Protein Levels Of GSK3β Research Articles

Glycogen synthase kinase-3 is an early determinant in the differentiation of pathogenic Th17 cells. (152.3)

Abstract CD4+ T cells are critical for host defense but also drive immune-mediated diseases. The classical view of Th1 and Th2 subtypes of CD4+ T cells was recently revised by the identification of the Th17 lineage that produces IL-17 and are critical in autoimmune disease pathogenesis. Mechanisms controlling the differentiation of Th17 cells have been described, but few feasible targets for therapeutically reducing Th17 cells are known. The generation of Th17 cells requires IL-6 and activation of STAT3. During polarization of CD4+ T cells to Th17 cells, we found that inhibition of glycogen synthase kinase-3 (GSK3) blocked IL-6 production, STAT3 activation, and polarization to Th17 cells. Polarization of CD4+ T cells to Th17 cells increased by 10-fold GSK3β protein levels in Th17 cells, while GSK3β was unaltered in Treg cells. Diminishing GSK3 activity pharmacologically or molecularly blocked Th17 cell production, and increasing GSK3 activity promoted polarization to Th17 cells. In vivo inhibition of GSK3 in mice depleted constitutive Th17 cells in intestinal mucosa, blocked Th17 cell generation in the lung following Francisella tularensis infection, and inhibited the increase in spinal cord Th17 cells and disease symptoms in the experimental autoimmune encephalomyelitis (EAE) mouse MS model. These findings identify GSK3 as a critical mediator of Th17 cell production, and indicate that GSK3 inhibitors provide a potential therapeutic intervention to control Th17-mediated diseases.

Thiazolidenediones induce tumour-cell apoptosis through the Akt-GSK3β pathway

Prostate cancer is a major health threat for men. Thiazolidenediones (TZDs) are synthetic ligands of the peroxisome proliferator-activated receptor γ (PPARγ), and previous studies have shown that TZDs induce apoptosis of prostate cancer cells independently of PPARγ activation. However, the exact mechanism of these effects remains unknown. Our objective was to investigate the effects of TZDs on apoptosis and on the serine/threonine kinase pathway, Akt, and glycogen synthase kinase 3β (GSK3β). LNCaP cells, a type of prostate cancer cells (derived from left supraclavicular lymph node of human prostrate carcinoma), were cultured in DMEM medium, and cell viability was evaluated with a colorimetric assay using MTT level. The total and phosphorylated protein level of Akt and GSK3β were detected by Western blotting. The apoptosis-inducing effect of TZDs on prostate cancer cells involves the inhibition of Akt phosphorylation. Furthermore, TZDs induce inactivation of GSK3β, a multifunctional kinase that mediates essential events promoting prostate cancer development and acquisition of androgen independence. In addition, the GSK3β inhibitor lithium chloride sensitizes prostate cancer cells to TZDs cytotoxicity. Our data suggest that modulation of Akt-GSK3β pathway is involved in the cell death pathway engaged by TZDs in prostate cancer cells. This reveals another possible mechanism of TZDs on apoptosis in prostate cancer. Inhibition of the Akt-GSK3β cascade may be a useful approach in prostate cancer.

Raf Kinase Inhibitor Protein RKIP Enhances Signaling by Glycogen Synthase Kinase-3β

Raf kinase inhibitory protein (RKIP) is a physiologic inhibitor of c-RAF kinase and nuclear factor κB signaling that represses tumor invasion and metastasis. Glycogen synthase kinase-3β (GSK3β) suppresses tumor progression by downregulating multiple oncogenic pathways including Wnt signaling and cyclin D1 activation. Here, we show that RKIP binds GSK3 proteins and maintains GSK3β protein levels and its active form. Depletion of RKIP augments oxidative stress-mediated activation of the p38 mitogen activated protein kinase, which, in turn, inactivates GSK3β by phosphorylating it at the inhibitory T390 residue. This pathway de-represses GSK3β inhibition of oncogenic substrates causing stabilization of cyclin D, which induces cell-cycle progression and β-catenin, SNAIL, and SLUG, which promote epithelial to mesenchymal transition. RKIP levels in human colorectal cancer positively correlate with GSK3β expression. These findings reveal the RKIP/GSK3 axis as both a potential therapeutic target and a prognosis-based predictor of cancer progression.

Glycogen Synthase Kinase-3 Is an Early Determinant in the Differentiation of Pathogenic Th17 Cells

CD4(+) T cells are critical for host defense but are also major drivers of immune-mediated diseases. The classical view of Th1 and Th2 subtypes of CD4(+) T cells was recently revised by the identification of the Th17 lineage of CD4(+) T cells that produce IL-17, which have been found to be critical in the pathogenesis of autoimmune and other diseases. Mechanisms controlling the differentiation of Th17 cells have been well described, but few feasible targets for therapeutically reducing Th17 cells are known. The generation of Th17 cells requires IL-6 and activation of STAT3. During polarization of CD4(+) T cells to Th17 cells, we found that inhibition of glycogen synthase kinase-3 (GSK3) blocked IL-6 production, STAT3 activation, and polarization to Th17 cells. Polarization of CD4(+) T cells to Th17 cells increased by 10-fold the expression of GSK3β protein levels in Th17 cells, whereas GSK3β was unaltered in regulatory T cells. Diminishing GSK3 activity either pharmacologically or molecularly blocked Th17 cell production, and increasing GSK3 activity promoted polarization to Th17 cells. In vivo inhibition of GSK3 in mice depleted constitutive Th17 cells in intestinal mucosa, blocked Th17 cell generation in the lung after Francisella tularensis infection, and inhibited the increase in spinal cord Th17 cells and disease symptoms in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. These findings identify GSK3 as a critical mediator of Th17 cell production and indicate that GSK3 inhibitors provide a potential therapeutic intervention to control Th17-mediated diseases.

Mechanical Stretch Up-regulates MicroRNA-26a and Induces Human Airway Smooth Muscle Hypertrophy by Suppressing Glycogen Synthase Kinase-3β

Airway smooth muscle hypertrophy is one of the hallmarks of airway remodeling in severe asthma. Several human diseases have been now associated with dysregulated microRNA (miRNA) expression. miRNAs are a class of small non-coding RNAs, which negatively regulate gene expression at the post-transcriptional level. Here, we identify miR-26a as a hypertrophic miRNA of human airway smooth muscle cells (HASMCs). We show that stretch selectively induces the transcription of miR-26a located in the locus 3p21.3 of human chromosome 3. The transcription factor CCAAT enhancer-binding protein α (C/EBPα) directly activates miR-26a expression through the transcriptional machinery upon stretch. Furthermore, stretch or enforced expression of miR-26a induces HASMC hypertrophy, and miR-26 knockdown reverses this effect, suggesting that miR-26a is a hypertrophic gene. We identify glycogen synthase kinase-3β (GSK-3β), an anti-hypertrophic protein, as a target gene of miR-26a. Luciferase reporter assays demonstrate that miR-26a directly interact with the 3'-untranslated repeat of the GSK-3β mRNA. Stretch or enforced expression of miR-26a attenuates the endogenous GSK-3β protein levels followed by the induction of HASMC hypertrophy. miR-26 knockdown reverses this effect, suggesting that miR-26a-induced hypertrophy occurs via its target gene GSK-3β. Overall, as a first time, our study unveils that miR-26a is a mechanosensitive gene, and it plays an important role in the regulation of HASMC hypertrophy.

Knockdown I2PP2A attenuates tau hyperphosphorylation and the underlying mechanisms

In Alzheimer's disease, increased inhibitor-2 of protein phosphatase-2A (I2PP-2A) level has been associated with decreased protein phosphatase-2A activity and hyperphosphorylation tau. Thus, reduction of I2PP-2A level could be important in the treatment of Alzheimer's disease. pSUPER-siI2PP-2A was constructed and transfected into HEK293/tau cells with administered wortmannin or okadaic acid. Western blots was used to detected the levels of hyperphosphrylated tau, GSK-3β and phospho-GSK-3β (Ser9). PP-2A activity was assayed using a serine/threonine phosphatse assay system (Promega, MA) according to the manufacturer's protocol. We found that I2PP-2A could only interact with the catalytic subunit of PP-2A and had no interaction with the regulatory subunit. Increased activity of PP-2A was for the associational level of I2PP-2A was decreased while transfection with pSUPER-siI2PP-2A. We also found that hyperphosphorylation tau levels induced by okadaic acid or wortmannin were decreased while transfection with DNA plasmid pSUPER-siI2PP-2A expressing siRNA targeting I2PP-2A. Knockdown I2PP-2A decreased the activity of GSK-3β, for it decreased the total levels of GSK-3β protein and mRNA, however increased the level of phospho-GSK-3β (Ser9), which was phosphorylated by PKA. All of these suggest that I2PP-2A may be a promising target for AD therapy.

Blockade of JAK2 activity suppressed accumulation of β‐catenin in leukemic cells

The Wnt/β-catenin pathway has been implicated in leukemogenesis. We found β-catenin abnormally accumulated in both human acute T cell leukemia Jurkat cells and human erythroleukemia HEL cells. β-Catenin can be significantly down-regulated by the Janus kinase 2 specific inhibitor AG490 in these two cells. AG490 also reduces the luciferase activity of a reporter plasmid driven by LEF/β-catenin promoter. Similar results were observed in HEL cells infected with lentivirus containing shRNA against JAK2 gene. After treatment with 50 µM AG490 or shRNA, the mRNA expression levels of β-catenin, APC, Axin, β-Trcp, GSK3α, and GSK3β were up-regulated within 12-16 h. However, only the protein levels of GSK3β and β-Trcp were found to have increased relative to untreated cells. Knockdown experiments revealed that the AG490-induced inhibition of β-catenin can be attenuated by shRNA targeting β-TrCP. Taken together; these results suggest that β-Trcp plays a key role in the cross-talk between JAK/STAT and Wnt/β-catenin signaling in leukemia cells.

Association of PI3-kinase and Wnt Signaling in Non-steroidal Anti-Inflammatory Drug-induced Apoptosis in Experimental Colon Cancer

In addition to having anti-inflammatory properties, non-steroidal anti-inflammatory drugs (NSAIDs) inhibit neoplastic cell proliferation by inducing apoptosis. Inhibition of cycloxygenase-2 (COX-2) seemed to be the principal target of NSAIDs, as it is overexpressed in several cancers and catalyzes the synthesis of prostaglandin E2 (PGE2), the critical proinflammatory molecule. A major role for phosphatidyl inositol 3kinase (PI3-kinase) pathway activation in human tumors has been more recently established. The present study explored the role of PI3-kinase and Wnt molecular pathways in NSAIDs’ chemopreventive effect in colon cancer. 1, 2-dimethylhydrazine was used for experimental colon cancer model and diclofenac as the chemopreventive agent. Protein expression of caspase-3 and 9 and fragmentation of DNA were checked in the colonic tissue. There was a decrease in all the three parameters, indicative of inhibition of apoptosis in the present experimental cancer model. Protein expression of PCNA and proliferation index as determined by both PCNA and BrdU immunostaining were also seen to be increased in the DMH treated animals. DMH upregulated the expression of PI3-kinase, Akt, Wnt and β-catenin, but reduced the GSK-3β levels. Coadministration with diclofenac, while decreasing PI3-kinase, Akt, Wnt and β-catenin, also increased the protein level of GSK-3β thus confirming that NSAIDs target PI3-kinase and Wnt signaling to exert the two important actions – induction of apoptosis and inhibition of cellular proliferation.

Beta-Catenin Activation Promotes Liver Regeneration after Acetaminophen-Induced Injury

Acute liver failure (ALF) remains a disease with poor patient outcome. Improved prognosis is associated with spontaneous liver regeneration, which supports the relevance of exploring 'regenerative' therapies. Therefore, the role of the Wnt/beta-catenin pathway in liver regeneration following ALF was investigated. ALF was induced in mice by acetaminophen overdose, which is also a leading cause of liver failure in patients. beta-catenin distribution was also studied in liver sections from acetaminophen-induced ALF patients. A nonlethal dose of acetaminophen, which induces liver regeneration, led to stabilization and activation of beta-catenin for 1 to 12 hours. These data were also verified by increased expression of the beta-catenin surrogate target glutamine synthetase. Beta-catenin activation occurred secondary to the inactivation of glycogen synthase kinase-3beta and an increase in levels of casein kinase 2alpha, and led to increased cyclin-D1, another known beta-catenin target. These observations were next substantiated in beta-catenin conditional-null mice (beta-catenin-null), which show dampened regeneration after acetaminophen injury following induction of CYP2e1/1a2 expression. In light of decreased acetaminophen injury in beta-catenin-null mice despite CYP induction, equitoxic studies in control mice were performed. Significant differences in regeneration persisted following comparable injury in beta-catenin-null and control animals. Retrospective analysis of liver samples from acetaminophen-overdose patients demonstrated a positive correlation between nuclear beta-catenin, proliferation, and spontaneous liver regeneration. Thus, our studies demonstrate early activation of beta-catenin signaling during acetaminophen-induced injury, which contributes to hepatic regeneration.

Cleavage of glycogen synthase kinase‐3beta by matrix metalloproteinase‐2 enhances its kinase activity

Matrix metalloproteinase (MMP)‐2 is localized to the sarcomere of cardiomyocytes and contributes to myocardial ischemia‐reperfusion injury by degrading sarcomeric and cytoskeletal proteins. Oxidative stress mediates its damaging effects through activation of MMP‐2 within the cardiomyocyte and subsequent proteolysis of susceptible intracellular targets. Glycogen synthase kinase (GSK)‐3β is involved in growth, death & metabolism and susceptible to proteolytic cleavage. We determined whether GSK‐3β is a proteolytic substrate of MMP‐2 and how this cleavage affects its kinase activity.Incubation of MMP‐2 and GSK‐3β resulted in a time‐ and concentration‐dependant cleavage of GSK‐3β with significant increase in GSK‐3β kinase activity. Dephosphorylation of GSK‐3β with λ‐phosphatase prevented MMP‐2 mediated augmented kinase activity. H2O2 treated H9c2 cardiomyoblasts showed increased levels and activity of MMP‐2 accompanied by significant reduction in GSK‐3β protein levels and increased kinase activity.The data indicate that GSK‐3β is a target of MMP‐2 and that its cleavage by MMP‐2 enhances GSK‐3β kinase activity. GSK‐3β dephosphorylation studies suggest that MMP‐2 may cleave off the inhibitory serine‐9 residue. This MMP‐2 mediated augmentation of GSK‐3β kinase activity may contribute to cardiac dysfunction as a result of myocardial oxidative stress injury. This work was supported by CIHR.

Antifolate/folate-activated HGF/c-Met signalling pathways in mouse kidneys—the putative role of their downstream effectors in cross-talk with androgen receptor

This in vivo study of mouse kidneys was focused on the identification of protein mediators involved in the cross-talk between two signalling pathways. One pathway was triggered by testosterone via an androgen receptor, AR, and the other induced by CB 3717/folate via HGF, and its membrane receptor c-Met. Sequential activation of these pathways leads to a drastic decrease of testosterone-induced ornithine decarboxylase, ODC, expression. We proved that CB 3717/folate-induced ODC expression is Akt-dependent. CB 3717/folate activates Akt and ERK1/2 kinases, PTEN phosphatase and also up-regulates cyclin D2 and PCNA, but decreases GSK3β and cyclin D1 protein levels. Testosterone activation of AR induces GSK3β and PTEN. Results of the sequential activation of the studied signalling pathways suggest that Akt, GSK3β and possibly ERK1/2 kinases may participate in the negative cross-talk and attenuation of AR transactivity, while the involvement of PTEN and cyclin D1 seems to be doubtful.

Cortical deficiency of laminin γ1 impairs the AKT/GSK-3β signaling pathway and leads to defects in neurite outgrowth and neuronal migration

Laminins have dramatic and varied actions on neurons in vitro. However, their in vivo function in brain development is not clear. Here we show that knockout of laminin γ1 in the cerebral cortex leads to defects in neuritogenesis and neuronal migration. In the mutant mice, cortical layer structures were disrupted, and axonal pathfinding was impaired. During development, loss of laminin expression impaired phosphorylation of FAK and paxillin, indicating defects in integrin signaling pathways. Moreover, both phosphorylation and protein levels of GSK-3β were significantly decreased, but only phosphorylation of AKT was affected in the mutant cortex. Knockout of laminin γ1 expression in vitro, dramatically inhibited neurite growth. These results indicate that laminin regulates neurite growth and neuronal migration via integrin signaling through the AKT/GSK-3β pathway, and thus reveal a novel mechanism of laminin function in brain development.

Activity‐dependent expression of ELAV/Hu RBPs and neuronal mRNAs in seizure and cocaine brain

Growing evidence indicates that both seizure (glutamate) and cocaine (dopamine) treatment modulate synaptic plasticity within the mesolimbic region of the CNS. Activation of glutamatergic neurons depends on the localized translation of neuronal mRNA products involved in modulating synaptic plasticity. In this study, we demonstrate the dendritic localization of HuR and HuD RNA-binding proteins (RBPs) and their association with neuronal mRNAs following these two paradigms of seizure and cocaine treatment. Both the ubiquitously expressed HuR and neuronal HuD RBPs were detected in different regions as well as within dendrites of the brain and in dissociated neurons. Quantitative analysis revealed an increase in HuR, HuD and p-glycogen synthase kinase 3beta (GSK3beta) protein levels as well as neuronal mRNAs encoding Homer, CaMKIIalpha, vascular early response gene, GAP-43, neuritin, and neuroligin protein products following either seizure or cocaine treatment. Inhibition of the Akt/GSK3beta signaling pathway by acute or chronic LiCl treatment revealed changes in HuR, HuD, pGSK3beta, p-Akt, and beta-catenin protein levels. In addition, a genetically engineered hyperdopaminergic mouse model (dopamine transporter knockout) revealed decreased expression of HuR protein levels, but no significant change was observed in HuD or fragile-X mental retardation protein RBPs. Finally, our data suggest that HuR and HuD RBPs potentially interact directly with neuronal mRNAs important for differentiation and synaptic plasticity.

Interleukin-18 increases expression of kinases involved in tau phosphorylation in SH-SY5Y neuroblastoma cells

Inflammatory cytokines, produced mainly by activated microglia in the brain, can enhance neuronal degeneration and the amyloid-β-plaque production involved in Alzheimer's disease (AD). We previously demonstrated that the expression of the pro-inflammatory cytokine interleukin-18 (IL-18) colocalizes with plaques and hyperphoshorylated tau containing neurons in AD patients. Here we exposed neuron-like, differentiated SH-SY5Y neuroblastomas to IL-18 and observed that the protein levels of p35, Cdk5, GSK-3β, and Ser15-phosphorylated p53 increased during 6 h-24 h. Tau phosphorylation and expression of cyclin G1, involved in neuronal regeneration, increased at 72 h. In vivo, over-expression of IL-18 may induce hyperphosphorylation of tau and induce cell cycle activators.

Makorin-2 Is a Neurogenesis Inhibitor Downstream of Phosphatidylinositol 3-Kinase/Akt (PI3K/Akt) Signal

Makorin-2 belongs to the makorin RING zinc finger gene family, which encodes putative ribonucleoproteins. Here we cloned the Xenopus makorin-2 (mkrn2) and characterized its function in Xenopus neurogenesis. Forced overexpression of mkrn2 produced tadpoles with dorso-posterior deficiencies and small-head/short-tail phenotype, whereas knockdown of mkrn2 by morpholino antisense oligonucleotides induced double axis in tadpoles. In Xenopus animal cap explant assay, mkrn2 inhibited activin, and retinoic acid induced animal cap neuralization, as evident from the suppression of a pan neural marker, neural cell adhesion molecule. Surprisingly, the anti-neurogenic activity of mkrn2 is independent of the two major neurogenesis signaling cascades, BMP-4 and Wnt8 pathways. Instead, mkrn2 works specifically through the phosphatidylinositol 3-kinase (PI3K) and Akt-mediated neurogenesis pathway. Overexpression of mkrn2 completely abrogated constitutively active PI3K- and Akt-induced, but not dominant negative glycogen synthase kinase-3beta (GSK-3beta)-induced, neural cell adhesion molecule expression, indicating that mkrn2 acts downstream of PI3K and Akt and upstream of GSK-3beta. Moreover, mkrn2 up-regulated the mRNA and protein levels of GSK-3beta. These results revealed for the first time the important role of mkrn2 as a new player in PI3K/Akt-mediated neurogenesis during Xenopus embryonic development.

STAT3 Sensitizes Insulin Signaling by Negatively Regulating Glycogen Synthase Kinase-3β

Glucose homeostasis is achieved by triggering regulation of glycogen synthesis genes in response to insulin when mammals feed, but the underlying molecular mechanism remains largely unknown. The aim of our study was to examine the role of the signal transducers and activators of transcription 3 (STAT3) in insulin signaling. We generated a strain of mice carrying a targeted disruption of Stat3 gene in the liver (L-Stat3(-/-) mice). Hepatocytes of the L-Stat3(-/-) mice were isolated to establish cell lines for mechanistic studies. Nuclear translocation and DNA-protein interaction of STAT3 was analyzed with immunofluorescent and chromatin immunoprecipitation methods, respectively. Levels of glucose, insulin, leptin, and glucagon were profiled, and putative downstream molecules of STAT3 were examined in the presence of various stimuli in L-Stat3(-/-) and control mice. STAT3 was found to sensitize the insulin signaling through suppression of GSK-3beta, a negative regulator of insulin signaling pathway. During feeding, both mRNA and protein levels of GSK-3beta decreased in Stat3(f/+) mice, which reflected the need of hepatocytes for insulin to induce glycogen synthesis. In contrast, the L-Stat3(-/-) mice lost this control and showed a monophasic increase in the GSK-3beta level in response to insulin. Administration of GSK-3beta inhibitors lithium chloride and L803-mts restored glucose homeostasis and rescued the glucose intolerance and impaired insulin response in L-Stat3(-/-) mice. These data indicate that STAT3 sensitizes insulin signaling by negatively regulating GSK-3beta. Inactivation of STAT3 in the liver contributes significantly to the pathogenesis of insulin resistance.

T-Darpp Promotes Cancer Cell Survival by Up-regulation of Bcl2 through Akt-Dependent Mechanism

t-Darpp is a cancer-related truncated isoform of Darpp-32 (dopamine and cyclic-AMP-regulated phosphoprotein of M(r) 32,000). We detected overexpression of t-Darpp mRNA in two thirds of gastric cancers compared with normal samples (P = 0.004). Using 20 micromol/L ceramide treatment as a model for induction of apoptosis in AGS cancer cells, we found that expression of t-Darpp led to an increase in Bcl2 protein levels and blocked the activation of caspase-3 and caspase-9. The MitoCapture mitochondrial apoptosis and cytochrome c release assays indicated that t-Darpp expression enforces the mitochondrial transmembrane potential and protects against ceramide-induced apoptosis. Interestingly, the expression of t-Darpp in AGS cells led to >or=2-fold increase in Akt kinase activity with an increase in protein levels of p-Ser(473) Akt and p-Ser(9) GSK3 beta. These findings were further confirmed using tetracycline-inducible AGS cells stably expressing t-Darpp. We also showed transcriptional up-regulation of Bcl2 using the luciferase assay with Bcl2 reporter containing P1 full promoter, quantitative reverse transcription-PCR, and t-Darpp small interfering RNA. The Bcl2 promoter contains binding sites for cyclic AMP-responsive element binding protein CREB/ATF1 transcription factors and using the electrophoretic mobility shift assay with a CREB response element, we detected a stronger binding in t-Darpp-expressing cells. The t-Darpp expression led to an increase in expression and phosphorylation of CREB and ATF-1 transcription factors that were required for up-regulating Bcl2 levels. Indeed, knockdown of Akt, CREB, or ATF1 in t-Darpp-expressing cells reduced Bcl2 protein levels. In conclusion, the t-Darpp/Akt axis underscores a novel oncogenic potential of t-Darpp in gastric carcinogenesis and resistance to drug-induced apoptosis.

Involvement of GSK-3β and DYRK1B in Differentiation-inducing Factor-3-induced Phosphorylation of Cyclin D1 in HeLa Cells

Differentiation-inducing factors (DIFs) are putative morphogens that induce cell differentiation in Dictyostelium discoideum. We previously reported that DIF-3 activates glycogen synthase kinase-3beta (GSK-3beta), resulting in the degradation of cyclin D1 in HeLa cells. In this study, we investigated the effect of DIF-3 on cyclin D1 mutants (R29Q, L32A, T286A, T288A, and T286A/T288A) to clarify the precise mechanisms by which DIF-3 degrades cyclin D1 in HeLa cells. We revealed that T286A, T288A, and T286A/T288A mutants were resistant to DIF-3-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr(286) and Thr(288) were critical for cyclin D1 degradation induced by DIF-3. Indeed, DIF-3 markedly elevated the phosphorylation level of cyclin D1, and mutations introduced to Thr(286) and/or Thr(288) prevented the phosphorylation induced by DIF-3. Depletion of endogenous GSK-3beta and dual-specificity tyrosine phosphorylation regulated kinase 1B (DYRK1B) by RNA interference attenuated the DIF-3-induced cyclin D1 phosphorylation and degradation. The effect of DIF-3 on DYRK1B activity was examined and we found that DIF-3 also activated this kinase. Further, we found that not only GSK-3beta but also DYRK1B modulates cyclin D1 subcellular localization by the phosphorylation of Thr(288). These results suggest that DIF-3 induces degradation of cyclin D1 through the GSK-3beta- and DYRK1B-mediated threonine phosphorylation in HeLa cells.

Glycogen synthase kinase-3β binds to E2F1 and regulates its transcriptional activity

GSK3β and E2F1 play an important role in the control of proliferation and apoptosis. Previous work has demonstrated that GSK3β indirectly regulates E2F activity through modulation of cyclin D1 levels. In this work we show that GSK3β phosphorylates human E2F1 in vitro at serine 403 and threonine 433, both residues localized at its transactivation domain. This phosphorylation was not detected in vivo. However, co-immunoprecipitation experiments do reveal in vivo binding of these proteins. Moreover, uninhibitable and catalitycally inactive GSK3β forms inhibit the transcriptional activity of a fusion protein containing E2F1 transactivation domain. Both forms of GSK3β inhibit E2F1 with similar efficiency. Interestingly the effect was independent of the mutation of serine 403 and threonine 433 to alanine. This suggests that this transcriptional modulation is independent of GSK3β kinase activity and phosphorylation state of serine 403 and threonine 433. The re-targeting of these GSK3β forms to the nucleus results in a higher capacity to regulate E2F1 transcriptional activity. Depletion of the levels of GSK3β protein using siRNA activates E2F1 transcriptional activity. The data presented in this study offer a new mechanism of regulation of E2F1 by direct binding of GSK3β to its transactivation domain.

Novel Anti-inflammatory Role for Glycogen Synthase Kinase-3β in the Inhibition of Tumor Necrosis Factor-α- and Interleukin-1β-induced Inflammatory Gene Expression

Glycogen synthase kinase-3beta (GSK-3beta) is a serine/threonine kinase with a broad array of cellular targets, such as cytoskeletal proteins and transcription factors. Recent studies with GSK-3beta-null mice showed impaired NFkappaB-mediated survival responses. Because NFkappaB serves a dual role as a key regulator of cytokine-induced inflammatory gene expression and apoptosis, we investigated whether modulation of GSK-3beta expression affects cytokine-induced and NFkappaB-mediated inflammatory gene expression. We observed that tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) treatment of primary cultures of human microvascular cells reduced net endogenous active GSK-3beta protein levels while inducing inflammatory cytokine (IL-6 and monocyte chemoattractant protein-1 (MCP-1)) expression. Interestingly, inhibition of GSK-3beta by antisense oligonucleotides or pharmacological agent (10 mm lithium) potentiated TNF-induced expression of IL-6 and MCP-1 by 2-6-fold suggesting that inhibition of GSK-3beta under inflammatory conditions (exposure to TNF-alpha and IL-1beta) may contribute to enhanced cytokine expression. Overexpression of GSK-3beta in endothelial cells, in contrast, significantly inhibited (by 70%, p < 0.01) both TNF-alpha and IL-1beta-induced expression of IL-6, MCP-1, and vascular cell adhesion molecule-1. Using adenoviruses in lipopolysaccharide-stimulated mice, overexpression of GSK-3beta significantly decreased TNF-alpha expression in lung and heart tissues (38 and 15%, respectively), further confirming the anti-inflammatory role of GSK-3beta. Overexpression of GSK-3beta did not affect the TNF-alpha-induced nuclear translocation of NFkappaB but reduced the nuclear half-life of TNF-alpha-induced NFkappaB considerably (by as much as 9 h) and enhanced phosphorylation (by as much as 33%). Interestingly, neither endothelial cell survival nor NFkappaB-mediated expression of anti-apoptotic genes was affected by GSK-3beta overexpression. We conclude that GSK-3beta selectively regulates NFkappaB-mediated inflammatory gene expression by controlling the flow of NFkappaB activity between transcription of inflammatory and survival genes.