast cancer.

  8. Gas6/Axl Axis Contributes to Chemoresistance and Metastasis in Breast Cancer through Akt/GSK-3β/β-catenin Signaling.

    Science.gov (United States)

    Wang, Cun; Jin, Haojie; Wang, Ning; Fan, Shaohua; Wang, Yanyan; Zhang, Yurong; Wei, Lin; Tao, Xuemei; Gu, Dishui; Zhao, Fangyu; Fang, Jingyuan; Yao, Ming; Qin, Wenxin

    2016-01-01

    Chemoresistance in breast cancer has been of great interest in past studies. However, the development of rational therapeutic strategies targeting chemoresistant cells is still a challenge in clinical oncology. By integrating data from global differences of gene expression and phospho-receptor tyrosine kinases between sensitive parental cells (MCF-7) and doxorubicin-resistant cells (MCF-7/ADR), we identified Axl as a potential target for chemoresistance and metastasis in multidrug resistant breast cancer cells. We analyzed Axl expression in 57 breast cancer cell lines and detected a dramatic increase in its expression level in mesenchymal breast cancer cell lines. Axl silencing suppressed invasive and metastatic potentials of chemoresistant breast cancer cells as well as increased elimination of cancer cells when combined with doxorubicin. Furthermore, in preclinical assays, an Axl inhibitor R428 showed increased cell death upon doxorubicin treatment. Additionally, using phospho-kinase array based proteomic analysis, we identified that Akt/GSK-3β/β-catenin cascade was responsible for Axl-induced cell invasion. Nuclear translocation of β-catenin then induced transcriptional upregulation of ZEB1, which in turn regulated DNA damage repair and doxorubicin-resistance in breast cancer cells. Most importantly, Axl was correlated with its downstream targets in tumor samples and was associated with poor prognosis in breast cancer patients. These results demonstrate that Gas6/Axl axis confers aggressiveness in breast cancer and may represent a therapeutic target for chemoresistance and metastasis.

  9. Autonomous and nonautonomous regulation of axis formation by antagonistic signaling via 7-span cAMP receptors and GSK3 in Dictyostelium.

    Science.gov (United States)

    Ginsburg, G T; Kimmel, A R

    1997-08-15

    Early during Dictyostelium development a fundamental cell-fate decision establishes the anteroposterior (prestalk/prespore) axis. Signaling via the 7-transmembrane cAMP receptor CAR4 is essential for creating and maintaining a normal pattern; car4-null alleles have decreased levels of prestalk-specific mRNAs but enhanced expression of prespore genes. car4- cells produce all of the signals required for prestalk differentiation but lack an extracellular factor necessary for prespore differentiation of wild-type cells. This secreted factor decreases the sensitivity of prespore cells to inhibition by the prestalk morphogen DIF-1. At the cell autonomous level, CAR4 is linked to intracellular circuits that activate prestalk but inhibit prespore differentiation. The autonomous action of CAR4 is antagonistic to the positive intracellular signals mediated by another cAMP receptor, CAR1 and/or CAR3. Additional data indicate that these CAR-mediated pathways converge at the serine/threonine protein kinase GSK3, suggesting that the anterior (prestalk)/posterior (prespore) axis of Dictyostelium is regulated by an ancient mechanism that is shared by the Wnt/Fz circuits for dorsoventral patterning during early Xenopus development and establishing Drosophila segment polarity.

  10. Fenugreek Seed Powder Nullified Aluminium Chloride Induced Memory Loss, Biochemical Changes, Aβ Burden and Apoptosis via Regulating Akt/GSK3β Signaling Pathway.

    Science.gov (United States)

    Prema, Asokan; Thenmozhi, Arokiasamy Justin; Manivasagam, Thamilarasan; Essa, Musthafa Mohamed; Akbar, Mohammed D; Akbar, Mohammed

    2016-01-01

    Alzheimer's disease (AD) is the most common form of dementia that mainly affects the cognitive functions of the aged populations. Trigonella foenum-graecum (L.) (fenugreek), a traditionally well utilized medicinal plant ubiquitously used as one of the main food additive worldwide, is known to have numerous beneficial health effects. Fenugreek seed extract could be able to inhibit the activity of acetylcholinesterase (AChE), a key enzyme involved in the pathogenesis of AD, and further shown to have anti-parkinsonic effect. The present study was aimed to explore the neuroprotective effect of fenugreek seed powder (FSP) against aluminium chloride (AlCl3) induced experimental AD model. Administration of germinated FSP (2.5, 5 and 10% mixed with ground standard rat feed) protected AlCl3 induced memory and learning impairments, Al overload, AChE hyperactivity, amyloid β (Aβ) burden and apoptosis via activating Akt/GSK3β pathway. Our present data could confirm the neuroprotective effect of fenugreek seeds. Further these results could lead a possible therapeutics for the management of neurodegenerative diseases including AD in future.

  11. Fenugreek Seed Powder Nullified Aluminium Chloride Induced Memory Loss, Biochemical Changes, Aβ Burden and Apoptosis via Regulating Akt/GSK3β Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Asokan Prema

    Full Text Available Alzheimer's disease (AD is the most common form of dementia that mainly affects the cognitive functions of the aged populations. Trigonella foenum-graecum (L. (fenugreek, a traditionally well utilized medicinal plant ubiquitously used as one of the main food additive worldwide, is known to have numerous beneficial health effects. Fenugreek seed extract could be able to inhibit the activity of acetylcholinesterase (AChE, a key enzyme involved in the pathogenesis of AD, and further shown to have anti-parkinsonic effect. The present study was aimed to explore the neuroprotective effect of fenugreek seed powder (FSP against aluminium chloride (AlCl3 induced experimental AD model. Administration of germinated FSP (2.5, 5 and 10% mixed with ground standard rat feed protected AlCl3 induced memory and learning impairments, Al overload, AChE hyperactivity, amyloid β (Aβ burden and apoptosis via activating Akt/GSK3β pathway. Our present data could confirm the neuroprotective effect of fenugreek seeds. Further these results could lead a possible therapeutics for the management of neurodegenerative diseases including AD in future.

  12. Erinacine A-Enriched Hericium erinaceus Mycelium Produces Antidepressant-Like Effects through Modulating BDNF/PI3K/Akt/GSK-3β Signaling in Mice

    Science.gov (United States)

    Chiu, Chun-Hung; Chyau, Charng-Cherng; Chen, Chin-Chu; Lee, Li-Ya; Chen, Wan-Ping; Liu, Jia-Ling; Lin, Wen-Hsin; Mong, Mei-Chin

    2018-01-01

    Antidepressant-like effects of ethanolic extract of Hericium erinaceus (HE) mycelium enriched in erinacine A on depressive mice challenged by repeated restraint stress (RS) were examined. HE at 100, 200 or 400 mg/kg body weight/day was orally given to mice for four weeks. After two weeks of HE administration, all mice except the control group went through with 14 days of RS protocol. Stressed mice exhibited various behavioral alterations, such as extending immobility time in the tail suspension test (TST) and forced swimming test (FST), and increasing the number of entries in open arm (POAE) and the time spent in the open arm (PTOA). Moreover, the levels of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) were decreased in the stressed mice, while the levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were increased. These changes were significantly inverted by the administration of HE, especially at the dose of 200 or 400 mg/kg body weight/day. Additionally, HE was shown to activate the BDNF/TrkB/PI3K/Akt/GSK-3β pathways and block the NF-κB signals in mice. Taken together, erinacine A-enriched HE mycelium could reverse the depressive-like behavior caused by RS and was accompanied by the modulation of monoamine neurotransmitters as well as pro-inflammatory cytokines, and regulation of BDNF pathways. Therefore, erinacine A-enriched HE mycelium could be an attractive agent for the treatment of depressive disorders. PMID:29364170

  13. Erinacine A-Enriched Hericium erinaceus Mycelium Produces Antidepressant-Like Effects through Modulating BDNF/PI3K/Akt/GSK-3β Signaling in Mice

    Directory of Open Access Journals (Sweden)

    Chun-Hung Chiu

    2018-01-01

    Full Text Available Antidepressant-like effects of ethanolic extract of Hericium erinaceus (HE mycelium enriched in erinacine A on depressive mice challenged by repeated restraint stress (RS were examined. HE at 100, 200 or 400 mg/kg body weight/day was orally given to mice for four weeks. After two weeks of HE administration, all mice except the control group went through with 14 days of RS protocol. Stressed mice exhibited various behavioral alterations, such as extending immobility time in the tail suspension test (TST and forced swimming test (FST, and increasing the number of entries in open arm (POAE and the time spent in the open arm (PTOA. Moreover, the levels of norepinephrine (NE, dopamine (DA and serotonin (5-HT were decreased in the stressed mice, while the levels of interleukin (IL-6 and tumor necrosis factor (TNF-α were increased. These changes were significantly inverted by the administration of HE, especially at the dose of 200 or 400 mg/kg body weight/day. Additionally, HE was shown to activate the BDNF/TrkB/PI3K/Akt/GSK-3β pathways and block the NF-κB signals in mice. Taken together, erinacine A-enriched HE mycelium could reverse the depressive-like behavior caused by RS and was accompanied by the modulation of monoamine neurotransmitters as well as pro-inflammatory cytokines, and regulation of BDNF pathways. Therefore, erinacine A-enriched HE mycelium could be an attractive agent for the treatment of depressive disorders.

  14. Inhibiting oncogenic signaling by sorafenib activates PUMA via GSK3β and NF-κB to suppress tumor cell growth

    Science.gov (United States)

    Dudgeon, Crissy; Peng, Rui; Wang, Peng; Sebastiani, Andrea; Yu, Jian; Zhang, Lin

    2011-01-01

    Aberrant Ras/Raf/MEK/ERK signaling is one of the most prevalent oncogenic alterations and confers survival advantage to tumor cells. Inhibition of this pathway can effectively suppress tumor cell growth. For example, sorafenib, a multi-kinase inhibitor targeting c-Raf and other oncogenic kinases, has been used clinically for treating advanced liver and kidney tumors, and also has shown efficacy against other malignancies. However, how inhibition of oncogenic signaling by sorafenib and other drugs suppresses tumor cell growth remains unclear. In this study, we found that sorafenib kills cancer cells by activating PUMA, a p53 target and a BH3-only Bcl-2 family protein. Sorafenib treatment induces PUMA in a variety of cancer cells irrespective of their p53 status. Surprisingly, the induction of PUMA by sorafenib is mediated by IκB-independent activation of NF-κB, which directly binds to the PUMA promoter to activate its transcription. NF-κB activation by sorafenib requires GSK3β activation, subsequent to ERK inhibition. Deficiency in PUMA abrogates sorafenib-induced apoptosis and caspase activation, and renders sorafenib resistance in colony formation and xenograft tumor assays. Furthermore, the chemosensitization effect of sorafenib is dependent on PUMA, and involves concurrent PUMA induction through different pathways. BH3 mimetics potentiate the anticancer effects of sorafenib, and restore sorafenib sensitivity in resistant cells. Together, these results demonstrate a key role of PUMA-dependent apoptosis in therapeutic inhibition of Ras/Raf/MEK/ERK signaling. They provide a rationale for manipulating the apoptotic machinery to improve sensitivity and overcome resistance to the therapies that target oncogenic kinase signaling. PMID:22286758

  15. SCD1 Confers Temozolomide Resistance to Human Glioma Cells via the Akt/GSK3β/β-Catenin Signaling Axis

    Directory of Open Access Journals (Sweden)

    Shuang Dai

    2018-01-01

    Full Text Available Resistance to temozolomide (TMZ, the standard chemotherapy agent for glioblastoma (GBM, poses a major clinical challenge to GBM prognosis. Understanding the mechanisms of TMZ resistance can help to identify novel drug targets and more effective therapies. Recent studies suggest that bioenergetic alterations of cancer cells play important roles in drug resistance. In our study, the altered metabolism of cancer cells was observed using a metabolic PCR array. We found that stearoyl-coenzyme A desaturase 1 (SCD1, a key rate-limiting enzyme for synthesis of monounsaturated fatty acids, was significantly upregulated in TMZ-resistant GBM cells compared to their parental counterparts. Overexpression of SCD1 promoted resistance to TMZ in parental GBM cells, whereas SCD1 downregulation by siRNA could re-sensitize TMZ-resistant cells in vitro. Combinational treatment of TMZ and an SCD1-specific inhibitor showed a combined inhibitory effect on TMZ-resistant glioma cells. We also observed that overexpression of SCD1 promoted Akt/GSK3β/β-catenin signaling, while silencing of SCD1 inhibited the signaling. The combination of an Akt activator with exogenous SCD1 or the combined inhibition of Akt and enforced expression of SCD1 resulted in the most significant changes of Akt signaling. Functionally, significantly lower viability and mobility rates were observed in TMZ-resistant cells when treated with Akt inhibitors and an SCD1 inhibitor simultaneously compared to when treated individually. In conclusion, our study identified SCD1 along with its functional pathway as a novel target in the development of TMZ resistance. SCD1 inhibition used alone or in combination with Akt inhibition could effectively overcome TMZ resistance in gliomas.

  16. Curcumin Decreases Hyperphosphorylation of Tau by Down-Regulating Caveolin-1/GSK-3β in N2a/APP695swe Cells and APP/PS1 Double Transgenic Alzheimer's Disease Mice.

    Science.gov (United States)

    Sun, Jieyun; Zhang, Xiong; Wang, Chen; Teng, Zhipeng; Li, Yu

    2017-01-01

    Caveolin-1, the marker protein of membranal caveolae, is not only involved in cholesterol regulation, but also participates in the cleavage of amyloid [Formula: see text]-protein precursor (APP) and the generation of [Formula: see text]-amyloid peptide. It has been reported to be tightly related with Tau. In our previous studies, curcumin has been confirmed to play a neuroprotective role in Alzheimer's disease (AD), but its effects on Caveolin-1, Tau and their correlation, and the mechanism is still unknown. As such, in the present study, N2a/WT cells, N2a/APP695swe cell and six-month-old APP/PS1 double transgenic mice were enrolled. After curcumin treatment, the expression of Caveolin-1, Tau and their relationship was detected, and the potential mechanisms were explored. The results showed that in the N2a/APP695swe cells, curcumin not only decreased the number of caveolae, but also made their membrane to be thinner; and curcumin could decreased the expression of phosphorylated Tau (P-Tau(ser404)/Tau) and Caveolin-1 ([Formula: see text]), but the expression of phosphorylated GSK-3[Formula: see text] (P-GSK-3[Formula: see text]/GSK-3[Formula: see text] was increased ([Formula: see text]). In APP/PS1 transgenic mice, the same results were observed. Taken together, our data suggest that curcumin may play an important role in AD via reducing Caveolin-1, inactivating GSK-3[Formula: see text] and inhibiting the abnormal excessive phosphorylation of Tau, which will provide a new theory for AD treatment with curcumin.

  17. Impaired insulin activation and dephosphorylation of glycogen synthase in skeletal muscle of women with polycystic ovary syndrome is reversed by pioglitazone treatment

    DEFF Research Database (Denmark)

    Glintborg, Dorte; Højlund, Kurt; Andersen, Nicoline Resen

    2008-01-01

    . No significant abnormalities in GSK-3alpha or -3beta were found in PCOS subjects. Pioglitazone treatment improved insulin-stimulated glucose metabolism and GS activity in PCOS (all P ...CONTEXT: Insulin resistance is a major risk factor for type 2 diabetes in women with polycystic ovary syndrome (PCOS). The molecular mechanisms underlying reduced insulin-mediated glycogen synthesis in skeletal muscle of patients with PCOS have not been established. SUBJECTS AND METHODS: We...... investigated protein content, activity, and phosphorylation of glycogen synthase (GS) and its major upstream inhibitor, GS kinase (GSK)-3 in skeletal muscle biopsies from 24 PCOS patients (before treatment) and 14 matched control subjects and 10 PCOS patients after 16 wk treatment with pioglitazone. All were...

  18. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  19. Inhibition of Glycogen Synthase Kinase or the Apoptotic Protein p53 Lowers the Threshold of Helium Cardioprotection In Vivo: The Role of Mitochondrial Permeability Transition

    Science.gov (United States)

    Pagel, Paul S.; Krolikowski, John G.; Pratt, Phillip F.; Shim, Yon Hee; Amour, Julien; Warltier, David C.; Weihrauch, Dorothee

    2008-01-01

    BACKGROUND Prosurvival signaling kinases inhibit glycogen synthase kinase-3β (GSK-3β) activity and stimulate apoptotic protein p53 degradation. Helium produces cardioprotection by activating prosurvival kinases, but whether GSK and p53 inhibition mediate this process is unknown. We tested the hypothesis that inhibition of GSK or p53 lowers the threshold of helium cardioprotection via a mitochondrial permeability transition pore (mPTP)-dependent mechanism. METHODS Rabbits (n = 85) instrumented for hemodynamic measurement and subjected to a 30 min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion received 0.9% saline (control), or 1, 3, or 5 cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture (fraction of inspired oxygen concentration = 0.30) before LAD occlusion. Other rabbits received the GSK inhibitor SB 216763 (SB21; 0.2 or 0.6 mg/kg), the p53 inhibitor pifithrin-α (PIF; 1.5 or 3.0 mg/kg), or SB21 (0.2 mg/kg) or PIF (1.5 mg/kg) plus helium (1 cycle) before LAD occlusion in the presence or absence of the mPTP opener atractyloside (5 mg/kg). RESULTS Helium reduced (P < 0.05) myocardial infarct size (35 ± 6 [n = 7], 25 ± 4 [n = 7], and 20 ± 3% [n = 6] of area at risk, 1, 3, and 5 cycles, respectively) compared with control (44 ± 6% [n = 7]). SB21 (0.6 [n = 7] but not 0.2 mg/kg [n = 6]) and PIF (3.0 [n = 6] but not 1.5 mg/kg [n = 7]) also reduced necrosis. SB21 (0.2 mg/kg) or 1.5 mg/kg PIF (1.5 mg/kg) plus helium (1 cycle; n = 6 per group) decreased infarct size to an equivalent degree as three cycles of helium alone, and this cardioprotection was blocked by atractyloside (n = 7 per group). CONCLUSIONS Inhibition of GSK or p53 lowers the threshold of helium-induced preconditioning via a mPTP-dependent mechanism in vivo. PMID:18713881

  20. Zanthoxylum avicennae extract enhances GSK-3β to attenuate β-catenin via phosphatase 2A to block metastatic effects of HA22T cells and hepatocellular carcinoma xenografted nude mice.

    Science.gov (United States)

    Wu, Hsi-Chin; Lay, Ing-Shiow; Shibu, Marthandam Asokan; Ho, Tsung-Jung; Cheng, Shiu-Min; Lin, Chih-Hao; Dung, Tran Duc; Jeng, Long-Bin; Viswanadha, Vijaya Padma; Huang, Chih-Yang

    2017-09-01

    Hepatocellular carcinoma (HCC) metastasis is often associated with the activation of Wnt/β-catenin signaling pathway. Zanthoxylum avicennae (Ying Bu Bo, YBB), a traditional herb with hepatoprotective effect, has been proven to inhibit human HCC in in vivo models however, the in vitro and in vivo effect of YBB on tumor metastasis is not clear yet. To determine whether YBB could inhibit HA22T human HCC cell by acting on β-catenin metastatic signaling in vitro and in vivo, HA22T cells were treated with different concentrations of YBB extracts (YBBE) and analyzed by Immunofluorescence staining assay, western blot analysis, siRNA mediated gene knock-down assays and co-immunoprecipitation assay. Additionally, the HA22T-implanted xenograft nude mice were used to confirm the assessed cellular effects. Mice treated with YBBEs showed a strong increasing trend in PP2Acα, GSK-3β, APC, and β-TrCP/HOS levels, however the expression of β-catenin, p-GSK-3β, TBX 3, and IL8 proteins showed a decreasing trend. YBBE significantly downregulated the nuclear and cytosolic β-catenin levels by facilitating the proteosomal degradation of β-catenin. Moreover, as observed by co-immunoprecipitation assay, YBBE directly promoted the protein interactions between GSK-3β, β-TrCP, APC, PP2A, and β-catenin. In conclusion, both in vitro and in vivo models clearly demonstrated that YBBE inhibits β-catenin involved metastatic signaling in highly metastatic HA22T cells through PP2A activation. © 2017 Wiley Periodicals, Inc.

  1. Topiramate via NMDA, AMPA/kainate, GABAAand Alpha2 receptors and by modulation of CREB/BDNF and Akt/GSK3 signaling pathway exerts neuroprotective effects against methylphenidate-induced neurotoxicity in rats.

    Science.gov (United States)

    Motaghinejad, Majid; Motevalian, Manijeh; Fatima, Sulail; Beiranvand, Tabassom; Mozaffari, Shiva

    2017-11-01

    Chronic abuse of methylphenidate (MPH) often causes neuronal cell death. Topiramate (TPM) carries neuroprotective effects, but its exact mechanism of action remains unclear. In the present study, the role of various doses of TPM and its possible mechanisms, receptors and signaling pathways involved against MPH-induced hippocampal neurodegeneration were evaluated in vivo. Thus, domoic acid (DOM) was used as AMPA/kainate receptor agonist, bicuculline (BIC) as GABA A receptor antagonist, ketamine (KET) as NMDA receptor antagonist, yohimbine (YOH) as α 2 adrenergic receptor antagonist and haloperidol (HAL) was used as dopamine D 2 receptor antagonist. Open field test (OFT) was used to investigate the disturbances in motor activity. Hippocampal neurodegenerative parameters were evaluated. Protein expressions of CREB/BDNF and Akt/GSK3 signaling pathways were also evaluated. Cresyl violet staining was performed to show and confirm the changes in the shape of the cells. TPM (70 and 100 mg/kg) reduced MPH-induced rise in lipid peroxidation, oxidized form of glutathione (GSSG), IL-1β and TNF-α levels, Bax expression and motor activity disturbances. In addition, TPM treatment increased Bcl-2 expression, the level of reduced form of glutathione (GSH) and the levels and activities of superoxide dismutase, glutathione peroxidase and glutathione reductase enzymes. TPM also inhibited MPH-induced hippocampal degeneration. Pretreatment of animals with DOM, BIC, KET and YOH inhibited TPM-induced neuroprotection and increased oxidative stress, neuroinflammation, neuroapoptosis and neurodegeneration while reducing CREB, BDNF and Akt protein expressions. Also pretreatment with DOM, BIC, KET and YOH inhibited TPM-induced decreases in GSK3. It can be concluded that the mentioned receptors by modulation of CREB/BDNF and Akt/GSK3 pathways, are involved in neuroprotection of TPM against MPH-induced neurodegeneration.

  2. The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis.

    Science.gov (United States)

    Ren, Hui; Koo, Junghui; Guan, Baoxiang; Yue, Ping; Deng, Xingming; Chen, Mingwei; Khuri, Fadlo R; Sun, Shi-Yong

    2013-11-22

    The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis. API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction. API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.

  3. Early postnatal exposure to lithium in vitro induces changes in ...

    Indian Academy of Sciences (India)

    2015-04-27

    Apr 27, 2015 ... DFMO, Di-fluoro-methyl-ornithine; GSK-3β, glycogen synthase kinase-3β; I-V curve, current–voltage curve; mEPSCs, miniature excitatory postsynaptic ..... (A) Sample raw traces of the AMPAR mediated mEPSCs recorded (in presence of TTX, APV and Picrotoxin in voltage clamp gap-free mode) at +50 and ...

  4. Fulltext PDF

    Indian Academy of Sciences (India)

    Ding Wu

    2017-07-04

    Jul 4, 2017 ... serem-free culture medium were added to each well instead. After incubation at 37°C for 2h, the ... 6-well plate with serum-free medium for 24 h, and the medium was collected and centrifuged to eliminate the cell debris. ..... Glycogen synthase kinase 3b (GSK-. 3b) generally acts as a downstream signalling ...

  5. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

    DEFF Research Database (Denmark)

    Jespersen, Jakob G; Nedergaard, Anders; Reitelseder, Søren

    2011-01-01

    Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3ß (GSK3ß) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors...

  6. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

    DEFF Research Database (Denmark)

    Jespersen, Jakob G; Nedergaard, Anders; Reitelseder, Søren

    2011-01-01

    Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors...

  7. De novo synthesis of beta-catenin via H-Ras and MEK regulates airway smooth muscle growth

    NARCIS (Netherlands)

    Gosens, Reinoud; Baarsma, Hoeke A.; Heijink, Irene; Oenema, Tjitske A.; Halayko, Andrew J.; Meurs, Herman; Schmidt, Martina

    beta-Catenin is a component of adherens junctions that also acts as a transcriptional coactivator when expressed in the nucleus. Growth factors are believed to regulate the nuclear expression of beta-catenin via inactivation of glycogen synthase kinase 3 (GSK-3) by phosphorylation, resulting in

  8. Roles of Endoplasmic Reticulum Stress in NECA-Induced Cardioprotection against Ischemia/Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Fengmei Xing

    2017-01-01

    Full Text Available Objective. This study aimed to investigate whether the nonselective A2 adenosine receptor agonist NECA induces cardioprotection against myocardial ischemia/reperfusion (I/R injury via glycogen synthase kinase 3β (GSK-3β and the mitochondrial permeability transition pore (mPTP through inhibition of endoplasmic reticulum stress (ERS. Methods and Results. H9c2 cells were exposed to H2O2 for 20 minutes. NECA significantly prevented H2O2-induced TMRE fluorescence reduction, indicating that NECA inhibited the mPTP opening. NECA blocked H2O2-induced GSK-3β phosphorylation and GRP94 expression. NECA increased GSK-3β phosphorylation and decreased GRP94 expression, which were prevented by both ERS inductor 2-DG and PKG inhibitor KT5823, suggesting that NECA may induce cardioprotection through GSK-3β and cGMP/PKG via ERS. In isolated rat hearts, both NECA and the ERS inhibitor TUDCA decreased myocardial infarction, increased GSK-3β phosphorylation, and reversed GRP94 expression at reperfusion, suggesting that NECA protected the heart by inhibiting GSK-3β and ERS. Transmission electron microscopy showed that NECA and TUDCA reduced mitochondrial swelling and endoplasmic reticulum expansion, further supporting that NECA protected the heart by preventing the mPTP opening and ERS. Conclusion. These data suggest that NECA prevents the mPTP opening through inactivation of GSK-3β via ERS inhibition. The cGMP/PKG signaling pathway is responsible for GSK-3β inactivation by NECA.

  9. Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

    Czech Academy of Sciences Publication Activity Database

    Procházka, Radek; Blaha, Milan

    2015-01-01

    Roč. 61, č. 6 (2015), s. 495-502 ISSN 0916-8818 R&D Projects: GA MZe(CZ) QJ1510138 Institutional support: RVO:67985904 Keywords : cumulus oocyte complexes * meiosis resumption * mitogen-activated protein kinase 3/1 (MAPK3/1) Subject RIV: GI - Animal Husbandry ; Breeding Impact factor: 1.453, year: 2015

  10. Increased phosphorylation of skeletal muscle glycogen synthase at NH2-terminal sites during physiological hyperinsulinemia in type 2 diabetes

    DEFF Research Database (Denmark)

    Højlund, Kurt; Staehr, Peter; Hansen, Bo Falck

    2003-01-01

    -hyperinsulinemic clamps. Analysis using phospho-specific antibodies revealed that insulin decreases phosphorylation of sites 3a + 3b in human muscle, and this was accompanied by activation of Akt and inhibition of glycogen synthase kinase-3alpha. In type 2 diabetic subjects these effects of insulin were fully intact...

  11. HIV-1 Tat promotes Kaposi's sarcoma-associated herpesvirus (KSHV vIL-6-induced angiogenesis and tumorigenesis by regulating PI3K/PTEN/AKT/GSK-3β signaling pathway.

    Directory of Open Access Journals (Sweden)

    Feng Zhou

    Full Text Available Kaposi's sarcoma (KS-associated herpesvirus (KSHV is etiologically associated with KS, the most common AIDS-related malignancy. KS is characterized by vast angiogenesis and hyperproliferative spindle cells. We have previously reported that HIV-1 Tat can trigger KSHV reactivation and accelerate Kaposin A-induced tumorigenesis. Here, we explored Tat promotion of KSHV vIL-6-induced angiogenesis and tumorigenesis. Tat promotes vIL-6-induced cell proliferation, cellular transformation, vascular tube formation and VEGF production in culture. Tat enhances vIL-6-induced angiogenesis and tumorigenesis of fibroblasts and human endothelial cells in a chicken chorioallantoic membrane (CAM model. In an allograft model, Tat promotes vIL-6-induced tumorigenesis and expression of CD31, CD34, SMA, VEGF, b-FGF, and cyclin D1. Mechanistic studies indicated Tat activates PI3K and AKT, and inactivates PTEN and GSK-3β in vIL-6 expressing cells. LY294002, a specific inhibitor of PI3K, effectively impaired Tat's promotion of vIL-6-induced tumorigenesis. Together, these results provide the first evidence that Tat might contribute to KS pathogenesis by synergizing with vIL-6, and identify PI3K/AKT pathway as a potential therapeutic target in AIDS-related KS patients.

  12. Nanoparticle Delivered Human Biliverdin Reductase-Based Peptide Increases Glucose Uptake by Activating IRK/Akt/GSK3 Axis: The Peptide Is Effective in the Cell and Wild-Type and Diabetic Ob/Ob Mice

    Directory of Open Access Journals (Sweden)

    Peter E. M. Gibbs

    2016-01-01

    Full Text Available Insulin’s stimulation of glucose uptake by binding to the IRK extracellular domain is compromised in diabetes. We have recently described an unprecedented approach to stimulating glucose uptake. KYCCSRK (P2 peptide, corresponding to the C-terminal segment of hBVR, was effective in binding to and inducing conformational change in the IRK intracellular kinase domain. Although myristoylated P2, made of L-amino acids, was effective in cell culture, its use for animal studies was unsuitable. We developed a peptidase-resistant formulation of the peptide that was efficient in both mice and cell culture systems. The peptide was constructed of D-amino acids, in reverse order, and blocked at both termini. Delivery of the encapsulated peptide to HepG2 and HSKM cells was confirmed by its prolonged effect on stimulation of glucose uptake (>6 h. The peptide improved glucose clearance in both wild-type and Ob/Ob mice; it lowered blood glucose levels and suppressed glucose-stimulated insulin secretion. IRK activity was stimulated in the liver of treated mice and in cultured cells. The peptide potentiated function of IRK’s downstream effector, Akt-GSK3-(α,β axis. Thus, P2-based approach can be used for improving glucose uptake by cells. Also, it allows for screening peptides in vitro and in animal models for treatment of diabetes.

  13. Low birth weight and zygosity status is associated with defective muscle glycogen and glycogen synthase regulation in elderly twins

    DEFF Research Database (Denmark)

    Poulsen, Pernille; Wojtaszewski, Jørgen; Richter, Erik

    2007-01-01

    OBJECTIVE: An adverse intrauterine environment indicated by both low birth weight and monozygosity is associated with an age- or time-dependent reduction in glucose disposal and nonoxidative glucose metabolism in twins, suggesting impaired regulation of muscle glycogen synthesis. RESEARCH DESIGN...... AND METHODS: We measured the activities of glycogen synthase (GS), GS kinase (GSK)3 alpha, GS phosphorylation, and glycogen levels in muscle biopsies obtained from 184 young and elderly twins before and after a euglycemic-hyperinsulinemic clamp. RESULTS: Elderly monozygotic twins had significantly lower...... fractional GS activity amidst higher glycogen and GS protein levels compared with dizygotic twins. In addition, we demonstrated strong nongenetic associations between birth weight and defect muscle glycogen metabolism in elderly--but not in younger--twins. Thus, for every 100 g increase in birth weight...

  14. Dicholine succinate, the neuronal insulin sensitizer, normalizes behavior, REM sleep, hippocampal pGSK3 beta and mRNAs of NMDA receptor subunits in mouse models of depression

    Directory of Open Access Journals (Sweden)

    Brandon H. Cline

    2015-02-01

    Full Text Available Central insulin receptor-mediated signalling is attracting the growing attention of researchers because of rapidly accumulating evidence implicating it in the mechanisms of plasticity, stress response and neuropsychiatric disorders including depression. Dicholine succinate (DS, a mitochondrial complex II substrate, was shown to enhance insulin-receptor mediated signaling in neurons and is regarded as a sensitizer of the neuronal insulin receptor. Compounds enhancing neuronal insulin receptor-mediated transmission exert an antidepressant-like effect in several pre-clinical paradigms of depression; similarly, such properties for DS were found with a stress-induced anhedonia model. Here, we additionally studied the effects of DS on several variables which were ameliorated by other insulin receptor sensitizers in mice. Pre-treatment with DS of chronically stressed C57BL6 mice rescued normal contextual fear conditioning, hippocampal gene expression of NMDA receptor subunit NR2A, the NR2A/NR2B ratio and increased REM sleep rebound after acute predation. In 18-month-old C57BL6 mice, a model of elderly depression, DS restored normal sucrose preference and activated the expression of neural plasticity factors in the hippocampus as shown by Illumina microarray. Finally, young naïve DS-treated C57BL6 mice had reduced depressive- and anxiety-like behaviours and, similarly to imipramine-treated mice, preserved hippocampal levels of the phosphorylated (inactive form of GSK3 beta that was lowered by forced swimming in pharmacologically naïve animals. Thus, DS can ameliorate behavioural and molecular outcomes under a variety of stress- and depression-related conditions. This further highlights neuronal insulin signalling as a new factor of pathogenesis and a potential pharmacotherapy of affective pathologies.

  15. Coagulation factor VIIa-mediated protease-activated receptor 2 activation leads to β-catenin accumulation via the AKT/GSK3β pathway and contributes to breast cancer progression.

    Science.gov (United States)

    Roy, Abhishek; Ansari, Shabbir A; Das, Kaushik; Prasad, Ramesh; Bhattacharya, Anindita; Mallik, Suman; Mukherjee, Ashis; Sen, Prosenjit

    2017-08-18

    Cell migration and invasion are very characteristic features of cancer cells that promote metastasis, which is one of the most common causes of mortality among cancer patients. Emerging evidence has shown that coagulation factors can directly mediate cancer-associated complications either by enhancing thrombus formation or by initiating various signaling events leading to metastatic cancer progression. It is well established that, apart from its distinct role in blood coagulation, coagulation factor FVIIa enhances aggressive behaviors of breast cancer cells, but the underlying signaling mechanisms still remain elusive. To this end, we investigated FVIIa's role in the migration and invasiveness of the breast cancer cell line MDA-MB-231. Consistent with previous observations, we observed that FVIIa increased the migratory and invasive potential of these cells. We also provide molecular evidence that protease-activated receptor 2 activation followed by PI3K-AKT activation and GSK3β inactivation is involved in these processes and that β-catenin, a well known tumor-regulatory protein, contributes to this signaling pathway. The pivotal role of β-catenin was further indicated by the up-regulation of its downstream targets cyclin D1, c-Myc, COX-2, MMP-7, MMP-14, and Claudin-1. β-Catenin knockdown almost completely attenuated the FVIIa-induced enhancement of breast cancer migration and invasion. These findings provide a new perspective to counteract the invasive behavior of breast cancer, indicating that blocking PI3K-AKT pathway-dependent β-catenin accumulation may represent a potential therapeutic approach to control breast cancer. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Tumor-derived CXCL5 promotes human colorectal cancer metastasis through activation of the ERK/Elk-1/Snail and AKT/GSK3β/β-catenin pathways.

    Science.gov (United States)

    Zhao, Jingkun; Ou, Baochi; Han, Dingpei; Wang, Puxiongzhi; Zong, Yaping; Zhu, Congcong; Liu, Di; Zheng, Minhua; Sun, Jing; Feng, Hao; Lu, Aiguo

    2017-03-29

    Metastasis is a major cause of death in human colorectal cancer patients. However, the contribution of chemokines in the tumor microenvironment to tumor metastasis is not fully understood. Herein, we examinined several chemokines in colorectal cancer patients using chemokine ELISA array. Immunohistochemistry was used to detect expression of CXCL5 in colorectal cancer patients tissues. Human HCT116 and SW480 cell lines stably transfected with CXCL5, shCXCL5 and shCXCR2 lentivirus plasmids were used in our in vitro study. Immunoblot, immunofluorescence and transwell assay were used to examine the molecular biology and morphological changes in these cells. In addition, we used nude mice to detect the influence of CXCL5 on tumor metastasis in vivo. We found that CXCL5 was overexpressed in tumor tissues and associated with advanced tumor stage as well as poor prognosis in colorectal cancer patients. We also demonstrated that CXCL5 was primarily expressed in the tumor cell cytoplasm and cell membranes, which may indicate that the CXCL5 was predominantly produced by cancer epithelial cells instead of fibroblasts in the tumor mesenchyme. Additionally, overexpression of CXCL5 enhanced the migration and invasion of colorectal cancer cells by inducing the epithelial-mesenchymal transition (EMT) through activation of the ERK/Elk-1/Snail pathway and the AKT/GSK3β/β-catenin pathway in a CXCR2-dependent manner. The silencing of Snail and β-catenin attenuated CXCL5/CXCR2-enhanced cell migration and invasion in vitro. The elevated expression of CXCL5 can also potentiate the metastasis of colorectal cancer cells to the liver in vivo in nude mice intrasplenic injection model. In conclusion, our findings support CXCL5 as a promoter of colorectal cancer metastasis and a predictor of poor clinical outcomes in colorectal cancer patients.

  17. AcEST: BP911847 [AcEST

    Lifescience Database Archive (English)

    Full Text Available YMU001_000009_H03 495 Adiantum capillus-veneris mRNA. clone: YMU001_000009_H03. BP911847 - Show BP91184...is mRNA. clone: YMU001_000009_H03. Accession BP911847 Tissue type prothallium Developmental stage - Contig I...otein database search programs, Nucleic Acids Res. 25:3389-3402. Query= BP911847|...protein database search programs, Nucleic Acids Res. 25:3389-3402. Query= BP911847|Adiantum capillus-veneris...ogen synthase kinase-3 beta OS=Homo sa... 80 7e-15 sp|P51136|GSK3_DICDI Glycogen synthase kinase-3 OS=Dictyo

  18. Recombinant adenylate kinase 3 from liver fluke Clonorchis sinensis for histochemical analysis and serodiagnosis of clonorchiasis.

    Science.gov (United States)

    Kwon, Soon Bin; Kim, Paul; Woo, Hae Sun; Kim, Tae Yun; Kim, Ju Yeong; Lee, Hye Min; Jang, Yun Soo; Kim, Eun-Min; Yong, Tai-Soon; Seong, Baik Lin

    2018-03-27

    Due to the lack of an effective prophylactic intervention and diagnosis, human liver fluke Clonorchis sinensis continues to afflict a large human population, causing a chronic inflammatory bile duct disease. With an aim to identify target antigens for sensitive serodiagnosis, adenylate kinase 3 of C. sinensis (CsAK3) was successfully expressed in soluble form in Escherichia coli by fusion to an RNA-interacting domain derived from human Lys-tRNA synthetase and purified by Ni2+-affinity chromatography. Anti-CsAK3 serum was raised by immunization of mice, and Western blotting confirmed that CsAK3 was expressed in adult-stage C. sinensis. Histochemical analysis showed that CsAK3 was localized to the subtegumental tissue of C. sinensis and was excreted into the bile duct of the host. When tested against sera from various parasite-infected patients by enzyme-linked immunosorbent assay, the recombinant CsAK3 elicited a specific response to C. sinensis-infected sera. The results suggest that CsAK3, either alone or in combination with other antigens, could be used for improving the clinical diagnosis of clonorchiasis.

  19. G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis.

    Directory of Open Access Journals (Sweden)

    Matthew J Billard

    Full Text Available Triple negative breast cancer (TNBC is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3 is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.

  20. G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis

    Science.gov (United States)

    Billard, Matthew J.; Fitzhugh, David J.; Parker, Joel S.; Brozowski, Jaime M.; McGinnis, Marcus W.; Timoshchenko, Roman G.; Serafin, D. Stephen; Lininger, Ruth; Klauber-Demore, Nancy; Sahagian, Gary; Truong, Young K.; Sassano, Maria F.; Serody, Jonathan S.; Tarrant, Teresa K.

    2016-01-01

    Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis. PMID:27049755

  1. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  2. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  3. Polyketide synthase from Fusarium

    DEFF Research Database (Denmark)

    Kvesel, Kasper; Wimmer, Reinhard; Sørensen, Jens Laurids

    Fungi produce a wide array of secondary metabolites, with interesting bioactivities by help of a number of enzyme complexes. Polyketide synthases (PKS) are a class of multidomain enzymes, producing a class of secondary metabolites called polyketides1,2. Only few structures of PKS’s have been...

  4. Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley.

    Science.gov (United States)

    Nakamura, Shingo; Pourkheirandish, Mohammad; Morishige, Hiromi; Kubo, Yuta; Nakamura, Masako; Ichimura, Kazuya; Seo, Shigemi; Kanamori, Hiroyuki; Wu, Jianzhong; Ando, Tsuyu; Hensel, Goetz; Sameri, Mohammad; Stein, Nils; Sato, Kazuhiro; Matsumoto, Takashi; Yano, Masahiro; Komatsuda, Takao

    2016-03-21

    Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Mixed lineage kinase 3 is required for matrix metalloproteinase expression and invasion in ovarian cancer cells

    International Nuclear Information System (INIS)

    Zhan, Yu; Abi Saab, Widian F.; Modi, Nidhi; Stewart, Amanda M.; Liu, Jinsong; Chadee, Deborah N.

    2012-01-01

    Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells. -- Highlights: ► Ovarian cancer cell lines have high levels of total and phosphorylated MLK3. ► MLK3 is required for MMP expression and activity in ovarian cancer cells. ► MLK3 is required for invasion of SKOV3 and HEY1B ovarian cancer cells. ► MLK3-dependent regulation of MMP-2 and MMP-9 activities requires ERK and JNK.

  6. Activity of glycogen synthase kinase-3á is down-regulated during transient differentiation of human colon cancer HT-29 cells

    Czech Academy of Sciences Publication Activity Database

    Tuháčková, Zdena; Šloncová, Eva; Hlaváček, Jan; Sovová, Vlasta; Velek, Jiří

    1999-01-01

    Roč. 6, č. 4 (1999), s. 827-832 ISSN 1021-335X R&D Projects: GA MZd IZ3517; GA ČR GV312/96/K205; GA ČR GA312/97/1188; GA AV ČR IPP2020702 Institutional research plan: CEZ:AV0Z5052915 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.659, year: 1999

  7. Altered Wnt Signaling Pathway in Cognitive Impairment Caused by Chronic Intermittent Hypoxia: Focus on Glycogen Synthase Kinase-3β and β-catenin

    Directory of Open Access Journals (Sweden)

    Yue-Ying Pan

    2016-01-01

    Conclusions: Wnt/β-catenin signaling pathway abnormalities possibly play an important role in the development of cognitive deficits among mice exposed to CIH and that LiCl might attenuate CIH-induced cognitive impairment via Wnt/β-catenin signaling pathway.

  8. Cleaning up polyketide synthases.

    Science.gov (United States)

    Kwan, Jason C; Schmidt, Eric W

    2012-03-23

    Complex biosynthetic enzymes such as polyketide synthases make mistakes. In this issue of Chemistry & Biology, Jensen et al. report that a discrete family of acyltransferases is responsible for error correction, hydrolyzing key biosynthetic intermediates from a multi-enzyme complex. This activity might find use in understanding polyketide biosynthesis, particularly in uncultivated organisms and in tailoring the synthesis of small molecules. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  10. Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing.

    Directory of Open Access Journals (Sweden)

    Andrew Godwin

    Full Text Available Wound healing consists of a complex, dynamic and overlapping process involving inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular level is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3 controls programmed necrosis in response to TNF-α during inflammation and has been shown to be highly induced during cutaneous wound repair. However, its role in wound healing remains to be demonstrated. To study this, we created dorsal cutaneous wounds on male wild-type (WT and RIPK3-deficient (Ripk3-/- mice. Wound area was measured daily until day 14 post-wound and skin tissues were collected from wound sites at various days for analysis. The wound healing rate in Ripk3-/- mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in Ripk3-/- mice was 53% larger than that of WT mice. H&E and Masson-Trichrome staining analysis showed impaired quality of wound closure in Ripk3-/- wounds with delayed re-epithelialization and angiogenesis and defected granulation tissue formation and collagen deposition compared to WT. The neutrophil infiltration pattern was altered in Ripk3-/- wounds with less neutrophils at day 1 and more neutrophils at day 3. This altered pattern was also reflected in the differential expression of IL-6, KC, IL-1β and TNF-α between WT and Ripk3-/- wounds. MMP-9 protein expression was decreased with increased Timp-1 mRNA in the Ripk3-/- wounds compared to WT. The microvascular density along with the intensity and timing of induction of proangiogenic growth factors VEGF and TGF-β1 were also decreased or delayed in the Ripk3-/- wounds. Furthermore, mouse embryonic fibroblasts (MEFs from Ripk3-/- mice migrated less towards chemoattractants TGF-β1 and PDGF than MEFs from WT mice. These results clearly demonstrate that RIPK3 is an essential molecule to maintain the temporal manner of the

  11. A novel, non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3)

    OpenAIRE

    Fuller, Stephen J; McGuffin, Liam J; Marshall, Andrew K; Giraldo, Alejandro; Pikkarainen, Sampsa; Clerk, Angela; Sugden, Peter

    2012-01-01

    The canonical pathway of regulation of the GCK (germinal centre kinase) III subgroup member, MST3 (mammalian Sterile20-related kinase 3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr178), induction of serine/threonine protein kinase activity and nuclear localization. We identified an alternative ‘non-canonical’ pathway of MST3 activation (regulated primarily thro...

  12. [Advances in isoprene synthase research].

    Science.gov (United States)

    Gou, Yan; Liu, Zhongchuan; Wang, Ganggang

    2017-11-25

    Isoprene emission can lead to significant consequence for atmospheric chemistry. In addition, isoprene is a chemical compound for various industrial applications. In the organisms, isoprene is produced by isoprene synthase that eliminates the pyrophosphate from the dimethylallyl diphosphate. As a key enzyme of isoprene formation, isoprene synthase plays an important role in the process of natural emission and artificial synthesis of isoprene. So far, isoprene synthase has been found in various plants. Isoprene synthases from different sources are of conservative structural and similar biochemical properties. In this review, the biochemical and structural characteristics of isoprene synthases from different sources were compared, the catalytic mechanism of isoprene synthase was discussed, and the perspective application of the enzyme in bioengineering was proposed.

  13. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...... expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant....

  14. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  15. Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr286 in squamous cell carcinoma

    International Nuclear Information System (INIS)

    Mori, Jun; Takahashi-Yanaga, Fumi; Miwa, Yoshikazu; Watanabe, Yutaka; Hirata, Masato; Morimoto, Sachio; Shirasuna, Kanemitsu; Sasaguri, Toshiyuki

    2005-01-01

    Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G 0 /G 1 phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3β (GSK-3β). Depletion of endogenous GSK-3β by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3β and found that DIF-1 dephosphorylated GSK-3β on Ser 9 and induced the nuclear translocation of GSK-3β, suggesting that DIF-1 activated GSK-3β. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr 286 was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3β-mediated phosphorylation of Thr 286

  16. Clonal evolution of AML on novel FMS-like tyrosine kinase-3 (FLT3 inhibitor therapy with evolving actionable targets

    Directory of Open Access Journals (Sweden)

    Pashtoon M. Kasi

    2016-01-01

    Full Text Available For acute myeloid leukemia (AML, identification of activating mutations in the FMS-like tyrosine kinase-3 (FLT3 has led to the development of several FLT3-inhibitors. Here we present clinical and next generation sequencing data at the time of progression of a patient on a novel FLT3-inhibitor clinical trial (ASP2215 to show that employing therapeutic interventions with these novel targeted therapies can lead to consequences secondary to selective pressure and clonal evolution of cancer. We describe novel findings alongside data on treatment directed towards actionable aberrations acquired during the process. (Clinical Trial: NCT02014558; registered at: 〈https://clinicaltrials.gov/ct2/show/NCT02014558〉

  17. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

    DEFF Research Database (Denmark)

    Ngo, HT; Pham, Long; Kim, JW

    2013-01-01

    and protein levels of MAPKAPK3 were elevated in both HCV subgenomic replicon cells and cell culture-derived HCV (HCVcc)-infected cells. Silencing of MAPKAPK3 expression resulted in decreases in both protein and HCV infectivity levels but not in the intracellular HCV RNA level. We showed that MAPKAPK3......Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray......, approximately 100 cellular proteins were identified as HCV core-interacting partners. Of these candidates, mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) was selected for further characterization. MAPKAPK3 is a serine/threonine protein kinase that is activated by stress and growth...

  18. Neurotrophic Effect of Asiatic acid, a Triterpene of Centella asiatica Against Chronic 1-Methyl 4-Phenyl 1, 2, 3, 6-Tetrahydropyridine Hydrochloride/Probenecid Mouse Model of Parkinson's disease: The Role of MAPK, PI3K-Akt-GSK3β and mTOR Signalling Pathways.

    Science.gov (United States)

    Nataraj, Jagatheesan; Manivasagam, Thamilarasan; Justin Thenmozhi, Arokiasamy; Essa, Musthafa Mohamed

    2017-05-01

    Regulation of various signalling (Ras-MAPK, PI3K and AKT) pathways by augmented activity of neurotrophic factors (NTFs) could prevent or halt the progress of dopaminergic loss in Parkinson's disease (PD). Various in vitro and in vivo experimental studies indicated anti-parkinsonic potential of asiatic acid (AA), a pentacyclic triterpene obtained from Centella asiatica. So the present study is designed to determine the neurotrophic effect of AA against 1-methyl 4-phenyl 1, 2, 3, 6-tetrahydropyridine hydrochloride/probenecid (MPTP/p) neurotoxicity in mice model of PD. AA treatment for 5 weeks significantly attenuated MPTP/p induced motor abnormalities, dopamine depletion and diminished expressions NTFs and tyrosine kinase receptors (TrKB). We further, revealed that AA treatment significantly inhibited the MPTP/p-induced phosphorylation of MAPK/P38 related proteins such as JNK and ERK. Moreover, AA treatment increased the phosphorylation of PI3K, Akt, GSK-3β and mTOR, suggesting that AA activated PI3K/Akt/mTOR signalling pathway, which might be the cause of neuroprotection offered by AA. The present findings provided more elaborate in vivo evidences to support the neuroprotective effect of AA on dopaminergic neurons of chronic Parkinson's disease mouse model and the potential of AA to be developed as a possible new therapeutic target to treat PD.

  19. Phosphoinositide Kinase-3 Status Associated With Presence or Absence of Human Papillomavirus in Head and Neck Squamous Cell Carcinomas

    International Nuclear Information System (INIS)

    Yarbrough, Wendell G.; Whigham, Amy; Brown, Brandee; Roach, Michael; Slebos, Robbert

    2007-01-01

    Purpose: To investigate phosphoinositide kinase-3 (PI3K) activation in relation to human papillomavirus (HPV) status in head and neck squamous cell carcinoma (HNSCC). Methods and Materials: Gene expression microarray data were analyzed to determine differentially expressed genes between HPV(+) and HPV(-) HNSCC. PIK3CA gene expression was confirmed by quantitative reverse transcriptase-polymerase chain reaction in seven HPV(+) and seven HPV(-) primary HNSCCs. PIK3CA mutation status in three HPV(+) and nine HPV(-) cell lines was determined by polymerase chain reaction amplification of hot spot exons (1, 9, 20) followed by direct sequencing. Results: PIK3CA was overexpressed in HPV(+)-associated HNSCC compared with the expression in HPV(-) HNSCC. Activation of PIK3CA by mutation was found in 1 of the 12 tested HNSCC cell lines. Conclusion: Activation of PI3K by mutation of PIK3CA is rare in HNSCC cell lines and was not found in three HPV(+) cell lines. One mechanism by which HPV-associated HNSCC might activate PI3K is increased expression of PIK3CA

  20. Proteomics data on MAP Kinase Kinase 3 knock out bone marrow derived macrophages exposed to cigarette smoke extract

    Directory of Open Access Journals (Sweden)

    Roshni Srivastava

    2017-08-01

    Full Text Available This data article reports changes in the phosphoproteome and total proteome of cigarette smoke extract (CSE exposed WT and MAP Kinase Kinase 3 knock out (MKK3−/− bone marrow derived macrophages (BMDM. The dataset generated is helpful for understanding the mechanism of CSE induced inflammation and the role of MAP kinase signaling pathway. The cellular proteins were labeled with isobaric tags for relative and absolute quantitation (iTRAQ® reagents and analyzed by LC-MS/MS. The standard workflow module for iTRAQ® quantification within the Proteome Discoverer was utilized for the data analysis. Ingenuity Pathway Analysis (IPA software and Reactome was used to identify enriched canonical pathways and molecular networks (Mannam et al., 2016 [1]. All the associated mass spectrometry data has been deposited in the Yale Protein Expression Database (YPED with the web-link to the data: http://yped.med.yale.edu/repository/ViewSeriesMenu.do;jsessionid=6A5CB07543D8B529FAE8C3FCFE29471D?series_id=5044&series_name=MMK3+Deletion+in+MEFs

  1. STRUCTURAL ENZYMOLOGY OF POLYKETIDE SYNTHASES

    OpenAIRE

    Tsai, Shiou-Chuan (Sheryl); Ames, Brian Douglas

    2009-01-01

    This chapter describes structural and associated enzymological studies of polyketide synthases, including isolated single domains and multidomain fragments. The sequence–structure–function relationship of polyketide biosynthesis, compared with homologous fatty acid synthesis, is discussed in detail. Structural enzymology sheds light on sequence and structural motifs that are important for the precise timing, substrate recognition, enzyme catalysis, and protein–protein interactions leading to ...

  2. A novel non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3).

    Science.gov (United States)

    Fuller, Stephen J; McGuffin, Liam J; Marshall, Andrew K; Giraldo, Alejandro; Pikkarainen, Sampsa; Clerk, Angela; Sugden, Peter H

    2012-03-15

    The canonical pathway of regulation of the GCK (germinal centre kinase) III subgroup member, MST3 (mammalian Sterile20-related kinase 3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr(178)), induction of serine/threonine protein kinase activity and nuclear localization. We identified an alternative 'non-canonical' pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein GOLGA2/gm130 (golgin A2/Golgi matrix protein 130). Activation of MST3 by calyculin A (a protein serine/threonine phosphatase 1/2A inhibitor) stimulated (auto)phosphorylation of MST3(Thr(178)) in the catalytic domain with essentially simultaneous cis-autophosphorylation of MST3(Thr(328)) in the regulatory domain, an event also requiring the MST3(341-376) sequence which acts as a putative docking domain. MST3(Thr(178)) phosphorylation increased MST3 kinase activity, but this activity was independent of MST3(Thr(328)) phosphorylation. Interestingly, MST3(Thr(328)) lies immediately C-terminal to a STRAD (Sterile20-related adaptor) pseudokinase-like site identified recently as being involved in binding of GCKIII/GCKVI members to MO25 scaffolding proteins. MST3(Thr(178)/Thr(328)) phosphorylation was concurrent with dissociation of MST3 from GOLGA2/gm130 and association of MST3 with MO25, and MST3(Thr(328)) phosphorylation was necessary for formation of the activated MST3-MO25 holocomplex.

  3. Metabolic Disorder, Inflammation, and Deregulated Molecular Pathways Converging in Pancreatic Cancer Development: Implications for New Therapeutic Strategies

    Energy Technology Data Exchange (ETDEWEB)

    Motoo, Yoshiharu, E-mail: motoo@kanazawa-med.ac.jp [Department of Medical Oncology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293 (Japan); Shimasaki, Takeo [Department of Medical Oncology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293 (Japan); Division of Translational & Clinical Oncology, Cancer Research Institute, Kanazawa University, Kanazawa (Japan); Ishigaki, Yasuhito [Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293 (Japan); Nakajima, Hideo [Department of Medical Oncology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293 (Japan); Kawakami, Kazuyuki; Minamoto, Toshinari [Division of Translational & Clinical Oncology, Cancer Research Institute, Kanazawa University, Kanazawa (Japan)

    2011-01-24

    Pancreatic cancer develops and progresses through complex, cumulative biological processes involving metabolic disorder, local inflammation, and deregulated molecular pathways. The resulting tumor aggressiveness hampers surgical intervention and renders pancreatic cancer resistant to standard chemotherapy and radiation therapy. Based on these pathologic properties, several therapeutic strategies are being developed to reverse refractory pancreatic cancer. Here, we outline molecular targeting therapies, which are primarily directed against growth factor receptor-type tyrosine kinases deregulated in tumors, but have failed to improve the survival of pancreatic cancer patients. Glycogen synthase kinase-3β (GSK3β) is a member of a serine/threonine protein kinase family that plays a critical role in various cellular pathways. GSK3β has also emerged as a mediator of pathological states, including glucose intolerance, inflammation, and various cancers (e.g., pancreatic cancer). We review recent studies that demonstrate the anti-tumor effects of GSK3β inhibition alone or in combination with chemotherapy and radiation. GSK3β inhibition may exert indirect anti-tumor actions in pancreatic cancer by modulating metabolic disorder and inflammation.

  4. Oxidative stress with tau hyperphosphorylation in memory impaired 1,2-diacetylbenzene-treated mice.

    Science.gov (United States)

    Kang, Sin-Woo; Kim, Sung Jin; Kim, Min-Sun

    2017-09-05

    Long-term exposure to organic solvent may be related to the incidence of neuronal diseases, such as, Alzheimer's disease, depression, multiple sclerosis, dementia, Parkinson's disease. Previously, the authors reported 1,2-diacetylbenzene (DAB; a neurotoxic metabolite of 1,2-diethylbenzene) causes central and peripheral neuropathies that lead to motor neuronal deficits. Furthermore, it is known DAB increases oxidative stress and protein adduct levels and impairs hippocampal neurogenesis in mice. The authors examined the relevance of oxidative stress and tau hyperphosphorylation in the hippocampus. Five-week-old male C57BL/6 mice were treated with 1 or 5mg/kg/day DAB for 2weeks. Neither overall body weight increases nor behavioral differences were observed after treatment, but kidney and liver weights decreased. Increased ROS production, activated glycogen synthase kinase-3β (GSK-3β) and tau hyperphosphorylation were observed in hippocampal homogenates. To assess memory impairment, the Morris Water Maze was used. Animals in the DAB-treated groups took longer to reach the platform. Movement patterns of DAB treated mice were more complicated and their swimming speeds were lower than those of controls. When SHSY5Y neuroblastoma cells were pretreated with NAC (an antioxidant) or a GSK-3β inhibitor, the expression of active GSK-3β and tau hyperphosphorylation were reduced. These results suggest ROS produced by DAB causes tau hyperphosphorylation via GSK-3β phosphorylation and it might be related to impaired memory deficit. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Human I-mfa domain proteins specifically interact with KSHV LANA and affect its regulation of Wnt signaling-dependent transcription

    Energy Technology Data Exchange (ETDEWEB)

    Kusano, Shuichi, E-mail: skusano@m2.kufm.kagoshima-u.ac.jp [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Eizuru, Yoshito [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

    2010-06-04

    Kaposi's sarcoma-associated herpes virus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein has been reported to interact with glycogen synthase kinase 3{beta} (GSK-3{beta}) and to negatively regulate its activity, leading to stimulation of GSK-3{beta}-dependent {beta}-catenin degradation. We show here that the I-mfa domain proteins, HIC (human I-mfa domain-containing protein) and I-mfa (inhibitor of MyoD family a), interacted in vivo with LANA through their C-terminal I-mfa domains. This interaction affected the intracellular localization of HIC, inhibited the LANA-dependent transactivation of a {beta}-catenin-regulated reporter construct, and decreased the level of the LANA.GSK-3{beta} complex. These data reveal for the first time that I-mfa domain proteins interact with LANA and negatively regulate LANA-mediated activation of Wnt signaling-dependent transcription by inhibiting the formation of the LANA.GSK-3{beta} complex.

  6. Tau Phosphorylation by GSK3 in Different Conditions

    Directory of Open Access Journals (Sweden)

    Jesús Avila

    2012-01-01

    Full Text Available Almost a 20% of the residues of tau protein are phosphorylatable amino acids: serine, threonine, and tyrosine. In this paper we comment on the consequences for tau of being a phosphoprotein. We will focus on serine/threonine phosphorylation. It will be discussed that, depending on the modified residue in tau molecule, phosphorylation could be protective, in processes like hibernation, or toxic like in development of those diseases known as tauopathies, which are characterized by an hyperphosphorylation and aggregation of tau.

  7. Glia and immune cell signaling in bipolar disorder: insights from neuropharmacology and molecular imaging to clinical application.

    Science.gov (United States)

    Watkins, C C; Sawa, A; Pomper, M G

    2014-01-21

    Bipolar disorder (BD) is a debilitating mental illness characterized by severe fluctuations in mood, sleep, energy and executive functioning. Pharmacological studies of selective serotonin reuptake inhibitors and the monoamine system have helped us to clinically understand bipolar depression. Mood stabilizers such as lithium and valproic acid, the first-line treatments for bipolar mania and depression, inhibit glycogen synthase kinase-3 beta (GSK-3β) and regulate the Wnt pathway. Recent investigations suggest that microglia, the resident immune cells of the brain, provide a physiological link between the serotonin system and the GSK-3β/Wnt pathway through neuroinflammation. We review the pharmacological, translational and brain imaging studies that support a role for microglia in regulating neurotransmitter synthesis and immune cell activation. These investigations provide a model for microglia involvement in the pathophysiology and phenotype of BD that may translate into improved therapies.

  8. Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

    OpenAIRE

    Karuppiah, Vijaykumar; Ranaghan, Kara E.; Leferink, Nicole G. H.; Johannissen, Linus O.; Shanmugam, Muralidharan; Ní Cheallaigh, Aisling; Bennett, Nathan J.; Kearsey, Lewis J.; Takano, Eriko; Gardiner, John M.; van der Kamp, Marc W.; Hay, Sam; Mulholland, Adrian J.; Leys, David; Scrutton, Nigel S.

    2017-01-01

    Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS)...

  9. Lithium prevents rat steroid-related osteonecrosis of the femoral head by β-catenin activation.

    Science.gov (United States)

    Yu, Zefeng; Fan, Lihong; Li, Jia; Ge, Zhaogang; Dang, Xiaoqian; Wang, Kunzheng

    2016-05-01

    This study explored the use of lithium to prevent rat steroid-related osteonecrosis of the femoral head (ONFH) through the modulation of the β-catenin pathway. ONFH was induced by methylprednisolone combined with lipopolysaccharide, and serum lipids were analyzed. ONFH was detected by hematoxylin-eosin staining. Micro-CT-based angiography and bone scanning were performed to analyze vessels and bone structure, respectively. Immunohistochemical staining for peroxisome proliferator-activated receptor gamma (PPARγ), bone morphogenetic protein-2 (BMP-2), and vascular endothelial growth factor (VEGF) was analyzed. Protein levels of phospho-glycogen synthase kinase-3β at Tyr-216 (p-Tyr(216) GSK-3β), total glycogen synthase kinase-3β (GSK-3β) and β-catenin, as well as mRNA levels of GSK-3β and β-catenin in femoral heads, were assessed. The rate of empty bone lacunae in the femoral heads was lower in the lithium and control groups than in the model group. The lithium group showed preventive effects against steroid-related vessel loss by micro-CT-based angiography and VEGF staining. Lithium treatment improved hyperlipidemia and reduced PPARγ expression. Moreover, lithium improved steroid-related bone loss in micro-CT bone scans and BMP-2 staining analyses. Furthermore, local β-catenin was reduced in steroid-related ONFH, and lithium treatment increased β-catenin expression while reducing p-Tyr(216) GSK-3β levels. The local β-catenin pathway was inhibited during steroid-related ONFH. Lithium may enhance angiogenesis and stabilize osteogenic/adipogenic homeostasis during steroid-related ONFH in rats by activating the β-catenin pathway.

  10. The Role of Janus Kinase 3 in the Regulation of Na+/K+ ATPase under Energy Depletion

    Directory of Open Access Journals (Sweden)

    Zohreh Hosseinzadeh

    2015-05-01

    Full Text Available Background/Aims: Janus kinase-3 (JAK3 is activated during energy depletion. Energy-consuming pumps include the Na+/K+-ATPase. The present study explored whether JAK3 regulates Na+/K+-ATPase in dendritic cells (DCs. Methods: Ouabain (100 µM-sensitive (Iouabain and K+-induced (Ipump outward currents were determined by utilizing whole cell patch-clamp, Na+/K+-ATPase α1-subunit mRNA levels by RT-PCR, Na+/K+-ATPase protein abundance by flow cytometry or immunofluorescence, and cellular ATP by luciferase-assay in DCs from bone marrow of JAK3-knockout (jak3-/- or wild-type mice (jak3+/+. Ipump was further determined by voltage clamp in Xenopus oocytes expressing JAK3, active A568VJAK3 or inactive K851AJAK3. Results: Na+/K+-ATPase α1-subunit mRNA and protein levels, as well as Ipump and Iouabain were significantly higher in jak3-/-DCs than in jak3+/+DCs. Energy depletion by 4h pre-treatment with 2,4-dinitro-phenol significantly decreased Ipump in jak3+/+ DCs but not in jak3-/-DCs. Cellular ATP was significantly lower in jak3-/-DCs than in jak3+/+DCs and decreased in both genotypes by 2,4-dinitro-phenol, an effect significantly more pronounced in jak3-/-DCs than in jak3+/+DCs and strongly blunted by ouabain in both jak3+/+ and jak3-/-DCs. Ipump and Iouabain in oocytes were decreased by expression of JAK3 and of A568VJAK3 but not of K851AJAK3. JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenylamino]-6,7-dimethoxyquinazoline, 22 μM enhanced Ipump and Iouabain in JAK3 expressing oocytes. The difference between A568VJAK3 and K851AJAK3 expressing oocytes was virtually abrogated by actinomycin D (50 nM. Conclusions: JAK3 down-regulates Na+/K+-ATPase activity, an effect involving gene expression and profoundly curtailing ATP consumption.

  11. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

    DEFF Research Database (Denmark)

    Jespersen, Jakob G; Nedergaard, Anders; Reitelseder, Søren

    2011-01-01

    involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls.......Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3ß (GSK3ß) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors...

  12. Producing biofuels using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  13. Exercise Training-Induced Adaptations Associated with Increases in Skeletal Muscle Glycogen Content

    Science.gov (United States)

    Manabe, Yasuko; Gollisch, Katja S.C.; Holton, Laura; Kim, Young–Bum; Brandauer, Josef; Fujii, Nobuharu L.; Hirshman, Michael F.; Goodyear, Laurie J.

    2012-01-01

    Chronic exercise training results in numerous skeletal muscle adaptations, including increases in insulin sensitivity and glycogen content. To understand the mechanism for increased muscle glycogen, we studied the effects of exercise training on glycogen regulatory proteins in rat skeletal muscle. Female Sprague Dawley rats performed voluntary wheel running for 1, 4, or 7 weeks. After 7 weeks of training, insulin-stimulated glucose uptake was increased in epitrochlearis muscle. Compared to sedentary control rats, muscle glycogen did not change after 1 week of training, but increased significantly after 4 and 7 weeks. The increases in muscle glycogen were accompanied by elevated glycogen synthase activity and protein expression. To assess the regulation of glycogen synthase, we examined its major activator, protein phosphatase 1 (PP1), and its major deactivator, glycogen synthase kinase 3 (GSK3). Consistent with glycogen synthase activity, PP1 activity was unchanged after 1 week of training but significantly increased after 4 and 7 weeks of training. Protein expression of RGL(GM), another regulatory PP1 subunit, significantly decreased after 4 and 7 weeks of training. Unlike PP1, GSK3 phosphorylation did not follow the pattern of glycogen synthase activity. The ~40% decrease in GSK-3α phosphorylation after 1 week of exercise training persisted until 7 weeks and may function as a negative feedback to elevated glycogen. Our findings suggest that exercise training-induced increases in muscle glycogen content could be regulated by multiple mechanisms including enhanced insulin sensitivity, glycogen synthase expression, allosteric activation of glycogen synthase and PP1activity. PMID:23206309

  14. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  15. Crystal structure of riboflavin synthase

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B. (DuPont); (NWU)

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  16. Heart 6-phosphofructo-2-kinase activation by insulin requires PKB (protein kinase B), but not SGK3 (serum- and glucocorticoid-induced protein kinase 3).

    OpenAIRE

    Mouton, Veronique; Toussaint, Louise; Vertommen, Didier; Gueuning, Marie-Agnes; Maisin, Liliane; Havaux, Xavier; Sanchez-Canedo, Cossette; Bertrand, Luc; Dequiedt, Franck; Hemmings, Brian A; Hue, Louis; Rider, Mark H

    2010-01-01

    On the basis of transfection experiments using a dominant-negative approach, our previous studies suggested that PKB (protein kinase B) was not involved in heart PFK-2 (6-phosphofructo2-kinase) activation by insulin. Therefore we first tested whether SGK3 (serum- and glucocorticoid-induced protein kinase 3) might be involved in this effect. Treatment of recombinant heart PFK-2 with [gamma-32P]ATP and SGK3 in vitro led to PFK-2 activation and phosphorylation at Ser466 and Ser483. However, in H...

  17. OncoPPi-informed discovery of mitogen-activated protein kinase kinase 3 as a novel binding partner of c-Myc | Office of Cancer Genomics

    Science.gov (United States)

    Mitogen-activated protein kinase kinase 3 (MKK3) is a dual threonine/tyrosine protein kinase that regulates inflammation, proliferation and apoptosis through specific phosphorylation and activation of the p38 mitogen-activated protein kinase. However, the role of MKK3 beyond p38-signaling remains elusive. Recently, we reported a protein-protein interaction (PPI) network of cancer-associated genes, termed OncoPPi, as a resource for the scientific community to generate new biological models. Analysis of the OncoPPi connectivity identified MKK3 as one of the major hub proteins in the network.

  18. Crosstalk between osteoprotegerin (OPG), fatty acid synthase (FASN) and, cycloxygenase-2 (COX-2) in breast cancer: implications in carcinogenesis.

    Science.gov (United States)

    Goswami, Sudeshna; Sharma-Walia, Neelam

    2016-09-13

    , GSK3β, and induced apoptosis by caspase-3/7 and caspase-9 activation. But a more efficient and effective decrease in lipid bodies/cell and survival kinase signaling was observed upon combining the drug treatments for the aggressive cancer cells. Collectively, the novel biological crosstalk between OPG, FASN, and COX-2 advocates for combinatorial drug treatment to block these players of carcinogenesis as a promising therapeutic target to treat highly invasive breast cancer.

  19. Inositol polyphosphates contribute to cellular circadian rhythms: Implications for understanding lithium's molecular mechanism.

    Science.gov (United States)

    Wei, Heather; Landgraf, Dominic; Wang, George; McCarthy, Michael J

    2018-01-11

    Most living organisms maintain cell autonomous circadian clocks that synchronize critical biological functions with daily environmental cycles. In mammals, the circadian clock is regulated by inputs from signaling pathways including glycogen synthase kinase 3 (GSK3). The drug lithium has actions on GSK3, and also on inositol metabolism. While it is suspected that lithium's inhibition of GSK3 causes rhythm changes, it is not known if inositol polyphosphates can also affect the circadian clock. We examined whether the signaling molecule inositol hexaphosphate (IP 6 ) has effects on circadian rhythms. Using a bioluminescent reporter (Per2::luc) to measure circadian rhythms, we determined that IP 6 increased rhythm amplitude and shortened period in NIH3T3 cells. The IP 6 effect on amplitude was attenuated by selective siRNA knockdown of GSK3B and pharmacological blockade of AKT kinase. However, unlike lithium, IP 6 did not induce serine-9 phosphorylation of GSK3B. The synthesis of IP 6 involves the enzymes inositol polyphosphate multikinase (IPMK) and inositol pentakisphosphate 2-kinase (IPPK). Knockdown of Ippk had effects opposite to those of IP 6 , decreasing rhythm amplitude and lengthening period. Ipmk knockdown had few effects on rhythm alone, but attenuated the effects of lithium on rhythms. However, lithium did not change the intracellular content of IP 6 in NIH3T3 cells or neurons. Pharmacological inhibition of the IP 6 kinases (IP6K) increased rhythm amplitude and shortened period, suggesting secondary effects of inositol pyrophosphates may underlie the period shortening effect, but not the amplitude increasing effect of IP 6 . Overall, we conclude that inositol phosphates, in particular IP 6 have effects on circadian rhythms. Manipulations affecting IP 6 and related inositol phosphates may offer a novel means through which circadian rhythms can be regulated. Published by Elsevier Inc.

  20. Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

    Science.gov (United States)

    2017-01-01

    Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids. PMID:28966840

  1. Fungal type III polyketide synthases.

    Science.gov (United States)

    Hashimoto, Makoto; Nonaka, Takamasa; Fujii, Isao

    2014-10-01

    This article covers the literature on fungal type III polyketide synthases (PKSs) published from 2005 to 2014. Since the first discovery of fungal type III PKS genes in Aspergillus oryzae, reported in 2005, putative genes for type III PKSs have been discovered in fungal genomes. Compared with type I PKSs, type III PKSs are much less abundant in fungi. However, type III PKSs could have some critical roles in fungi. This article summarizes the studies on fungal type III PKS functional analysis, including Neurospora crassa ORAS, Aspergillus niger AnPKS, Botrytis cinerea BPKS and Aspergillus oryzae CsyA and CsyB. It is mostly in vitro analysis using their recombinant enzymes that has revealed their starter and product specificities. Of these, CsyB was found to be a new kind of type III PKS that catalyses the coupling of two β-keto fatty acyl CoAs. Homology modelling reported in this article supports the importance of the capacity of the acyl binding tunnel and active site cavity in fungal type III PKSs.

  2. Terpene synthases from Cannabis sativa.

    Directory of Open Access Journals (Sweden)

    Judith K Booth

    Full Text Available Cannabis (Cannabis sativa plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E-β-ocimene, (--limonene, (+-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  3. Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits).

    Science.gov (United States)

    Borejsza-Wysocki, W.; Hrazdina, G.

    1996-03-01

    p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response.

  4. Estrogen increases Nrf2 activity through activation of the PI3K pathway in MCF-7 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Juanjuan, E-mail: jwu32@emory.edu [Department of Gynecology and Obstetrics, Emory University School of Medicine, 101 Woodruff Circle, Suite 4211 WMB, Atlanta, GA 30322 (United States); Williams, Devin [Department of Obstetrics and Gynecology, Morehouse School of Medicine, Atlanta, GA 30310 (United States); Walter, Grant A. [Department of Gynecology and Obstetrics, Emory University School of Medicine, 101 Woodruff Circle, Suite 4211 WMB, Atlanta, GA 30322 (United States); Thompson, Winston E. [Department of Obstetrics and Gynecology, Morehouse School of Medicine, Atlanta, GA 30310 (United States); Sidell, Neil [Department of Gynecology and Obstetrics, Emory University School of Medicine, 101 Woodruff Circle, Suite 4211 WMB, Atlanta, GA 30322 (United States)

    2014-11-01

    The actions of the transcription factor Nuclear factor erythroid 2-related factor (Nrf2) in breast cancer have been shown to include both pro-oncogenic and anti-oncogenic activities which is influenced, at least in part, by the hormonal environment. However, direct regulation of Nrf2 by steroid hormones (estrogen and progesterone) has received only scant attention. Nrf2 is known to be regulated by its cytosolic binding protein, Kelch-like ECH-associated protein 1 (Keap1), and by a Keap1-independent mechanism involving a series of phosphorylation steps mediated by phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase 3 beta (GSK3β). Here, we report that estrogen (E2) increases Nrf2 activity in MCF7 breast cancer cells through activation of the PI3K/GSK3β pathway. Utilizing antioxidant response element (ARE)-containing luciferase reporter constructs as read-outs for Nrf2 activity, our data indicated that E2 increased ARE activity >14-fold and enhanced the action of the Nrf2 activators, tertiary butylhydroquinone (tBHQ) and sulforaphane (Sul) 4 to 9 fold compared with cells treated with tBHQ or Sul as single agents. This activity was shown to be an estrogen receptor-mediated phenomenon and was antagonized by progesterone. In addition to its action on the reporter constructs, mRNA and protein levels of heme oxygenase 1, an endogenous target gene of Nrf2, was markedly upregulated by E2 both alone and in combination with tBHQ. Importantly, E2-induced Nrf2 activation was completely suppressed by the PI3K inhibitors LY294002 and Wortmannin while the GSK3β inhibitor CT99021 upregulated Nrf2 activity. Confirmation that E2 was, at least partly, acting through the PI3K/GSK3β pathway was indicated by our finding that E2 increased the phosphorylation status of both GSK3β and Akt, a well-characterized downstream target of PI3K. Together, these results demonstrate a novel mechanism by which E2 can regulate Nrf2 activity in estrogen receptor-positive breast cancer

  5. Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells

    Directory of Open Access Journals (Sweden)

    Zhang Chen-Ou

    2010-12-01

    Full Text Available Abstract Background Urinary bladder cancer is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. Antiproliferative factor (APF is a potent glycopeptide inhibitor of epithelial cell proliferation that was discovered in the urine of patients with interstitial cystitis, a disorder with bladder epithelial thinning and ulceration. APF mediates its antiproliferative activity in primary normal bladder epithelial cells via cytoskeletal associated protein 4 (CKAP4. Because synthetic asialo-APF (as-APF has also been shown to inhibit T24 bladder cancer cell proliferation at nanomolar concentrations in vitro, and because the peptide segment of APF is 100% homologous to part of frizzled 8, we determined whether CKAP4 mediates as-APF inhibition of proliferation and/or downstream Wnt/frizzled signaling events in T24 cells. Methods T24 cells were transfected with double-stranded siRNAs against CKAP4 and treated with synthetic as-APF or inactive control peptide; cells that did not undergo electroporation and cells transfected with non-target (scrambled double-stranded siRNA served as negative controls. Cell proliferation was determined by 3H-thymidine incorporation. Expression of Akt, glycogen synthase kinase 3β (GSK3β, β-catenin, p53, and matrix metalloproteinase 2 (MMP2 mRNA was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR. Akt, GSK-3β, MMP2, β-catenin, and p53 protein expression, plus Akt, GSK-3β, and β-catenin phosphorylation, were determined by Western blot. Results T24 cell proliferation, MMP2 expression, Akt ser473 and thr308 phosphorylation, GSK3β tyr216 phosphorylation, and β-catenin ser45/thr41 phosphorylation were all decreased by APF, whereas p53 expression, and β-catenin ser33,37/thr41 phosphorylation, were increased by APF treatment in non-electroporated and non-target siRNA-transfected cells. Neither mRNA nor total protein expression of Akt, GSK3β, or

  6. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  7. Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

    LENUS (Irish Health Repository)

    Manser, C

    2012-05-31

    A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3β (GSK3β). KLC2 phosphorylation by GSK3β induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3β on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-β (TGFβ) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFβ-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling.

  8. Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle Tribolium castaneum

    Science.gov (United States)

    Fraga, Amanda; Ribeiro, Lupis; Lobato, Mariana; Santos, Vitória; Silva, José Roberto; Gomes, Helga; da Cunha Moraes, Jorge Luiz; de Souza Menezes, Jackson

    2013-01-01

    Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen. PMID:23750237

  9. Piperine Attenuates Pathological Cardiac Fibrosis Via PPAR-γ/AKT Pathways

    Directory of Open Access Journals (Sweden)

    Zhen-Guo Ma

    2017-04-01

    Full Text Available Mitogen-activated protein kinases (MAPKs and AMP­activated protein kinase α (AMPKα play critical roles in the process of cardiac hypertrophy. Previous studies have demonstrated that piperine activates AMPKα and reduces the phosphorylation of extracellular signal-regulated kinase (ERK. However, the effect of piperine on cardiac hypertrophy remains completely unknown. Here, we show that piperine-treated mice had similar hypertrophic responses as mice treated with vehicle but exhibited significantly attenuated cardiac fibrosis after pressure overload or isoprenaline (ISO injection. Piperine inhibited the transformation of cardiac fibroblasts to myofibroblasts induced by transforming growth factor-β (TGF-β or angiotensin II (Ang II in vitro. This anti-fibrotic effect was independent of the AMPKα and MAPK pathway. Piperine blocked activation of protein kinase B (AKT and, downstream, glycogen synthase kinase 3β (GSK3β. The overexpression of constitutively active AKT or the knockdown of GSK3β completely abolished the piperine-mediated protection of cardiac fibroblasts. The cardioprotective effects of piperine were blocked in mice with constitutively active AKT. Pretreatment with GW9662, a specific inhibitor of peroxisome proliferator activated receptor-γ (PPAR-γ, reversed the effect elicited by piperine in vitro. In conclusion, piperine attenuated cardiac fibrosis via the activation of PPAR-γ and the resultant inhibition of AKT/GSK3β.

  10. Lithium blocks ethanol-induced modulation of protein kinases in the developing brain

    International Nuclear Information System (INIS)

    Chakraborty, Goutam; Saito, Mitsuo; Mao, Rui-Fen; Wang, Ray; Vadasz, Csaba; Saito, Mariko

    2008-01-01

    Lithium has been shown to be neuroprotective against various insults including ethanol exposure. We previously reported that ethanol-induced apoptotic neurodegeneration in the postnatal day 7 (P7) mice is associated with decreases in phosphorylation levels of Akt, glycogen synthase kinase-3β (GSK-3β), and AMP-activated protein kinase (AMPK), and alteration in lipid profiles in the brain. Here, P7 mice were injected with ethanol and lithium, and the effects of lithium on ethanol-induced alterations in phosphorylation levels of protein kinases and lipid profiles in the brain were examined. Immunoblot and immunohistochemical analyses showed that lithium significantly blocked ethanol-induced caspase-3 activation and reduction in phosphorylation levels of Akt, GSK-3β, and AMPK. Further, lithium inhibited accumulation of cholesterol ester (ChE) and N-acylphosphatidylethanolamine (NAPE) triggered by ethanol in the brain. These results suggest that Akt, GSK-3β, and AMPK are involved in ethanol-induced neurodegeneration and the neuroprotective effects of lithium by modulating both apoptotic and survival pathways

  11. Differentiation-inducing factor-1 suppresses gene expression of cyclin D1 in tumor cells

    International Nuclear Information System (INIS)

    Yasmin, Tania; Takahashi-Yanaga, Fumi; Mori, Jun; Miwa, Yoshikazu; Hirata, Masato; Watanabe, Yutaka; Morimoto, Sachio; Sasaguri, Toshiyuki

    2005-01-01

    To determine the mechanism by which differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium discoideum, inhibits tumor cell proliferation, we examined the effect of DIF-1 on the gene expression of cyclin D1. DIF-1 strongly reduced the expression of cyclin D1 mRNA and correspondingly decreased the amount of β-catenin in HeLa cells and squamous cell carcinoma cells. DIF-1 activated glycogen synthase kinase-3β (GSK-3β) and inhibition of GSK-3β attenuated the DIF-1-induced β-catenin degradation, indicating the involvement of GSK-3β in this effect. Moreover, DIF-1 reduced the activities of T-cell factor (TCF)/lymphoid enhancer factor (LEF) reporter plasmid and a reporter gene driven by the human cyclin D1 promoter. Eliminating the TCF/LEF consensus site from the cyclin D1 promoter diminished the effect of DIF-1. These results suggest that DIF-1 inhibits Wnt/β-catenin signaling, resulting in the suppression of cyclin D1 promoter activity

  12. Resibufogenin Induces G1-Phase Arrest through the Proteasomal Degradation of Cyclin D1 in Human Malignant Tumor Cells.

    Directory of Open Access Journals (Sweden)

    Masami Ichikawa

    Full Text Available Huachansu, a traditional Chinese medicine prepared from the dried toad skin, has been used in clinical studies for various cancers in China. Resibufogenin is a component of huachansu and classified as bufadienolides. Resibufogenin has been shown to exhibit the anti-proliferative effect against cancer cells. However, the molecular mechanism of resibufogenin remains unknown. Here we report that resibufogenin induces G1-phase arrest with hypophosphorylation of retinoblastoma (RB protein and down-regulation of cyclin D1 expression in human colon cancer HT-29 cells. Since the down-regulation of cyclin D1 was completely blocked by a proteasome inhibitor MG132, the suppression of cyclin D1 expression by resibufogenin was considered to be in a proteasome-dependent manner. It is known that glycogen synthase kinase-3β (GSK-3β induces the proteasomal degradation of cyclin D1. The addition of GSK-3β inhibitor SB216763 inhibited the reduction of cyclin D1 caused by resibufogenin. These effects on cyclin D1 by resibufogenin were also observed in human lung cancer A549 cells. These findings suggest that the anti-proliferative effect of resibufogenin may be attributed to the degradation of cyclin D1 caused by the activation of GSK-3β.

  13. MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3

    International Nuclear Information System (INIS)

    Xu, Guangxian; Zhang, Yilin; Wei, Jun; Jia, Wei; Ge, Zhaohui; Zhang, Zhaobo; Liu, Xiaoming

    2013-01-01

    microRNA 21 (miR-21) has been demonstrated to be significantly elevated in many types of cancers, including the hepatocellular carcinoma (HCC). In the present study, we investigated the role of miR-21 in HCC by identifying its novel targets, as well as its underlying molecular mechanism. The expression of mitogen-activated protein kinase-kinase 3 (MAP2K3) in human HCC tumor tissues and adjacent non-tumor tissues was determined by immunohistochemistry staining (IHC) analysis. The 3’-untranslated region (3’-UTR) of MAP2K3 combined with miR-21 was experimentally verified by a miRNA luciferase reporter approach. Moreover, the role of miR-21 in regulating HCC cell proliferation was analyzed by an MTT assay infected with miR-21mimics/sponge inhibitor Adenoviral viral vectors. By immunohistochemistry staining analysis, we found that mitogen-activated protein kinase-kinase 3 (MAP2K3) was strikingly repressed in the human HCC tumor tissues, in comparison with the adjacent non-tumor tissues in clinical settings. More importantly, the repression of MAP2K3 was inversely correlated with the expression of miR-21 in HCC. Further study demonstrated that the MAP2K3 was a novel direct target of miR-21, which was experimentally validated by a miRNA luciferase reporter approach. In HepG2 cells, inhibition of miR-21 expression with an adenoviral miR-21 sponge vector profoundly suppressed cell proliferation by up-regulating MAP2K3 expression at both mRNA and protein levels. These results provide a clinical evidence that MAP2K3 may be a tumor repressor gene, and it is a direct target of miR-21 in HCC, indicating an underlying mechanism by which miR-21 is able to directly target MAP2K3 and inhibit its expression during the carcinogenesis of HCC, at both transcriptional and post-translational levels. This study also suggests that targeting miR-21-MAP2K3 pathway may be a promising strategy in the prevention and treatment of HCC

  14. Class II recombinant phosphoribosyl diphosphate synthase from spinach

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows...

  15. Evolutionary and mechanistic insights from the reconstruction of (+)-humulene synthases from a modern (+)-Germacrene A Synthase

    OpenAIRE

    Gonzalez Gonzalez, Veronica; Touchet, Sabrina; Grundy, Daniel J.; Faraldos, Juan A.; Allemann, Rudolf Konrad

    2014-01-01

    Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and

  16. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    Endothelial nitric oxide synthase (NOS3) is involved in key steps of immune response. Genetic factors predispose individuals to periodontal disease. This study's aim was to explore the association between NOS3 gene polymorphisms and clinical parameters in patients with periodontal disease. Genomic DNA was obtained ...

  17. Glutamate synthase: An archaeal horizontal gene transfer?

    Indian Academy of Sciences (India)

    (GOGAT) which is a key enzyme in ammonia assimilation in bacteria, algae and plants. It catalyzes the reductive transamidation of amido nitrogen from glutamine to 2-oxoglutarate to form two molecules of glutamate (Temple et al 1998). Glutamate synthases differ according to their molecular weights, subunit compositions, ...

  18. Relationship between endothelial nitric oxide synthase gene ...

    African Journals Online (AJOL)

    Introduction: Endothelial nitric oxide synthase (eNOS), the enzyme in charge of nitric oxide production, plays a crucial role in vascular biology. However, the impact of single nucleotide polymorphisms (SNPs) affecting the gene encoding for eNOS (eNOS) on coronary artery diseases remains under debate and no data were ...

  19. Producing alpha-olefins using polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Katz, Leonard; Steen, Eric J.; Keasling, Jay D.

    2018-01-02

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an .alpha.-olefin, such as 1-hexene or butadiene. The present invention also provides for a host cell comprising the PKS and when cultured produces the .alpha.-olefin.

  20. Expression of Deinococcus geothermalis trehalose synthase gene ...

    African Journals Online (AJOL)

    A novel trehalose synthase gene from Deinococcus geothermalis (DSMZ 11300) containing 1692 bp reading-frame encoding 564 amino acids was amplified using polymerase chain reaction (PCR). The gene was ligated into pET30Ek/LIC vector and expressed after isopropyl β-D-thiogalactopyranoside induction in ...

  1. Cloning and expression of pineapple sucrosephosphate synthase ...

    African Journals Online (AJOL)

    A 1132-base pairs (bp) polymerase-chain-reaction product of sucrose-phosphate synthase (SPS) (EC 2.3.1.14) from pineapple (Ananas comosus cv. Comte de paris) fruit was cloned and nominated as Ac- SPS1. The sequence encodes a putative 377 amino acids protein containing two serine conserved features that had ...

  2. Chronic active Epstein-Barr virus infection as the initial symptom in a Janus kinase 3 deficiency child: Case report and literature review.

    Science.gov (United States)

    Zhong, Linqing; Wang, Wei; Ma, Mingsheng; Gou, Lijuan; Tang, Xiaoyan; Song, Hongmei

    2017-10-01

    With the progress of sequencing technology, an increasing number of atypical primary immunodeficiency (PID) patients have been discovered, including Janus kinase 3 (JAK3) gene deficiency. We report a patient who presented with chronic active Epstein-Barr virus (CAEBV) infection but responded poorly to treatment with ganciclovir. Next-generation sequencing (NGS) was performed, including all known PID genes, after which Sanger sequencing was performed to verify the results. Genetic analysis revealed that our patient had 2 novel compound heterozygous mutations of JAK3, a gene previously reported to cause a rare form of autosomal recessive severe combined immunodeficiency with recurrent infections. The p.H27Q mutation came from his father, while p. R222H from his mother. Thus, his diagnosis was corrected for JAK3-deficiency PID and CAEBV. Maintenance treatment of subcutaneous injection of recombinant human interferon α-2a was given to our patient with 2 MU, 3 times a week. Interferon alpha was applied and the EBV infection was gradually controlled and his symptoms ameliorated remarkably. Our patient is in good health now and did not have relapses. The diagnoses of PID should be taken into consideration when CAEBV patients respond poorly to conventional treatments. Good results of our patient indicate that interferon α-2a may be an alternative treatment for those who are unwilling to accept hematopoietic stem cell transplantation (HSCT) like our patient. Literature review identified 59 additional cases of JAK3 deficiency with various infections.

  3. Engineering of plant type III polyketide synthases.

    Science.gov (United States)

    Wakimoto, Toshiyuki; Morita, Hiroyuki; Abe, Ikuro

    2012-01-01

    Members of the chalcone synthase superfamily of type III polyketide synthases (PKSs) catalyze iterative condensations of CoA thioesters to produce a variety of polyketide scaffolds with remarkable structural diversity and biological activities. The homodimeric type III PKSs share a common three-dimensional overall fold with a conserved Cys-His-Asn catalytic triad; notably, only a slight modification of the active site dramatically expands the catalytic repertoire of the enzymes. In addition, the enzymes exhibit extremely promiscuous substrate specificities, and accept a variety of nonphysiological substrates, making the type III PKSs an excellent platform for the further production of unnatural, novel polyketide scaffolds with promising biological activities. This chapter summarizes recent advances in the engineering of plant type III PKS enzymes in our laboratories, using approaches combining structure-based enzyme engineering and precursor-directed biosynthesis with rationally designed substrate analogs. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Priming phosphorylation of Chk2 by polo-like kinase 3 (Plk3) mediates its full activation by ATM and a downstream checkpoint in response to DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Bahassi, El Mustapha [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States); Myer, David L. [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States); McKenney, Richard J. [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States); Hennigan, Robert F. [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States); Stambrook, Peter J. [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States)]. E-mail: peter.stambrook@uc.edu

    2006-04-11

    The tumor suppressor gene Chk2 encodes a serine/threonine kinase that signals DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 is phosphorylated on threonine 68 (T68) by ataxia-telangiectasia mutated (ATM) protein leading to its activation. We have previously shown that polo-like kinase 3 (Plk3), a protein involved in DNA damage checkpoint and M-phase functions, interacts with and phosphorylates Chk2. When Chk2 was immunoprecipitated from Daudi cells (Plk3-deficient), it had weak kinase activity towards Cdc25C compared with Chk2 derived from T47D cells (Plk3-expressing cells). This activity was restored by addition of recombinant Plk3 in a dose-dependent manner. Plk3 phosphorylates Chk2 at two residues, serine 62 (S62) and serine 73 (S73) in vitro, and this phosphorylation facilitates subsequent phosphorylation of Chk2 on T68 by ATM in response to DNA damage. When the Chk2 mutant construct GFP-Chk2 S73A (serine 73 mutated to alanine) is transfected into cells, it no longer associates with a large complex in vivo, and manifests a significant reduction in kinase activity. It is also inefficiently activated by ATM by phosphorylation at T68 and, in turn, is unable to phosphorylate the Cdc25C peptide 200-256, which contains the inhibitory S216 target phosphorylation residue. As a consequence, tyrosine 15 (Y15) on Cdc2 remains hypophosphorylated, and there is a loss of the G2/M checkpoint. These data describe a functional role for Plk3 in a pathway linking ATM, Plk3, Chk2, Cdc25C and Cdc2 in cellular response to DNA damage.

  5. Loss of serum and glucocorticoid-regulated kinase 3 (SGK3 does not affect proliferation and survival of multiple myeloma cell lines.

    Directory of Open Access Journals (Sweden)

    Stefan Hausmann

    Full Text Available Multiple myeloma (MM is a generally fatal plasma cell cancer that often shows activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt pathway. Targeted pharmacologic therapies, however, have not yet progressed beyond the clinical trial stage, and given the complexity of the PI3K/Akt signalling system (e.g. multiple protein isoforms, diverse feedback regulation mechanisms, strong variability between patients it is mandatory to characterise its ramifications in order to better guide informed decisions about the best therapeutic approaches. Here we explore whether serum and glucocorticoid-regulated kinase 3 (SGK3, a potential downstream effector of PI3K, plays a role in oncogenic signalling in MM cells--either in concert with or independent of Akt. SGK3 was expressed in all MM cell lines and in all primary MM samples tested. Four MM cell lines representing a broad range of intrinsic Akt activation (very strong: MM.1s, moderate: L 363 and JJN-3, absent: AMO-1 were chosen to test the effects of transient SGK3 knockdown alone and in combination with pharmacological inhibition of Akt, PI3K-p110α, or in the context of serum starvation. Although the electroporation protocol led to strong SGK3 depletion for at least 5 days its absence had no substantial effect on the activation status of potential downstream substrates, or on the survival, viability or proliferation of MM cells in all experimental contexts tested. We conclude that it is unlikely that SGK3 plays a significant role for oncogenic signalling in multiple myeloma.

  6. Engineering cotton (+)-delta-cadinene synthase to an altered function: germacrene D-4-ol synthase.

    Science.gov (United States)

    Yoshikuni, Yasuo; Martin, Vincent J J; Ferrin, Thomas E; Keasling, Jay D

    2006-01-01

    The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-delta-cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-delta-cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-delta-cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-delta-cadinene synthase suggested that the G helix plays a very important role in (+)-delta-cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).

  7. Donepezil, an acetylcholine esterase inhibitor, and ABT-239, a histamine H3 receptor antagonist/inverse agonist, require the integrity of brain histamine system to exert biochemical and procognitive effects in the mouse.

    Science.gov (United States)

    Provensi, Gustavo; Costa, Alessia; Passani, M Beatrice; Blandina, Patrizio

    2016-10-01

    Histaminergic H3 receptors (H3R) antagonists enhance cognition in preclinical models and modulate neurotransmission, in particular acetylcholine (ACh) release in the cortex and hippocampus, two brain areas involved in memory processing. The cognitive deficits seen in aging and Alzheimer's disease have been associated with brain cholinergic deficits. Donepezil is one of the acetylcholinesterase (AChE) inhibitor approved for use across the full spectrum of these cognitive disorders. We addressed the question if H3R antagonists and donepezil require an intact histamine neuronal system to exert their procognitive effects. The effect of the H3R antagonist ABT-239 and donepezil were evaluated in the object recognition test (ORT), and on the level of glycogen synthase kinase 3 beta (GSK-3β) phosphorylation in normal and histamine-depleted mice. Systemic administration of ABT-239 or donepezil ameliorated the cognitive performance in the ORT. However, these compounds were ineffective in either genetically (histidine decarboxylase knock-out, HDC-KO) or pharmacologically, by means of intracerebroventricular (i.c.v.) injections of the HDC irreversible inhibitor a-fluoromethylhistidine (a-FMHis), histamine-deficient mice. Western blot analysis revealed that ABT-239 or donepezil systemic treatments increased GSK-3β phosphorylation in cortical and hippocampal homogenates of normal, but not of histamine-depleted mice. Furthermore, administration of the PI3K inhibitor LY294002 that blocks GSK-3β phosphorylation, prevented the procognitive effects of both drugs in normal mice. Our results indicate that both donepezil and ABT-239 require the integrity of the brain histaminergic system to exert their procognitive effects and strongly suggest that impairments of PI3K/AKT/GSK-3β intracellular pathway activation is responsible for the inefficacy of both drugs in histamine-deficient animals. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Neuroprotective effects of JGK-263 in transgenic SOD1-G93A mice of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Ahn, Suk-Won; Jeon, Gye Sun; Kim, Myung-Jin; Shon, Jee-Heun; Kim, Jee-Eun; Shin, Je-Young; Kim, Sung-Min; Kim, Seung Hyun; Ye, In-Hae; Lee, Kwang-Woo; Hong, Yoon-Ho; Sung, Jung-Joon

    2014-05-15

    Glycogen synthase kinase-3β (GSK-3β) activity plays a central role in motor neuron degeneration. GSK-3β inhibitors have been shown to prolong motor neuron survival and suppress disease progression in amyotrophic lateral sclerosis (ALS). In this study, we evaluated the therapeutic effects of a new GSK-3b inhibitor, JGK-263, on ALS in G93A SOD1 transgenic mice. Previously, biochemical efficacy of JGK-263 was observed in normal and mutant (G93A) hSOD1-transfected motor neuronal cell lines (NSC34). Based on these previous results, we administered JGK-263 orally to 93 transgenic mice with the human G93A-mutated SOD1 gene. The mice were divided into three groups: a group administered 20mg/kg JGK-263, a group administered 50mg/kg JGK-263, and a control group not administered with JGK-263. Clinical status, rotarod test, and survival rates of transgenic mice with ALS were evaluated. Sixteen mice from each group were selected for further biochemical study that involved examination of motor neuron count, apoptosis, and cell survival signals. JGK-263 administration remarkably improved motor function and prolonged the time until symptom onset, rotarod failure, and death in transgenic mice with ALS compared to control mice. In JGK-263 groups, choline acetyltransferase (ChAT) staining in the ventral horn of the lower lumbar spinal cord showed a large number of motor neurons, suggesting normal morphology. The neuroprotective effects of JGK-263 in ALS mice were also suggested by western blot analysis of spinal cord tissues in transgenic mice. These results suggest that JGK-263, an oral GSK-3β inhibitor, is promising as a novel therapeutic agent for ALS. Still, further biochemical studies on the underlying mechanisms and safety of JGK-263 are necessary. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. [Effects of electroacupuncture on Wnt-β-catenin signal pathway in annulus fibrosus cells in intervertebral disc in rats with cervical spondylosis].

    Science.gov (United States)

    Liao, Jun; Xie, Qiao-Yu; Zhang, Le; Ke, Mei-Gui

    2014-12-01

    To observe the effects of electroacupuncture (EA) at "Dazhui" (GV 14) on Wnt-β-catenin signal pathway in annulus fibrosus cells in intervertebral disc in rats with cervical spondylosis. Forty SD rats were randomized into a control group, a model group, an EA group and a medication group, 10 rats in each one. Rats in the control group were treated with sham operation, only incision on local skin; rats in the remaining groups were made into cervical spondylosis models. After model establishment, rats in the control group and model group received fixed treatment under identical condition; rats in the EA group were treated with EA at "Dazhui" (GV 14), 30 min per treatment; rats in the medication group were treated with intragastric administration of meloxicam tablets. Treatments were both given once a day, and 14 days were taken as one session; there was an interval of 2 days between two sessions, and totally two sessions were given. After the treatments, immunohistochemistry was applied to measure the expression of Wnt, glycogen synthase kinase-3β (GSK-3β) and Axin in annulus fibrosus cells; western blot was used to test the expression of P-β-catenin. In the control group, there were more positive cells of Wnt, GSK-3β and Axin, which were intensively distributed, deeply colored, and strongly positive; In the model group, there were less positive cells of Wnt, GSK-3β and Axin, which were sparsely distributed and weakly positive. The expression of Wnt, GSK-3β, Axin and P-β-catenin in the model group was less than that in the control group (all P 0.05). EA could delay the degeneration of intervertebral disc, which may be related to EA inhibiting signal pathway of Wnt-β-catenin.

  10. Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals.

    Science.gov (United States)

    Nicholson-Fish, Jessica C; Cousin, Michael A; Smillie, Karen J

    2016-03-01

    The efficient retrieval of synaptic vesicle membrane and cargo in central nerve terminals is dependent on the efficient recruitment of a series of endocytosis modes by different patterns of neuronal activity. During intense neuronal activity the dominant endocytosis mode is activity-dependent endocytosis (ADBE). Triggering of ADBE is linked to calcineurin-mediated dynamin I dephosphorylation since the same stimulation intensities trigger both. Dynamin I dephosphorylation is maximised by a simultaneous inhibition of its kinase glycogen synthase kinase 3 (GSK3) by the protein kinase Akt, however it is unknown how increased neuronal activity is transduced into Akt activation. To address this question we determined how the activity-dependent increases in intracellular free calcium ([Ca(2+)]i) control activation of Akt. This was achieved using either trains of high frequency action potentials to evoke localised [Ca(2+)]i increases at active zones, or a calcium ionophore to raise [Ca(2+)]i uniformly across the nerve terminal. Through the use of either non-specific calcium channel antagonists or intracellular calcium chelators we found that Akt phosphorylation (and subsequent GSK3 phosphorylation) was dependent on localised [Ca(2+)]i increases at the active zone. In an attempt to determine mechanism, we antagonised either phosphatidylinositol 3-kinase (PI3K) or calmodulin. Activity-dependent phosphorylation of both Akt and GSK3 was arrested on inhibition of PI3K, but not calmodulin. Thus localised calcium influx in central nerve terminals activates PI3K via an unknown calcium sensor to trigger the activity-dependent phosphorylation of Akt and GSK3.

  11. Alteration of 11β-Hydroxysteroid Dehydrogenase Type 1 and Glucocorticoid Receptor by Ethanol in Rat Liver and Mouse Hepatoma Cells

    Directory of Open Access Journals (Sweden)

    Zhaojie Meng

    2013-01-01

    for 3 months and 100 mM for 48 h, respectively. Glucose and insulin tolerance tests in vivo were performed, and protein levels of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1 and glucocorticoid receptor (GR in liver and Hepa 1–6 cells were measured. Alterations of key enzymes of gluconeogenesis phosphoenolpyruvate carboxykinase (PEPCK and glucose 6 phosphatase (G6Pase, as well as glycogen synthase kinase 3a (GSK3α, were also examined. The results revealed that glucose levels were increased, and insulin sensitivity was impaired accompanied with liver injury in rats exposed to ethanol compared with controls. The 11β-HSD1, GR, PEPCK, G6Pase, and GSK3α proteins were increased in the liver of rats treated with ethanol compared with controls. Ethanol-exposed Hepa 1–6 cells also showed higher expression of 11β-HSD1, GR, PEPCK, G6Pase, and GSK3α proteins than control cells. After treatment of Hepa 1–6 cells exposed to ethanol with the GR inhibitor RU486, the expression of 11β-HSD1 and GR was significantly decreased. At the same time the increases in PEPCK, G6Pase, and GSK3α levels induced by ethanol in Hepa 1–6 cells were also attenuated by RU486. The results indicate that ethanol causes glucose intolerance by increasing hepatic expression of 11β-HSD1 and GR, which leads to increased expression of gluconeogenic and glycogenolytic enzymes.

  12. Lithium trial in Alzheimer's disease: a randomized, single-blind, placebo-controlled, multicenter 10-week study.

    LENUS (Irish Health Repository)

    Hampel, Harald

    2012-02-01

    OBJECTIVE: Lithium, a first-line drug for the treatment of bipolar depression, has recently been shown to regulate glycogen synthase kinase-3 (GSK-3), a kinase that is involved in the phosphorylation of the tau protein. Since hyperphosphorylation of tau is a core pathological feature in Alzheimer\\'s disease, lithium-induced inhibition of GSK-3 activity may have therapeutic effects in Alzheimer\\'s disease. In the current study, we tested the effect of short-term lithium treatment in patients with Alzheimer\\'s disease. METHOD: A total of 71 patients with mild Alzheimer\\'s disease (Mini-Mental State Examination score > or = 21 and < or = 26) were successfully randomly assigned to placebo (N = 38) or lithium treatment (N = 33) at 6 academic expert memory clinics. The 10-week treatment included a 6-week titration phase to reach the target serum level of lithium (0.5-0.8 mmol\\/L). The primary outcome measures were cerebrospinal fluid (CSF) levels of phosphorylated tau (p-tau) and GSK-3 activity in lymphocytes. Secondary outcome measures were CSF concentration of total tau and beta-amyloid(1-42) (Abeta(1-42)), plasma levels of Abeta(1-42), Alzheimer\\'s Disease Assessment Scale (ADAS)-Cognitive summary scores, MMSE, and Neuropsychiatric Inventory (NPI). Patients were enrolled in the study from November 2004 to July 2005. RESULTS: No treatment effect on GSK-3 activity or CSF-based biomarker concentrations (P > .05) was observed. Lithium treatment did not lead to change in global cognitive performance as measured by the ADAS-Cog subscale (P = .11) or in depressive symptoms. CONCLUSIONS: The current results do not support the notion that lithium treatment may lead to reduced hyperphosphorylation of tau protein after a short 10-week treatment in the Alzheimer\\'s disease target population. TRIAL REGISTRATION: (Controlled-Trials.com) Identifier: ISRCTN72046462.

  13. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  14. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed...... in the sarcolemma as well as the cytoplasm of type I muscle fibres. NADPH diaphorase activity confirmed a higher level of NO synthase activity in the sarcolemma as well as the cytoplasm of type I muscle fibers. Histochemical staining for cytochrome oxidase showed a staining pattern similar to that observed for type...... I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different...

  15. Functional plasticity of paralogous diterpene synthases involved in conifer defense

    OpenAIRE

    Keeling, Christopher I.; Weisshaar, Sabrina; Lin, Roy P. C.; Bohlmann, Jörg

    2008-01-01

    The diversity of terpenoid compounds produced by plants plays an important role in mediating various plant–herbivore, plant–pollinator, and plant–pathogen interactions. This diversity has resulted from gene duplication and neofunctionalization of the enzymes that synthesize and subsequently modify terpenes. Two diterpene synthases in Norway spruce (Picea abies), isopimaradiene synthase and levopimaradiene/abietadiene synthase, provide the hydrocarbon precursors for most of the diterpene resin...