Overexpression Of GSK3β Research Articles

The Roles of GSK-3β in Regulation of Retinoid Signaling and Sorafenib Treatment Response in Hepatocellular Carcinoma

Background: Glycogen synthase kinase-3β (GSK-3β) is recently demonstrated to be a tumor promoter and a potential drug target in several tumors. However, the implication and mechanism of GSK-3β in hepatocellular carcinoma (HCC) remain unexplored. Methods: We firstly clarified the expression of GSK-3β in human HCC samples. Given that deviated retinoid signalling is critical for HCC development, we investigated where GSK-3β could be involved in the regulation. Since sorafenib is currently used to treat HCC, the involvement of GSK-3β in sorafenib treatment response was determined. Coimmunoprecipitation, GST pull down, in vitro kinase assay, luciferase reporter and chromatin immunoprecipitation were used to study the molecular mechanism. The biological readouts were examined with MTT, flow cytometry and animal experiments. Findings: We demonstrated that GSK-3β is highly expressed in HCC and associated with shorter overall survival (OS). Tumor-associated GSK-3β is correlated with reduced expression of retinoic acid receptor-β (RARβ), which is caused by GSK-3β-mediated phosphorylation and heterodimerization abrogation of retinoid X receptor (RXRα) with RARα on RARβ promoter. Overexpression of functional GSK-3β impairs retinoid response and represses sorafenib anti-HCC effect. Inactivation of GSK-3β by tideglusib can potentiate 9-cis-RA enhancement of sorafenib sensitivity (tumor inhibition: from 48.3% to 93.4%). Efficient induction of RARβ by tideglusib/9-cis-RA is required for enhanced therapeutic outcome of sorafenib, which effect is greatly inhibited by knocking down RARβ. Interpretation: Our findings suggest that overexpression of GSK-3β may confer HCC growth through interfering retinoid signalling. Targeting GSK3β can be a promising strategy for designing improved treatment of sorafenib in HCC. Funding Statement: This work is supported by grants from Natural Science Foundation of China (No. 81673467, 31471273, 31461163002/RGC N_HKU 740/14), the 10th Singapore-China Joint Research Program (S2014GR0448), Natural Science Foundation of Fujian Province (2019I0002) and Leading Talents in Scientific and Technological Innovation, Double Hundred Talents Program of Fujian Province. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All the use of human samples and study protocols were approved by the Hospital Ethics Committee. All patients signed an informed consent form in prior to sample collection.

Lithium as a Treatment for Alzheimer's Disease: The Systems Pharmacology Perspective.

Systems pharmacology is a novel framework for drug research that models traditional and innovative pharmacological parameters and provides the overall efficacy and safety profile of a drug across body systems and complex, non-linear, molecular interactions. Lithium chloride, a pharmacological compound approved for the therapy of psychiatric disorders, represents a poorly explored compound for the treatment of Alzheimer's disease (AD). Lithium has been shown to reduce downstream effects associated with the aberrant overactivation of certain molecular pathways, such as glycogen synthase kinase 3 subunit β (GSK3-β)-related pathways, involved in AD-related pathophysiology. It seems that overactivation and overexpression of GSK3-β lead to an impairment of long-term potentiation and amyloid-β induced neurotoxicity that can be normalized using lithium. Moreover, a growing body of evidence has demonstrated that lithium's GSK3-β inhibitory effect prevents tau phosphorylation in mouse models of tauopathies. Clinical data have been inconclusive, partly due to methodological limitations. The lack of studies exploring the dynamics of protein misfolding in AD and investigating the specific tau-isoforms appearing prior to the accumulation of neurofibrillary tangles calls for new and optimized clinical trials. Advanced computer modeling based on a formal implementation of quantitative parameters and basic enzymatic insights into a mechanism-based model would present a good start to tackle these non-linear interactions. This innovative approach will pave the way for developing "molecularly" biomarker-guided targeted therapies, i.e., treatments specifically adapted ("tailored") to the individual, consistently with the primary objectives and key conceptual points of precision medicine and precision pharmacology.

Rational design, cognition and neuropathology evaluation of QTC-4-MeOBnE in a streptozotocin-induced mouse model of sporadic Alzheimer\u2019s disease

Alzheimer’s disease (AD) is a multifactorial pathology characterized by amyloid deposits, neurofibrillary formation, oxidative stress and cholinergic system dysfunction. In this sense, here we report the rational design of a multi-target directed ligand (MTDL) for AD based on virtual screening and bioinformatic analyses, exploring the molecular targets β-secretase (BACE-1), glycogen synthase kinase-3β (GSK-3β) and acetylcholinesterase (AChE). After this screening, the compound with higher molecular docking affinity was selected, the 1-(7-chloroquinolin-4-yl)-N-(4-methoxybenzyl)-5-methyl-1H-1,2,3-triazole-4 carboxamide(QTC-4-MeOBnE). To further our studies, the protective effect of QTC-4-MeOBnE (0.1 and 1 mg/kg for 20 days) on STZ-induced sporadic AD mice was determined. QTC-4-MeOBnE pretreatment attenuated cognitive and memory deficit induced by STZ in an object recognition test, Y-maze, social recognition test and step-down passive avoidance. The mechanisms underlying this action might be attributed to the reduction of lipid peroxidation and reactive species formation in the prefrontal cortex and hippocampus of mice submitted to STZ. In addition, QTC-4-MeOBnE pretreatment abolished the up-regulation of AChE activity and the overexpression of GSK 3β and genes involved in amyloid cascade such as BACE-1, protein precursor amyloid, у-secretase, induced by STZ. Moreover, toxicological parameters were not modified by QTC-4-MeOBnE chronic treatment. This evidence suggests that QTC-4-MeOBnE exerts its therapeutic effect through multiple pathways involved in AD.

SLC34A2 promotes neuroblastoma cell stemness via enhancement of miR-25/Gsk3β-mediated activation of Wnt/β-catenin signaling.

Cancer stem cells contribute to cancer progression, but the mechanisms underlying neuroblastoma stem cell development are unclear. Here, we examined the roles of the transcription factor SLC34A2 in regulating the stemness of neuroblastoma cells. We found that SLC34A2 expression was negatively correlated with the overall survival and relapse‐free survival probability of neuroblastoma patients. Additionally, SLC34A2 expression was observed to be remarkably increased in spheroids derived from neuroblastoma cells. Knockdown of SLC34A2 attenuated the expression of stemness markers and spheroid formation capacity of neuroblastoma cell‐derived spheroids, and overexpression of SLC34A2 exerted the opposite effects in neuroblastoma cells. Mechanistically, SLC34A2 was found to directly bind to the promoter of MIR25, which targets glycogen synthesis kinase 3β (Gsk3β), an antagonist of Wnt signaling. Transfection of miR‐25 inhibitor or a Gsk3β overexpression plasmid attenuated the effects of SLC34A2 overexpression on the stemness of neuroblastoma cells. Our results demonstrate that miR‐25/Gsk3β‐mediated activation of Wnt signaling is responsible for SLC34A2‐induced enhancement of neuroblastoma cell stemness.

GSK3β overexpression driven by GFAP promoter improves rotarod performance

We have studied the consequences of in vivo GSK3β overexpression in the cerebellum using transgenic mice with conditional expression where the transactivator tTA protein expression is driven by GFAP promoter. We demonstrate an increase in GSK3β in Bergmann cells. To study cerebellar dysfunctions and evaluate motor coordination we analysed the latency to fall in the accelerating rotarod test. GSK3β transgenic mice performed significantly better than wild-type mice and transgene shutdown with doxycycline normalizes the values in latency to fall in rotarod test. We had previously demonstrated using the same transgenic model, that overexpression of GSK3β in the hippocampus results in an increase in neural precursor cells. However, we did not observe that increase in the number of Sox2+ cells in the cerebellum. All the same, we observed an increase in cerebellar glutamate transporters GLT1 and GLAST. These data show that GSK3β can be a crucial kinase in cerebellum and especially in Bergmann glial cells.

Cis pT231-Tau Drives Neurodegeneration in Bipolar Disorder.

Bipolar disorder is a complex neuropsychiatric disorder, characterized by intermittent episodes of mania and depression. Recent studies have indicated argyrophilic grains, composed of hyperphosphorylated tau, are observable in postmortem brains of bipolar patients. It remains uncertain how tau hyperphosphorylation results in neurodegeneration upon the disease. Recent studies have demonstrated that phosphorylated tau at Thr231 exists in two distinct cis and trans conformations, in which cis pT231-tau is highly neurotoxic and acts as an early driver of tauopathy in several neurodegenerative diseases. We herein employed an in vitro model, which resembles some aspects of bipolar disorder, to study the cis p-tau mediatory role. We established GSK3β overexpressing SH-SY5Y cells and examined cell viability, cis p-tau formation, and lithium effects by immunofluorescence and flow cytometry. We found an increase in cis p-tau levels as well as viability decrease in the cell model. Furthermore, we discovered that lithium treatment inhibits cis p-tau formation, resulting in diminished cell death. We also examined BD and healthy human brain samples and detected cis p-tau in the patients' brains. Our results show that tauopathy, observed in bipolar disorder, is being mediated through cis p-tau and that a conformer could be the cause of neurodegeneration upon the disease. Our findings would suggest novel therapeutic target to fight the devastating disorder.

Transcriptional Regulation of NAMPT Gene by Glycogen Synthase Kinase 3β in Goat Adipocytes.

Nicotinamide phosphoribosyl transferase (NAMPT) is a cytokine which is secreted by adipose tissue and involved in the process of immunoreactions, inflammation, and lipid metabolism through the endocrine or paracrine. However, the information regarding NAMPT and its transcriptional regulation in goats remains unknown. In this study, the inactivation of glycogen synthase kinase 3β (GSK3β) decreased the supernatant NAMPT productions in goat adipocytes. The luciferase activities were kept in high levels in pGL3 (-735/+34) and the deletion from -735 bp to -486 bp significantly decreased the luciferase activity (p < 0.01). In addition, a peroxisome proliferator-activated receptor gamma (PPARγ) element in this region was responsible for NAMPT promoter activity. The transcription activity of PPARγ was activated by SB216763 stimulation and failed to induce luciferase activity of the pGL3 (Mut-PPARγ-735/+34). In contrast, the GSK3β overexpression repressed luciferase activity of the NAMPT promoter. Besides, chromatin immunoprecipitation assay indicated that PPARγ was capable of binding to the NAMPT promoter, and inhibition of GSK3β increased the binding capacity of PPARγ to the NAMPT promoter. These results confirm the role of GSK3β as a negative regulator of NAMPT transcription in goat adipocytes, and GSK3β-regulated expression of NAMPT is mediated by PPARγ.

Glycogen Synthase Kinase 3β Enhances Hepatitis C Virus Replication by Supporting miR-122

Hepatitis C virus (HCV) infection is associated with alterations in host lipid and insulin signaling cascades, which are partially explained by a dependence of the HCV life cycle on key molecules in these metabolic pathways. Yet, little is known on the role in the HCV life cycle of glycogen synthase kinase 3 (GSK3), one of the most important kinases in cellular metabolism. Therefore, the impact of GSK3 on the HCV life cycle was assessed in human hepatoma cell lines harboring subgenomic genotype 1b and 2a replicons or producing cell culture-derived HCV genotype 2a by exposure to synthetic GSK3 inhibitors, GSK3 gene silencing, overexpression of GSK3 constructs and immunofluorescence analyses. In addition, the role of GSK3 in hepatitis E virus (HEV) replication was investigated to assess virus specificity of the observed findings. We found that both inhibition of GSK3 function by synthetic inhibitors as well as silencing of GSK3β gene expression resulted in a decrease of HCV replication and infectious particle production, whereas silencing of the GSK3α isoform had no relevant effect on the HCV life cycle. Conversely, overexpression of GSK3β resulted in enhanced HCV replication. In contrast, GSK3β had no effect on replication of subgenomic HEV replicon. The pro-viral effect of GSK3β on HCV replication was mediated by supporting expression of microRNA-122 (miR-122), a micro-RNA which is mandatory for wild-type HCV replication, as GSK3 inhibitors suppressed miR-122 levels and as inhibitors of GSK3 had no antiviral effect on a miR-122-independent HCV mutant. In conclusion, we have identified GSK3β is a novel host factor supporting HCV replication by maintaining high levels of hepatic miR-122 expression.

Bi-directional genetic modulation of GSK-3\u03b2 exacerbates hippocampal neuropathology in experimental status epilepticus

Glycogen synthase kinase-3 (GSK-3) is ubiquitously expressed throughout the brain and involved in vital molecular pathways such as cell survival and synaptic reorganization and has emerged as a potential drug target for brain diseases. A causal role for GSK-3, in particular the brain-enriched GSK-3β isoform, has been demonstrated in neurodegenerative diseases such as Alzheimer’s and Huntington’s, and in psychiatric diseases. Recent studies have also linked GSK-3 dysregulation to neuropathological outcomes in epilepsy. To date, however, there has been no genetic evidence for the involvement of GSK-3 in seizure-induced pathology. Status epilepticus (prolonged, damaging seizure) was induced via a microinjection of kainic acid into the amygdala of mice. Studies were conducted using two transgenic mouse lines: a neuron-specific GSK-3β overexpression and a neuron-specific dominant-negative GSK-3β (GSK-3β-DN) expression in order to determine the effects of increased or decreased GSK-3β activity, respectively, on seizures and attendant pathological changes in the hippocampus. GSK-3 inhibitors were also employed to support the genetic approach. Status epilepticus resulted in a spatiotemporal regulation of GSK-3 expression and activity in the hippocampus, with decreased GSK-3 activity evident in non-damaged hippocampal areas. Consistent with this, overexpression of GSK-3β exacerbated status epilepticus-induced neurodegeneration in mice. Surprisingly, decreasing GSK-3 activity, either via overexpression of GSK-3β-DN or through the use of specific GSK-3 inhibitors, also exacerbated hippocampal damage and increased seizure severity during status epilepticus. In conclusion, our results demonstrate that the brain has limited tolerance for modulation of GSK-3 activity in the setting of epileptic brain injury. These findings caution against targeting GSK-3 as a treatment strategy for epilepsy or other neurologic disorders where neuronal hyperexcitability is an underlying pathomechanism.

MiR-199b-5p Regulates Immune-Mediated Allograft Rejection after Lung Transplantation Through the GSK3β and NF-κB Pathways.

Emerging evidence indicates that acute rejection mainly associated with the inflammatory response is an independent risk factor for chronic rejection after lung transplantation. Monocytes are the main pro-inflammatory leukocytes infiltrating around the lesions and play vital roles in triggering the acute rejection. In the rat lung transplantation model, lipopolysaccharide (LPS)-induced severe acute rejection was strongly associated with advanced chronic rejection. The exact regulatory mechanism of pro-inflammation in monocytes is not yet clear. In this study, we identified a novel anti-inflammatory effect of miR-199b-5p (miR-199b) through the GSK3β and NF-κB pathways. THP-1 monocytes treated with LPS showed a significant decrease in miR-199b that is inversely correlated to GSK3β expression and NF-κB activation. Furthermore, the NF-κB-associated inflammatory response was reduced due to the overexpression of miR-199b targeting GSK3β, which was rescued by the inhibition of miR-199b. These results indicated that miR-199b attenuated the inflammatory response at least partly through the GSK3β/NF-κB signaling pathways in monocytes. Our data point toward a potentially important role for miR-199b in the inhibition of rejection after lung transplantation.

MicroRNA-135a alleviates oxygen-glucose deprivation and reoxygenation-induced injury in neurons through regulation of GSK-3β/Nrf2 signaling.

MicroRNAs (miRNAs) have been suggested as pivotal regulators in the pathological process of cerebral ischemia and reperfusion injury. In this study, we aimed to investigate the role of miR-135a in regulating neuronal survival in cerebral ischemia and reperfusion injury using an in vitro cellular model induced by oxygen-glucose deprivation and reoxygenation (OGD/R). Our results showed that miR-135a expression was significantly decreased in neurons with OGD/R treatment. Overexpression of miR-135a significantly alleviated OGD/R-induced cell injury and oxidative stress, whereas inhibition of miR-135a showed the opposite effects. Glycogen synthase kinase-3β (GSK-3β) was identified as a potential target gene of miR-135a. miR-135a was found to inhibit GSK-3β expression, but promote the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and downstream signaling. However, overexpression of GSK-3β significantly reversed miR-135a-induced neuroprotective effect. Overall, our results suggest that miR-135a protects neurons against OGD/R-induced injury through downregulation of GSK-3β and upregulation of Nrf2 signaling.

Modulation of GSK-3β/β-Catenin Signaling Contributes to Learning and Memory Impairment in a Rat Model of Depression.

BackgroundIt is widely accepted that cognitive processes, such as learning and memory, are affected in depression, but the molecular mechanisms underlying the interactions of these 2 disorders are not clearly understood. Recently, glycogen synthase kinase-3 beta (GSK-3β)/β-catenin signaling was shown to play an important role in the regulation of learning and memory.MethodsThe present study used a rat model of depression, chronic unpredictable stress, to determine whether hippocampal GSK-3β/β-catenin signaling was involved in learning and memory alterations.ResultsOur results demonstrated that chronic unpredictable stress had a dramatic influence on spatial cognitive performance in the Morris water maze task and reduced the phosphorylation of Ser9 of GSK-3β as well as the total and nuclear levels of β-catenin in the hippocampus. Inhibition of GSK3β by SB216763 significantly ameliorated the cognitive deficits induced by chronic unpredictable stress, while overexpression of GSK3β by AAV-mediated gene transfer significantly decreased cognitive performance in adult rats. In addition, chronic unpredictable stress exposure increased the expression of the canonical Wnt antagonist Dkk-1. Furthermore, chronic administration of corticosterone significantly increased Dkk-1 expression, decreased the phosphorylation of Ser9 of GSK-3β, and resulted in the impairment of hippocampal learning and memory.ConclusionsOur results indicate that impairment of learning and memory in response to chronic unpredictable stress may be attributed to the dysfunction of GSK-3β/β-catenin signaling mediated by increased glucocorticoid signaling via Dkk-1.

The Role of Insulin/IGF-1/PI3K/Akt/GSK3β Signaling in Parkinson's Disease Dementia.

Dementia, a condition that frequently afflicts patients in advanced stages of Parkinson's disease (PD), results in decreased quality of life and survival time. Nevertheless, the pathological mechanisms underlying Parkinson's disease dementia (PDD) are not completely understood. The symptoms characteristic of PDD may be the result of functional and structural deficiencies. The present study implicates the accumulation of Lewy bodies in the cortex and limbic system as a potent trigger in the development of PDD. In addition, significant Alzheimer-type pathologies, including amyloid-β (Aβ) plaques and NFTs, are observed in almost half of PDD patients. Interestingly, links between PDD pathogenesis and the mechanisms underlying the development of insulin resistance have begun to emerge. Furthermore, previous studies have demonstrated that insulin treatment reduces amyloid plaques in Alzheimer's disease (AD), and normalizes the production and functionality of dopamine and ameliorates motor impairments in 6-OHDA-induced rat PD models. GSK3β, a downstream substrate of PI3K/Akt signaling following induction by insulin and IGF-1, exerts an influence on AD and PD physiopathology. The genetic overexpression of GSK3β in cortex and hippocampus results in signs of neurodegeneration and spatial learning deficits in in vivo models (Lucas et al., 2001), whereas its inhibition results in improvements in cognitive impairment in these rodents, including AD and PD. Accordingly, insulin- or IGF-1-activated PI3K/Akt/GSK3β signaling may be involved in PDD pathogenesis, at least in the pathology of PD-type + AD-type.

NONO ubiquitination is mediated by FBW7 and GSK3 β via a degron lost upon chromosomal rearrangement in cancer.

NONO is an RNA-binding protein involved in transcription, mRNA splicing, DNA repair, and checkpoint activation in response to UV radiation. NONO expression has been found altered in several tumor types, including prostate, colon, breast, melanoma, and in papillary renal carcinoma, in which an X chromosome inversion generates a NONO-TFE3 fusion protein. Upon such rearrangement, NONO loses its C-terminal domain. Through bioinformatics analysis, we identified a putative degron motif, known to be recognized by the Skp1-Cul1-F-box-protein (SCF) complex. Here, we evaluated how this domain could affect NONO protein biology. We showed that NONO interacts with the nuclear FBW7α isoform and its ubiquitination is regulated following modulation of the GSK3β kinase. Mutation of T428A/T432A within the degron impaired polyubiquitination upon FBW7α and GSK3β overexpression. Overall, our data suggest that NONO is likely subjected to proteasome-mediated degradation and add NONO to the list of proteins targeted by FBW7, which is itself often deregulated in cancer.

Post-translational modification of the interferon-gamma receptor alters its stability and signaling.

The IFN gamma receptor 1 (IFNGR1) binds IFN-γ and activates gene transcription pathways crucial for controlling bacterial and viral infections. Although decreases in IFNGR1 surface levels have been demonstrated to inhibit IFN-γ signaling, little is known regarding the molecular mechanisms controlling receptor stability. Here, we show in epithelial and monocytic cell lines that IFNGR1 displays K48 polyubiquitination, is proteasomally degraded, and harbors three ubiquitin acceptor sites at K277, K279, and K285. Inhibition of glycogen synthase kinase 3 beta (GSK3β) destabilized IFNGR1 while overexpression of GSK3β increased receptor stability. We identified critical serine and threonine residues juxtaposed to ubiquitin acceptor sites that impacted IFNGR1 stability. In CRISPR-Cas9 IFNGR1 generated knockout cell lines, cellular expression of IFNGR1 plasmids encoding ubiquitin acceptor site mutations demonstrated significantly impaired STAT1 phosphorylation and decreased STAT1-dependent gene induction. Thus, IFNGR1 undergoes rapid site-specific polyubiquitination, a process modulated by GSK3β. Ubiquitination appears to be necessary for efficient IFNGR1-dependent gamma gene induction and represents a relatively uncharacterized regulatory mechanism for this receptor.

Elucidating the functions of brain GSK3α: Possible synergy with GSK3β upregulation and reversal by antidepressant treatment in a mouse model of depressive-like behaviour

Glycogen synthase kinase 3 (GSK3) has been linked to the mechanisms of stress, mood regulation, and the effects of antidepressants. The functions of the GSK3β isoform have been extensively investigated, but little is known about the α-isoform, although they may functionally related. In a recently established modified swim test with a third delayed swim exposure, brain GSK3β mRNA expression positively correlated with floating behaviour on the third test. A two-week-long pretreatment regime with imipramine (7.5mg/kg/day) or thiamine (200mg/kg/day), which is known to have antidepressant properties, reduced the GSK3β over-expression and decreased floating behaviour on Day 5. GSK3α mRNA levels were measured in the hippocampus and prefrontal cortex on Days 1, 2 and 5. GSK3α expression was decreased in the prefrontal cortex on Day 2 and increased on Day 5. In this model, GSK3α mRNA changes were prevented by imipramine or thiamine treatment. There was a significant correlation between the expression of the two isoforms in the prefrontal cortex on Day 2 in untreated group. These results provide the first evidence for the potential involvement of GSK3α in depressive-like behaviours and as a target of anti-depressant therapy. Furthermore, the correlations suggest some cross-talk may exist between the two GSK3 isoforms.

Role of GSK-3β in Regulation of Canonical Wnt/β-catenin Signaling and PI3-K/Akt Oncogenic Pathway in Colon Cancer

ABSTRACTNon-steroidal anti-inflammatory drugs (NSAIDs) are emerging as novel chemopreventive agents because of their ability in blocking cellular proliferation, and thereby tumor development, and also by promoting apoptosis. GSK-3β, a serine threonine kinase and a negative regulator of the oncogenic Wnt/β-catenin signaling pathway, plays a critical role in the regulation of oncogenesis. Celecoxib and etoricoxib, the two cyclooxygenase-2 (COX-2) selective NSAIDs, and Diclofenac, a preferential COX-2 inhibitory NSAID, had shown uniformly the chemopreventive and anti-neoplastic effects in the early stage of colon cancer by promoting apoptosis as well as an over-expression of GSK-3β while down-regulating the PI3-K/Akt oncogenic pathway.

糖原合成激酶-3β/β-连环蛋白信号通路在贫铀致人肾近曲小管上皮细胞损伤中的作用

Objective To investigate the effects of glycogen synthase kinase-3β (GSK-3β) and β-catenin signaling on the human renal proximal tubular epithelial HK-2 cell injury induced by depleted uranium(DU), and provide a new enlightenment for the development of DU antidotes. Methods HK-2 cells were exposed to different concentrations of DU for 3-24 h, then the protein expressions of kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and nuclear β-catenin were detected by immunofluorescence staining. The protein expressions of p-GSK-3β(S9), GSK-3β and c-myc were detected by Western blot assay. HK-2 cells were transiently transfected by GSK-3β (KD) plasmid or treated by TDZD-8 to inhibit the activity of GSK-3β specifically. Other HK-2 cells were transiently transfected by β-catenin plasmid to overexpress the β-catenin protein. Results The percentages of KIM-1 and NGAL-positive cells increased with DU exposure time and concentrations from 300 and 600 μmol/L, and they were significantly higher than those of the blank control at 6-24 h of DU exposure (KIM-1-positive cells: t=11.06, 18.97, 30.49, P<0.05; t=6.79, 16.02, 85.45, P<0.05; NGAL-positive cells: t=11.78, 11.37, 34.29, P<0.05; t=7.34, 21.63, 36.84, P<0.05). In contrast, the ratio of p-GSK-3β(S9) to GSK-3β and percentage of nuclear β-catenin-positive cells were significantly higher than that of the blank control at 3-24 h of DU exposure (p-GSK-3β(S9)/GSK-3β: t=3.95, 4.69, 5.40, 3.34, P<0.05; nuclear β-catenin-positive cells: t=4.61, 6.52, 36.64, 14.93, P<0.05) with a maximum response at 9 h of DU exposure accompanied with corresponding increase of protein level of c-myc, a downstream target gene of β-catenin. Transient transfection of HK-2 cells with GSK-3β(KD) plasmid significantly inhibited the activity of GSK-3β (t=8.07, P<0.05) and reduced the DU-increased percentage of KIM-1-positive cells (t=24.77, P<0.05). Treatment cells with TDZD-8 inhibited the activity of GSK-3β and enhanced the percentage of nuclear β-catenin-positive cells, and it also significantly reduced the percentage of KIM-1-positive cells in HK-2 cells exposed to DU (t=6.25, 6.73, P<0.05). Moreover, overexpression of β-catenin significantly reduced DU-induced cell injury (t=7.48, P<0.05). Conclusions GSK-3β/β-catenin signaling plays a key role in regulating the DU-induced cytotoxicity of HK-2 cells. Inhibition of GSK-3β activity and overexpression of β-catenin can protect the HK-2 cells from DU-induced damage. Key words: Depleted uranium; GSK-3β; β-catenin; KIM-1; HK-2 cell

Correlation between PKB/Akt, GSK-3β expression and tubular epithelial-mesenchymal transition in renal allografts with chronic active antibody-mediated rejection.

Chronic antibody-mediated rejection (ABMR) is a major cause of the transplant renal interstitial fibrosis and transplanted kidney epithelial cell transdifferentiation is one of the main mechanisms. The transforming growth factor (TGF)-β1/integrin-linked kinase (ILK) signaling pathway has a significant role in the epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells; however, the molecular mechanisms of this process have remained elusive. The present study confirmed that Akt and glycogen synthase kinase (GSK)-3β, as TGF-β1 downstream signaling factors, are involved in fibrotic processes caused by kidney disease, which, however, has been rarely reported in the kidney transplant field. Based on the Banff 2009 standard, transplanted kidney specimens were classified according to the fibrosis level. The results showed that with the reduction of the interstitial fibrosis level, E-cadherin expression was gradually reduced, while α-smooth muscle actin expression progressively increased. The expression of Akt and GSK-3β in normal human kidney tissue was not obvious but showed a marked increase with the aggravation of the interstitial fibrosis level, which confirmed the occurrence of EMT during the fibrosis process, and that phosphorylated (p)-Akt and GSK-3β have an important role in the EMT process in the transplanted kidney. A correlation analysis of p-Akt, GSK-3β, TGF-β1 and ILK suggested that overexpression of p-Akt and GSK-3β may induce and mediate the transdifferentiation of renal tubular epithelial cells to myofibroblasts and that this proceeds via TGFβ1/ILK signaling pathways.

GSK3β attenuates TGF-β1 induced epithelial–mesenchymal transition and metabolic alterations in ARPE-19 cells

While TGF-β1 is known to induce epithelial–mesenchymal transition (EMT), a major factor in the pathogenesis of proliferative vitreoretinopathy (PVR), in ARPE-19 cells. The molecular pathways involved in EMT formation have not yet to be fully characterized. In this study, we have found that TGF-β1-mediated induction of EMT in ARPE-19 cells varied in a dose- and time-dependent manner. Specifically, TGF-β1 inhibited GSK-3β by accelerating phosphorylation at ser9. GSK-3β inhibitor or knockdown of GSK-3β resulted in enhanced TGF-β1-mediated EMT, migration and collagen contraction in ARPE-19 cells, which were then abrogated by GSK-3β overexpression and PI3K/AKT inhibitor. Importantly, GSK-3β also mediated metabolic reprogramming in TGF-β1-treated cells. Our results indicate that GSK-3β plays a pivotal role in TGF-β1-mediated EMT in ARPE-19 cells.