Regulation Of GSK-3β Activity Research Articles

Inhibition of Dickkopf-1 enhances the anti-tumor efficacy of sorafenib via inhibition of the PI3K/Akt and Wnt/β-catenin pathways in hepatocellular carcinoma

BackgroundSorafenib improves the overall survival in patients with advanced hepatocellular carcinoma (HCC). Dickkopf-1 (DKK1) is commonly overexpressed in HCC. In this study, we investigated whether the inhibition of DKK1 enhances the anti-tumor efficacy of sorafenib in HCC.MethodsHCC cells were treated with sorafenib and WAY-262611, which is an inhibitor of DKK1. Transgenic mouse models were also developed using hydrodynamic tail vein injection. Mice were orally administered with sorafenib (32 mg/kg), WAY-262611 (16 mg/kg), or sorafenib + WAY-262611 for 10 days. Mechanisms of sorafenib and WAY-262611 were explored via western blotting, immunostaining, and RNA sequencing.ResultsDKK1 was significantly overexpressed in patients with HCC than in the healthy controls and patients with liver diseases except HCC (all P < 0.05). Compared with sorafenib alone, sorafenib + WAY-262611 significantly inhibited the cell viability, invasion, migration, and colony formation by promoting apoptosis and altering the cell cycles in HCC cells (all P < 0.05). Moreover, sorafenib + WAY-262611 decreased the p110α, phospho-Akt (all P < 0.05), active β-catenin (all P < 0.05) and phospho-GSK-3β (Ser9) expression levels, while increasing the phospho-GSK-3β (Tyr216) expression levels compared with those in the sorafenib alone in vitro and in vivo. In addition, sorafenib + WAY-262611 inhibited tumor progression by regulating cell proliferation and apoptosis, significantly better than sorafenib alone in mouse models.ConclusionsOur results indicate that DKK1 inhibition significantly enhances the anti-tumor efficacy of sorafenib by inhibiting the PI3K/Akt and Wnt/β-catenin pathways via regulation of GSK3β activity, suggesting a novel therapeutic strategy for HCC.7yYqh-521_R5UtWAF2iJ8yVideo

ANXA1-GSK3β interaction and its involvement in NSCLC metastasis.

Although initially discovered and extensively studied for its role in inflammation, Annexin A1 (ANXA1) has been reported to be closely related to cancer in recent years, and its role in cancer is specific to tumor types and tissues. In the present study, we identified ANXA1 as an interaction partner of glycogen synthase kinase 3 beta (GSK3β), a multi-functional serine/threonine kinase tightly associated with cell fate determination and cancer, and assessed the functional significance of GSK3β-ANXA1 interaction in the metastasis of non-small cell lung cancer (NSCLC). We confirmed the interaction between GSK3β and ANXA1 in vitro and in H1299 and A549 cells by Glutathione-S-transferase (GST) pull-down assay and co-immunoprecipitation. We found that ANXA1 negatively regulated the phosphorylation of GSK3β and inhibited the epithelial-mesenchymal transformation (EMT) process and migration and invasion of NSCLC cells. By functional rescue assay, we confirmed that ANXA1 inhibited EMT through the regulation of GSK3β activity and thereby inhibited the migration and invasion of NSCLC cells. Our study sheds light on the function of ANXA1 and GSK3β and provides new elements for the understanding of NSCLC pathogenesis.

Identification of antisense lncRNAs targetingGSK3β as a regulator in major depressive disorder.

Aim: To identify lncRNAs targeting GSK3β in MDD. Materials & methods: The levels of GSK3β and its three targeting lncRNAs (gsk3β antisense AS1, AS2 and AS3) were detected in 52 patients with major depressive disorder (MDD)before and after 8weeks of escitalopram treatment. The functional study was evaluated using the silence of lncR-gsk3βAS2/3. The correlation between lncRNA-gsk3β and 89 MDD patients was analyzed. Human neuron progenitor cellswere used to investigate the functional role of lncRNA-gsk3β in MDD. Results: All three lncRNAs were downregulated in MDD patients but upregulated after treatment. Inhibition of gsk3βAS2/3 reduced GSK3β expression and its phosphorylation levels in the neuron progenitor cells. Conclusion: Our findings suggestthat lncRNA-gsk3βAS3 regulates GSK3β activity in MDD and has potential as a novel therapeutic target.

Aspartate β-hydroxylase targeting in castration-resistant prostate cancer modulates the NOTCH/HIF1α/GSK3β crosstalk.

Castration-resistant prostate cancer (CRPC) is an incurable stage of the disease. A multivariate principal component analysis on CRPC in vitro models identified aspartyl (asparaginyl) β hydrolase (ASPH) as the most relevant molecule associated with the CRPC phenotype. ASPH is overexpressed in various malignant neoplasms and catalyzes the hydroxylation of aspartyl and asparaginyl residues in the epidermal growth factor (EGF)-like domains of proteins like NOTCH receptors and ligands, enhancing cell motility, invasion and metastatic spread. Bioinformatics analyses of ASPH in prostate cancer (PCa) and CRPC datasets indicate that ASPH gene alterations have prognostic value both in PCa and CRPC patients. In CRPC cells, inhibition of ASPH expression obtained through specific small interfering RNA or culturing cells in hypoxic conditions, reduced cell proliferation, invasion and cyclin D1 expression through modulation of the NOTCH signaling. ASPH and HIF1α crosstalk, within a hydroxylation-regulated signaling pathway, might be transiently driven by the oxidative stress evidenced inside CRPC cells. In addition, increased phosphorylation of GSK3β by ASPH silencing demonstrates that ASPH regulates GSK3β activity inhibiting its interactions with upstream kinases. These findings demonstrate the critical involvement of ASPH in CRPC development and may represent an attractive molecular target for therapy.

Hypoxia-Induced miR-675-5p Supports β-Catenin Nuclear Localization by Regulating GSK3-β Activity in Colorectal Cancer Cell Lines.

The reduction of oxygen partial pressure in growing tumors triggers numerous survival strategies driven by the transcription factor complex HIF1 (Hypoxia Inducible Factor-1). Recent evidence revealed that HIF1 promotes rapid and effective phenotypic changes through the induction of non-coding RNAs, whose contribution has not yet been fully described. Here we investigated the role of the hypoxia-induced, long non-coding RNA H19 (lncH19) and its intragenic miRNA (miR-675-5p) into HIF1-Wnt crosstalk. During hypoxic stimulation, colorectal cancer cell lines up-regulated the levels of both the lncH19 and its intragenic miR-675-5p. Loss of expression experiments revealed that miR-675-5p inhibition, in hypoxic cells, hampered β-catenin nuclear localization and its transcriptional activity, while lncH19 silencing did not induce the same effects. Interestingly, our data revealed that miRNA inhibition in hypoxic cells restored the activity of Glycogen Synthase Kinase 3β (GSK-3β) reducing the amount of P-Ser9 kinase, thus unveiling a role of the miR-675-5p in controlling GSK-3β activity. Bioinformatics analyses highlighted the serine/threonine-protein phosphatases PPP2CA, responsible for GSK-3β activation, among the miR-675-5p targets, thus indicating the molecular mediator through which miR-675-5p may control β-catenin nuclear localization. In conclusion, here we demonstrated that the inhibition of the hypoxia-induced non-coding RNA miR-675-5p hampered the nuclear localization of β-catenin by regulating GSK-3β activity, thus proposing the miR-675-5p as a new therapeutic target for the treatment of colorectal cancer.

Imine stilbene analog ameliorate isoproterenol-induced cardiac hypertrophy and hydrogen peroxide-induced apoptosis

Cardiac hypertrophy is an adaptive response to stress, in order to maintain proper cardiac function. However, sustained stress leads to pathological hypertrophy accompanied by maladaptive responses and ultimately heart failure. At the cellular level, cardiomyocyte hypertrophy is characterized by an increase in myocyte size, reactivation of the fetal gene markers, disassembly of the sarcomere and transcriptional remodelling which are regulated by heart-specific transcription factors like MEF2, GATA4 and immediate early genes like c-jun and c-fos.2. It has been explored and established that the hypertrophic process is associated by oxidative stress and mediated by pathways involving several terminal stress kinases like P38, JNK and ERK1/2. Stilbenoids are bioactive polyphenols and earlier studies have shown that imine stilbene exert cardioprotective and anti aging effects by acting as modulators of Sirt1. The present study was aimed at designing and synthesizing a series of imine stilbene analogs and investigate its anti hypertrophic effects and regulatory mechanism in cardiac hypertrophy and apoptosis. Interestingly one of the analog, compound 3e (10 μM) alleviated isoproterenol (ISO, 25 μM) induced hypertrophy in rat cardiomyocyte (H9c2) cells by showing a marked decrease in the myocyte size. Further, compound 3e also restored the cardiac function by activating the metabolic stress sensor, AMPK. Moreover, molecular docking studies showed stable binding between compound 3e and GSK3β suggesting that compound 3e may directly regulate GSK3β activity and ameliorate ISO-induced cardiac hypertrophy. In agreement with this, compound 3e also modulated the crosstalk of all the hypertrophy inducing terminal Kinases by bringing down the expression to near control conditions. The compound also relieved H2O2 (100 μM) mediated ROS and normalized abnormal mitochondrial oxygen demand in hypertrophic conditions indicating the possibility of the compound to show promise in playing a role in cardiac hypertrophy.

Schisantherin A Improves the Learning and Memory by Reducing the Phosphorylation of Tau Protein of the Hippocampus in AD Mice

Memory disorders are the main symptoms of aging and Alzheimer’s disease and seriously affect the quality of life. Schisandra, as a famous traditional Chinese medicine, has been used for modulating “the internal organs” for a thousand years. The total lignans from Schisandra have been scientifically proved to improve learning and memory ability. Since it is unclear which monomer in Schisandra total lignans exerts such a function, we evaluated the potential effects of Schisantherin A (SCA), the main monomer from Schisandra, on improving learning ability and memory in amyloid β-protein (Aβ1-42)-induced Alzheimer’s disease (AD) model mice. We found that SCA (5 mg/kg) significantly prolonged the latency and reduced the number of errors in a step-through test. SCA significantly shortened the time of finding the platform and increased the number of crossing the platform and the residence time in a Morris water maze test. SCA increased superoxide dismutase activities and reduced the Malondialdehyde level of the hippocampal tissue, suggesting its role in reducing oxidative stress in the AD mice. Furthermore, we found that SCA significantly decreased the hyperphosphorylation of Tau by altering glycogen synthase kinase-3β (GSK-3β) phosphorylation on Tyr216 and Ser9. Our results revealed the mechanism underlying SCA-mediated learning and memory improvement by regulating GSK-3β activity and lowering the hyperphosphorylation of Tau protein in the hippocampus of AD mice.

Activation of GSK3β induced by recall of cocaine reward memories is dependent on GluN2A/B NMDA receptor signaling

Glycogen synthase kinase-3β (GSK3β) is a critical regulator of the balance between long-term depression and long-term potentiation which is essential for learning and memory. Our previous study demonstrated that GSK3β activity is highly induced during cocaine memory reactivation, and that reconsolidation of cocaine reward memory is attenuated by inhibition of GSK3β. NMDA receptors and protein phosphatase 1 (PP1) are activators of GSK3β. Thus, this study investigated the roles of NMDA receptor subtypes and PP1in the reconsolidation of cocaine contextual reward memory. Cocaine contextual memories were established and evaluated using cocaine conditioned place preference methods. The regulation of GSK3β activity in specific brain areas was assessed by measuring its phosphorylation state using immunoblot assays. Mice underwent cocaine place conditioning for 8days and were tested for place preference on day 9. Twenty-four hours later, mice were briefly confined to the compartment previous paired with cocaine to reactivate cocaine-associated memories. Administration of the GluN2A- and GluN2B-NMDA receptor antagonists, NVP-AAM077 and ifenprodil, respectively, immediately following recall abrogated an established cocaine place preference, while preventing the activation of GSK3β in the amygdala, nucleus accumbens, and hippocampus during cocaine memory reactivation. PP1 inhibition with okadaic acid also blocked the activation of GSK3β and attenuated a previously established cocaine place preference. These findings suggest that the dephosphorylation of GSK3β that occurred upon activation of cocaine-associated reward memories may be initiated by the activation of PP1 during the induction of NMDA receptor-dependent reconsolidation of cocaine mnemonic traces. Moreover, the importance of NMDA receptors and PP1 in reconsolidation of cocaine memory makes them potential therapeutic targets in treatment of cocaine use disorder and prevention of relapse.

Effect of phosphoglycogen synthase kinase-3 β induced by low dose of tunicamycin on mitochondrial permeability transition pore in ischemia-reperfusion myocardium

Objective To evaluate the effect of glycogen synthase kinase-3beta (GSK-3β) activity on mitochondrial permeability transition pore (mPTP) in the protective effects of preconditioning with endoplasmic reticulum stress (ERS) induced by low dose of tunicamycin (TM) against myocardial ischemia reperfusion (I/R) injury. Methods Forty adult male sprague-dawley rats were randomly divided into four groups: Sham, TM (Rats were treatmented by TM 0.6 mg/kg at 30 min before model established), I/R, and TM+ LY [Rats were treatmented by TM 0.6 mg/kg and protein kinase B (Akt) inhibitor LY294002 10 mg/kg]. The model of I/R were established by occlusion of left anterior descending coronary artery for 0.5 h followed by reperfusion for 2 h. Hemodynamic parameters were recorded after coronary artery perforation, 1 h and 2 h after reperfusion. After 2 hours of reperfusion, the expression of phosphorylated Akt (p-Akt), p-GSK-3β and the openness of mPTP were detected in the hearts. Results Compared with base line, left ventricular systolic pressure (LVSP), maximum rate of increase or decrease of left ventricular pressure (±dp/dt max) decreased, and left ventricular end-diastolic pressure (LVEDP)increased in I/R group, TM group and TM+ LY group, while the change was more significantly in I/R group. The expression of p-Akt and p-GSK-3 beta protein in TM and TM+ LY group was higher than that in I/R group, and the expression of p-Akt and p-GSK-3β protein in TM+ LY group was lower than that in TM group. At 2 hours after reperfusion, degree of variation of absorbance value (ΔA) of the mPTP of Sham, I/R, TM and TM+ LY group were (78.9±9.2)%, (36.1±4.8)%, (61.4±8.3)% and (46.3 ±5.2)%. The ΔA of TM group was larger than that of I/R group (t=3.280, P<0.01). Compared with TM group, the ΔA of TM+ LY group was relatively small (t=2.720, P<0.01). Conclusion The protective effect of preconditioning with ERS induced by low dose TM is related to the inhibition of mPTP opening by regulating GSK-3β activity. Key words: Myocardial ischemia reperfusion injury; Endoplasmic reticulum stress; Preconditioning; Glycogen synthase kinase-3β; Mitochondrial permeability transition pore

Aerobic Exercise Regulates GSK3β Activity to Attenuate the Neuropathology In APP/PS1 Transgenic Mice

Aerobic Exercise Regulates GSK3β Activity to Attenuate the Neuropathology In APP/PS1 Transgenic Mice

Analysis of the role of GSK3 in the mitotic checkpoint

The mitotic checkpoint ensures proper chromosome segregation; defects in this checkpoint can lead to aneuploidy, a hallmark of cancer. The mitotic checkpoint blocks progression through mitosis as long as chromosomes remain unattached to spindle microtubules. Unattached kinetochores induce the formation of a mitotic checkpoint complex (MCC) composed of Mad2, BubR1, Bub1 and Bub3 which inhibits anaphase onset. Spindle toxins induce prolonged mitotic arrest by creating persistently unattached kinetochores which trigger MCC formation. We find that the multifunctional ser/thr kinase, glycogen synthase kinase 3 (GSK3) is required for a strong mitotic checkpoint. Spindle toxin-induced mitotic arrest is relieved by GSK3 inhibitors SB 415286 (SB), RO 318220 (RO) and lithium chloride. Similarly, targeting GSK3β with knockout or RNAi reduced mitotic arrest in the presence of Taxol. GSK3 was required for optimal localization of Mad2, BubR1, and Bub1 at kinetochores and for optimal assembly of the MCC in spindle toxin-arrested cells. The WNT- and PI3K/Akt signaling pathways negatively regulate GSK3β activity. Inhibition of WNT and PI3K/Akt signaling, in the presence of Taxol, induced a longer mitotic arrest compared to Taxol alone. Our observations provide novel insight into the regulation of the mitotic checkpoint and its connection to growth-signaling pathways.

Long non-coding RNA LINC00222 regulates GSK3β activity and promotes cell apoptosis in lung adenocarcinoma

Recent evidence indicates that long noncoding RNAs (lncRNAs) play a critical role in the regulating cellular processes such as differentiation, proliferation, metastasis and apoptosis. These lncRNAs are found to be dysregulated in a variety of cancers. However, the underlying mechanisms of lncRNAs action in lung adenocarcinoma remain unclear. We investigated expression of the lncRNA LINC00222 in 76 lung adenocarcinoma tissues and paired normal lung tissues and found that LINC00222 is downregulated in lung adenocarcinoma tissues. Enforced expression of LINC00222 significantly inhibited the proliferation, migration and invasive ability of lung adenocarcinoma cells, and also induced apoptosis. Furthermore, overexpression of LINC00222 enhanced the activity of glycogen synthase kinase-3β (GSK3β), which is a key element of the Wnt signaling pathway. Taken together, our findings suggest that LINC00222 acts as a tumor suppressor in lung adenocarcinoma and, therefore, could serve as a valuable prognostic biomarker and therapeutic target for lung adenocarcinoma patients.

Abstract 4293: Glycogen synthase kinase 3 b maintains mitotic arrest by regulating mitotic checkpoint complex levels

Abstract The cell cycle is regulated by checkpoints that ensure genomic integrity and proper cell division. We are investigating the role of a multifunctional ser/thr kinase, glycogen synthase kinase 3 (GSK3), in the regulation of the mitotic checkpoint. Our data show that spindle toxin-induced mitotic arrest is relieved in multiple cancer cell lines by the GSK3 inhibitors, SB 415286 (SB), RO-81220 (RO) and lithium chloride. Additionally, the mitotic index is reduced in CRISPR-generated GSK3β knockout human cells and GSK3β knockout mouse embryo fibroblasts compared to wild type cells in the presence of taxol. Mitotic arrest is dependent on the mitotic checkpoint complex (MCC), composed of Mad2, BubR1, Cdc20 and Bub1. GSK3 appears to target the MCC to regulate mitotic arrest. For example, Mad2, BubR1 and Bub1 localization at kinetochores and MCC assembly were both reduced by co-treatment of GSK3 inhibitors and spindle toxin compared to spindle toxin alone. These data imply that GSK3β plays a role in maintaining the mitotic checkpoint. The strength of the checkpoint was determined to act like a rheostat rather than a toggle-switch, as cells undergo mitotic slippage after prolonged arrest in spindle toxins. Our data indicates that GSK3 regulates the strength of the checkpoint, as illustrated by time lapse-imaging. GSK3-inhibition induced exit was observed to be dependent on how long the cells were arrested in mitosis prior to GSK3-inhibitor addition. GSK3 inhibition in taxol also decreases the amount of KnL1, part of the KMN network forming the kinetochore, at the centromeres. This indicates a regulatory role of GSK3 in kinetochore assembly and function. The Wnt-signaling pathway and the PI3K/Akt signaling pathway are known upstream regulators of GSK3β, negatively regulating GSK3β activity. Our data shows that inhibiting Wnt-signaling and PI3K/Akt-signaling in the presence of taxol, induces a longer mitotic arrest compared to taxol alone. This suggests that GSK3β is regulated upstream by the Wnt- and the PI3K/Akt signaling archs to control mitosis. Finally, our data implicated that de-acetylase sirtuin2 is a downstream target of GSK3 in its mitotic regulatory role. Our observations indicate a novel regulator of the checkpoint and novel insight in connecting growth-signaling pathways with mitosis. Citation Format: Maisha S. Rashid, William R. Taylor. Glycogen synthase kinase 3 b maintains mitotic arrest by regulating mitotic checkpoint complex levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4293.

Guanosine monophosphate reductase 1 is a potential therapeutic target for Alzheimer\u2019s disease

Alzheimer’s disease (AD) is a severe neurodegenerative disorder for which identification of differentially expressed genes is one way to find new therapeutic targets. Here, we conducted analysis to identify age-independent, AD-specific genes. We found that the MET, WIF1, and NPTX2 genes are downregulated in AD. WIF1 and MET are implicated in Wnt and MET signaling and regulate GSK3β activity and are thus linked with AD. Importantly, we found that the GMPR gene exhibited a gradual increase in AD progression. A logistic model based on GMPR has good ability to classify AD cases. GMPR’s product GMPR1 is in the AMPK and adenosine receptor pathways and is thus associated with Tau phosphorylation in AD. This allows GMPR1 to be a therapeutic target. Therefore, we screened five possible inhibitors to GMPR1 by docking GMPR1 with 1,174 approved drugs. Among them, lumacaftor is ideal. We then tested the effects of lumacaftor on AD model mice. After 20 days of oral administration, we observed that β-Amyloid accumulation was slowed down, and phosphorylation of Tau was almost eliminated in the treated mice. We highlight the elevated expression level of GMPR in AD and propose a therapeutic strategy of inhibiting GMPR1 with lumacaftor.

P 222 - Regulation of Nrf2 signaling during the antitumoral activity of Sorafenib in hepatoma cells

Introduction Sorafenib is the accepted treatment for patients in advanced stage of hepatocarcinoma. Goal: The study evaluated the regulation of the endoplasmic reticulum stress (ERS), Nrf2, oxidative/nitrosative stress, autophagy, cell proliferation and apoptosis by Sorafenib. Methods: The study includes in vitro and in vivo experimental designs. Results: Sorafenib induced ERS, Thr183JNK/JNK and transient autophagy that was followed by apoptosis, and reduction of cell proliferation. Sorafenib reduced dose-dependent nitric oxide, O2.- and H2O2 generation, and S-nitrosylated, carbonylated and tyrosine nitrated proteins. Sorafenib reduced S-nitrosylation of cell death receptors that shifted caspase-8- to caspase-3-related apoptosis. Sorafenib decreased luciferase activity in control, VEGF- and PDGF-stimulated ARE-Luc-transfected HepG2, and went down the expression of Nrf2-related redox genes. The reduction of Nrf2 signaling was related to increased Ser9GSK3β/Tyr216GSK3β. The in vivo study confirmed the antitumoral properties and molecular signaling of Sorafenib in xenograft mice model. Conclusions: 1) Sorafenib induced ERS, autophagy and apoptosis in Sorafenib-treated HepG2. 2) Sorafenib reduced Nrf2-dependent signaling through regulation of GSK3β activity. 3) Nrf2 signaling and thioredoxin were involved in the reduction of S-nitrosylation of cell death receptor by Sorafenib.

Transfection of tumor metastasis suppressor gene nm23-H1 up-regulates the activity of glycogen synthase kinase -3β in lung cancer cell line L9981

Objective To investigate the influence of tumor metastasis suppressor gene nm23-H1 on the activity of glycogen synthase kinase -3β (GSK-3β) in lung cancer cell line L9981. Methods The levels of GSK-3β expression in cytoplasm and nucleus were determined with anti-GSK-3β antibody in lung cancer cell line control group (cell line with nm23-H1 gene deletion), nm23-H1 transfection group (cell line with nm23-H1 transfected) and blank transfection group (cell line with vector transfected) by Western blotting. The activity of GSK-3β among those three groups was detected by immunoprecipitation and analyzed by a radioactive isotope scintillation counter before and after treatment with 20 mmol/L LiCl. Results (1) The expression indensity of GSK-3β in cytoplasm and nucleus was (6 639±503) and (4 481±446) A in nm23-H1 transfection group, (645±352) and (726±68) A in blank transfection group, and (763±267) and (683±49) A in control group, respectively. There was very significance in GSK-3β expressive indensity of both cytoplasm and nucleus among nm23-H1 transfection group, blank transfection group and control group (P=0.025). There was significant difference between nm23-H1 transfection group and blank transfection group or control group (P=0.017), but no significant difference was observed between blank transfection group and control group (P=0.094). (2) The GSK-3β activity in cytoplasm and nucleus was (29 371±2 492) and (9 647±859) counts per minute in nm23-H1 transfection group, (11 241±1 495) and (1 492±176) counts per minute in blank transfection group, and (14 271±1 137) and (1 853±113) counts per minute in control group, respectively. There was very significant difference in GSK-3β activity of both cytoplasm and nucleus among nm23-H1 transfection group, blank transfection group and control group (P=0.032). The GSK-3β activity in nm23-H1 transfection group was significantly higher than that in blank transfection group and control group (P=0.027), but no significant difference was observed between the blank transfection group and control group (P=0.131). (3) After treatment with 20 mmol/L LiCl, the expression indensity of GSK-3β in cytoplasm and nucleus was (5 037±427) and (823±350) A in nm23-H1 transfection group, (2 174±126) and (248±12) A in blank transfection group, and (2 273±128) and (253±14) A in control group. No significant difference in GSK-3β expression indensity existed before and after treatment with LiCl in nm23-H1 transfection group (P=0.168). However, the GSK-3β expression indensity in cytoplasm and nucleus before treatment was remarkably higher than that after treatment in both blank transfection group and control group (P=0.012, 0.015). (4) After treatment with 20 mmol/L LiCl, the GSK-3β activity in cytoplasm and nucleus was (12 837±1 042) and (748±215) counts per minute in nm23-H1 transfection group, (4 739±401) and (947±126) counts per minute in control group, and (4 638±401) and (1 162±127) counts per minute in blank transfection group, respectively. The GSK-3β activity in cytoplasm and nucleus after treatment with LiCl was remarkably lower than that before treatment in nm23-H1 transfection group, blank transfection group and control group (P=0.006, 0.029, 0.010). Conclusion Transfection of nm23-H1 gene can significantly up-regulate the expression level and activity of GSK-3β in lung cancer cell line. The inhibitory effects of nm23-H1 gene on the signal transduction of Wnt pathway might be carried out through up regulating GSK-3β expression and activity in lung cancer cell line. Key words: Lung cancer cell lines; nm23-H1 gene; Wnt signal pathway; Glycogen synthase kinase -3β

Calreticulin Is Required for TGF-β-Induced Epithelial-to-Mesenchymal Transition during Cardiogenesis in Mouse Embryonic Stem Cells.

SummaryCalreticulin, a multifunctional endoplasmic reticulum resident protein, is required for TGF-β-induced epithelial-to-mesenchymal transition (EMT) and subsequent cardiomyogenesis. Using embryoid bodies (EBs) derived from calreticulin-null and wild-type (WT) embryonic stem cells (ESCs), we show that expression of EMT and cardiac differentiation markers is induced during differentiation of WT EBs. This induction is inhibited in the absence of calreticulin and can be mimicked by inhibiting TGF-β signaling in WT cells. The presence of calreticulin in WT cells permits TGF-β-mediated signaling via AKT/GSK3β and promotes repression of E-cadherin by SNAIL2/SLUG. This is paralleled by induction of N-cadherin in a process known as the cadherin switch. We show that regulated Ca2+ signaling between calreticulin and calcineurin is critical for the unabated TGF-β signaling that is necessary for the exit from pluripotency and the cadherin switch during EMT. Calreticulin is thus a key mediator of TGF-β-induced commencement of cardiomyogenesis in mouse ESCs.

Gsk3β and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson's disease.

Fbxo7 is a clinically relevant F-box protein, associated with both cancer and Parkinson's disease (PD). Additionally, SNPs within FBXO7 are correlated with alterations in red blood cell parameters. Point mutations within FBXO7 map within specific functional domains, including near its F-box domain and its substrate recruiting domains, suggesting that deficiencies in SCFFbxo7/PARK15 ubiquitin ligase activity are mechanistically linked to early-onset PD. To date, relatively few substrates of the ligase have been identified. These include HURP (hepatoma up-regulated protein), whose ubiquitination results in proteasome-mediated degradation, and c-IAP1 (inhibitor of apoptosis protein 1), TNF receptor-associated factor 2 (TRAF2), and NRAGE, which are not destabilized as a result of ubiquitination. None of these substrates have been linked directly to PD, nor has it been determined whether they would directly engage neuronal cell death pathways. To discover ubiquitinated substrates of SCFFbxo7 implicated more directly in PD aetiology, we conducted a high-throughput screen using protein arrays to identify new candidates. A total of 338 new targets were identified and from these we validated glycogen synthase kinase 3β (Gsk3β), which can phosphorylate α-synuclein, and translocase of outer mitochondrial membrane 20 (Tomm20), a mitochondrial translocase that, when ubiquitinated, promotes mitophagy, as SCFFbxo7 substrates both in vitro and in vivo. Ubiquitin chain restriction analyses revealed that Fbxo7 modified Gsk3β using K63 linkages. Our results indicate that Fbxo7 negatively regulates Gsk3β activity, rather than its levels or localization. In addition, Fbxo7 ubiquitinated Tomm20, and its levels correlated with Fbxo7 expression, indicating a stabilizing effect. None of the PD-associated mutations in Fbxo7 impaired Tomm20 ubiquitination. Our findings demonstrate that SCFFbxo7 has an impact directly on two proteins implicated in pathological processes leading to PD.

Spinal CXCL5 contributes to nerve injury-induced neuropathic pain via modulating GSK-3β phosphorylation and activity in rats

BackgroundNeuroinflammation is identified to be crucial in the development of neuropathic pain, whereas definite molecular mechanisms remain obscure. Recently, chemokine CXCL5 is manifested to participate in the inflammatory process of central nervous system, however, little is known about the potential effect of spinal CXCL5 on pathogenesis of pain. This study investigated whether and how CXCL5 and its receptor CXCR2 regulated neuropathic pain in a rat model of chronic constriction injury (CCI) of the sciatic nerves. MethodsRecombinant CXCL5, a neutralizing antibody against CXCL5, selective CXCR2 antagonist SB225002 and GSK-3β inhibitor TDZD-8 were injected intrathecally. PWT and PWL were documented to assess mechanical allodynia and thermal hyperalgesia. Simultaneously, levels of CXCL5 and CXCR2 in spinal dorsal horn were measured by RT-qPCR after nociceptive testing. Western blot was utilized to evaluate spinal GSK-3β expression and phosphorylation. ResultsWe found that CCI engendered rapid and long-lasting mechanical allodynia and thermal hyperalgesia, which was accompanied by dramatical rise of spinal CXCL5 and CXCR2 expression. CCI also caused an increase of pGSK-3β (Tyr216) and a decrease of pGSK-3β (Ser9) without affecting total protein level of GSK-3β. Moreover, spinal blockage of CXCL5/CXCR2 pathway attenuated neuropathic pain and inhibited the enhancement of GSK-3β activity. Also, intrathecal delivery of exogenous CXCL5 dose-dependently induced nociceptive hypersensitivity in naïve rats, which was prevented by the supplemental addition of TDZD-8. ConclusionThese present findings indicate that up-regulation of spinal CXCL5 and CXCR2 is involved in neuropathic pain after nerve injury, through regulating GSK-3β activity in rats.

Glycogen Synthase Kinase 3β Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists.

The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3β (GSK-3β) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3β interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3β redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3β at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3β in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3β. In vitro activity assays demonstrated that GSK-3β phosphorylation mediated by PKCζ enhanced GSK-3β activity. We mapped Ser147 of GSK-3β as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3β activity, resulting in β-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3β basal activity. Thus, we found that PKCζ phosphorylates GSK-3β at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3β activity in opposite manners in normal and malignant colon cells.