Effect of l-carnitine supplementation on lead acetate-induced liver cell apoptosis and inflammation: role of caspase-3 and glycogen synthase kinase-3ß enzymes.

AIM: The study aimed at studying the hepatoprotective effect of l-carnitine against lead (Pb) acetate-induced hepatocellular injury, emphasizing the role of caspase-3 and glycogen synthase kinase-3ß in hepatocellular apoptosis and inflammation. MATERIALS AND METHODS: Male Wistar rats were used. The experimental approach involved estimation of the liver enzymes' serum levels. Oxidative and inflammatory biomarkers were measured in hepatic tissue homogenates. Paraffin-embedded hepatic sections were prepared for histopathology and immunohistochemistry. Quantitative determination of the phosphorylated glycogen synthase kinase-3 beta was performed. KEY FINDINGS: The serum showed a significant elevation in ALT, AST, and LDH; tissue homogenates showed significant elevation in lipid peroxide and inflammatory biomarkers with significant reduction in reduced glutathione in the Pb acetate-treated group. Co-administration of l-carnitine with Pb acetate produced significant reduction in liver enzymes with significant improvement in oxidant, antioxidant and inflammatory markers. Lead acetate treatment significantly reduced the phosphorylated glycogen synthase kinase-3 beta, while l-carnitine enhanced its phosphorylation. Histopathological examination showed inflammatory reaction around blood vessels with fatty degeneration in hepatocytes of the Pb acetate intoxicated group. l-Carnitine caused a decrease in hepatic damage with minimal vascular alterations in central vein. Caspase-3 expression in hepatocytes was decreased in Pb-treated group supplemented with l-carnitine. SIGNIFICANCE: Our study reveals that oxidative stress and inflammation participate in Pb acetate-induced hepatocellular injury. Glycogen synthase kinase-3ß and caspase-3 play role in Pb acetate-induced hepatic damage. l-Carnitine shows significant protective effects against hepatocellular apoptosis and inflammation induced by Pb acetate through antioxidant, anti-inflammatory and anti-apoptotic pathways in part mediated by GSK-3ß inhibition.

Aurora kinase A regulates liver regeneration through macrophages polarization and Wnt/ß-catenin signalling.

BACKGROUND AND AIMS: Liver regeneration is a complex process regulated by a variety of cells, cytokines and biological pathways. Aurora kinase A (AURKA) is a serine/threonine kinase that plays a role in centrosome maturation and spindle formation during the cell division cycle. The purpose of this study was to further explore the mechanism of AURKA on liver regeneration and to identify new possible targets for liver regeneration. METHODS: The effect and mechanism of AURKA on liver regeneration were studied using a 70% hepatectomy model. Human liver organoids were used as an in vitro model to investigate the effect of AURKA on hepatocyte proliferation. RESULTS: AURKA inhibition significantly reduced the level of ß-catenin protein by reducing the phosphorylation level of glycogen synthase kinase-3ß (GSK-3ß), leading to the inhibition of liver regeneration. Further studies showed that AURKA co-localized and interacted with GSK-3ß in the cytoplasm of hepatocytes. When phosphorylation of GSK-3ß was enhanced, the total GSK-3ß level remained unchanged, while AURKA was not affected, and ß-catenin protein levels were increased. In addition, AURKA inhibition affected the formation and proliferation of human liver organoids. Furthermore, AURKA inhibition led to the polarization of M1 macrophages and the release of interleukin-6 and Tumour necrosis factor α, which also led to reduced liver regeneration and increased liver injury. CONCLUSIONS: These results provide more details on the mechanism of liver regeneration and suggest that AURKA is an important regulator of this mechanism.

The Akt/GSK3ß/ß-catenin signaling regulated by ZCCHC14 is responsible for accelerating the proliferation of hepatocellular carcinoma.

PURPOSE: To clarify how ZCCHC14 affects the development of hepatocellular carcinoma (HCC). METHODS: Differential levels of ZCCHC14 in HCC tissues and cells were examined. Proliferative and migratory changes in HCC cells with overexpression or knockdown of ZCCHC14 were detected using 5-Ethynyl-2'- deoxyuridine (EdU) and Transwell assay, respectively. Expression changes of p-Akt/Akt, p-GSK3ß/GSK3ß and ß-catenin in HCC cells mediated by ZCCHC14 were determined. Intervened by the p-Akt activator SC79 or overexpression of ß-catenin, further validated the involvement of the Akt/GSK3ß/ß-catenin signaling in HCC cell phenotypes mediated by ZCCHC14. RESULTS: Upregulated ZCCHC14 in HCC accelerated in vitro proliferative potential of HCC cells. Knockdown of ZCCHC14 inactivated the Akt/GSK3ß/ß-catenin signaling and inhibited malignant phenotypes of HCC, which were partially reversed by SC79 induction or overexpression of ß-catenin. CONCLUSIONS: By activating the Akt/GSK3ß/ß-catenin signaling, ZCCHC14 accelerates HCC cells proliferation.

CHIR99021, trough GSK-3ß Targeting, Reduces Epithelioid Sarcoma Cell Proliferation by Activating Mitotic Catastrophe and Autophagy.

Epithelioid sarcoma (ES) is a rare disease representing <1% of soft tissue sarcomas. Current therapies are based on anthracycline alone or in combination with ifosfamide or other cytotoxic drugs. ES is still characterized by a poor prognosis with high rates of recurrence. Indeed, for years, ES survival rates have remained stagnant, suggesting that conventional treatments should be revised and improved. New therapeutic approaches are focused to target the key regulators of signaling pathways, the causative markers of tumor pathophysiology. To this end, we selected, among the drugs to which an ES cell line is highly sensitive, those that target signaling pathways known to be dysregulated in ES. In particular, we found a key role for GSK-3ß, which results in up-regulation in tumor versus normal tissue samples and associated to poor prognosis in sarcoma patients. Following this evidence, we evaluated CHIR99021, a GSK-3 inhibitor, as a potential drug for use in ES therapy. Our data highlight that, in ES cells, CHIR99021 induces cell cycle arrest, mitotic catastrophe (MC) and autophagic response, resulting in reduced cell proliferation. Our results support the potential efficacy of CHIR99021 in ES treatment and encourage further preclinical and clinical studies.

Tideglusib, a Non-ATP Competitive Inhibitor of GSK-3ß as a Drug Candidate for the Treatment of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is the most common degenerative motor neuron disease in adults. About 97% of ALS patients present TDP-43 aggregates with post-translational modifications, such as hyperphosphorylation, in the cytoplasm of affected cells. GSK-3ß is one of the protein kinases involved in TDP-43 phosphorylation. Up-regulation of its expression and activity is reported on spinal cord and cortex tissues of ALS patients. Here, we propose the repurposing of Tideglusib, an in-house non-ATP competitive GSK-3ß inhibitor that is currently in clinical trials for autism and myotonic dystrophy, as a promising therapeutic strategy for ALS. With this aim we have evaluated the efficacy of Tideglusib in different experimental ALS models both in vitro and in vivo. Moreover, we observed that GSK-3ß activity is increased in lymphoblasts from sporadic ALS patients, with a simultaneous increase in TDP-43 phosphorylation and cytosolic TDP-43 accumulation. Treatment with Tideglusib decreased not only phospho-TDP-43 levels but also recovered its nuclear localization in ALS lymphoblasts and in a human TDP-43 neuroblastoma model. Additionally, we found that chronic oral treatment with Tideglusib is able to reduce the increased TDP-43 phosphorylation in the spinal cord of Prp-hTDP-43A315T mouse model. Therefore, we consider Tideglusib as a promising drug candidate for ALS, being proposed to start a clinical trial phase II by the end of the year.

Keratin 19 interacts with GSK3ß to regulate its nuclear accumulation and degradation of cyclin D3.

Cyclin D3 regulates the G1/S transition and is frequently overexpressed in several cancer types including breast cancer, where it promotes tumor progression. Here we show that a cytoskeletal protein keratin 19 (K19) physically interacts with a serine/threonine kinase GSK3ß and prevents GSK3ß-dependent degradation of cyclin D3. The absence of K19 allowed active GSK3ß to accumulate in the nucleus and degrade cyclin D3. Specifically, the head (H) domain of K19 was required to sustain inhibitory phosphorylation of GSK3ß Ser9, prevent nuclear accumulation of GSK3ß, and maintain cyclin D3 levels and cell proliferation. K19 was found to interact with GSK3ß and K19-GSK3ß interaction was mapped out to require Ser10 and Ser35 residues on the H domain of K19. Unlike wildtype K19, S10A and S35A mutants failed to maintain total and nuclear cyclin D3 levels and induce cell proliferation. Finally, we show that the K19-GSK3ß-cyclin D3 pathway affected sensitivity of cells toward inhibitors to cyclin-dependent kinase 4 and 6 (CDK4/6). Overall, these findings establish a role for K19 in the regulation of GSK3ß-cyclin D3 pathway and demonstrate a potential strategy for overcoming resistance to CDK4/6 inhibitors.

Microglia: A Double-Edged Sword in Intracerebral Hemorrhage From Basic Mechanisms to Clinical Research.

Microglia are the resident immune cells of the central nervous system (CNS). It is well established that microglia are activated and polarized to acquire different inflammatory phenotypes, either pro-inflammatory or anti-inflammatory phenotypes, which act as a critical component in the neuroinflammation following intracerebral hemorrhage (ICH). Microglia produce pro-inflammatory mediators at the early stages after ICH onset, anti-inflammatory microglia with neuroprotective effects appear to be suppressed. Previous research found that driving microglia towards an anti-inflammatory phenotype could restrict inflammation and engulf cellular debris. The principal objective of this review is to analyze the phenotypes and dynamic profiles of microglia as well as their shift in functional response following ICH. The results may further the understanding of the body's self-regulatory functions involving microglia following ICH. On this basis, suggestions for future clinical development and research are provided.

Glycogen Synthase Kinase 3ß Modulates the Inflammatory Response Activated by Bacteria, Viruses, and Parasites.

Knowledge of glycogen synthase kinase 3ß (GSK3ß) activity and the molecules identified that regulate its function in infections caused by pathogenic microorganisms is crucial to understanding how the intensity of the inflammatory response can be controlled in the course of infections. In recent years many reports have described small molecular weight synthetic and natural compounds, proteins, and interference RNA with the potential to regulate the GSK3ß activity and reduce the deleterious effects of the inflammatory response. Our goal in this review is to summarize the most recent advances on the role of GSK3ß in the inflammatory response caused by bacteria, bacterial virulence factors (i.e. LPS and others), viruses, and parasites and how the regulation of its activity, mainly its inhibition by different type of molecules, modulates the inflammation.

Investigating function of long noncoding RNA of HOTAIRM1 in progression of SKOV3 ovarian cancer cells.

Ovarian cancer is one of the most heterogeneous malignancies in the field of gynecologic oncology. Deregulation of long noncoding RNAs (lncRNAs) is implicated in carcinogenesis. Therefore, the present study was conducted to investigate the possible role of lncRNA of HOXA transcript antisense intergenic RNA myeloid-specific 1(HOTAIRM1) in progression of SKOV3 cells in ovarian cancer and also its underlying molecular mechanisms. HOTAIRM1 expression level will be measured by real-time polymerase chain reaction (PCR) in SKOV3 cells. For determining the effect of HOTAIRM1 silencing on progression of SKOV3 cells, siHOTAIRM1 will be designed and transfected into cells using a liposomal approach. MTT and trypan blue assays will be used to determine the effect of HOTAIRM1 silencing on cell proliferation. Apoptosis of the cells will be detected by flow cytometry. Furthermore, expressions of apoptosis-related genes and Wnt pathway-related proteins and genes will be analyzed by Western blot and real-time PCR. HOTAIRM1 was overexpressed in SKOV3 cells. Silencing of HOTAIRM1 alleviated cell proliferation, and increased cell apoptosis of SKOV3 cells. Moreover, siHOTAIRM1 significantly increased expression of pro-apoptotic agents, such as Bad and Bax, while it decreased expressions of Bid and Bcl-2 (anti-apoptotic agents). Also, silencing of HOTAIRM1 resulted in a suppressed expression of Wnt pathway-related proteins and also expression of its downstream target gene, matrix metalloproteinase 9(MMP9). Our findings provided new insights into function of lncRNA of HOTAIRM1 in progression of ovarian cancer by modulating Wnt pathway and its downstream target gene, MMP9.

A Crosstalk between the Biorhythms and Gatekeepers of Longevity: Dual Role of Glycogen Synthase Kinase-3.

This review discusses genetic and molecular pathways that link circadian timing with metabolism, resulting in the emergence of positive and negative regulatory feedback loops. The Nrf2 pathway is believed to be a component of the anti-aging program responsible for the healthspan and longevity. Nrf2 enables stress adaptation by activating cell antioxidant defense and other metabolic processes via control of expression of over 200 target genes in response to various types of stress. The GSK3 system represents a "regulating valve" that controls fine oscillations in the Nrf2 level, unlike Keap1, which prevents significant changes in the Nrf2 content in the absence of oxidative stress and which is inactivated by the oxidative stress. Furthermore, GSK3 modifies core circadian clock proteins (Bmal1, Clock, Per, Cry, and Rev-erbα). Phosphorylation by GSK3 leads to the inactivation and degradation of circadian rhythm-activating proteins (Bmal1 and Clock) and vice versa to the activation and nuclear translocation of proteins suppressing circadian rhythms (Per and Rev-erbα) with the exception of Cry protein, which is likely to be implicated in the fine tuning of biological clock. Functionally, GSK3 appears to be one of the hubs in the cross-regulation of circadian rhythms and antioxidant defense. Here, we present the data on the crosstalk between the most powerful cell antioxidant mechanism, the Nrf2 system, and the biorhythm-regulating system in mammals, including the impact of GSK3 overexpression and knockout on the Nrf2 signaling. Understanding the interactions between the regulatory cascades linking homeostasis maintenance and cell response to oxidative stress will help in elucidating molecular mechanisms that underlie aging and longevity.

GSK3ß Interacts With CRMP2 and Notch1 and Controls T-Cell Motility.

The trafficking of T-cells through peripheral tissues and into afferent lymphatic vessels is essential for immune surveillance and an adaptive immune response. Glycogen synthase kinase 3ß (GSK3ß) is a serine/threonine kinase and regulates numerous cell/tissue-specific functions, including cell survival, metabolism, and differentiation. Here, we report a crucial involvement of GSK3ß in T-cell motility. Inhibition of GSK3ß by CHIR-99021 or siRNA-mediated knockdown augmented the migratory behavior of human T-lymphocytes stimulated via an engagement of the T-cell integrin LFA-1 with its ligand ICAM-1. Proteomics and protein network analysis revealed ongoing interactions among GSK3ß, the surface receptor Notch1 and the cytoskeletal regulator CRMP2. LFA-1 stimulation in T-cells reduced Notch1-dependent GSK3ß activity by inducing phosphorylation at Ser9 and its nuclear translocation accompanied by the cleaved Notch1 intracellular domain and decreased GSK3ß-CRMP2 association. LFA-1-induced or pharmacologic inhibition of GSK3ß in T-cells diminished CRMP2 phosphorylation at Thr514. Although substantial amounts of CRMP2 were localized to the microtubule-organizing center in resting T-cells, this colocalization of CRMP2 was lost following LFA-1 stimulation. Moreover, the migratory advantage conferred by GSK3ß inhibition in T-cells by CHIR-99021 was lost when CRMP2 expression was knocked-down by siRNA-induced gene silencing. We therefore conclude that GSK3ß controls T-cell motility through interactions with CRMP2 and Notch1, which has important implications in adaptive immunity, T-cell mediated diseases and LFA-1-targeted therapies.

Downregulation of GSK-3ß Expression via Ultrasound-Targeted Microbubble Destruction Enhances Atherosclerotic Plaque Stability in New Zealand Rabbits.

Accumulating evidence suggests that atherosclerosis (AS) is the underlying cause of vascular diseases, including heart disease and stroke. Ultrasound-targeted microbubble destruction (UTMD) technology provides a tolerable, efficient and effective system for drug delivery and gene transfection, which has broad application prospects in the treatment of AS. In addition, glycogen synthase kinase (GSK)-3ß has been implicated as a potentially valuable therapeutic agent for AS treatment; however, the specific molecular mechanisms remain unknown. Therefore, this study was conducted to explore the effect of downregulation of GSK-3ß expression via UTMD on atherosclerotic plaque stability. We established a THP-1 macrophage-derived foam cell model in vitro and an atherosclerotic plaque model in the right common carotid artery of New Zealand rabbits. We determined levels of the relevant vulnerable plaque stability elements. The results indicate that GSK-3ß was upregulated in the foam cells and in atherosclerotic rabbits. Downregulation of GSK-3ß expression by UTMD suppressed vulnerable plaque factors and inflammation in vitro and in vivo, changed the cytoskeleton of the foam cells in vitro, increased Young's modulus and decreased the peak intensity of atherosclerotic plaque in vivo. Moreover, GSK-3ß inhibition by UTMD did not influence the viability of the foam cells. Collectively, our results indicate that GSK-3ß could be a potential target for anti-atherogenic interventions and, in particular, can improve the stability of AS plaques in combination with UTMD.

GSK3ß-Mediated Expression of CUG-Translated WT1 Is Critical for Tumor Progression.

The Wilms' tumor 1 (WT1) gene is well known as a chameleon gene. It plays a role as a tumor suppressor in Wilms' tumor but also acts as an oncogene in other cancers. Previously, our group reported that a canonical AUG starting site for the WT1 protein (augWT1) acts as a tumor suppressor, whereas a CUG starting site for the WT1 protein (cugWT1) functions as an oncogene. In this study, we report an oncogenic role of cugWT1 in the AOM/DSS-induced colon cancer mouse model and in a urethane-induced lung cancer model in mice lacking cugWT1. Development of chemically-induced tumors was significantly depressed in cugWT1-deficient mice. Moreover, glycogen synthase kinase 3ß promoted phosphorylation of cugWT1 at S64, resulting in ubiquitination and degradation of the cugWT1 associated with the F-box-/- WD repeat-containing protein 8. Overall, our findings suggest that inhibition of cugWT1 expression provides a potential candidate target for therapy. SIGNIFICANCE: These findings demonstrate that CUG-translated WT1 plays an oncogenic role in vivo, and GSK3ß-mediated phosphorylation of cugWT1 induces its ubiquitination and degradation in concert with FBXW8.

Glycogen Synthase Kinase 3ß Promotes Postoperative Cognitive Dysfunction by Inducing the M1 Polarization and Migration of Microglia.

Postoperative cognitive dysfunction (POCD) is a common postoperative central nervous system complication, especially in the elderly. It has been consistently reported that the pathological process of this clinical syndrome is related to neuroinflammation and microglial proliferation. Glycogen synthase kinase 3ß (GSK-3ß) is a widely expressed kinase with distinct functions in different types of cells. The role of GSK-3ß in regulating innate immune activation has been well documented, but as far as we know, its role in POCD has not been fully elucidated. Lithium chloride (LiCl) is a widely used inhibitor of GSK-3ß, and it is also the main drug for the treatment of bipolar disorder. Prophylactic administration of lithium chloride (2 mM/kg) can inhibit the expression of proinflammatory mediators in the hippocampus, reduce the hippocampal expression of NF-κB, and increase both the downregulation of M1 microglial-related genes (inducible nitric oxide synthase and CD86) and upregulation of M2 microglial-related genes (IL-10 and CD206), to alleviate the cognitive impairment caused by orthopedic surgery. In vitro, LiCl reversed LPS-induced production of proinflammatory mediators and M1 polarization of microglia. To sum up these results, GSK-3ß is a key contributor to POCD and a potential target of neuroprotective strategies.

RPN2 is targeted by miR-181c and mediates glioma progression and temozolomide sensitivity via the wnt/ß-catenin signaling pathway.

Accumulating evidence indicates that the dysregulation of the miRNAs/mRNA-mediated carcinogenic signaling pathway network is intimately involved in glioma initiation and progression. In the present study, by performing experiments and bioinformatics analysis, we found that RPN2 was markedly elevated in glioma specimens compared with normal controls, and its upregulation was significantly linked to WHO grade and poor prognosis. Knockdown of RPN2 inhibited tumor proliferation and invasion, promoted apoptosis, and enhanced temozolomide (TMZ) sensitivity in vitro and in vivo. Mechanistic investigation revealed that RPN2 deletion repressed ß-catenin/Tcf-4 transcription activity partly through functional activation of glycogen synthase kinase-3ß (GSK-3ß). Furthermore, we showed that RPN2 is a direct functional target of miR-181c. Ectopic miR-181c expression suppressed ß-catenin/Tcf-4 activity, while restoration of RPN2 partly reversed this inhibitory effect mediated by miR-181c, implying a molecular mechanism in which TMZ sensitivity is mediated by miR-181c. Taken together, our data revealed a new miR-181c/RPN2/wnt/ß-catenin signaling axis that plays significant roles in glioma tumorigenesis and TMZ resistance, and it represents a potential therapeutic target, especially in GBM.

Glycogen synthase kinase-3ß participates in acquired resistance to gemcitabine in pancreatic cancer.

Acquisition of resistance to gemcitabine is a challenging clinical and biological hallmark property of refractory pancreatic cancer. Here, we investigated whether glycogen synthase kinase (GSK)-3ß, an emerging therapeutic target in various cancer types, is mechanistically involved in acquired resistance to gemcitabine in human pancreatic cancer. This study included 3 gemcitabine-sensitive BxPC-3 cell-derived clones (BxG30, BxG140, BxG400) that acquired stepwise resistance to gemcitabine and overexpressed ribonucleotide reductase (RR)M1. Treatment with GSK3ß-specific inhibitor alone attenuated the viability and proliferation of the gemcitabine-resistant clones, while synergistically enhancing the efficacy of gemcitabine against these clones and their xenograft tumors in rodents. The gemcitabine-resensitizing effect of GSK3ß inhibition was associated with decreased expression of RRM1, reduced phosphorylation of Rb protein, and restored binding of Rb to the E2 transcription factor (E2F)1. This was followed by decreased E2F1 transcriptional activity, which ultimately suppressed the expression of E2F1 transcriptional targets including RRM1, CCND1 encoding cyclin D1, thymidylate synthase, and thymidine kinase 1. These results suggested that GSK3ß participates in the acquisition of gemcitabine resistance by pancreatic cancer cells via impairment of the functional interaction between Rb tumor suppressor protein and E2F1 pro-oncogenic transcription factor, thereby highlighting GSK3ß as a promising target in refractory pancreatic cancer. By providing insight into the molecular mechanism of gemcitabine resistance, this study identified a potentially novel strategy for pancreatic cancer chemotherapy.

Inhibition of GSK-3ß restores delayed gastric emptying in obesity-induced diabetic female mice.

Diabetic gastroparesis (DG) is a clinical syndrome characterized by delayed gastric emptying (DGE). Loss of nuclear factor erythroid 2-related factor 2 (Nrf2) is associated with reduced neuronal nitric oxide synthase-α (nNOSα)-mediated gastric motility and DGE. Previous studies have shown that nuclear exclusion and inactivation of Nrf2 is partly regulated by glycogen synthase kinase 3ß (GSK-3ß). In the current study, the molecular signaling of GSK-3ß-mediated Nrf2 activation and its mechanistic role on DG were investigated in high-fat diet (HFD)-induced obese/Type 2 diabetes (T2D) female mice. Adult female C57BL/6J mice were fed with HFD or normal diet (ND) with or without GSK-3ß inhibitor (SB 216763, 10 mg/kg body wt ip) start from the 14th wk and continued feeding mice for an additional 3-wk time period. Our results show that treatment with GSK-3ß inhibitor SB attenuated DGE in obese/T2D mice. Treatment with SB restored impaired gastric 1) Nrf2 and phase II antioxidant enzymes through PI3K/ERK/AKT-mediated pathway, 2) tetrahydrobiopterin (BH4, cofactor of nNOS) biosynthesis enzyme dihydrofolate reductase, and 3) nNOSα dimerization in obese/T2 diabetic female mice. SB treatment normalized caspase 3 activity and downstream GSK-3ß signaling in the gastric tissues of the obese/T2 diabetic female mice. In addition, GSK-3ß inhibitor restored impaired nitrergic relaxation in hyperglycemic conditions. Finally, SB treatment reduced GSK3 marker, pTau in adult primary enteric neuronal cells. These findings emphasize the importance of GSK-3ß on regulating gastric Nrf2 and nitrergic mediated gastric emptying in obese/diabetic rodents.NEW & NOTEWORTHY Inhibition of glycogen synthase kinase 3ß (GSK-3ß) with SB 216763 attenuates delayed gastric emptying through gastric nuclear factor erythroid 2-related factor 2 (Nrf2)-phase II enzymes in high-fat diet-fed female mice. SB 216763 restored impaired gastric PI3K/AKT/ ß-catenin/caspase 3 expression. Inhibition of GSK-3ß normalized gastric dihydrofolate reductase, neuronal nitric oxide synthase-α expression, dimerization and nitrergic relaxation. SB 216763 normalized both serum estrogen and nitrate levels in female obese/Type 2 diabetes mice. SB 216763 reduced downstream signaling of GSK-3ß in enteric neuronal cells in vitro.

Suppression of G6PD induces the expression and bisecting GlcNAc-branched N-glycosylation of E-Cadherin to block epithelial-mesenchymal transition and lymphatic metastasis.

BACKGROUND: As the rate-limit enzyme of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD) plays important roles in tumour progression, but the exact mechanism through which G6PD controls cancer metastasis remains unclear. METHODS: G6PD expression in resected oral squamous cell carcinoma (OSCC) samples was analysed by immunohistochemistry. The effects and mechanism of G6PD suppression on OSCC cell lines were measured by transwell assay, wound healing assay, western and lectin blot, mass spectrometer analysis, ChIP-PCR, and luciferase reporter assay. BALB/c-nude mice were used to establish orthotopic xenograft model. RESULTS: G6PD expression in the tumours of 105 OSCC patients was associated with lymphatic metastasis and prognosis. In vitro cellular study suggested that G6PD suppression impaired cell migration, invasion, and epithelial-mesenchymal transition. Furtherly, G6PD knockdown activated the JNK pathway, which then blocked the AKT/GSK-3ß/Snail axis to induce E-Cadherin expression and transcriptionally regulated MGAT3 expression to promote bisecting GlcNAc-branched N-glycosylation of E-Cadherin. An orthotopic xenograft model further confirmed that dehydroepiandrosterone reduced lymphatic metastatic rate of OSCC, which was partially reversed by JNK inhibition. CONCLUSIONS: Suppression of G6PD promoted the expression and bisecting GlcNAc-branched N-glycosylation of E-Cadherin via activating the JNK pathway, which thus acted on OSCC metastasis.

Glycogen synthase kinase-3ß promotes radiation-induced lung fibrosis by regulating ß-catenin/lin28 signaling network to determine type II alveolar stem cell transdifferentiation state.

The role of type II alveolar epithelial stem cells (AEC II) for alveolar repair in radiation-induced lung fibrosis (RILF) remains largely unknown, mainly because of AEC II phenotype's spontaneous change in vitro. Cell differentiation status is determined by Lin28 and let-7 miRNAs in see-saw-pattern. Lin28, a repressor of let-7 and a stem cell marker, is activated by ß-catenin. The expression of ß-catenin is regulated by GSK-3ß/TGF-ß1 signaling. To understand the true role of AEC II in RILF, we freshly isolated primary AEC II directly from thoracically irradiated lungs. We then explored the expressions of cell phenotype markers and differentiation regulators in these isolated AEC II to analyze the correlation between GSK-3ß/TGF-ß1/ß-catenin signaling pathway, lin28/let-7 balance, and AEC II phenotypes at different injury phases following irradiation. Results showed that isolated single primary cells displayed AEC II ultrastructural features and proSP-C positive. The gene expressions of prosp-c (an AEC II biomarker) and hopx (an AEC I marker) significantly increased in isolated AEC II during injury repair phase (P < .001 and P < .05) but decreased at end-stage of injury, while mesenchymal markers increased in both isolated AEC II and irradiated lungs. mRNA levels of gsk-3ß, tgf-ß1, and ß-catenin increased in all irradiated AEC II, but more pronounced in the second half of injury phase (P < .05-P < .001). Similarly, the expression of lin28 was also significantly elevated in isolated AEC II at the late phase (P < .05-P < .001). Four let-7 miRNAs were significantly upregulated in all irradiated AEC II groups (P < .05-P < .001). The time-dependent and highly consistent uptrends for four lin28/let-7 ratios in sorted AEC II contrasted to downtrends in irradiated lungs. In conclusion, RILF occurred when GSK-3ß/TGF-ß1 signaling increased ß-catenin levels, which led to the augmentation of AEC II population by elevated lin28/let-7 ratio and the transcription of profibrotic cytokines and factors, thereby inducing AEC II to undergo transdifferentiation into mesenchymal cells.

Toxoplasma gondii Chinese I genotype Wh6 strain infection induces tau phosphorylation via activating GSK3ß and causes hippocampal neuron apoptosis.

Toxoplasma gondii (T. gondii) is a neurophilic and intracellular parasite that can affect plenty of vertebrate animals, including humans. Recent researches indicate that T. gondii infection is associated with neurodegenerative diseases such as Alzheimer's disease(AD). In addition, tau hyper-phosphorylation is a crucial event leading to the formation of nerve fiber tangles in AD. Despite the efforts to understand the interactions between T. gondii and AD, there are no clear results available so far. Here, we infected mice with the T. gondii of the Chinese 1 genotype Wh6 strain (TgCtwh6) for 60 days. Then we observed the formation of tissue cysts in the brain, the damage of neuron and the increased expression of phosphorylated tau (p-tau) in the hippocampal tissue of the mice. Similarly, we also found that p-tau, glycogen synthase kinase 3 beta (GSK3ß), and phosphorylated GSK3ß (p-GSK3ß) were upregulated in vitro in TgCtwh6 challenged hippocampal neuron cell strain, HT22 cells. We noted a down-regulated expression of GSK3ß,p-GSK3ß, and p-tau in HT22 cells, which were pretreated with LiCl, an inhibitor of GSK3ß. These data suggested that p-GSK3ß may mediate tau phosphorylation after TgCtwh6 infection. Furthermore, TgCtwh6 infection also caused the increased expression of Bax and Caspase3, the decreased expression of Bcl-XL in HT22 cells, which had both early and late apoptosis. In all, our results indicated that TgCtwh6 infection not only led to phosphorylation of tau via activating GSK3ß but also promoted hippocampal neuron apoptosis. Our research may partially reveal the mechanism with which TgCtwh6 induce neurofibrillary pathology.