Effect Of GSK-3β Inhibitor Research Articles

PM2.5-Induced Cardiac Structural Modifications and Declined Pro-Survival Signalling Pathways Are Responsible for the Inefficiency of GSK-3β Inhibitor in Attenuating Myocardial Ischemia-Reperfusion Injury in Rats.

Circulatory GSK3β is recognized as a biomarker and therapeutic target for diseases, including myocardial diseases. However, its potential as a target for myocardial ischemia-reperfusion injury (IR) in the presence of PM2.5 exposure is unclear. Wistar rats underwent IR following either a 21-day or single exposure to PM2.5 at a concentration of 250 µg/m3. The effects of GSK3β inhibitor on cardiac physiology, tissue injury, mitochondrial function, and the PI3K/AKT/GSK3β signalling axis were examined. The inhibitor was not effective in improving hemodynamics or reducing IR-induced infarction in the myocardium exposed to PM2.5 for 21 days. However, for a single-day exposure, the inhibitor showed potential in mitigating cardiac injury. In normal hearts undergoing IR, the inhibitor activated the PI3K/AKT signalling pathway, improved mitochondrial function, and reduced oxidative stress. These positive effects were not observed in PM2.5-exposed rats. Furthermore, the inhibitor stimulated autophagy in hearts exposed to PM2.5 for 21 days and subjected to IR, resulting in increased mTOR expression and decreased AMPK expression. In normal hearts and those exposed to a single dose of PM2.5, the inhibitor effectively activated the PI3K/Akt/AMPK axis. These findings suggest that GSK3β may not be a reliable therapeutic target for IR in the presence of chronic PM2.5 exposure.

GSK-3β inhibition protects human nucleus pulposus cell against oxidative stress-inducing apoptosis through mitochondrial pathway.

Oxidative stress in the intervertebral disc leads to nucleus pulposus (NP) degeneration by inducing cell apoptosis. However, the molecular mechanisms underlying this process remain unclear. Increasing evidence indicates that GSK-3β is related to cell apoptosis induced by oxidative stress. In this study, we explored whether GSK-3β inhibition protects human NP cell against apoptosis under oxidative stress. Immunofluorescence staining was used to show the expression of GSK-3β in human NP cells (NPCs). Flow cytometry, mitochondrial staining and western blot (WB) were used to detect apoptosis of treated NPCs, changes of mitochondrial membrane potential and the expression of mitochondrial apoptosis-related proteins using GSK-3β specific inhibitor SB216763. Co-Immunoprecipitation (Co-IP)was used to demonstrate the interaction between GSK-3β and Bcl-2. We delineated the protective effect of GSK-3β specific inhibitor SB216763 on human NPCs apoptosis induced by oxidative stress in vitro. Further, we showed SB216763 exert the protective effect by preservation of the mitochondrial membrane potential and inhibition of caspase 3/7 activity during oxidative injury. The detailed mechanism underlying the antiapoptotic effect of GSK-3β inhibition was also studied by analyzing mitochondrial apoptosis pathway in vitro. We concluded that the GSK-3β inhibitor SB216763 protected mitochondrial membrane potential to delay nucleus pulposus cell apoptosis by inhibiting the interaction between GSK-3β and Bcl-2 and subsequently reducing cytochrome c(Cyto-C) release and caspase-3 activation. Together, inhibition of GSK-3β using SB216763 in NPCs may be a favorable therapeutic strategy to slow intervertebral disc degeneration.

Effects of GSK3β inhibition in the regeneration of Syllis malaquini (Syllidae, Annelida).

The Wnt/β-catenin signaling pathway has been widely associated to the reestablishment of anteroposterior body polarities in the embryonic development and regeneration in animals. For instance, in annelids, cellular proliferation, wound healing, and blastema development can be affected when this pathway is disrupted. However, very little is known about the genetic regulatory processes involved in these anomalies. Here, we investigate the morphological effects of 1-azakenpaullone, a pharmacological inhibitor of GSK3β that is supposed to over-activate the Wnt/β-catenin pathway, during the anterior and posterior regeneration of the annelid Syllis malaquini. The results showed that high concentrations of 1-azakenpaullone affect the stages of blastema differentiation and resegmentation. Therefore, GSK3β-associated gene regulatory networks are candidate to investigate the genetic mechanisms involved in the regular course of S. malaquini regeneration.

Epibrassinolide prevents tau hyperphosphorylation via GSK3β inhibition in vitro and improves Caenorhabditis elegans lifespan and motor deficits in combination with roscovitine.

Glycogen synthase kinase 3β (GSK3β) is considered an important element of glycogen metabolism; however, it has many other regulatory roles. Changes in the GSK3β signaling mechanism have been associated with various disorders, such as Alzheimer's disease (AD), type II diabetes, and cancer. Although the effects of GSK3β inhibitors on reducing the pathological effects of AD have been described, an effective inhibitor has not yet been developed. Epibrassinolide (EBR), a brassinosteroid (BR), is structurally similar to mammalian steroid hormones. Our studies have shown that EBR has an inhibitory effect on GSK3β in different cell lines. Roscovitine (ROSC), a cyclin-dependent kinase (CDK) inhibitor, has also been identified as a potential GSK3 inhibitor. Within the scope of this study, we propose that EBR and/or ROSC might have mechanistic action in AD models. To test this hypothesis, we used in vitro models and Caenorhabditis elegans (C. elegans) AD strains. Finally, EBR treatment successfully protected cells from apoptosis and increased the inhibitory phosphorylation of GSK3β. In addition, EBR and/or ROSC treatment had a positive effect on the survival rates of C. elegans strains. More interestingly, the paralysis phenotype of the C. elegans AD model due to Aβ42 toxicity was prevented by EBR and/or ROSC. Our findings suggest that EBR and ROSC administration have neuroprotective effects on both in vitro and C. elegans models via inhibitory GSK3β phosphorylation at Ser9.

Abstract 5217: 9-ING-41, a GSK-3β-selective small molecule inhibitor, in combination with ruxolitinib in JAK2617F primary myelofibrosis

Abstract Introduction: Primary myelofibrosis (MF) is a chronic clonal malignancy which is compounded by the presence of inflammation, bone marrow fibrosis/scarring and risk of transformation to acute myeloid leukemia (AML). Ruxolitinib (Rux), a JAK2 inhibitor, is beneficial for reduction of symptoms and splenomegaly, however, significant myelosuppression is also a toxic side effect of this treatment, limiting optimal drug dosage in many patients. 9-ING-41 is a small molecule GSK-3β inhibitor which has significant clinical activity in patients with advanced malignancies and causes no myelosuppression. We thus assessed the effect of 9-ING-41in MF alone or in combination with Rux. We performed ex-vivo colony assays with primary cells from cases with untreated MF along with normal bone marrow (BM). The experiments were designed to assess the effect of drug on the ability of MF stem/progenitor cells to proliferate/differentiate by evaluating the number, size, and the morphology of colonies, both with and without treatment. Methods: Primary peripheral blood mononuclear cells from MF patients and BM from controls were plated in methylcellulose in duplicate, containing cytokines promoting hematopoietic growth, in the presence of either no drug, Rux only (0.05µM), 9-ING-41 only (range: 0.125µM - 10µM), or a combination of 9-ING-41 (range: 0.125µM - 10µM) and Rux (0.05uM). Colonies were counted ten days later, and colony growth frequency, distribution, and morphology were calculated. Results: We observed an increasing presence of large primitive granulocytic/erythroid/macrophage/monocyte (GEMM) colonies proportional to no drug treatment and linear to increasing concentrations of 9-ING-41 in both MF cases relative to other colony types and normal BM, suggestive of a selective primitive proliferative and/or differentiation effect of GSK-3β inhibition on MF hematopoietic progenitor growth. We did not observe this effect in the combinatory treatments (9-ING-41 + Rux), and there was no significant difference on MF colony number or size in colonies grown without treatment or with Rux alone (Figure 1 A/C/E). However, the morphology of the colonies within different treatments was remarkable. In both MF cases, either without treatment or addition of Rux only (0.05µM), the colonies appeared similar to each other -irregular and disorganized relative to normal bone marrow (Figure 1, B/D/F). In the presence of 9-ING-41 alone, we observed morphologically very large, fully differentiated primitive colonies in both MF cases. However, the most striking observation was the comparison of colonies after combination of both inhibitors. These colonies appeared normal and healthy-looking -discreet, round size and similar in morphology to normal bone marrow colonies. Additional studies are currently underway to look at the signaling consequences, including proliferation, differentiation, and cell cycle effect. Our preliminary results suggest this same mechanism might be occurring in MF cases and could explain the selective presence of large differentiated primitive colonies in MF vs. normal BM in the presence of 9-ING-41. Combining 9-ING-41 with Rux may reduce the dose needed for optimal therapeutic response and/or reverse myelosuppression. A Phase 2 study of 9-ING-41 in patients with advanced MF will be conducted. Citation Format: Terra L. Lasho, Francis Giles, Christy Finke, Naseema Gangat, Ayalew Tefferi. 9-ING-41, a GSK-3β-selective small molecule inhibitor, in combination with ruxolitinib in JAK2617F primary myelofibrosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5217.

Neuronal protective effect of GSK3β inhibition in Drosophila ALS model

Neuronal protective effect of GSK3β inhibition in Drosophila ALS model

GSK3\u03b2 regulates epithelial-mesenchymal transition and cancer stem cell properties in triple-negative breast cancer

BackgroundTriple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC.MethodsA library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3β inhibitors were identified as EMT inhibitors. The effects of GSK3β inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3β correlates with the overall survival of breast cancer patients.ResultsWe identified a GSK3β inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3β inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3β reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3β inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24− in cells after exposure to GSK3β inhibitors. We found that GSK3β inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3β inhibitors and found that GSK3β inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3β correlated with poorer overall patient survival.ConclusionsTaken together, our data demonstrate that GSK3β is a potential target for TNBCs and suggest that GSK3β inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3β inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.

Effect of GSK-3β inhibitor on the expression of RANK-RANKL in rats kidney tissue with diabetic nephropathy

Objective: To investigate the effect and significance of GSK-3β inhibitor(LiCl)and RANK-RANKL on the renal tissue of diabetic nephropathy(DN) rats. Methods: SD rats were divided into normal control group (NC), DN model group (DN) and GSK-3β inhibitor intervention group (LiCl). Twenty-four hour urine protein of rats were determined by Coomassie brilliant blue. Kidney tissue sections were stained by HE. The expression of GSK-3β, RANK and RANKL protein were determined by immunohistochemistry staining. The mRNA of GSK-3β, RANK, RANKL was detected by RT-qPCR. Results: Compared with NC group[(14.72±3.37)g], the level of 24-hour urinary protein[(154.17±20.65)g] increased significantly in DN group; compared with DN Group, the level of 24-hour urinary protein [(107.22±31.15)g]decreased in LiCl group(P<0.05). Compared with NC group(2.10±0.60, 1.10±0.20, 1.21±0.20; 19.52±3.20, 1.80±1.10, 1.81±0.50), the pathological changes of renal tissues of DN group aggravated, the mRNA and expression of protein of GSK-3β, RANK and RANKL increased(9.10±2.15, 8.95±2.40, 9.90±2.60; 32.70±7.20, 19.20±4.32, 20.92±5.90); compared with DN group, the pathological changes of renal tissues of LiCl group alleviated, mRNA and the expression of protein of factors above declined(2.70±0.80, 2.32±0.65, 3.58±1.10; 22.35±3.25, 4.20±2.42, 5.90±2.36; P<0.05). Conclusion: RANK and RANKL play an important role in the development of DN, LiCl influence Wnt and NF-κB signal pathway down-regulating RANK and RANKL to suspend development of diabetic nephropathy.

Inhibition of GSK-3β Alleviates Collagen II-Induced Rheumatoid Arthritis in Rats.

BackgroundGlycogen synthase kinase-3β (GSK-3β) inhibitor is a serine/threonine kinase with an inhibitory role in glycogen synthesis, which is essential in inflammatory and immunological diseases. The purpose of our study was to determine if TDZD-8 can alleviate collagen II-induced rheumatoid arthritis in rats.Material/MethodsTwenty collagen II-induced rheumatoid arthritis rats were treated with selective GSK-3β inhibitor. The effects of GSK-3β inhibition on collagen II-induced rheumatoid arthritis in the rats were evaluated by paw edema, histological examination of arthritic synovium, radiographic examination of knee joint, and the level of inflammation mediators such as prostaglandin E2, 5-hydroxytryptamin, and histamine. The level of cytokines such as IL-6, IL-12, IL-10, and TNF-α, was examined by Elisa.ResultsGSK-3β inhibitor significantly reduced the development of rheumatoid arthritis in rats. The levels of inflammation mediators such as prostaglandin E2, 5-hydroxytryptamin, and histamine were decreased in the TDZD-8 group. Serum levels of IL-6, IL-12, and TNF-α were significantly reduced in the TDZD-8 group compared with the RA group.ConclusionsTreatment with GSK-3β inhibitor suppressed inflammatory response in RA rats. These findings suggest that the inhibition of GSK-3β can be an effective treatment for RA.

Potential Role of Glycogen Synthase Kinase-3β in Regulation of Myocardin Activity in Human Vascular Smooth Muscle Cells.

Glycogen synthase kinase (GSK)-3β, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK-3β results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co-activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study, therefore, aimed to examine whether and how inhibition of GSK-3β regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK-3β inhibitors AR-A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22), and ACTA2 (SM α-actin). In human SMCs overexpressing myocardin through the T-REx system, treatment with either GSK-3β inhibitor also inhibited the expression of CNN1, TAGLN1, and ACTA2. These effects of GSK-3β inhibitors were mimicked by transfection with GSK-3β siRNA. Notably, both AR-A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR-A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant-negative GSK-3β mutant in myocardin-overexpressing SMCs reduced the expression of calponin, SM22, and SM α-actin. As expected, overexpression of constitutively active or wild-type GSK-3β in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK-3β reduces myocardin transcriptional activity, suggesting a role for GSK-3β in myocardin transcriptional activity and smooth muscle differentiation.

Inhibition of GSK3β Stimulates BMP Signaling and Decreases SOST Expression Which Results in Enhanced Osteoblast Differentiation.

Both bone morphogenetic protein (BMP) and Wnt signaling have significant roles in osteoblast differentiation and the interaction between BMP and Wnt signaling is well known. Sclerostin is an important inhibitor of bone formation, inhibiting Wnt signaling and downstream effects of BMP such as alkaline phosphatase activity and matrix mineralization in vitro. However, little is known about the effect of BMP and Wnt signaling interaction on the regulation of SOST, the gene encoding sclerostin. Possibly, uncoupling of osteoblast differentiation regulators and SOST expression could increase osteoblast differentiation. Therefore, we investigated the effect of BMP and Wnt signaling interaction on the expression of SOST and the subsequent effect on osteoblast differentiation. Human osteosarcoma cells (SaOS-2) and murine pre-osteoblast cells (KS483) were treated with different concentrations of Wnt3a, a specific GSK3β inhibitor (GIN) and BMP4. Both Wnt3a and GIN increased BMP4-induced BMP signaling and BMP4 increased Wnt3a and GIN-induced Wnt signaling. However, the effect of GIN was much stronger. Quantitative RT-PCR analysis showed that SOST expression dose-dependently decreased with increasing Wnt signaling, while BMP4 induced SOST expression. GIN significantly decreased the BMP4-induced SOST expression. This resulted in an increased osteoblast differentiation as measured by ALP activity in the medium and matrix mineralization. We conclude that GSK3β inhibition by GIN caused an uncoupling of BMP signaling and SOST expression, resulting in an increased BMP4-induced osteoblast differentiation. This effect can possibly be used in clinical practice to induce local bone formation, for example, fracture healing or osseointegration of implants.

The mechanisms and effects of GSK-3β inhibitor on cellular multidrug resistance in colon carcer cells.

e13592 Background: GSK-3β participates in a variety of important physiological processes. However, the roles of GSK-3 β in the biological properties and drug resistance of tumor cells remains controversial. In this study, the effects of GSK-3β inhibitor (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO) on cellular multidrug resistance of Colon cancer cells, and the roles of Wnt/β-catenin pathway and cancer stem cells (CSCs) in this process were investigated. Methods: The colon cancer SW480 and SW620 cells were treated with BIO, 5-fluorouracil (5-FU), BIO/5-FU, Cisplatin (DDP) and BIO/DDP separately. The intracellular ATP concentration was detected by luminometer. Cell apoptosis level was detected by flow cytometry. The expressions of GSK-3β, p-GSK-3β, β-catenin, E-cadherin and CSCs markers EpCAM, TERT, and DCAMKL-1 protein were detected by Western blot. The expressions of CD133 protein were detected by Flow Cytometry. Results: Compared with those colon cancer cells untreated with BIO, The intracellular ATP concentration is slightly elevated in SW480 and SW620 cells After BIO treatment. The apoptosis induced by 5-Fu and DDP were reduced significantly in SW480 cells after BIO treatment (P < 0.05), whereas the effects were slight in SW620 cells (P > 0.05). BIO significantly reduces the expression of p-GSK-3β protein in SW480 and SW620 cells (P < 0.05), but the change of GSK-3β protein were not obvious (P > 0.05). BIO significantly increased the expression of β-catenin and E-cadherin protein in SW480 and SW620 cells (P < 0.05), and promoted nuclear translocation of β-catenin protein. BIO significantly increased the expression of EpCAM, TERT and DCAMKL-1 protein in SW480 cells (P < 0.05), but the change of CD133 expression was not obvious (P > 0.05). BIO significantly up-regulated the expression of EpCAM protein in SW620 cells (P < 0.05), but the change of CD133, TERT and DCAMKL-1 protein expression were not significant (P > 0.05). Conclusions: The GSK-3β inhibitor BIO could affect cellular multidrug resistance of colon cancer cells in vitro, and the Wnt/β-catenin pathway and CSCs may play some roles in this process. GSK-3β may be a regulating target of drug resistance in colon cancer.

Glycogen synthase kinase 3β sustains invasion of glioblastoma via the focal adhesion kinase, Rac1, and c-Jun N-terminal kinase-mediated pathway.

The failure of current treatment options for glioblastoma stems from their inability to control tumor cell proliferation and invasion. Biologically targeted therapies offer great hope and one promising target is glycogen synthase kinase-3β (GSK3β), implicated in various diseases, including cancer. We previously reported that inhibition of GSK3β compromises the survival and proliferation of glioblastoma cells, induces their apoptosis, and sensitizes them to temozolomide and radiation. Here, we explore whether GSK3β also contributes to the highly invasive nature of glioblastoma. The effects of GSK3β inhibition on migration and invasion of glioblastoma cells were examined by wound-healing and Transwell assays, as well as in a mouse model of glioblastoma. We also investigated changes in cellular microarchitectures, cytoskeletal components, and proteins responsible for cell motility and invasion. Inhibition of GSK3β attenuated the migration and invasion of glioblastoma cells in vitro and that of tumor cells in a mouse model of glioblastoma. These effects were associated with suppression of the molecular axis involving focal adhesion kinase, guanine nucleotide exchange factors/Rac1 and c-Jun N-terminal kinase. Changes in cellular phenotypes responsible for cell motility and invasion were also observed, including decreased formation of lamellipodia and invadopodium-like microstructures and alterations in the subcellular localization, and activity of Rac1 and F-actin. These changes coincided with decreased expression of matrix metalloproteinases. Our results confirm the potential of GSK3β as an attractive therapeutic target against glioblastoma invasion, thus highlighting a second role in this tumor type in addition to its involvement in chemo- and radioresistance.

GSK3β Overexpression Indicates Poor Prognosis and Its Inhibition Reduces Cell Proliferation and Survival of Non-Small Cell Lung Cancer Cells

BackgroundGlycogen synthase kinase 3 beta (GSK3β) is centrally involved in diverse cellular processes, including proliferation and apoptosis. This study aimed to investigate the influence of GSK3β expression on the prognosis of human non-small cell lung cancer (NSCLC) and the effects of GSK3β inhibition in NSCLC cell lines.MethodsImmunohistochemical and western blot assays were used to evaluate the GSK3β expression level in human NSCLC tissues. Lentiviral RNA interference was performed to inhibit the expression of GSK3β in the A549, H292, H1299 and SK-MES-1 cell lines. Cell survival, apoptosis and motility were evaluated in vivo and in vitro.ResultsThe levels of GSK3β were greater in NSCLC tissues (n = 211) than in control tissues (n = 194) (P<0.001). The 5-year follow-up analysis showed that positive GSK3β expression was indicative of poor prognosis (P = 0.006). Furthermore, knockdown of GSK3β in NSCLC cell lines suppressed cell proliferation, arrested tumor cells in G0/G1 phase, induced apoptosis and reduced cell motility. A xenograft model showed that the deregulation of GSK3β attenuated tumorigenesis, as confirmed by reduced cell proliferation based on Ki-67 and significantly increased apoptotic cell death. The inhibition of GSK3β had inconsistent effects on the expression of β-catenin, depending on the cell type examined.ConclusionAberrant expression of GSK3β serves as an independent marker of poor prognosis for NSCLC. The inhibition of GSK3β suppressed tumorigenesis by attenuating cell proliferation, increasing apoptosis and restraining cell motility. These results identify GSK3β as a tumor promoter and a potential therapeutic target in NSCLC.

GSK3β regulates oligodendrogenesis in the dorsal microdomain of the subventricular zone via Wnt-β-catenin signaling

Oligodendrocytes, the myelinating cells of the CNS, are derived postnatally from oligodendrocyte precursors (OPs) of the subventricular zone (SVZ). However, the mechanisms that regulate their generation from SVZ neural stem cells (NSC) are poorly understood. Here, we have examined the role of glycogen synthase kinase 3β (GSK3β), an effector of multiple converging signaling pathways in postnatal mice. The expression of GSK3β by rt-qPCR was most prominent in the SVZ and in the developing white matter, around the first 1–2 weeks of postnatal life, coinciding with the peak periods of OP differentiation. Intraventricular infusion of the GSK3β inhibitor ARA-014418 in mice aged postnatal day (P) 8–11 significantly increased generation of OPs in the dorsal microdomain of the SVZ, as shown by expression of cell specific markers using rt-qPCR and immunolabelling. Analysis of stage specific markers revealed that the augmentation of OPs occurred via increased specification from earlier SVZ cell types. These effects of GSK3β inhibition on the dorsal SVZ were largely attributable to stimulation of the canonical Wnt/β-catenin signaling pathway over other pathways. The results indicate GSK3β is a key endogenous factor for specifically regulating oligodendrogenesis from the dorsal SVZ microdomain under the control of Wnt-signaling.

Abstract T P223: Role of GSK-3ß and Transforming Growth Factor-ß-activated Kinase in Ischemic Neuroprotection

Objectives: Glycogen synthase kinase-3β (GSK-3β) is involved in several essential intracellular signaling pathways involved in both cell survival and cell death. GSK-3β activation promotes inflammation and apoptosis which are detrimental in stroke. GSK-3β inhibition enhances cognition in many animal models; however the role of GSK-3β in stroke and the mechanism by which activation is detrimental remains to be elucidated. It was recently found that GSK inhibits the function of the master energy sensor AMP-activated kinase (AMPK). We have also recently shown that TAK1, an upstream kinase that activates AMPK, is also involved in regulating ischemic injury. Inhibition of TAK leads to dramatic protection, even when treatment is delayed, and this effect is independent of AMPK signaling. We hypothesize that TAK1 regulates GSK-3β. The aim of this study was to determine whether GSK-3β inhibition confers ischemic neuroprotection via TAK1 signaling. Methods: C57BL/6 male mice were randomly assigned to treatment groups and subjected to right middle cerebral artery occlusion The GSK-3β inhibitor VIII (4 mg/kg); and/or the TAK1 inhibitor, 5Z-7-oxozeaenol (5 mg/kg) were administered i.p. either immediately or 2h after stroke. Control mice were treated vehicle. Western blotting was performed for on brains obtained 6h after stroke. Data were presented as mean ± SEM. Results: GSK-3β inhibition was protective when delivered immediately after stroke (Total hemisphere: 44.2±3.2 vs 36.2±3.1; n=8, p&lt;0.05) or if treatment was delayed for 2h (Total: 46.2±3.0 vs 32.2±4.3; n=7, p&lt;0.05). Beneficial effects of GSK-3β inhibition in NDS (p&lt;0.05) was also seen. TAK inhibitor reduced pTAK1 in parallel to GSK-3β inhibition. But, GSK-3β inhibitor treated brains has no effect on pTAK1 levels p&gt;0.05. Finally, compared to GSK-3β inhibition alone, simultaneous GSK-3β+TAK1 inhibition did not offer any additional neuroprotection in stroke (Total: 33.6±2.8 vs 30.7±3.9; p&gt;0.05). Conclusion: Our findings indicate that GSK-3β inhibition is protective even with delayed administration after stroke. In this study, we identified TAK1 as a potential upstream regulator of GSK-3β. Directly targeting TAK1 and GSK-3β inhibition may develop into a novel and powerful neuroprotective strategy.

Inhibition of glycogen synthase kinase-3β suppresses inflammatory responses in rheumatoid arthritis fibroblast-like synoviocytes and collagen-induced arthritis

ObjectivesGlycogen synthase kinase (GSK)-3β, a serine/threonine protein kinase, has been implicated as a regulator of the inflammatory response. This study was performed to evaluate the effect of selective GSK-3β inhibitors in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and collagen-induced arthritis (CIA). MethodFLS from RA patients were treated with selective GSK-3β inhibitors, including lithium chloride, 6-bromoindirubin-3′-oxime (BIO), or 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8). The effects of GSK-3β inhibition on pro-inflammatory mediators were determined by real-time PCR and ELISA. The levels of NF-κB, phosphorylated JNK, c-jun, ATF-2 and p-38 proteins were evaluated by western blot analysis. The in vivo effects of GSK-3β inhibitors were examined in mice with CIA. ResultsTreatment of RA FLS with GSK-3β inhibitors induced dose-dependent reductions in gene expression and the production of pro-inflammatory mediators. The levels of NF-κB, phosphorylated JNK, c-jun, ATF-2 and p-38 were decreased following treatment with GSK-3β inhibitors. GSK-3β inhibitors treatment attenuated clinical and histological severities of CIA in mice. Infiltration of T-cells, macrophages, and tartrate-resistant acid phosphatase positive cells was decreased in joint sections of CIA mice by GSK-3β inhibitors treatment. Serum levels of IL-1β, IL-6, TNF-α and IFN-γ in CIA mice were also significantly decreased in dose-dependent manners by treatment with GSK-3β inhibitors. ConclusionTreatment with GSK-3β inhibitors suppressed inflammatory responses in RA FLS and CIA mice. These findings suggest that the inhibition of GSK-3β can be used as an effective therapeutic agent for RA.

Inhibition of Glycogen Synthase Kinase-3β Prevents Remifentanil-Induced Hyperalgesia via Regulating the Expression and Function of Spinal N-Methyl-D-Aspartate Receptors In Vivo and Vitro

A large number of experimental and clinical studies have confirmed that brief remifentanil exposure can enhance pain sensitivity presenting as opioid-induced hyperalgesia (OIH). N-methyl-D-aspartate (NMDA) receptor antagonists have been reported to inhibit morphine analgesic tolerance in many studies. Recently, we found that glycogen synthase kinase-3β (GSK-3β) modulated NMDA receptor trafficking in a rat model of remifentanil-induced postoperative hyperalgesia. In the current study, it was demonstrated that GSK-3β inhibition prevented remifentanil-induced hyperalgesia via regulating the expression and function of spinal NMDA receptors in vivo and in vitro. We firstly investigated the effects of TDZD-8, a selective GSK-3β inhibitor, on thermal and mechanical hyperalgesia using a rat model of remifentanil-induced hyperalgesia. GSK-3β activity as well as NMDA receptor subunits (NR1, NR2A and NR2B) expression and trafficking in spinal cord L4-L5 segments were measured by Western blot analysis. Furthermore, the effects of GSK-3β inhibition on NMDA-induced current amplitude and frequency were studied in spinal cord slices by whole-cell patch-clamp recording. We found that remifentanil infusion at 1 μg·kg-1·min-1 and 2 μg·kg-1·min-1 caused mechanical and thermal hyperalgesia, up-regulated NMDA receptor subunits NR1 and NR2B expression in both membrane fraction and total lysate of the spinal cord dorsal horn and increased GSK-3β activity in spinal cord dorsal horn. GSK-3β inhibitor TDZD-8 significantly attenuated remifentanil-induced mechanical and thermal hyperalgesia from 2 h to 48 h after infusion, and this was associated with reversal of up-regulated NR1 and NR2B subunits in both membrane fraction and total lysate. Furthermore, remifentanil incubation increased amplitude and frequency of NMDA receptor-induced current in dorsal horn neurons, which was prevented with the application of TDZD-8. These results suggest that inhibition of GSK-3β can significantly ameliorate remifentanil-induced hyperalgesia via modulating the expression and function of NMDA receptors, which present useful insights into the mechanistic action of GSK-3β inhibitor as potential anti-hyperalgesic agents for treating OIH.

Aberrant Glycogen Synthase Kinase 3β Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

Background and PurposeThe major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer.MethodsPancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined.ResultsPancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts.ConclusionThe targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.

O3-001 - Aberrant Glycogen Synthase Kinase 3βIS Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

ABSTRACT Purpose The major obstacles to effective treatment of pancreatic cancer are the highly invasive nature of this tumor type and its resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) is a serine/threonine protein kinase that regulates multiple cellular pathways and has been implicated in various diseases including cancer. Here we investigate a putative pathological role for aberrant GSK3β in the highly invasive and treatment resistant phenotype of pancreatic cancer. Methods Pancreatic cancer cell lines were examined for GSK3β expression, phosphorylation and activity using western bloting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Results Pancreatic cancer cells showed increased expression and deregulated activity of GSK3β that were associated with changes in its differential phosphorylation. Inhibition of GSK3β reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their chemotactic migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac-1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the attenuation of cancer cell migration and invasion, reduced secretion of matrix metalloproteinase-2 and decreased phosphorylation of focal adhesion kinase. Conclusion The targeting of GSK3β may represent a novel and effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.