GSK3 Inhibitor SB216763 Research Articles

Lithium inhibits tryptophan catabolism via the inflammation-induced kynurenine pathway in human microglia.

Despite its decades' long therapeutic use in psychiatry, the biological mechanisms underlying lithium's mood-stabilizing effects have remained largely elusive. Here, we investigated the effect of lithium on tryptophan breakdown via the kynurenine pathway using immortalized human microglia cells, primary human microglia isolated from surgical specimens, and microglia-like cells differentiated from human induced pluripotent stem cells. Interferon (IFN)-γ, but not lipopolysaccharide, was able to activate immortalized human microglia, inducing a robust increase in indoleamine-2,3-dioxygenase (IDO1) mRNA transcription, IDO1 protein expression, and activity. Further, chromatin immunoprecipitation verified enriched binding of both STAT1 and STAT3 to the IDO1 promoter. Lithium counteracted these effects, increasing inhibitory GSK3βS9 phosphorylation and reducing STAT1S727 and STAT3Y705 phosphorylation levels in IFN-γ treated cells. Studies in primary human microglia and hiPSC-derived microglia confirmed the anti-inflammatory effects of lithium, highlighting that IDO activity is reduced by GSK3 inhibitor SB-216763 and STAT inhibitor nifuroxazide via downregulation of P-STAT1S727 and P-STAT3Y705 . Primary human microglia differed from immortalized human microglia and hiPSC derived microglia-like cells in their strong sensitivity to LPS, resulting in robust upregulation of IDO1 and anti-inflammatory cytokine IL-10. While lithium again decreased IDO1 activity in primary cells, it further increased release of IL-10 in response to LPS. Taken together, our study demonstrates that lithium inhibits the inflammatory kynurenine pathway in the microglia compartment of the human brain.

The indirubin derivative 6-bromoindirubin-3′-glycerol-oxime ether (6BIGOE) potently modulates inflammatory cytokine and prostaglandin release from human monocytes through GSK-3 interference

Indirubin is a natural bis-indole alkaloid contained as active ingredient in the traditional Chinese remedy Danggui Longhui Wan. Indirubin and its 3′-oxime derivatives exhibit anti-cancer and anti-inflammatory properties and they inhibit glycogen synthase kinase (GSK)-3 in cell-free assays where 6-bromoindirubin-3′-oxime (6BIO) is among the most potent analogs. Here, we reveal 6-bromoindirubin-3′-glycerol-oxime ether (6BIGOE) as highly potent derivative able to inhibit pro-inflammatory cytokine, chemokine and prostaglandin (PG) release in human primary monocytes while increasing anti-inflammatory interleukin (IL)-10 levels. 6BIGOE suppressed lipopolysaccharide (LPS)-induced IL-1β and PGE2 release with IC50 of 0.008 and 0.02 µM, respectively, being ≥ 12-fold more potent than 6BIO. The effects of 6BIGOE are mediated via intracellular inhibition of GSK-3, where 6BIGOE again surpassed the effectiveness of 6BIO despite the higher potency of the latter in cell-free GSK-3 activity assays. Side-by-side comparison of 6BIGOE (0.1 µM) with the selective GSK-3 inhibitor SB216763 (5 µM) revealed congruent properties such as enrichment of β-catenin and suppression of cyclooxygenase (COX)-2 protein levels due to GSK–3 inhibition. Metabololipidomics using ultra-performance liquid chromatography-tandem mass spectrometry showed that 6BIGOE selectively decreases pro-inflammatory COX-derived product formation without marked modulation of other lipid mediators. In summary, 6BIGOE is a highly potent indirubin derivative in the cellular context that favorably modulates pro- and anti-inflammatory cytokines as well as COX-2-derived PG via interference with GSK-3.

Dopamine D3 receptor and GSK3\u03b2 signaling mediate deficits in novel object recognition memory within dopamine transporter knockdown mice

BackgroundOver-stimulation of dopamine signaling is thought to underlie the pathophysiology of a list of mental disorders, such as psychosis, mania and attention-deficit/hyperactivity disorder. These disorders are frequently associated with cognitive deficits in attention or learning and memory, suggesting that persistent activation of dopamine signaling may change neural plasticity to induce cognitive or emotional malfunction.MethodsDopamine transporter knockdown (DAT-KD) mice were used to mimic a hyper-dopamine state. Novel object recognition (NOR) task was performed to assess the recognition memory. To test the role of dopamine D3 receptor (D3R) on NOR, DAT-KD mice were treated with either a D3R antagonist, FAUC365 or by deletion of D3R. Total or phospho-GSK3 and –ERK1/2 signals in various brain regions were measured by Western blot analyses. To examine the impact of GSK3 signal on NOR, wild-type mice were systemically treated with GSK3 inhibitor SB216763 or, micro-injected with lentiviral shRNA of GSK3β or GSK3α in the medial prefrontal cortex (mPFC).ResultsWe confirmed our previous findings that DAT-KD mice displayed a deficit in NOR memory, which could be prevented by deletion of D3R or exposure to FAUC365. In WT mice, p-GSK3α and p-GSK3β were significantly decreased in the mPFC after exposure to novel objects; however, the DAT-KD mice exhibited no such change in mPFC p-GSK3α/β levels. DAT-KD mice treated with FAUC365 or with D3R deletion exhibited restored novelty-induced GSK3 dephosphorylation in the mPFC. Moreover, inhibition of GSK3 in WT mice diminished NOR performance and impaired recognition memory. Lentiviral shRNA knockdown of GSK3β, but not GSK3α, in the mPFC of WT mice also impaired NOR.ConclusionThese findings suggest that D3R acts via GSK3β signaling in the mPFC to play a functional role in NOR memory. In addition, treatment with D3R antagonists may be a reasonable approach for ameliorating cognitive impairments or episodic memory deficits in bipolar disorder patients.

CDP-choline accumulation in breast and colorectal cancer cells treated with a GSK-3-targeting inhibitor

PurposeGlycogen synthase kinase 3 (GSK3) is a key controlling element of many cellular processes including cell-cycle progression and recent studies suggest that GSK3 is a potential anticancer target. Changes in glucose metabolism associated with GSK3 inhibition may impact on lipid synthesis, whilst lipid metabolites can act as molecular response markers.MethodsHere, SKBr3 breast and HCT8 colorectal cancer cells were treated with the GSK3 inhibitor SB216763, and [14C (U)] glucose and [3H] choline incorporation into lipids was determined. Cell extracts from treated cells were subject to 31P NMR spectroscopy.ResultsSB216763 treatment decreased choline incorporation into lipids and caused an accumulation of CDP-choline which was accompanied by decreased conversion of glucose into lipid components.ConclusionSB216763 profoundly inhibits phospholipid synthesis in cancer cells which demonstrate accumulation of CDP-choline detectable by 31P NMR spectroscopy. Metabolic changes in lipid metabolism present potential response markers to drugs targeting GSK3.

Transcriptional regulation, stabilization, and subcellular redistribution of multidrug resistance-associated protein 1 (MRP1) by glycogen synthase kinase 3αβ: novel insights on modes of cadmium-induced cell death stimulated by MRP1.

Cadmium (Cd) resistance is associated with the suppression of autophagy in H460 lung cancer cells, which is regulated by phospho(p)serine-glycogen synthase kinase (GSK) 3αβ. However, the involvement of multidrug resistance (MDR) in this signaling pathway and its underlying mechanisms remain to be elucidated. In this study, we used Cd-resistant cells (RH460), developed from H460 lung cancer cells, to demonstrate that the induction of MDR-associated protein (MRP1) in response to Cd is enhanced in H460 cells compared to RH460. Treating RH460 cells with Cd induced large cytoplasmic vacuoles, which was inhibited by the autophagy inhibitor 3-methyladenine. MRP1 was detected in the nuclear-rich membrane fractions and redistributed from the perinuclear to the cytoplasmic compartment following exposure to Cd. Cd-induced MRP1, p-Ser/p-Tyr GSK3αβ, and LC3-II were all suppressed by the GSK3 inhibitor SB216763, but increased by lithium. Furthermore, MRP1 was upregulated by the Ser/Thr phosphatase inhibitor okadaic acid and downregulated by the tyrosine phosphatase inhibitor vanadate, suggesting that MRP1 protein was stabilized by p-Ser GSK3αβ. In addition, co-immunoprecipitation and co-localization analyzes revealed a physical interaction between MRP1 and p-Ser GSK3αβ. Genetic knockdown of GSK3β decreased Cd-induced MRP1 mRNA and protein levels, whereas its overexpression upregulated MRP1 protein expression. MRP1 also co-localized with lysosomal membrane protein-2, which may cause lysosomal membrane permeabilization and the subsequent release of cathepsins into the cytosol. In mice chronically injected with Cd, MRP1 localized to the perinuclear region of bronchial and alveolar epithelial cells. Collectively, these data suggest that Cd toxicity is regulated by the transcriptional regulation, stabilization, and subcellular redistribution of MRP1 via the posttranslational modification of GSK3αβ. Therefore, the serine phosphorylation of GSK3αβ plays a critical role in MRP1-induced cell death.

The inhibition effects of insulin on BMP2-induced muscle heterotopic ossification

Bone morphogenetic proteins (BMPs) play an important role in regulating osteoblastic differentiation and bone formation. But the diffuse of BMPs into muscle tissues around bone injury sites often leads to heterotopic ossification, which has been regarded as one of major side-effects of BMP implementation in bone defect patients. It raises great demands for exploring effective methods that preventing BMP-induced heterotopic ossification while not interrupting the osteoinductive activity of BMPs for in situ bone defect repair. Here we found insulin, a positive regulator for bone regeneration, inhibited BMP2-induced muscle heterotopic ossification by suppressing the expression of bone transcription factor Osterix. By analyzing downstream molecules of insulin pathway, we found AKT/mTOR/GSK3 signaling was responsible for the inhibition of insulin on BMP2-induced ossification, and GSK3 inhibitor SB216763 attenuated BMP2-induced muscle heterotopic ossification. The data might shed light on developing effective clinical therapy for inhibiting muscle heterotopic ossification when BMPs were used bone defect repair.

Hepatocyte Gsk3 Inactivation Via Phosphorylations Is Essential for Ischemia Reperfusion Induced Inflammatory Cell Death.

Background: Glycogen synthase 3 α and β are ubiquitously expressed and play roles in various aspects of cellular responses, such as proliferation, apoptosis, and immune activation. We have documented previously that IR induced liver Gsk3b phosphorylation and Gsk3 inhibition protected livers from IRI via an IL-10 mediated immune regulatory mechanism. As Gsk3 is generally constitutively active and inhibited via phosphorylation (at serine 21 and 9 in Gsk3α and β respectively) upon stimulation, the question of what the biological significance of this inhibitory phosphorylation process is in liver cells has yet to be defined. Methods: In a murine liver partial warm ischemia model, we investigated the effect of phosphorylation-resistant Gsk3αβ in both immune activation and hepatocyte death against IR by using dual Gsk3αS21A and Gsk3βS9A knock-in (KI) mice. Macrophages and hepatocytes isolated from the KI animals were studied in vitro. Results: Dual Gsk3 KI mice were protected from liver IRI, as measured by sALT levels and liver histological examination at both 6 and 24h post reperfusion. However, liver inflammatory gene inductions by IR, including TNF-α, IL-6, IL-10, IL-12p40, and CXCL10, measured by qRT-PCR, were significantly enhanced in these KI mice, as compared with those in WT ones. This was in parallel with our in vitro finding that bone marrow derived macrophages from these KI mice responded to TLR stimulation more vigorously that inflammatory cytokine productions, TNF-α, IL-12p40, as well as IL-10, were all increased, measured by ELISA, as compared with cells from WT mice. Hepatocytes Gsk3 became phosphorylated in response to inflammatory stimuli, including TNF-α. Although Gsk3 inhibitor SB216763 had no significant effect in regulating WT hepatocyte death in their response to TNF-α, Gsk3 KI hepatocytes became significantly more resistant to TNF-α induced cell death (in combination with either Actinomycin D or Tunicamycin), as measured by LDH assay, and the Gsk3 inhibitor SB216763 readily reversed their resistance. Conclusions: Gsk3 has distinctive roles in different liver cell types. Its inhibitory phosphorylation limits inflammatory immune activation in macrophages, while promotes inflammatory cell death in hepatocytes in their response to IR.

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

BackgroundGlycogen synthase kinase-3 (GSK-3) is a constitutively active kinase that regulates multiple signalling proteins and transcription factors involved in a myriad of cellular processes. The kinase acts as a negative regulator in β-catenin signalling and is critically involved in the smad pathway. Activation of both pathways may contribute to pulmonary features of chronic obstructive pulmonary disease (COPD).MethodsIn the present study, we investigated the effect of the selective GSK-3 inhibitor SB216763 on pulmonary pathology in a guinea pig model of lipopolysaccharide (LPS)-induced COPD. Guinea pigs were instilled intranasally with LPS or saline twice weekly for 12 weeks and pre-treated with either intranasally instilled SB216763 or corresponding vehicle 30 min prior to each LPS/saline challenge.ResultsRepeated LPS exposures activated β-catenin signalling, primarily in the airway epithelium and submucosa. LPS also induced pulmonary inflammation and tissue remodelling as indicated by inflammatory cell influx, increased pulmonary fibronectin expression and enhanced small airway collagen content. Inhibition of GSK-3 by SB216763 did not affect LPS-induced inflammatory cell influx, but prevented the small airway remodelling and, unexpectedly, inhibited the activation of β-catenin in vivo. LPS or SB216763 treatment had no effect on the airway smooth muscle content and alveolar airspace size. However, GSK-3 inhibition prevented LPS-induced right ventricle hypertrophy.ConclusionsOur findings indicate that GSK-3 inhibition prevents LPS-induced pulmonary pathology in guinea pigs, and that locally reduced LPS-induced β-catenin activation appears in part to underlie this effect.

PKB/SGK-dependent GSK3-phosphorylation in the regulation of LPS-induced Ca2+ increase in mouse dendritic cells

The function of dendritic cells (DCs) is modified by glycogen synthase kinase GSK3 and GSK3 inhibitors have been shown to protect against inflammatory disease. Regulators of GSK3 include the phosphoinositide 3 kinase (PI3K) pathway leading to activation of protein kinase B (PKB/Akt) and serum and glucocorticoid inducible kinase (SGK) isoforms, which in turn phosphorylate and thus inhibit GSK3. The present study explored, whether PKB/SGK-dependent inhibition of GSK3 contributes to the regulation of cytosolic Ca2+ concentration following stimulation with bacterial lipopolysaccharides (LPS). To this end DCs from mutant mice, in which PKB/SGK-dependent GSK3α,β regulation was disrupted by replacement of the serine residues in the respective SGK/PKB-phosphorylation consensus sequence by alanine (gsk3KI), were compared to DCs from respective wild type mice (gsk3WT). According to Western blotting, GSK3 phosphorylation was indeed absent in gsk3KI DCs. According to flow cytometry, expression of antigen-presenting molecule major histocompatibility complex II (MHCII) and costimulatory molecule CD86, was similar in unstimulated and LPS (1μg/ml, 24h)-stimulated gsk3WT and gsk3KI DCs. Moreover, production of cytokines IL-6, IL-10, IL-12 and TNFα was not significantly different in gsk3KI and gsk3WT DCs. In gsk3WT DCs, stimulation with LPS (1μg/ml) within 10min led to transient phosphorylation of GSK3. According to Fura2 fluorescence, LPS (1μg/ml) increased cytosolic Ca2+ concentration, an effect significantly more pronounced in gsk3KI DCs than in gsk3WT DCs. Conversely, GSK3 inhibitor SB216763 (3-[2,4-Dichlorophenyl]-4-[1-methyl-1H-indol-3-yl]-1H-pyrrole-2,5-dione, 10μM, 30min) significantly blunted the increase of cytosolic Ca2+ concentration following LPS exposure. In conclusion, PKB/SGK-dependent GSK3α,β activity participates in the regulation of Ca2+ signaling in dendritic cells.

PTEN regulation by the Akt/GSK-3β axis during RANKL signaling

Phosphatase and tensin homolog (PTEN) negatively regulates phosphoinositide 3-kinase (PI3K)/Akt signaling as a lipid phosphatase for the second messenger phosphatidylinositol 3,4,5-triphosphate. We discovered recently that inactivating glycogen synthase kinase-3β (GSK-3β) via Akt plays an important role in receptor activator of nuclear factor κb ligand (RANKL)-induced osteoclastogenesis. However, the signaling link between GSK-3β and PTEN in RANKL signaling has not been revealed. Downregulating PTEN by RNA interference increases Akt and GSK-3β phosphorylation levels by RANKL, thereby promoting the formation of osteoclasts. PTEN phosphorylation at threonine 366 (T366) decreased gradually during RANKL-induced osteoclastogenesis, whereas PTEN protein levels were unaffected. Interestingly, the PTEN phosphorylation defective mutant (T366A) showed increased osteoclastogenesis, which is consistent with its lower phosphatase activity, compared to that of wild-type PTEN. Moreover, treatment with the GSK-3 inhibitor SB216763 suppressed PTEN phosphorylation levels and phosphatase activity and enhanced Akt phosphorylation. These data suggest that inhibiting GSK-3β during RANKL-induced osteoclastogenesis decreases PTEN phosphorylation, leading to enhanced osteoclast differentiation through Akt activation.

Long-Lasting Antidepressant Action of Ketamine, but Not Glycogen Synthase Kinase-3 Inhibitor SB216763, in the Chronic Mild Stress Model of Mice

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

Destabilization of Heterologous Proteins Mediated by the GSK3β Phosphorylation Domain of the β-Catenin Protein

Background and Aims: Wnt/β-catenin signaling plays important roles in development and cellular processes. The hallmark of canonical Wnt signaling activation is the stabilization of β-catenin protein in cytoplasm and/or nucleus. The stability of β-catenin is the key to its biological functions and is controlled by the phosphorylation of its amino-terminal degradation domain. Aberrant activation of β-catenin signaling has been implicated in the development of human cancers. It has been recently suggested that GSK3βmay play an essential role in regulating global protein turnover. Here, we investigate if the GSK3β phosphorylation site-containing degradation domain of β-catenin is sufficient to destabilize heterologous proteins. Methods and Results: We engineer chimeric proteins by fusing β-catenin degradation domain at the N- and/or C-termini of the enhanced green fluorescent protein (eGFP). In both transient and stable expression experiments, the chimeric GFP proteins exhibit a significantly decreased stability, which can be effectively antagonized by lithium and Wnt1. An activating mutation in the destruction domain significantly stabilizes the fusion protein. Furthermore, GSK3 inhibitor SB-216763 effectively increases the GFP signal of the fusion protein. Conversely, the inhibition of Wnt signaling with tankyrase inhibitor XAV939 results in a decrease in GFP signal of the fusion proteins, while these small molecules have no significant effects on the mutant destruction domain-GFP fusion protein. Conclusion: Our findings strongly suggest that the β-catenin degradation domain may be sufficient to destabilize heterologous proteins in Wnt signaling-dependent manner. It is conceivable that the chimeric GFP proteins may be used as a functional reporter to measure the dynamic status of β-catenin signaling, and to identify potential anticancer drugs that target β-catenin signaling.

Structural and Functional Characterization of Nrf2 Degradation by the Glycogen Synthase Kinase 3/β-TrCP Axis

The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3β (GSK-3β) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of β-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of β-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3β exhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/β-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3.

Phloroglucinol derivative MCPP induces cell apoptosis in human colon cancer

This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (3,6-bis(3-chlorophenylacetyl)phloroglucinol; MCPP) in human colon cancer cells. MCPP induced cell death and antiproliferation in three human colon cancer, HCT-116, SW480, and Caco-2 cells, but not in primary human dermal fibroblast cells. MCPP-induced concentration-dependent apoptotic cell death in colon cancer cells was measured by fluorescence-activated cell sorter (FACS) analysis. Treatment of HCT-116 human colon cancer cells with MCPP was found to induce a number of signature endoplasmic reticulum (ER) stress markers; and up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein (GRP)-78, phosphorylation of eukaryotic initiation factor-2α (eIF-2α), suggesting the induction of ER stress. MCPP also increased GSK3α/β(Tyr270/216) phosphorylation and reduced GSK3α/β(Ser21/9) phosphorylation time-dependently. Transfection of cells with GRP78 or CHOP siRNA, or treatment of GSK3 inhibitor SB216163 reduced MCPP-mediated cell apoptosis. Treatment of MCPP also increased caspase-7, caspase-9, and caspase-3 activity. The inhibition of caspase activity by z-DEVE-FMK or z-VAD-FMK significantly reduced MCPP-induced apoptosis. Furthermore, treatment of GSK3 inhibitor SB216763 also dramatically reversed MCPP-induced GRP and CHOP up-regulation, and pro-caspase-3 and pro-caspase-9 degradation. Taken together, the present study provides evidences to support that GRP78 and CHOP expression, and GSK3α/β activation in mediating the MCPP-induced human colon cancer cell apoptosis.