Glycogen synthase kinase-3β regulates anti-inflammatory property of fluoxetine

A selective serotonin reuptake inhibitor fluoxetine not only is widely used in the treatment of depression but also has an anti-inflammatory property. Glycogen synthase kinase-3beta (GSK-3β) is a vital factor in the inflammation process. How fluoxetine interferes with inflammation via a GSK-3β-dependent pathway remains unclear. The aim of this study is to investigate the effects of fluoxetine on lipopolysaccharide (LPS)-induced inflammation. Results showed that fluoxetine decreased mortality rate of the mice. It also inhibited LPS-induced release of nitric oxide (NO) and prostaglandin E2 (PGE2) in serum and RAW264.7 murine macrophages and expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Upon LPS stimulation, fluoxetine caused a delay but increased in the phosphorylated levels of GSK-3β (ser9), whereas it did not affect LPS-induced activation of mitogen-activated protein kinase (MAPK) and generation of reactive oxygen species (ROS). Fluoxetine in combination with phosphatidylinositol 3-kinases/Akt inhibitors (LY294002 and Wortmannin) did not have a synergistic inhibition on LPS-induced NO release and PGE2 production. In addition, peroxisome proliferator-activated receptor γ (PPARγ) antagonist GW9622 showed no reverse effects of this inhibition of fluoxetine. GSK-3β knockdown blocked the inhibitory effects of fluoxetine on LPS-induced iNOS/NO release and COX-2/PGE2 production. These results indicated that GSK-3β regulated anti-inflammatory property of fluoxetine. However, Akt activation, ROS generation, and altered PPARγ activity were not involved in this inhibition of fluoxetine.     

Glycogen synthase kinase-3β regulates astrocytic differentiation of U87-MG human glioblastoma cells

Aim:

To evaluate the role of glycogen synthase kinase-3β (GSK-3β) in the induced differentiation of human glioblastoma cells.

Methods:

Cell proliferation was determined by bromodeoxyuridine (BrdU) incorporation assay. The protein level of p-GSK-3β, GSK-3β, glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) were determined using Western blots. The overexpression of mutant GSK-3β was analyzed by immunocytochemistry.

Results:

The biotoxin cholera toxin is capable of inducing differentiation of U87-MG human glioblastoma cells, which is characterized by morphological changes to astrocytic phenotype, increase in differentiation marker protein GFAP and decrease in proliferation. GSK-3β activation is induced during this differentiation. Small interfering RNA against GSK-3β suppresses the induced-differentiation in U87-MG cells. Conversely, overexpression of a constitutively active form of human GSK-3β (pcDNA3-GSK-3β-S9A) mutant leads to differentiation of U87-MG cells.

Conclusion:

Our findings suggest that GSK-3β plays an important role in astrocytic differentiation of human glioblastoma cells and may be a novel therapeutic target in the malignant tumor.     

Glycogen synthase kinase-3β2 has lower phosphorylation activity to tau than glycogen synthase kinase-3β1

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase that phosphorylate protein substrates involved in Alzheimer's disease (AD), such as microtubule-associated protein tau and amyloid precursor protein (APP). GSK-3β consists of two splice variants; the major short form (GSK-3β1) distributes in many organs and the minor long form (GSK-3β2), whose structural difference is the insert of only 13 amino acid residues to the C-terminal side of the catalytic site of GSK-3β1, is present in central nervous system. However, the physiological significances of the two variants are unclear. Here we examined whether the phosphorylation activities of two variants to tau and APP are different in cells. We found that GSK-3β2 has lower phosphorylation activity to tau at AD-relevant epitope (Ser396) than GSK-3β1 in cells, whereas the two variants exhibit equivalent levels of phosphorylation activities to APP. Recombinant GSK-3β2 has also lower phosphorylation activity to tau than GSK-3β1 in vitro, although the phosphorylation activities of the two variants to a synthetic peptide substrate pGS-2 are comparable. Furthermore, the deletion of the C-terminal tail (CT) of GSK-3β2 resulted in considerable reduction of tau phosphorylation activity as compared with GSK-3β1, suggesting that the lower phosphorylation activity of GSK-3β2 to tau is attributed to weak interaction with tau through its unique higher-order structure of CT constructed by the 13 amino acids insertion. Such information may provide a clue for understanding of the physiological significance of the two splice variants of GSK-3β and a new insight into the regulation of tau phosphorylation in central nervous system.     

Glycogen synthase kinase-3β is a functional modulator of serotonin-1B receptors

Glycogen synthase kinase-3 (GSK3) is a constitutively active protein kinase that is involved in neuronal regulation and is a potential pharmacological target of neurological disorders. We found previously that GSK3β selectively interacts with 5-hydroxytryptamine-1B receptors (5-HT1BR) that have important functions in serotonin neurotransmission and behavior. In this study, we provide new information supporting the importance of GSK3β in 5-HT1BR-regulated signaling, physiological function, and behaviors. Using molecular, biochemical, pharmacological, and behavioral approaches, we tested 5-HT1BR's interaction with G(i)α(2) and β-arrestin2 and 5-HT1BR-regulated signaling in cells, serotonin release in mouse cerebral cortical slices, and behaviors in wild-type and β-arrestin2 knockout mice. Molecular ablation of GSK3β and GSK3 inhibitors abolished serotonin-induced change of 5-HT1BR coupling to G(i)α(2) and associated signaling but had no effect on serotonin-induced recruitment of β-arrestin2 to 5-HT1BR. This effect is specific for 5-HT1BR because GSK3 inhibitors did not change the interaction between serotonin 1A receptors and G(i)α(2). Two GSK3 inhibitors, N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418) and 3-(5-bromo-1-methyl-1H-indol-3-yl)-4-(benzofuran-3-yl)pyrrole-2,5-dione (BIP-135), efficiently abolished the inhibitory effect of the 5-HT1BR agonist anpirtoline on serotonin release in mouse cerebral cortical slices. GSK3 inhibitors also facilitated the 5-HT1BR agonist anpirtoline-induced behavioral effect in the tail suspension test but spared anpirtoline-induced locomotor activity. These results suggest that GSK3β is a functional selective modulator of 5-HT1BR-regulated signaling, and GSK3 inhibitors fine-tune the physiological and behavioral actions of 5-HT1BR. Future studies may elucidate the significant roles of GSK3 in serotonin neurotransmission and implications of GSK3 inhibitors as functional selective modulators of 5-HT1BR.     

Glycogen synthase kinase-3β inhibition alleviates NLRP3 inflammasome activation in myocardial infarction

OBJECTIVE To investigate the regulatory mechanisms of glycogen synthase kinase-3β(GSK-3β)on NLRP3 inflammasome activation. METHODS We conducted myocardial infarction(MI) model in male Sprague-Dawley(SD) rat during days 2-28. An in vitro investigation was performed using new-born rat/human cardiomyocyte and fibroblast cultures under typical inflammasome stimulation and hypoxia treatment. Further identification for possibility of GSK-3β active NLRP3 inflammasomes, GSK-3β immunoprecipitation was performed from the lysate of inflammasome stimulation-treated rat neonatal fibroblasts(RCFs) with or without GSK-3β inhibitor pretreatment. RESULTS Assessments of cardiac function, histochemical and biochemical assays for cardiac tissues, as well as detection of protein and m RNA expressions in heart tissues, showed that GSK-3β inhibition remarkably improves myocardial dysfunction and prevents remodeling with parallel reduction of the parameters of NLRP3 inflammasome activation after MI. The measurement of primary rats/human cells expounded that GSK-3β inhibition reduce NLRP3 inflammasome activation happens in cardiac fibroblasts, but not in cardiomyocytes. Futhermore, GSK-3β interacts with ASC and GSK-3β inhibition reduces cytoplasmic aggregates of ASC, NLRP3 and caspase-1 formation. CONCLUSION GSK-3β directly mediates NLRP3 inflammasome activation causing cardiac dysfunction in MI.     

Identification of glycogen synthase kinase-3 inhibitors with a selective sting for glycogen synthase kinase-3α

The glycogen synthase kinase-3 (GSK-3) has been linked to the pathogenesis of colorectal cancer, diabetes, cardiovascular disease, acute myeloid leukemia (AML), and Alzheimer's disease (AD). The debate on the respective contributions of GSK-3α and GSK-3β to AD pathology and AML is ongoing. Thus, the identification of potent GSK-3α-selective inhibitors, endowed with favorable pharmacokinetic properties, may elucidate the effect of GSK-3α inhibition in AD and AML models. The analysis of all available crystallized GSK-3 structures provided a simplified scheme of the relevant hot spots responsible for ligand binding and potency. This resulted in the identification of novel scorpion shaped GSK-3 inhibitors. It is noteworthy, compounds 14d and 15b showed the highest GSK-3α selectivity reported so far. In addition, compound 14d did not display significant inhibition of 48 out of 50 kinases in the test panel. The GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish embryo assay.     

Effect of IFN-γ and dexamethasone on TGF-β1-induced human fetal lung fibroblast-myofibroblast differentiation

AIM: To study whether Smads signaling pathway was involved in human fetal lung fibroblast-myofibroblast differentiation induced by transforming growth factor (TGF)-beta1 and the role of interferon (IFN)-gamma, dexamethasone (DEX) in the fibroblast-myofibroblast differentiation. METHODS: Alpha-smooth muscle actin (alpha-SMA), Smad2/3, and Smad7 protein were assessed by Western blot. Collagen protein was analyzed by measuring hydroxyproline. Alpha-SMA and collagen III mRNA were assessed by RT-PCR. Myofibroblasts morphology and Smad2/3 nuclear translocation were assessed by immunohistochemistry. The overexpression of Smad7, a negative mediator of Smads signaling pathway, was acquired by transfection of Smad7 vector. RESULTS: During fibroblast-myofibroblast differentiation induced by TGF-beta1, IFN-gamma 200 microg/L markedly blocked TGF-beta1-induced alpha-SMA protein expression (P<0.01), collagen protein (P<0.01) and mRNA (P<0.05) expression, and myofibroblasts morphological transformation, but DEX 10 micromol/L augmented TGF-beta1-induced alpha-SMA expression (P<0.01). For myofibroblasts, both IFN-gamma 200 microg/L and DEX 10 micromol/L did not inhibit alpha-SMA expression (P>0.05) and collagen protein (P>0.05) and mRNA expression (P>0.05) and did not change myofibroblasts morphology. Transient transfection of Smad7 vector resulted in significant inhibition of TGF-beta1-induced alpha-SMA expression (P<0.01). IFN-gamma 200 microg/L did not block TGF-beta1-stimulated Smad2/3 phosphorylation and their nuclear translocation. CONCLUSION: TGF-beta1 induced fibroblast-myofibroblast differentiation in a Smad proteins-dependent manner. IFN-gamma could block this process but it was not mediated by interrupting smad2/3 phosphorylation and their nuclear translocation and DEX played a synergism with TGF-beta1. Differentiated myofibroblasts, however, were resistant to both IFN-gamma and DEX.     

Glycogen synthase kinase-3 – a promising therapeutic target: interview with Hagit Eldar-Finkelman

Dr Hagit Eldar-Finkelman (Sackler School of Medicine, Israel) was interviewed by Emma Quigley (Commissioning Editor, Expert Opinion on Therapeutic Targets) on 16th February 2006.

Born in Jerusalem, Dr Eldar-Finkelman received her BSc in Chemistry in 1984 and both her MSc in Physical Chemistry (1986) and PhD in Life Science (1993) from the Weizmann Institute of Science. She was a recipient of the British Council Award, which allowed her to conduct research in biological nuclear magnetic resonance at the University of Oxford in the laboratory of Professor George K Radda. Following postdoctoral work at the School of Medicine of the University of Washington with Nobel Laureate Professor Edwin G Krebs, she became an Assistant Professor in the Department of Medicine at Harvard Medical School. Dr Eldar-Finkelman joined the Sackler School of Medicine at Tel Aviv University in 1999. Dr Eldar-Finkelman’s research focuses on the molecular mechanisms regulating the protein kinase glycogen synthase kinase-3 (GSK-3), and their implications in negative regulation of signalling pathways. In particular, her work aims to develop specific inhibitors for GSK-3 and to test their functions in vitro and in vivo, considering the concept that such inhibitors may be useful in insulin resistance and Type 2 diabetes. These studies provide a conceptual basis for development of GSK-3 inhibitors and may lead to design of small molecules for treatment of diabetes and or neurodegenerative disorders.     

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

Cognitive deficits are common in patients with bipolar disorder (BD) in remission and may be associated with glycogen synthase kinase-3 (GSK-3) activity, which is inhibited by lithium. GSK-3 may be a relevant treatment target for interventions tailored at cognitive disturbances in BD but the relation between GSK-3 activity, cognition and lithium treatment is unknown. We therefore investigated the possible association between GSK-3 activity and cognition and whether lithium treatment moderates this association in patients with BD. In a prospective 6–12 month follow-up study, GSK- 3β activity in peripheral blood mononuclear cells was measured concurrently with cognitive performance assessed using a comprehensive test battery in 27 patients with BD-I in early and late remission following a manic or mixed episode. The GSK-3β activity, measured as serine-9 phosphorylated GSK-3β (pGSK-3β) and the GSK-3β ratio (serine-9-pGSK-3β /total GSK-3β), was negatively associated with sustained attention (p = 0.009 and p = 0.042, respectively), but not with other cognitive domains or global cognition. A crossover interaction between lithium treatment and the GSK activity was observed, indicating that lower pGSK-3β levels (p = 0.015) and GSK ratio (p = 0.010) were associated with better global cognition in lithium users whereas the opposite association was observed in non-lithium treated patients. Findings were not statistically significant after Bonferroni correction. In conclusion, cognitive functioning may be associated with GSK-3 activity in patients with BD-I and lithium treatment may modulate this relationship. Future studies in larger sample sizes are warranted to confirm these associations.     

Effect of efonidipine on TGF-β1-induced cardiac fibrosis through Smad2-dependent pathway in rat cardiac fibroblasts

Transforming growth factor beta-1 (TGF-β1) plays a critical role in progression of cardiac fibrosis, which may involve intracellular calcium change. We examined effects of efonidipine, a dual T-type and L-type calcium channel blocker (CCB), on TGF-β1-induced fibrotic changes in neonatal rat cardiac fibroblast. T-type and L-type calcium channel mRNAs were highly expressed in cultured cardiac fibroblasts. TGF-β1 (5 ng/mL) significantly increased Smad2 phosphorylation and [(3)H]-leucine incorporation, which were attenuated by pretreatment with efonidipine (10 μM). Neither R(-)efonidipine (10 μM), selective T-type CCB, nor nifedipine (10 μM), selective L-type CCB, efficaciously inhibited both TGF-β1-induced Smad2 phosphorylation and [(3)H]-leucine incorporation. However, both were markedly attenuated by combination of R(-)efonidipine and nifedipine, EDTA, or calcium-free medium. Pretreatment with Smad2 siRNA significantly attenuated [(3)H]-leucine incorporation induced by TGF-β1. These data suggest that efonidipine elicits inhibitory effects on TGF-β1- and Smad2-dependent protein synthesis through both T-type and L-type calcium channel-blocking actions in cardiac fibroblasts.     

The anti-tumoral drug enzastaurin inhibits natural killer cell cytotoxicity via activation of glycogen synthase kinase-3β

Enzastaurin is a selective protein kinase Cβ inhibitor which is shown to have direct antitumor effect as well as suppress glycogen synthase kinase-3β (GSK-3β) phosphorylation (resulting in its activation) in both tumor tissues and peripheral blood mononuclear cells (PBMC). It is currently used in phase II trials for the treatment of colon cancer, refractory glioblastoma and diffuse large B cell lymphoma. In this study, the direct effect of enzastaurin on effector function of human natural killer (NK) cells was investigated. The results obtained showed that enzastaurin suppressed both natural and antibody-dependent cellular cytotoxicity (ADCC) of NK cells against different tumor targets. This inhibition was associated with a specific down-regulation of surface expression of NK cell activating receptor NKG2D and CD16 involved in natural cytotoxicity and ADCC respectively, as well as the inhibition of perforin release. Analysis of signal transduction revealed that enzastaurin activated GSK-3β by inhibition of GSK-3β phosphorylation. Treatment of NK cells with GSK-3β-specific inhibitor TDZD-8 prevented enzastaurin-induced inhibition of NK cell cytotoxicity. Apart from the known antitumor and antiangiogenic effects, these results demonstrate that enzastaurin suppresses NK cell activity and may therefore interfere with NK cell-mediated tumor control in enzastaurin-treated cancer patients.     

Alpha-pinene promotes osteoblast differentiation and attenuates TNFα-induced inhibition of differentiation in MC3T3-E1 pre-osteoblasts

Alpha-pinene (α-pinene) is an organic compound, found in the oils of many species of coniferous trees, especially pine. α-Pinene reportedly has antioxidant and anti-inflammatory activities; however, its effects on osteoblasts are unknown. This study investigated the effects of α-pinene on osteoblast differentiation and tumour necrosis factor-alpha (TNFα)-induced inhibition of osteogenesis. Culture in control or osteogenic medium containing α-pinene increased osteogenic marker expression. Alkaline phosphatase staining and alizarin red S staining confirmed that α-pinene enhanced osteoblast differentiation. Also, α-pinene attenuated TNFα-induced inhibition of Smad1/5/9 phosphorylation and extracellular matrix mineralization. Taken together, our findings suggest that α-pinene enhances osteoblast differentiation and mineralization in MC3T3-E1 pre-osteoblasts.     

Up-regulation of microRNA-93 inhibits TGF-β1-induced EMT and renal fibrogenesis by down-regulation of Orai1

TGF-β1-induced excessive deposition of ECM and EMT process of tubular epithelial cells play critical roles in the development and progression of fibrosis in diabetic nephropathy (DN). Orai1 has been demonstrated to be involved in TGF-β1-induced EMT via TGF-β/Smad3 pathway. We are aimed to explore the effects of miR-93 on TGF-β1-induced EMT process in HK2 cells. In this study, our data showed that miR-93 was dramatically decreased in renal tissues of patients with DN and TGF-β1-stimulated HK2 cells. Moreover, the decreased level of miR-93 was closely associated with the increased expression of Orai1. Overexpression of miR-93 decreased Orai1 expression, and then suppressed TGF-β1-mediated EMT and fibrogenesis. Next, we predicted that the Orai1 was a potential target gene of miR-93, and demonstrated that miR-93 could directly target Orai1. SiRNA targeting Orai1 was sufficient to suppress TGF-β1-induced EMT and fibrogenesis in HK2 cells. Furthermore, Overexpression of Orai1 partially reversed the protective effect of miR-93 overexpression on TGF-β1-mediated EMT and fibrogenesis in HK2 cells. Taken together, Orai1 and miR-93 significantly impact on the progression of TGF-β1-mediated EMT and fibrogenesis in HK2 cells, and they may represent novel targets for the prevention strategies of fibrosis in the context of DN.     

Drug Development Targeting the Glycogen Synthase Kinase-3β (GSK-3β)-Mediated Signal Transduction Pathway: Inhibitors of the Wnt/β-Catenin Signaling Pathway as Novel Anticancer Drugs

Accumulating evidence suggests that the Wnt/β-catenin signaling pathway is often involved in oncogenesis and cancer development. Accordingly, a novel anticancer drug can be developed using inhibitors of this pathway. However, at present, there is no selective inhibitor of this pathway available as a therapeutic agent. Although all the components of the Wnt/β-catenin signaling pathway can be a target for drug development, glycogen synthase kinase-3β (GSK-3β), in particular, may be a good target because GSK-3β is an essential component of the pathway, and activation of this kinase results in the inhibition of the Wnt signaling pathway. We found that the differentiation-inducing factors (DIFs), putative morphogens for Dictyostelium discoideum, inhibit the Wnt/β-catenin signaling pathway via the activation of GSK-3β, resulting in the cell-cycle arrest of human cancer cell lines. In this review, we summarize our recent findings on the antiproliferative effect of DIFs and show the possibility for development of a novel anticancer drug from DIFs and their derivatives.     

Effect of TGF-β/Smad signaling pathway on lung myofibroblast differentiation

AIM: Myofibroblasts play important roles in the pathogenesis of lung fibrosis. Transforming growth factor (TGF)-beta 1 has been widely recognized as a key fibrogenic cytokine. The major signaling pathway of (TGF)-beta(1) is through cytoplasmic Smad proteins. Our study investigated the role of individual (TGF)-beta(1)/Smad signal proteins in mediating alpha-smooth muscle actin (alpha-SMA) gene expression, which is a well-known key marker of myofibroblast differentiation. METHODS: We transiently cotransfected alpha-SMA promoter-luciferase fusion plasmid (p895-Luc) and Smad expression plasmids and measured Luc activity in (TGF)-beta(1)-treated human fetal lung fibroblasts. We induced Smad3 knockout mice lung fibrosis by bleomycin. alpha-SMA protein expression was assessed by Western blotting. Collagen protein was analyzed by measuring hydroxyprolin. Myofibroblast morphology was assessed by immunohistochemistry. RESULTS: We found that the overexpression of Smad3, not Smad2 markedly increased (TGF)-beta(1)-induced alpha-SMA promoter activity and alpha-SMA protein expression in vitro, whereas the overexpression of dominant negative mutant Smad3 and Smad7 repressed (TGF)-beta(1)-induced alpha-SMA gene expression. Compared to wild-type mice, Smad3 knockout mice showed attenuated lung fibrosis after bleomycin treatment, manifested by lower collagen production and myofibroblast differentiation. CONCLUSION: Our study suggested (TGF)-beta(1)/Smad3 is a major pathway which regulated the myofibroblast differentiation. This result indicates a potential significance for future attempts of attenuating the progression of human lung fibrosis by the inhibition of the Smad3 cascade.     

Cornel iridoid glycoside induces autophagy to protect against tau oligomer neurotoxicity induced by activation of glycogen synthase kinase-3β

Tau oligomers are the etiologic molecules of Alzheimer disease(AD), and correlate strongly with neuronal loss and exhibit neurotoxicity. Recent evidence indicates that small tau oligomers are the most relevant toxic aggregate species. The aim of the present study was to investigate the mechanisms of cornel iridoid glycoside(CIG) on tau oligomers and cognitive functions. We injected wortmannin and GF-109203 X(WM/GFX, 200 μmol·L-1 each) into the lateral ventricles to induce tau oligomer and memory impairment in rats. When oral y administered with CIG at 60 and 120 mg·kg~(-1) per day for 14 d, CIG decreased the escape latency in Morris water maze test. We also found that CIG restored the expression of presynaptic p-synapsin, synaptophysin, and postsynaptic density-95(PSD-95) decreased by WM/GFX in rat cortex. CIG reduced the accumulation of tau oligomers in the brain of WM/GFX rats and in cells transfected with wild type glycogen synthase kinase-3β(wt GSK-3β). In addition, CIG up-regulated the levels of ATG7, ATG12, Beclin-1, and LC3 II in vivo and in vitro, suggesting the restoration of autophagy function. These results suggest that CIG could ameliorate memory deficits and regulate memory-associated synaptic proteins through the clearance of tau oligomers accumulation. Moreover, CIG clears tau oligomers by restoring autophagy function.     

TGF-β1 as a therapeutic target for pulmonary fibrosis and COPD

TGF-β1 is a multifunctional molecule that is expressed in an exaggerated fashion during injury, inflammation and repair. Its expression is dysregulated in lung tissues from patients with pulmonary fibrosis and chronic obstructive pulmonary disease. In animal models, introduction of TGF-β1 expression in the lung causes prominent tissue fibrosis and alveolar destruction. On the other hand, the exaggerated production of TGF-β1, an inability to activate TGF-β1 or a block in TGF-β1 signaling have all been associated with the development of emphysematous pulmonary lesions. A number of studies have demonstrated that TGF-β1 is a major player in the pathogenesis of pulmonary fibrosis and emphysema. In this review, we discuss how TGF-β1 expression is regulated and mechanistically related to the development of tissue fibrosis and emphysema in experimental animal models and humans. We further highlight potential therapeutic options that control TGF-β1-associated genes or signals to restore extracellular matrix homeostasis in which TGF-β1 plays a central role.