Total GSK-3β Research Articles

ADAMTS16 Suppression by MicroRNA-25 Leads to Oncogenic Properties in Renal Cell Carcinoma.

Increasing evidence indicates that microRNAs (miRNAs) play a crucial role in modulating tumor growth. This study is centered on investigating the contribution of miR-25 to the progression of Renal Cell Carcinoma (RCC). The investigators examined the expression levels of miR-25 and ADAMTS16 in RCC samples and cell lines. The association between miR-25 and ADAMTS16 was validated via a luciferase reporter assay. Cell viability, apoptosis, migration, and invasion were evaluated utilizing CCK-8 and flow cytometry techniques, while the expression levels of ADAMTS16, β-catenin, GSK-3β, and p-GSK-3β were assessed through western blot analysis. The investigation revealed elevated expression levels of miR-25 in RCC tissues. Subsequently, ADAMTS16 was identified as a target of miR-25. Increased miR-25 levels were associated with decreased expression of ADAMTS16, resulting in enhanced cell viability and diminished apoptosis. Conversely, inhibition of miR-25 led to decreased cell viability, proliferation, and migration. Additionally, the researchers observed that miR-25 triggered the phosphorylation of GSK-3β and β-catenin while leaving the total GSK-3β level unaffected. This study suggests that miR-25 regulates the expression of ADAMTS16 through the Wnt/β-catenin signaling pathway, providing new insights into the cause and potential treatment of RCC.

KAT7/HMGN1 signaling epigenetically induces tyrosine phosphorylation-regulated kinase 1A expression to ameliorate insulin resistance in Alzheimer's disease.

Epidemiological studies have revealed a correlation between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2D). Insulin resistance in the brain is a common feature in patients with T2D and AD. KAT7 is a histone acetyltransferase that participates in the modulation of various genes. To determine the effects of KAT7 on insulin patients with AD. APPswe/PS1-dE9 double-transgenic and db/db mice were used to mimic AD and diabetes, respectively. An in vitro model of AD was established by Aβ stimulation. Insulin resistance was induced by chronic stimulation with high insulin levels. The expression of microtubule-associated protein 2 (MAP2) was assessed using immunofluorescence. The protein levels of MAP2, Aβ, dual-specificity tyrosine phosphorylation-regulated kinase-1A (DYRK1A), IRS-1, p-AKT, total AKT, p-GSK3β, total GSK3β, DYRK1A, and KAT7 were measured via western blotting. Accumulation of reactive oxygen species (ROS), malondialdehyde (MDA), and SOD activity was measured to determine cellular oxidative stress. Flow cytometry and CCK-8 assay were performed to evaluate neuronal cell death and proliferation, respectively. Relative RNA levels of KAT7 and DYRK1A were examined using quantitative PCR. A chromatin immunoprecipitation assay was conducted to detect H3K14ac in DYRK1A. KAT7 expression was suppressed in the AD mice. Overexpression of KAT7 decreased Aβ accumulation and MAP2 expression in AD brains. KAT7 overexpression decreased ROS and MDA levels, elevated SOD activity in brain tissues and neurons, and simultaneously suppressed neuronal apoptosis. KAT7 upregulated levels of p-AKT and p-GSK3β to alleviate insulin resistance, along with elevated expression of DYRK1A. KAT7 depletion suppressed DYRK1A expression and impaired H3K14ac of DYRK1A. HMGN1 overexpression recovered DYRK1A levels and reversed insulin resistance caused by KAT7 depletion. We determined that KAT7 overexpression recovered insulin sensitivity in AD by recruiting HMGN1 to enhance DYRK1A acetylation. Our findings suggest that KAT7 is a novel and promising therapeutic target for the resistance in AD.

A Complex Interplay between Notch Effectors and β-Catenin Signaling in Morular Differentiation of Endometrial Carcinoma Cells

Notch signaling contributes to tissue development and homeostasis, but little is known about its roles in morular differentiation of endometrial carcinoma (Em Ca) cells. The current study focused on crosstalk between Notch and β-catenin signaling in Em Ca with morules. Promoters of hairy and enhancer of split 1 (Hes1) and mastermind-like 2 (MAML2) were activated by Notch intracellular domain 1 (NICD1) but not β-catenin; we also observed a positive feedback loop between Hes1 and MAML2. Immunoreactivities for nuclear β-catenin, Hes1, and MAML2, as well as the interaction between β-catenin and Hes1 or MAML2, were significantly higher in morular lesions as compared to surrounding carcinoma (Sur Ca) in Em Ca. Inhibition of glycogen synthase kinase-3β (GSK-3β) increased expression of total nuclear and cytoplasmic GSK-3β and its phosphorylated forms, as well as NICD1, Hes1, and active β-catenin. GSK-3β inhibition also decreased proliferation and migration, consistent with the response of cells stably overexpressing Hes1. Finally, the nuclear/cytoplasmic GSK-3β score was significantly higher in morules compared to Sur Ca in Em Ca, and was positively correlated with nuclear β-catenin, Hes1, and MAML2 scores. This complex interplay between Notch effectors and β-catenin signaling through GSK-3β inhibition contributes to the establishment and maintenance of β-catenin-mediated morular differentiation, which is in turn associated with reduced proliferation and inhibition of migration in Em Ca.

Abstract 18545: GSK-3β Prevents Lipotoxicity in Obesity Cardiomyopathy Through Upregulation of ATGL

Introduction: Obesity induces accumulation of lipids in cardiomyocytes, leading to cardiac dysfunction, termed lipotoxic cardiomyopathy. We have shown previously that GSK-3α promotes fatty acid uptake in the heart. In contrast, GSK-3β may mediate lipid catabolism. In this study, we aimed to elucidate the role of GSK-3β in lipolysis, primarily focusing on the post-translational regulation of FoxO1 and its association with ATGL, which is the first lipase to hydrolyze lipids. Methods: Wild-type (WT) and homozygous cardiac-specific GSK-3β knock-out (GSK-3β-cKO) mice were fed a normal diet (ND) or a high fat diet (HFD) for 3 months. Results: HFD consumption in WT mice activated GSK-3β in the heart, as indicated by decreases in Ser9-phosphorylated GSK-3β/total GSK-3β (0.5-fold, p<0.05 compared to ND consumption), and upregulated ATGL (2.4-fold, p<0.05) in the heart. Echocardiographic analyses showed preserved systolic and diastolic function in WT mice at this time point (LVEF: 56.3 ± 1.5 %, E/A: 2.06 ± 0.22, n = 13). However, ATGL was not increased (1.6-fold) and both systolic and diastolic dysfunction were observed (LVEF: 45.8 ± 1.5 %, p<0.01 compared to WT, E/A: 1.43 ± 0.20, p<0.05, n =12) in GSK-3β-cKO mice fed a HFD. GSK-3β overexpression in neonatal rat ventricular cardiomyocytes increased ATGL in a dose-dependent manner (p<0.05). FoxO1 knock-down on this background inhibited upregulation of ATGL, suggesting that GSK-3β-induced upregulation of ATGL is mediated through a FoxO1-dependent mechanism. Immunoprecipitation showed that GSK-3β interacts with FoxO1, while mass spectrometry analyses revealed that GSK-3β phosphorylates FoxO1 at 8 Serine/Threonine residues. A FoxO1 mutant in which Serine/Threonine residues at the GSK-3β phosphorylation sites are mutated to Alanine was excluded from the nucleus, and its overexpression prevented upregulation of ATGL in the presence of GSK-3β overexpression. Conclusions: GSK-3β is activated in the presence of HFD consumption in the heart, which may, in turn, prevent lipotoxic cardiomyopathy through phosphorylation of FoxO1 and upregulation of ATGL.

Lithium Cholesterol Sulfate: A Novel and Potential Drug for Treating Alzheimer's Disease and Autism Spectrum Disorder.

Recent studies have shown that lithium treatment can reduce symptoms of Alzheimer's disease (AD) and Autism Spectrum Disorder (ASD). However, the present lithium salts clinically available have serious short-term and long-term side effects which requires frequent monitoring of blood chemistry and plasma lithium levels so as to avoid toxicity. Consequently, there is a demand for a safer and more effective lithium formulation to treat these diseases. Hence, we firstly synthesized lithium cholesterol sulfate (LiCS) and compared its pharmacological effects with that of lithium chloride (LiCl) and sodium cholesterol sulfate (NaCS) on markers of neurodegenerative disease in cell cultures. LiCS was more potent than LiCl in increasing inhibitory GSK3β (Ser9) phosphorylation (pGSK3β) in both CHO and SH-SY5Y cells. These agents dose-dependently increased pGSK3β, starting at 10 µM for LiCS and 60µM for LiCl and maximally by approximately 100% at 60 µM for LiCS and 1.25 mM for LiCl, without altering total GSK3β levels. In HEK293/tau cells, LiCS reduced tau (Thr231) phosphorylation (ptau) starting at 10 µM and maximally by 63% at 40 µM without altering total tau levels, but ptau levels were not altered by LiCl at any dose between 60 µM and 1.25 mM. In BV2 cells, LiCS and LiCl decreased LPS-induced TNFα levels, starting at 20 µM for LiCS and 5 mM for LiCl, and maximally by approximately 30% at 80 µM for LiCS and 20 mM for LiCl. NaCS at any dose between 5 and 90 µM did not alter pGSK3β, ptau or LPS-induced TNFα. LiCS may become a new drug with good pharmacological potential for the treatment of neurodegenerative disorders such as AD and ASD by allowing lithium to more readily access intracellular pathological processes.

Curcumin promotes apoptosis of liver cancer cells by down-regulating WNT/β-catenin

Curcumin (C21H20O6) is considered to exert anticancer potential. However, the relationship between curcumin and β-catenin has not been fully clarified and its regulatory role in human liver cancer has not yet been confirmed. In this study, human liver cancer cells were exposed to curcumin and the proliferation of HepG2 cells was detected by MTT method along with analysis of cell cycle and apoptosis, as well as Wnt/β-catenin signaling proteins expression. Curcumin shows a time-dependent and dose-dependent effect on HepG2 cell proliferation. The IC50 at 24 h, 48 h and 72 h were 12.8±0.67) μmol/L, 8.8±0.43 μmol/L and 4.6±0.42 μmol/L, respectively. Curcumin could dose-dependently (5, 10 and 20 μmol/L) arrest HepG2 cells in the G2/M phase (p < 0.05) and increase apoptosis (p < 0.05). In addition, curcumin down-regulated total, cytoplasmic and nuclear β-catenin proteins in HepG2 cells, indicating inhibited Wnt/β-catenin signaling. Curcumin reduced the expression of Dvl-2, Dvl-3, GSK-3β (p-ser9), C-myc, and Survivin, and increased the expression of GSK-3 (p-tyr216) and Axin-2 without affecting total GSK-3β levels. Curcumin is able to inhibit liver cancer cell activities possibly through inhibition of Wnt/β-catenin signaling.

Trimethylamine-N-oxide paradoxically depresses contractile function while activating mitochondrial respiration in mouse hearts.

Trimethylamine-N-oxide (TMAO) is an end-product of gut-microbiome metabolism linked to cardiovascular disease (CVD). However, precise cardiovascular influences of the TMAO concentrations reported in early or severe disease remain to be detailed. We investigated acute effects of TMAO on cardiac contractile, coronary and mitochondrial function. Male C57Bl/6 mouse hearts were Langendorff perfused to assess concentration-dependent effects of TMAO (1-300 µM) on left ventricular (LV) function, coronary flow and select protein expression. Effects of 10 and 100 µM TMAO on LV mitochondrial function were examined via respirometry. TMAO at 10-300 µM concentration-dependently depressed LV contractile function, with coronary flow paralleling changes in isovolumic pressure development. Direct coronary effects were evident at >30 µM TMAO in hearts performing minimal isovolumic work, though this response was reduced by >65%. In contrast, exposure to 10 or 100 µM TMAO increased mitochondrial complex I, II and maximal respiratory fluxes while appearing to reduce outer membrane integrity. Expression of phospho-AMPKα and total GSK-3β declined. Acute exposure to TMAO levels reported in advanced CVD significantly inhibits cardiac contractility and induces modest coronary constriction while paradoxically over-activating mitochondrial respiration.

Upregulation of endocytic protein expression in the Alzheimer’s disease male human brain

Amyloid-beta (Aβ) is produced from amyloid precursor protein (APP) primarily after APP is internalised by endocytosis and clathrin-mediated endocytic processes are altered in Alzheimer’s disease (AD). There is also evidence that cholesterol and flotillin affect APP endocytosis. We hypothesised that endocytic protein expression would be altered in the brains of people with AD compared to non-diseased subjects which could be linked to increased Aβ generation. We compared protein expression in frontal cortex samples from men with AD compared to age-matched, non-diseased controls. Soluble and insoluble Aβ40 and Aβ42, the soluble Aβ42/Aβ40 ratio, βCTF, BACE1, presenilin-1 and the ratio of phosphorylated:total GSK3β were significantly increased while the insoluble Aβ42:Aβ40 ratio was significantly decreased in AD brains. Total and phosphorylated tau were markedly increased in AD brains. Significant increases in clathrin, AP2, PICALM isoform 4, Rab-5 and caveolin-1 and 2 were seen in AD brains but BIN1 was decreased. However, using immunohistochemistry, caveolin-1 and 2 were decreased. The results obtained here suggest an overall increase in endocytosis in the AD brain, explaining, at least in part, the increased production of Aβ during AD.

Intrarenal and Urinary Glycogen Synthase Kinase-3 Beta Levels in Diabetic and Nondiabetic Chronic Kidney Disease

Background: Renal glycogen synthase kinase-3 beta (GSK3β) overactivity has been associated with a diverse range of kidney diseases. GSK3β activity in urinary exfoliated cells was reported to predict the progression of diabetic kidney disease (DKD). We compared the prognostic value of urinary and intrarenal GSK3β levels in DKD and nondiabetic chronic kidney disease (CKD). Methods: We recruited 118 consecutive biopsy-proved DKD patients and 115 nondiabetic CKD patients. Their urinary and intrarenal GSK3β levels were measured. They were then followed for dialysis-free survival and rate of renal function decline. Results: DKD group had higher intrarenal and urinary GSK3β levels than nondiabetic CKD (p < 0.0001 for both), but their urinary GSK3β mRNA levels were similar. Urinary p-GSK3β level is statistically significantly correlated with the baseline estimated glomerular filtration rate (eGFR), but urinary GSK3β level by ELISA, its mRNA level, the p-GSK3β level, or the p-GSK3β/GSK3β ratio had no association with dialysis-free survival or the slope of eGFR decline. In contrast, the intrarenal pY216-GSK3β/total GSK3β ratio significantly correlated with the slope of eGFR decline (r = −0.335, p = 0.006) and remained an independent predictor after adjusting for other clinical factors. Conclusion: Intrarenal and urinary GSK3β levels were increased in DKD. The intrarenal pY216-GSK3β/total GSK3β ratio was associated with the rate of progression of DKD. The pathophysiological roles of GSK3β in kidney diseases deserve further studies.

MicroRNA-186 suppresses cell proliferation and metastasis in bladder cancer.

Bladder cancer (BCa) is a common malignancy in the urinary system. This study aims to explore the role of miR-186 in BCa tumorigenesis. The expression of miR-186 and ADAMTS12 in clinical BCa tissues and cell lines was detected. BCa cell lines T24, 5637 and EJ were used to transfect miR-186 mimics or inhibitors. Luciferase reporter gene detection confirmed the correlation between miR-186 and ADAMTS12. MTT method and flow cytometry were used to detect cell viability and apoptosis. Cell migration and invasion ability was detected by transwell assay. The protein level of ADAMTS12, β-catenin, GSK-3β and p-GSK-3β was determined using western blot analysis. MiR-186 was negatively correlated with the expression of ADAMTS12 in BCa tissues. Further research confirmed that ADAMTS12 is the direct target of miR-186. In addition, overexpression of miR-186 down-regulated the expression of ADAMTS12, inhibiting cell viability and apoptosis, while knockout of miR-186 led to the opposite result. miR-186 also inhibits the phosphorylation of GSK-3 β and β-catenin without changing the total GSK-3β level. Our study shows that miR-186 has a negative regulatory effect on the expression of ADAMTS12 in clinical specimens and in vitro. miR-186 can inhibit the proliferation and invasion of BCa cells. miR-186 has the potential to be used as a biomarker in the early detection of BCa.

The Tumor Suppressor Protein TRAF3 Modulates GSK3 Activity and Susceptibility of B Lymphoma Cells to GSK3 Inhibition.

Simple SummaryTRAF3 is an adapter protein that regulates signals through many receptors important for B cell differentiation and function. Loss-of-function mutations or deletions of TRAF3 are common in B cell malignancies. Glycogen Synthase Kinase 3 (GSK3) regulates signaling in growth, survival, and metabolism pathways. GSK3 inhibitors have been effective against many solid tumors; the inhibitor used in this work is currently being tested for efficacy against BCLs. We found that TRAF3 and GSK3 associate in multiple BCL cell lines, and that BCLs with low TRAF3 have a higher susceptibility to GSK3 inhibition. In contrast to BCL cell lines, GSK3 inhibition has little effect on TRAF3-sufficient and deficient resting primary B cells. These results suggest TRAF3 level as a predictor of BCL responsiveness to GSK3 inhibitor therapy.TNF receptor-associated factor 3 (TRAF3) is an adapter protein that inhibits many signals that promote B cell survival and activation. Mice with a B cell-specific TRAF3 deficiency and humans with a rare haploinsufficiency in TRAF3 have enhanced development of BCLs as they age. Loss-of-function mutations in TRAF3 are common in B cell malignancies. Recent studies show that pharmacological inhibition of the enzyme glycogen synthase kinase 3 (GSK3), which regulates cellular growth, survival, and metabolism, inhibits growth and survival of BCL-derived B cells. In this study, we found that TRAF3 and GSK3 associated in B cells. The relative levels of TRAF3 in BCL cell lines correlated positively with the ratio of inactive to total GSK3β, and negatively correlated with susceptibility to GSK3 inhibition by the GSK3 inhibitory drug 9-ING-41, currently in clinical trials. Uniquely in BCLs with low TRAF3, GSK3 inhibition caused increased loss of the TRAF3-regulated, anti-apoptotic protein Mcl-1. GSK3 inhibition also blocked hyperresponsiveness to IL-6 receptor signaling in TRAF3-deficient BCL cells. Together, these results support the utility of 9-ING-41 as a treatment for BCL, and suggest that a decrease or loss of TRAF3 in BCLs could act as a biomarker for increased susceptibility to GSK3 inhibitor treatment.

Helicobacter pylori Urease: Potential Contributions to Alzheimer's Disease.

Alzheimer’s disease (AD) causes dementia and memory loss in the elderly. Deposits of beta-amyloid peptide and hyperphosphorylated tau protein are present in a brain with AD. A filtrate of Helicobacter pylori’s culture was previously found to induce hyperphosphorylation of tau in vivo, suggesting that bacterial exotoxins could permeate the blood–brain barrier and directly induce tau’s phosphorylation. H. pylori, which infects ~60% of the world population and causes gastritis and gastric cancer, produces a pro-inflammatory urease (HPU). Here, the neurotoxic potential of HPU was investigated in cultured cells and in rats. SH-SY5Y neuroblastoma cells exposed to HPU (50–300 nM) produced reactive oxygen species (ROS) and had an increased [Ca2+]i. HPU-treated BV-2 microglial cells produced ROS, cytokines IL-1β and TNF-α, and showed reduced viability. Rats received daily i.p., HPU (5 µg) for 7 days. Hyperphosphorylation of tau at Ser199, Thr205 and Ser396 sites, with no alterations in total tau or GSK-3β levels, and overexpression of Iba1, a marker of microglial activation, were seen in hippocampal homogenates. HPU was not detected in the brain homogenates. Behavioral tests were performed to assess cognitive impairments. Our findings support previous data suggesting an association between infection by H. pylori and tauopathies such as AD, possibly mediated by its urease.

Effect of N-cadherin on Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells through Wnt Signaling Pathway.

This study aimed to compare and analyze the effect of N-cadherin on chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and to explore the related mechanism, so as to provide a novel theoretical basis for the clinical work of articular cartilage injury regeneration and repair. For this purpose, the experimental animals were clean grade SD rats (aged 5-6 weeks, weighing 180-250g). Alcian blue staining was carried out to observe the induced chondrogenesis following N-cadherin inhibition. The specific role of N-cadherin in the Wnt signaling pathway and chondrogenic differentiation of BMSCs was detected by Western blot; while the effect of N-cadherin on the molecular level changes of β-catenin in the cytoplasm was evaluated by fluorescence quantitative real-time PCR (qRT-PCR). In addition, immunoprecipitation (IP) was used for the verification of the interaction between N-cadherin and β-catenin. Results showed that under the light microscope, 90% of the BMSCs at the third generation, 90% of the cells were fused. Alcian blue staining showed that the green staining area in the BMP2 induction group was large and dense, while that in the N-cadherin inhibition group and blank control group was small and sparse. Western blot revealed that N-cadherin and SOX9 were significantly developed in the BMP2 induction group, but Wnt3a was not significantly developed. While in the N-cadherin inhibition group, the development of Wnt3a was obvious, yet without evident development of N-cadherin and SOX9. The qRT-PCR indicated that the relative mRNA expression of Wnt3a was significantly increased in the N-cadherin inhibition group (P<0.05). However, no obvious difference was observed in the mRNA expression of β-catenin between the BMP2 induction group and the N-cadherin inhibition group (P>0.05). Western blot indicated that in the BMP2 induction group; there existed the development of β-catenin, significant development of phos-GSK-3β and total GSK-3β, but no obvious development of Wnt3a. In the N-cadherin inhibition group, there was significantly enhanced development of Wnt3a and β-catenin than that before, blurred development of phos-GSK-3β than that before, and also obvious development of total GSK-3β with little change from before. N-cadherin promoted the expression of β-catenin mostly in the cell membrane, but only a few in the cytoplasm and nucleus. Additionally, verification by IP showed that N-cadherin and β-catenin were developed on N-cadherin and β-catenin bands, suggesting an interaction between N-cadherin and β-catenin. According to these results, N-cadherin can ultimately promote chondrogenic differentiation of BMSCs by inhibiting the Wnt signaling pathway.

Exercise with 40-Hz light flicker improves hippocampal insulin signaling in Alzheimer disease mice.

We examined whether exercise is associated with hippocampus-mediated improvement in insulin signaling and cell differentiation in the triple transgenic mouse model of Alzheimer disease (3xTg AD) murine model following exposure to 40-Hz light flickering and exercise. We subjected 12-month-old 3xTg AD mice to exercise and 40-Hz light flickering for 3 months. The exercise session was proceeded for 12 consecutive weeks with gradual increase of intensity. To investigate insulin signaling proteins, western blot was conducted to detect the ratio of phosphorylated insulin receptor β (p-IRβ)/total IRβ (t-IRβ), phosphorylated insulin receptor substrate 1 (p-IRS-1)/total IRS-1 (t-IRS-1), phosphorylated phosphatidylinositide-3-kinase (p-PI3K)/total PI3K (t-PI3K), phosphorylated 3-phosphoinositide dependent protein kinase-1 (p-PDK1)/total PDK-1 (t-PDK1), phosphorylated protein kinase B (p-Akt)/total-Akt (t-Akt), and phosphorylated glycogen synthase kinase 3 beta (p-GSK3β)/total GSK3β (t-GSK3β). Doublecortin immunohistochemistry was performed for assessing cell differentiation in the hippocampus. Treatments exerted a positive effect. The combination of exercise and 40-Hz light flickering exposure was the most effective treatment enhancing insulin signaling. Increased ratio of p-IRβ/t-IRβ, p-IRS-1/t-IRS-1, p-PI3K/t-PI3K, p-PDK1/t-PDK1, p-Akt/t-Akt, and p-GSK3β/t-GSK3β and enhanced cell differentiation were observed in the 3xTg AD with exercise under 40-Hz light flickering group. Our results indicate that exercise under 40-Hz light flickering most potently improved insulin signaling, thereby promoted cell differentiation.

MiR-30a Serves as a Tumor Suppressor for Hepatocellular Carcinoma by Downregulating ADAMTS14.

Hepatocellular carcinoma (HCC) is a very common malignancy in the world, but the effect of therapies on advanced HCC has not improved for decades. The present study aimed to investigate the role of miR-30a in the tumorigenesis of HCC. The expression of miR-30a and ADAMTS14 in HCC tissues were determined. Luciferase reporter gene detection confirmed the correlation between miR-30a and ADAMTS14. Cell viability and apoptosis rate were examined using an MTT assay and flow cytometry. Cell migration and invasion ability were detected by a transwell assay. The protein expression of ADAMTS14, β-catenin, GSK-3β, and p-GSK-3β were determined using western blotting. miR-30a was negatively correlated with the expression of ADAMTS14 in HCC tissues. Further research confirmed that ADAMTS14 is the direct target of miR-30a. In addition, the expression of ADAMTS14, cell viability and apoptosis were suppressed by miR-30a overexpression, while knockdown of miR-30a led to the opposite result. miR-30a also inhibits the phosphorylation of GSK-3β and β-catenin, without changing the total GSK-3β level. miR-30a acts as a tumor suppressor in the progression of HCC and can be used as a biomarker for early prediction and diagnosis.

Glycogen synthase kinase-3β inactivation promotes cervical cancerprogression, invasion, and drug resistance.

Human papillomavirus (HPV) infection-dependent cervical cancer is one of the most common gynecological cancers and often becomes aggressive, with rapid proliferation, invasion/migration, and drug resistance. Here, 135 fresh human cervical squamous cell carcinoma (CSCC) tissue specimens, comprising 21 adjacent normal (AN), 30 cervical intraepithelial neoplasia (CIN1-3 ), 45 CSCC, and 39 drugs (chemo-radiation)-resistant cervical tumor (DRCT) tissues were included. HPV-positive (HeLa, SiHa), HPV-negative (C33A), and cisplatin-resistant (CisR-HeLa/-SiHa/-C33A) cell lines were used for in vitro studies. HPV16/18 oncoproteins E6/E7, pERK1/2, and glycogen synthase kinase-3 (GSK3) and the matrix metalloproteinases (MMPs) MMP-9/-2 were assessed using immunohistochemistry, WB, and gelatin zymography. HPV16/18 infection was observed in 16.7% of the CIN1-3 , 77.8% of the CSCC, and 89.7% of DRCT samples. Total and inactive GSK3β expressions were associated with overall CSCC progression (p=0.039 and p=0.024, respectively) and chemoresistance (p=0.004 and p=0.014, respectively). Positive correlations were observed, between the expression of E6 and pGSK3β expression (p=0.013); E6 and CSCC progression (p<0.0001)/drug resistance (p=0.0001). CisR-HeLa/-SiHa was more dependent on pGSK3β, and activation of GSK3 by SMIs (iAkt), treatment with nimbolide, or knockdown of E6/E7 reduced cisplatin resistance and promoted apoptosis. Hence, the activation of GSK3β with nimbolide and iAkt can be exploited for therapeutic interventions of cervical cancer.

The extracellular HDAC6 ZnF UBP domain modulates the actin network and post-translational modifications of Tau

BackgroundMicrotubule-associated protein Tau undergoes aggregation in Alzheimer`s disease (AD) and a group of other related diseases collectively known as Tauopathies. In AD, Tau forms aggregates, which are deposited intracellularly as neurofibrillary tangles. Histone deacetylase-6 (HDAC6) plays an important role in aggresome formation, where it recruits polyubiquitinated aggregates to the motor protein dynein.MethodsHere, we have studied the effects of HDAC6 ZnF UBP on Tau phosphorylation, ApoE localization, GSK-3β regulation and cytoskeletal organization in neuronal cells by immunocytochemical analysis. This analysis reveals that the cell exposure to the UBP-type zinc finger domain of HDAC6 (HDAC6 ZnF UBP) can modulate Tau phosphorylation and actin cytoskeleton organization.ResultsHDAC6 ZnF UBP treatment to cells did not affect their viability and resulted in enhanced neurite extension and formation of structures similar to podosomes, lamellipodia and podonuts suggesting the role of this domain in actin re-organization. Also, HDAC6 ZnF UBP treatment caused increase in nuclear localization of ApoE and tubulin localization in microtubule organizing centre (MTOC). Therefore, our studies suggest the regulatory role of this domain in different aspects of neurodegenerative diseases. Upon HDAC6 ZnF UBP treatment, inactive phosphorylated form of GSK-3β increases without any change in total GSK-3β level.ConclusionsHDAC6 ZnF UBP was found to be involved in cytoskeletal re-organization by modulating actin dynamics and tubulin localization. Overall, our study suggests that ZnF domain of HDAC6 performs various regulatory functions apart from its classical function in aggresome formation in protein misfolding diseases.BbeLHN_zgyG8Hf4zoSkS1nVideo abstract

Glycogen Synthase Kinase 3 activation status in soleus and EDL muscles from space‐flown mice aboard the BION‐M1 biosatellite

Background A significant limitation to long-term spaceflight and space habitation is the deterioration of human musculoskeletal health. It is well known that prolonged exposure to microgravity leads to pronounced muscle and bone loss, with consequences mimicking several major effects observed in human aging (i.e. osteoporosis and sarcopenia). It is therefore critical to elucidate the underlying molecular mechanisms of these processes to develop meaningful intervention strategies. Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase initially defined in its role of regulating glycogen homeostasis, though it's now known to interact with more than 100 target proteins. GSK3 has notably proven to negatively regulate muscle mass by inhibiting hypertrophy-related mechanisms, myoblast fusion, and muscle regeneration, while also promoting ubiquitin-mediated protein degradation and muscle inflammation. In this study, we explored GSK3 expression and its serine phosphorylation status, known to control its activation/inactivation, in soleus and extensor digitorum longus (EDL) muscles obtained from the BION-M1 mission. Methods Male C57BL6/J mice (3 months old) were flown in space for 1 month aboard the BION-M1 biosatellite (F) and were compared to a ground control (GC, space housing and diet conditions) and a flight control (FC, standard housing conditions) group. Soleus and EDL muscles were extracted and homogenized in protein extraction buffer. Western blotting was used to examine total and phosphorylated (i.e. inactivated) forms of GSK3 (ser21 GSK3α; ser9 GSK3β). Results In the soleus muscle, total GSK3α and GSK3β were reduced in the F group. Though we did not observe any changes in ser21 (GSK3α) or ser9 (GSK3β) phosphorylation, the ratio of phosphorylated:total GSK3β was significantly elevated in the F group compared with GC and FC. We did not observe any differences in soleus phosphorylated:total GSK3α. Further, we did not observe any differences in total, phosphorylated or phosphorylated:total GSK3α/β content in the EDL F group compared to GC and FC. Conclusions Altogether, our data suggest that in response to 1 month of spaceflight, GSK3 is less active in the soleus muscle through changes in expression and phosphorylation. GSK3 may therefore not be involved in the later stages of soleus atrophy in flown mice. This may be explained by a slow-to-fast fibre shift in soleus muscle, as GSK3 presence and activity is typically reduced in fast-twitch muscle fibres compared to slow-twitch. Future studies with soleus muscles from two other NASA-flown missions (RR1 and RR9) will be conducted to confirm these effects.

N-glycosylated IgG in patients with kidney transplants increases calcium/calmodulin kinase IV in podocytes and causes injury.

Transplant glomerulopathy (TG) is a major cause of late allograft loss. Increased urine podocin/creatinine ratio in TG signifies accelerated podocyte loss. The mechanisms that lead to podocyte injury in TG remain unclear. We report that IgG from kidney transplant recipients with TG, but not from those without TG, cause a reduction in the expression of nephrin, significant podocyte actin cytoskeleton, and motility changes. These changes are preceded by increased expression of calcium/calmodulin kinase IV (CAMK4). Mechanistically, we found that CAMK4 phosphorylates GSK3β (glycogen synthase kinase 3 beta), activates the Wnt pathway and stabilizes the nephrin transcriptional repressor SNAIL. Silencing neonatal Fc Receptor (FcRn) or CAMK4 prevented the podocyte-damaging effects of IgG from patients with TG. Furthermore, we show that removal of N-linked glycosyl residues from these IgG did not interfere with its entry into the podocytes but eliminated its ability to upregulate CAMK4 and cause podocyte injury. The translational value of these findings is signified by the fact that CAMK4 is increased in podocytes of patients with TG but not in those without TG despite other forms of renal dysfunction. Our results offer novel considerations to limit podocyte injury in patients with kidney transplants, which may lead to eventual glomerular destabilization and transplant glomerulopathy.

Farrerol Directly Targets GSK-3β to Activate Nrf2-ARE Pathway and Protect EA.hy926 Cells against Oxidative Stress-Induced Injuries.

Oxidative stress-mediated endothelial injury is considered to be involved in the pathogenesis of various cardiovascular diseases. Farrerol, a typical natural flavanone from the medicinal plant Rhododendron dauricum L., has been reported to show protective effects against oxidative stress-induced endothelial injuries in our previous study. However, its action molecular mechanisms and targets are still unclear. In the present study, we determined whether farrerol can interact with glycogen synthase kinase 3β- (GSK-3β-) nuclear factor erythroid 2-related factor 2- (Nrf2-) antioxidant response element (ARE) signaling, which is critical in defense against oxidative stress. Our results demonstrated that farrerol could specifically target Nrf2 negative regulator GSK-3β and inhibit its kinase activity. Mechanistic studies proved that farrerol could induce an inhibitory phosphorylation of GSK-3β at Ser9 without affecting the expression level of total GSK-3β protein and promote the nuclear translocation of Nrf2 as well as the mRNA and protein expression of its downstream target genes heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in EA.hy926 cells. Further studies performed with GSK-3β siRNA and specific inhibitor lithium chloride (LiCl) confirmed that GSK-3β inhibition was involved in farrerol-mediated endothelial protection and Nrf2 signaling activation. Moreover, molecular docking and molecular dynamics studies revealed that farrerol could bind to the ATP pocket of GSK-3β, which is consistent with the ATP-competitive kinetic behavior. Collectively, our results firstly demonstrate that farrerol could attenuate endothelial oxidative stress by specifically targeting GSK-3β and further activating the Nrf2-ARE signaling pathway.