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5-Fluorouracil (5-FU) is widely used in the treatment of gastric cancer, and the emergence of drug resistance and toxic effects has limited its application. Therefore, there is an urgent need for safe and effective novel drugs or new therapies. β-Ionone (BI) is found in vegetables and fruits and possesses an inhibitory proliferation of tumor cells in vitro and in vivo. In this study, we investigated whether BI could enhance the inhibitory effects of 5-FU on the proliferation of gastric adenocarcinoma cells and the growth of gastric cancer cell xenografts in a mouse model. The effects of BI and 5-FU alone or their combination on the cell viability, apoptosis, and mitochondrial membrane potential, the cell cycle, and its related proteins-Cyclin D1, and CDK4 as well as PCNA and GSK-3β were evaluated in SGC-7901 cells and MKN45 cells by MTT, MB, flow cytometry and Western blot. In addition, the effects of BI and 5-FU alone or their combination on the growth of SGC-7901 cell xenografts in nude mice were investigated. The results showed that BI significantly enhanced the sensitivity of gastric adenocarcinoma cells to 5-FU in vitro and in vivo, i.e. proliferation inhibited, apoptosis induced and GSK-3β protein activated. Therefore, our results suggest that BI increases the antitumor effect of 5-FU on gastric adenocarcinoma cells, at least partly from an activated GSK-3β signaling pathway., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Salidroside (SAL) is a phenol glycoside compound found in plants of the Rhodiola genus which has natural antioxidant and free radical scavenging properties. SAL are able to protect against manganese-induced ototoxicity. However, the molecular mechanism by which SAL reduces levels of reactive oxygen species (ROS) is unclear. Here, we established an in vitro gentamicin (GM) ototoxicity model to observe the protective effect of SAL on GM-induced hair cells (HC) damage. Cochlear explants of postnatal day 4 rats were obtained and randomly divided into six groups: two model groups (treatment with 0.2 mM or 0.4 mM GM for 24 h); two 400 μmol/L SAL-pretreated groups pretreatment with SAL for 3 h followed by GM treatment (0.2 mM or 0.4 mM) for 24 h; 400 μmol/L SAL group (treatment with SAL for 24 h); control group (normal cultured cochlear explants). The protective effects of SAL on GM-induced HC damage, and on mRNA and protein levels of antioxidant enzymes were observed. HC loss occurred after 24 h of GM treatment. Pretreatment with SAL significantly reduced GM-induced OHC loss. In cochlear tissues, mRNA and protein levels of NRF2 and HO-1 were enhanced in the GM alone group compared with the SAL pretreatment GM treatment group. SAL may protect against GM-induced ototoxicity by regulating the antioxidant defense system of cochlear tissues; SAL can activate NRF2/HO-1 signaling, inhibit NF-κB activation, activate AKT, and increase inhibitory phosphorylation of GSK3β to decrease GSK3 activity, all of which exert antioxidant effects., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Tauopathies are a class of neurodegenerative disorders characterized by abnormal deposition of post-translationally modified tau protein in the human brain. Tauopathies are associated with Alzheimer's disease (AD), chronic traumatic encephalopathy (CTE), and other diseases. Hyperphosphorylation increases tau tendency to aggregate and form neurofibrillary tangles (NFT), a pathological hallmark of AD. In this study, okadaic acid (OA, 100 nM), a protein phosphatase 1/2A inhibitor, was treated for 24h in mouse neuroblastoma (N2a) and differentiated rat primary neuronal cortical cell cultures (CTX) to induce tau-hyperphosphorylation and oligomerization as a cell-based tauopathy model. Following the treatments, the effectiveness of different kinase inhibitors was assessed using the tauopathy-relevant tau antibodies through tau-immunoblotting, including the sites: pSer202/pThr205 (AT8), pThr181 (AT270), pSer202 (CP13), pSer396/pSer404 (PHF-1), and pThr231 (RZ3). OA-treated samples induced tau phosphorylation and oligomerization at all tested epitopes, forming a monomeric band (46-67 kDa) and oligomeric bands (170 kDa and 240 kDa). We found that TBB (a casein kinase II inhibitor), AR and LiCl (GSK-3 inhibitors), cyclosporin A (calcineurin inhibitor), and Saracatinib (Fyn kinase inhibitor) caused robust inhibition of OA-induced monomeric and oligomeric p-tau in both N2a and CTX culture. Additionally, a cyclin-dependent kinase 5 inhibitor (Roscovitine) and a calcium chelator (EGTA) showed contrasting results between the two neuronal cultures. This study provides a comprehensive view of potential drug candidates (TBB, CsA, AR, and Saracatinib), and their efficacy against tau hyperphosphorylation and oligomerization processes. These findings warrant further experimentation, possibly including animal models of tauopathies, which may provide a putative Neurotherapy for AD, CTE, and other forms of tauopathy-induced neurodegenerative diseases., Competing Interests: The authors have declared that no competing interests exist.

Sterol regulatory element-binding protein (SREBP), a highly conserved family of membrane-bound transcription factors, is an essential regulator for cellular cholesterol and lipid homeostasis in mammalian cells. Sre1, the homolog of SREBP in the fission yeast Schizosaccharomyces pombe (S. pombe), regulates genes involved in the transcriptional responses to low sterol as well as low oxygen. Previous study reported that casein kinase 1 family member Hhp2 phosphorylated the Sre1 N-terminal transcriptional factor domain (Sre1N) and accelerated Sre1N degradation, and other kinases might exist for regulating the Sre1 function. To gain insight into the mechanisms underlying the Sre1 activity and to identify additional kinases involved in regulation of Sre1 function, we developed a luciferase reporter system to monitor the Sre1 activity through its binding site called SRE2 in living yeast cells. Here we showed that both ergosterol biosynthesis inhibitors and hypoxia-mimic CoCl2 caused a dose-dependent increase in the Sre1 transcription activity, concurrently, these induced transcription activities were almost abolished in Δsre1 cells. Surprisingly, either AMPKα Subunit Ssp2 deletion or Glycogen Synthase Kinases Gsk3/Gsk31 double deletion significantly suppressed ergosterol biosynthesis inhibitors- or CoCl2-induced Sre1 activity. Notably, the Δssp2Δgsk3Δgsk31 mutant showed further decreased Sre1 activity when compared with their single or double deletion. Consistently, the Δssp2Δgsk3Δgsk31 mutant showed more marked temperature sensitivity than any of their single or double deletion. Moreover, the fluorescence of GFP-Sre1N localized at the nucleus in wild-type cells, but significantly weaker nuclear fluorescence of GFP-Sre1N was observed in Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells. On the other hand, the immunoblot showed a dramatic decrease in GST-Sre1N levels in the Δgsk3Δgsk31 or the Δssp2Δgsk3Δgsk31 cells but not in the Δssp2 cells. Altogether, our findings suggest that Gsk3/Gsk31 may regulate Sre1N degradation, while Ssp2 may regulate not only the degradation of Sre1N but also its translocation to the nucleus., Competing Interests: The authors have declared that no competing interests exist.

Glycogen synthase kinase-3 (GSK3) is over-expressed and hyperactivated in non-small cell lung carcinoma (NSCLC) and plays a role in ensuring the correct alignment of chromosomes on the metaphase plate during mitosis through regulation of microtubule stability. This makes the enzyme an attractive target for cancer therapy. We examined the effects of a selective cell-permeant GSK3 inhibitor (CHIR99021), used alone or in combination with paclitaxel, using an in vitro cell growth assay, a quantitative chromosome alignment assay, and a tumor xenograft model. CHIR99021 inhibits the growth of human H1975 and H1299 NSCLC cell lines in a synergistic manner with paclitaxel. CHIR99021 and paclitaxel promoted a synergistic defect in chromosomal alignment when compared to each compound administered as monotherapy. Furthermore, we corroborated our in vitro findings in a mouse tumor xenograft model. Our results demonstrate that a GSK3 inhibitor and paclitaxel act synergistically to inhibit the growth of NSCLC cells in vitro and in vivo via a mechanism that may involve converging modes of action on microtubule spindle stability and thus chromosomal alignment during metaphase. Our findings provide novel support for the use of the GSK3 inhibitor, CHIR99021, alongside taxol-based chemotherapy in the treatment of human lung cancer., Competing Interests: The authors have declared that no competing interests exist.

Sodium dodecyl sulfate is a detergent that disrupts cell membranes, activates cell wall integrity signaling and restricts cell growth in Saccharomyces cerevisiae. However, the underlying mechanism of how sodium dodecyl sulfate inhibits cell growth is not fully understood. Previously, we have shown that deletion of the MCK1 gene leads to sensitivity to sodium dodecyl sulfate; thus, we implemented a suppressor gene screening revealing that the overexpression of TAT2 tryptophan permease rescues cell growth in sodium dodecyl sulfate-treated Δmck1 cells. Therefore, we questioned the involvement of tryptophan in the response to sodium dodecyl sulfate treatment. In this work, we show that trp1-1 cells have a disadvantage in the response to sodium dodecyl sulfate compared to auxotrophy for adenine, histidine, leucine or uracil when cells are grown on rich media. While also critical in the response to tea tree oil, TRP1 does not avert growth inhibition due to other cell wall/membrane perturbations that activate cell wall integrity signaling such as Calcofluor White, Congo Red or heat stress. This implicates a distinction from the cell wall integrity pathway and suggests specificity to membrane stress as opposed to cell wall stress. We discovered that tyrosine biosynthesis is also essential upon sodium dodecyl sulfate perturbation whereas phenylalanine biosynthesis appears dispensable. Finally, we observe enhanced tryptophan import within minutes upon exposure to sodium dodecyl sulfate indicating that these cells are not starved for tryptophan. In summary, we conclude that internal concentration of tryptophan and tyrosine makes cells more resistant to detergent such as sodium dodecyl sulfate., Competing Interests: The authors declared that no competing interests exist.

Serotonin-1B receptors (5-HT1BRs) modulate perseverative behaviors and prepulse inhibition (PPI) in humans and mice. These inhibitory G-protein-coupled receptors signal through a canonical G-protein-coupled pathway that is modulated by GSK-3β, and a noncanonical pathway mediated by the adaptor protein β-arrestin2 (Arrb2). Given the development of biased ligands that differentially affect canonical versus noncanonical signaling, we examined which signaling pathway mediates 5-HT1BR agonist-induced locomotor perseveration and PPI deficits, behavioral phenotypes observed in both obsessive-compulsive disorder (OCD) and autism spectrum disorder (ASD). To assess the role of canonical 5-HT1BR signaling, mice received acute pretreatment with a GSK-3 inhibitor (SB216763 or AR-A014418) and acute treatment with the 5-HT1A/1B receptor agonist RU24969 prior to assessing perseverative locomotor behavior in the open field, and PPI. To determine the role of noncanonical 5-HT1BR signaling, Arrb2 wild-type (WT), heterozygous (HT), and knockout (KO) mice received acute RU24969 treatment prior to behavioral testing. GSK-3 inhibition increased locomotor perseveration overall, and also failed to influence the RU24969-induced perseverative locomotor pattern in the open field. Yet, GSK-3 inhibition modestly reduced RU24969-induced PPI deficits. On the other hand, Arrb2 HT and KO mice showed reduced locomotion and no changes in perseveration overall, in addition to modest reductions in RU24969-induced locomotion and PPI deficits. In conclusion, our data do not support use of either GSK-3 inhibitors or β-arrestin2 inhibition in treatment of perseverative behaviors., Competing Interests: The authors have declared that no competing interests exist.

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis has been reported in some cancer cells, including AGS human gastric adenocarcinoma cells. Reducing this resistance might shed light on the treatment of human gastric adenocarcinoma. In this study, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors can restore TRAIL responsiveness in gastric adenocarcinoma cells. The effect of two GSK-3 inhibitors, SB-415286, and LiCl, on apoptosis signaling of TRAIL in human gastric adenocarcinoma cell lines and primary gastric epithelial cells was analyzed. Both inhibitors can sensitize gastric adenocarcinoma cells, but not primary gastric epithelial cells, to TRAIL-induced apoptosis by increasing caspase-8 activity and its downstream signal transmission. Adding p53 siRNA can downregulate GSK-3 inhibitor-related sensitization to TRAIL-induced apoptosis and caspase-3 activity. GSK-3 inhibitors strongly activate the phosphorylation of JNK. Inhibition of JNK leads to earlier and more intense apoptosis, showing that the activation of JNK may provide anti-apoptotic equilibrium of pro-apoptotic cells. Our observations indicate that GSK-3 inhibitors can sentize AGS gastric adenocarcinoma cells to TRAIL-induced apoptosis. Therefore, in certain types of gastric adenocarcinoma, GSK-3 inhibitor might enhance the antitumor activity of TRAIL and mightbe a promising candidate for the treatment of certain types of gastric adenocarcinoma., Competing Interests: The authors have declared that no competing interests exist.

Countless neurodegenerative diseases are associated with perverse multiple targets of cyclic nucleotide signalling, hastening neuronal death. Cilostazol, a phosphodiesterase-III inhibitor, exerts neuroprotective effects against sundry models of neurotoxicity, however, its role against Huntington's disease (HD) has not yet been tackled. Hence, its modulatory effect on several signalling pathways using the 3-nitropropionic acid (3-NP) model was conducted. Animals were injected with 3-NP (10 mg/kg/day, i.p) for two successive weeks with or without the administration of cilostazol (100 mg/kg/day, p.o.). Contrary to the 3-NP effects, cilostazol largely preserved striatal dopaminergic neurons, improved motor coordination, and enhanced the immunohistochemical reaction of tyrosine hydroxylase enzyme. The anti-inflammatory effect of cilostazol was documented by the pronounced reduction of the toll like receptor-4 (TLR-4) protein expression and the inflammatory cytokine IL-6, but with a marked elevation in IL-10 striatal contents. As a consequence, cilostazol reduced IL-6 downstream signal, where it promoted the level of suppressor of cytokine signalling 3 (SOCS3), while abated the phosphorylation of Janus Kinase 2 (JAK-2) and Signal transducers and activators of transcription 3 (STAT-3). Phosphorylation of the protein kinase B/glycogen synthase kinase-3β/cAMP response element binding protein (Akt/GSK-3β/CREB) cue is another signalling pathway that was modulated by cilostazol to further signify its anti-inflammatory and antiapoptotic capacities. The latter was associated with a reduction in the caspase-3 expression assessed by immunohistochemical assay. In conclusion the present study provided a new insight into the possible mechanisms by which cilostazol possesses neuroprotective properties. These intersecting mechanisms involve the interference between TLR-4, IL-6-IL-10/JAK-2/STAT-3/SOCS-3, and Akt/GSK-3β/CREB signalling pathways., Competing Interests: The authors have declared that no competing interests exist.

Aims: The aims of this study were to determine the effects of four weeks of intermittent exposure to a moderate hypoxia environment (15% oxygen), and compare with the effects of exercise in normoxia or hypoxia, on glucose homeostasis, insulin sensitivity, GLUT4 translocation, insulin receptor phosphorylation, Akt-dependent GSK3 phosphorylation and Akt activity in skeletal muscle of obese mice with type 2 diabetes., Methods: C57BL/6J mice that developed type 2 diabetes with a high-fat-diet (55% fat) (fasting blood glucose, FBG = 13.9 ± 0.69 (SD) mmol/L) were randomly allocated into diabetic control (DC), rest in hypoxia (DH), exercise in normoxia (DE), and exercise in hypoxia (DHE) groups (n = 7, each), together with a normal-diet (4% fat) control group (NC, FBG = 9.1 ± 1.11 (SD) mmol/L). The exercise groups ran on a treadmill at intensities of 75-90% VO2max. The interventions were applied one hour per day, six days per week for four weeks. Venous blood samples were analysed for FBG, insulin (FBI) and insulin sensitivity (QUICKI) pre and post the intervention period. The quadriceps muscle samples were collected 72 hours post the last intervention session for analysis of GLUT4 translocation, insulin receptor phosphorylation, Akt expression and phosphorylated GSK3 fusion protein by western blot. Akt activity was determined by the ratio of the phosphorylated GSK3 fusion protein to the total Akt protein., Results: The FBG of the DH, DE and DHE groups returned to normal level (FBG = 9.4 ± 1.50, 8.86 ± 0.94 and 9.0 ± 1.13 (SD) mmol/L for DH, DE and DHE respectively, P < 0.05), with improved insulin sensitivity compared to DC (P < 0.05), after the four weeks treatment, while the NC and DC showed no significant changes, as analysed by general linear model with repeated measures. All three interventions resulted in a significant increase of GLUT4 translocation to cell membrane compared to the DC group (P < 0.05). The DE and DH showed a similar level of insulin receptor phosphorylation compared with NC that was significantly lower than the DC (P < 0.05) post intervention. The DH and DHE groups showed a significantly higher Akt activity compared to the DE, DC and NC (P < 0.05) post intervention, as analysed by one-way ANOVA., Conclusions: This study produced new evidence that intermittent exposure to mild hypoxia (0.15 FiO2) for four weeks resulted in normalisation of FBG, improvement in whole body insulin sensitivity, and a significant increase of GLUT4 translocation in the skeletal muscle, that were similar to the effects of exercise intervention during the same time period, in mice with diet-induced type 2 diabetes. However, exercise in hypoxia for four weeks did not have additive effects on these responses. The outcomes of the research may contribute to the development of effective, alternative and complementary interventions for management of hyperglycaemia and type 2 diabetes, particularly for individuals with limitations in participation of physical activity., Competing Interests: All authors have declared that no competing interests exist.

Glycogen synthase kinase 3 (GSK3) plays an important role in the development of diabetes mellitus and renal injury. GSK3 inhibition increases glucose uptake in insulin-insensitive muscle and adipose tissue, while it reduces albuminuria and glomerulosclerosis in acute kidney injury. The effect of chronic GSK3 inhibition in diabetic nephropathy is not known. We tested the effect of lithium, the only clinical GSK3 inhibitor, on the development of diabetes mellitus and kidney injury in a mouse model of diabetic nephropathy. Twelve-week old female BTBR-ob/ob mice were treated for 12 weeks with 0, 10 and 40 mmol LiCl/kg after which the development of diabetes and diabetic nephropathy were analysed. In comparison to BTBR-WT mice, ob/ob mice demonstrated elevated bodyweight, increased blood glucose/insulin levels, urinary albumin and immunoglobulin G levels, glomerulosclerosis, reduced nephrin abundance and a damaged proximal tubule brush border. The lithium-10 and -40 diets did not affect body weight and resulted in blood lithium levels of respectively <0.25 mM and 0.48 mM. The Li-40 diet fully rescued the elevated non-fasting blood glucose levels. Importantly, glomerular filtration rate was not affected by lithium, while urine albumin and immunoglobulin G content were further elevated. While lithium did not worsen the glomerulosclerosis, proximal tubule function seemed affected by lithium, as urinary NGAL levels were significantly increased. These results demonstrate that lithium attenuates non-fasting blood glucose levels in diabetic mice, but aggravates urinary albumin and immunoglobulin G content, possibly resulting from proximal tubule dysfunction.

Myo-inositol, the precursor of all inositol compounds, is essential for the viability of eukaryotes. Identifying the factors that regulate inositol homeostasis is of obvious importance to understanding cell function and the pathologies underlying neurological and metabolic resulting from perturbation of inositol metabolism. The current study identifies Mck1, a GSK3 homolog, as a novel positive regulator of inositol de novo synthesis in yeast. Mck1 was required for normal activity of myo-inositol phosphate synthase (MIPS), which catalyzes the rate-limiting step of inositol synthesis. mck1Δ cells exhibited a 50% decrease in MIPS activity and a decreased rate of incorporation of [13C6]glucose into [13C6]-inositol-3-phosphate and [13C6]-inositol compared to WT cells. mck1Δ cells also exhibited decreased growth in the presence of the inositol depleting drug valproate (VPA), which was rescued by supplementation of inositol. However, in contrast to wild type cells, which exhibited more than a 40% decrease in MIPS activity in the presence of VPA, the drug did not significantly decrease MIPS activity in mck1Δ cells. These findings indicate that VPA-induced MIPS inhibition is Mck1-dependent, and suggest a model that unifies two current hypotheses of the mechanism of action of VPA-inositol depletion and GSK3 inhibition.

Background/aims: The therapeutic options for metastatic neuroendocrine tumors (NETs) are limited. As PI3K signaling is often activated in NETs, we have assessed the effects of selective PI3Kp110α inhibition by the novel agent BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and pulmonary (H727) NET cell lines., Methods: Cell viability was investigated by WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7 assay, the cell cycle by FACS, cell signaling by Western blot analysis, expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and chromogranin A secretion by ELISA., Results: BYL719 dose-dependently decreased cell viability and colony formation with the highest sensitivity in BON-1, followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced apoptosis and G0/G1 cell cycle arrest associated with increased p27 expression. Western blots showed inhibition of PI3K downstream targets to a varying degree in the different cell lines, but IGF1R activation. The most sensitive BON-1 cells displayed a significant, and H727 cells a non-significant, GSK3 inhibition after BYL719 treatment, but these effects do not appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1 cells showed no GSK3 inhibition, but a modest activation, which would partially counteract the other anti-proliferative effects. Accordingly, BYL719 enhanced neuroendocrine differentiation with the strongest effect in BON-1, followed by H727 cells indicated by induction of chromogranin A and somatostatin receptor 1/2 mRNA-synthesis, but not in QGP-1 cells. In BON-1 and QGP-1 cells, the BYL719/everolimus combination was synergistic through simultaneous AKT/mTORC1 inhibition, and significantly increased somatostatin receptor 2 transcription compared to each drug separately., Conclusion: Our results suggest that the agent BYL719 could be a novel therapeutic approach to the treatment of NETs that may sensitize NET cells to somatostatin analogs, and that if there is resistance to its action this may be overcome by combination with everolimus.

Type 2 diabetes (T2D) is a high-risk factor for Alzheimer's disease (AD) due to impaired insulin signaling pathway in brain. Capsaicin is a specific transient receptor potential vanilloid 1 (TRPV1) agonist which was proved to ameliorate insulin resistance. In this study, we investigated whether dietary capsaicin could reduce the risk of AD in T2D. T2D rats were fed with capsaicin-containing high fat (HF) diet for 10 consecutive days (T2D+CAP). Pair-fed T2D rats (T2D+PF) fed with the HF-diet of average dose of T2D+CAP group were included to control for the effects of reduced food intake and body weight. Capsaicin-containing standard chow was also introduced to non-diabetic rats (NC+CAP). Blood glucose and insulin were monitored. The phosphorylation level of tau at individual sites, the activities of phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) and glycogen synthase kinase-3β (GSK-3β) were analyzed by Western blots. The results revealed that the levels of phosphorylated tau protein at sites Ser199, Ser202 and Ser396 in hippocampus of T2D+CAP group were decreased significantly, but these phospho-sites in T2D+PF group didn't show such improvements compared with T2D group. There were almost no changes in non-diabetic rats on capsaicin diet (NC+CAP) compared with the non-diabetic rats with normal chow (NC). Increased activity of PI3K/AKT and decreased activity of GSK-3β were detected in hippocampus of T2D+CAP group compared with T2D group, and these changes did not show in T2D+PF group either. These results demonstrated that dietary capsaicin appears to prevent the hyperphosphorylation of AD-associated tau protein by increasing the activity of PI3K/AKT and inhibiting GSK-3β in hippocampus of T2D rats, which supported that dietary capsaicin might have a potential use for the prevention of AD in T2D.

Potential benefits of statins in the treatment of chronic kidney disease beyond lipid-lowering effects have been described. However, molecular mechanisms involved in renoprotective actions of statins have not been fully elucidated. We questioned whether statins influence development of diabetic nephropathy through reactive oxygen species, RhoA and Akt/GSK3 pathway, known to be important in renal pathology. Diabetic mice (db/db) and their control counterparts (db/+) were treated with atorvastatin (10 mg/Kg/day, p.o., for 2 weeks). Diabetes-associated renal injury was characterized by albuminuria (albumin:creatinine ratio, db/+: 3.2 ± 0.6 vs. db/db: 12.5 ± 3.1*; *P<0.05), increased glomerular/mesangial surface area, and kidney hypertrophy. Renal injury was attenuated in atorvastatin-treated db/db mice. Increased ROS generation in the renal cortex of db/db mice was also inhibited by atorvastatin. ERK1/2 phosphorylation was increased in the renal cortex of db/db mice. Increased renal expression of Nox4 and proliferating cell nuclear antigen, observed in db/db mice, were abrogated by statin treatment. Atorvastatin also upregulated Akt/GSK3β phosphorylation in the renal cortex of db/db mice. Our findings suggest that atorvastatin attenuates diabetes-associated renal injury by reducing ROS generation, RhoA activity and normalizing Akt/GSK3β signaling pathways. The present study provides some new insights into molecular mechanisms whereby statins may protect against renal injury in diabetes., Competing Interests: The authors have declared that no competing interests exist.

Porcine epidemic diarrhea virus (PEDV) has recently caused high mortality in suckling piglets with subsequent large economic losses to the swine industry. Many intracellular signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, are activated by viral infection. The PI3K/Akt pathway is an important cellular pathway that has been shown to be required for virus replication. In the present study, we found that the PEDV JS-2013 strain activated Akt in Vero cells at early (5-15 min) and late stages (8-10 h) of infection. Inhibiting PI3K, an upstream activator of Akt, enhanced PEDV replication. Inhibiting GSK-3α/β, one of the downstream effectors of PI3K/Akt pathway and regulated by Akt during PEDV infected Vero cells, also enhanced PEDV replication. Collectively, our data suggest that PI3K/Akt/GSK-3α/β signaling pathway is activated by PEDV and functions in inhibiting PEDV replication., Competing Interests: The authors have declared that no competing interests exist.

The WNT signalling pathway controls many developmental processes and plays a key role in maintenance of intestine renewal and homeostasis. Glycogen Synthase Kinase 3 (GSK3) is an important component of the WNT pathway and is involved in regulating β-catenin stability and expression of WNT target genes. The mechanisms underpinning GSK3 regulation in this context are not completely understood, with some evidence suggesting this occurs through inhibitory N-terminal serine phosphorylation in a similar way to GSK3 inactivation in insulin signaling. To investigate this in a physiologically relevant context, we have analysed the intestinal phenotype of GSK3 knockin mice in which N-terminal serines 21/9 of GSK3α/β have been mutated to non-phosphorylatable alanine residues. We show that these knockin mutations have very little effect on overall intestinal integrity, cell lineage commitment, β-catenin localization or WNT target gene expression although a small increase in apoptosis at villi tips is observed. Our results provide in vivo evidence that GSK3 is regulated through mechanisms independent of N-terminal serine phosphorylation in order for β-catenin to be stabilised.

Background: The regulation of microRNAs (miRNAs) at different stages of the progression of type 2 diabetes mellitus (T2DM) and their role in glucose homeostasis was investigated., Methods: Microarrays were used to assess miRNA expression in skeletal muscle biopsies taken from healthy individuals and patients with pre-diabetes or T2DM, and insulin resistant offspring of rat dams fed a high fat diet during pregnancy., Results: Twenty-three miRNAs were differentially expressed in patients with T2DM, and 7 in the insulin resistant rat offspring compared to their controls. Among these, only one miRNA was similarly regulated: miR-194 expression was significantly reduced by 25 to 50% in both the rat model and in human with pre-diabetes and established diabetes. Knockdown of miR-194 in L6 skeletal muscle cells induced an increase in basal and insulin-stimulated glucose uptake and glycogen synthesis. This occurred in conjunction with an increased glycolysis, indicated by elevated lactate production. Moreover, oxidative capacity was also increased as we found an enhanced glucose oxidation in presence of the mitochondrial uncoupler FCCP. When miR-194 was down-regulated in vitro, western blot analysis showed an increased phosphorylation of AKT and GSK3β in response to insulin, and an increase in expression of proteins controlling mitochondrial oxidative phosphorylation., Conclusions: Type 2 diabetes mellitus is associated with regulation of several miRNAs in skeletal muscle. Interestingly, miR-194 was a unique miRNA that appeared regulated across different stages of the disease progression, from the early stages of insulin resistance to the development of T2DM. We have shown miR-194 is involved in multiple aspects of skeletal muscle glucose metabolism from uptake, through to glycolysis, glycogenesis and glucose oxidation, potentially via mechanisms involving AKT, GSK3 and oxidative phosphorylation. MiR-194 could be down-regulated in patients with early features of diabetes as an adaptive response to facilitate tissue glucose uptake and metabolism in the face of insulin resistance.

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin's lymphoma with a still undefined etiology. Several lines of evidence are consistent with the possible involvement of peculiar microenvironmental stimuli sustaining tumor cell growth and survival, as the activation of Toll-like receptors (TLR) 4 and 9. However, little is known about the contribution of other TLRs of pathogenic relevance in the development of MCL. This study reports evidence that MCL cell lines and primary MCL cells express different levels of TLR2 and TLR5, and that their triggering is able to further activate the Akt, MAPK, and NF-κB signaling cascades, known to be altered in MCL cells. This leads to the enhancement of cyclin D1 and D3 over-expression, occurring at post-translational level through a mechanism that likely involves the Akt/GSK-3α/β pathway. Interestingly, in primary B cells, TLR1/2 or TLR5 ligands increase protein level of cyclin D1, which is not usually expressed in normal B cells, and cyclin D3 when associated with CD40 ligand (CD40L), IL-4, and anti-human-IgM co-stimulus. Finally, the activation of TLR1/2 and TLR5 results in an increased proliferation of MCL cell lines and, in the presence of co-stimulation with CD40L, IL-4, and anti-human-IgM also of primary MCL cells and normal B lymphocytes. These effects befall together with an enhanced IL-6 production in primary cultures. Overall, our findings suggest that ligands for TLR1/2 or TLR5 may provide critical stimuli able to sustain the growth and the malignant phenotype of MCL cells. Further studies aimed at identifying the natural source of these TLR ligands and their possible pathogenic association with MCL are warranted in order to better understand MCL development, but also to define new therapeutic targets for counteracting the tumor promoting effects of lymphoma microenvironment.

Overexpression or activation of cyclic AMP-response element-binding protein (CREB) has been known to be involved in several human malignancies, including lung cancer. Genes regulated by CREB have been reported to suppress apoptosis, induce cell proliferation, inflammation, and tumor metastasis. However, the critical target genes of CREB in lung cancer have not been well understood. Here, we identified GSK-3α as one of the CREB target genes which is critical for the viability of lung cancer cells. The CREB knockdown significantly reduced the expression of GSK-3α and the direct binding of CREB on the promoter of GSK3A was identified. Kaplan-Meier analysis with a public database showed a prognostic significance of aberrant GSK-3α expression in lung cancer. Inhibition of GSK-3α suppressed cell viability, colony formation, and tumor growth. For the first time, we demonstrated that GSK-3α is regulated by CREB in lung cancer and is required for the cell viability. These findings implicate CREB-GSK-3α axis as a novel therapeutic target for lung cancer treatment.

Increasing evidence shows that oxidative stress and the hyperphosphorylation of tau protein play essential roles in the progression of Alzheimer's disease (AD). Quercetin is a major flavonoid that has anti-oxidant, anti-cancer and anti-inflammatory properties. We investigated the neuroprotective effects of quercetin to HT22 cells (a cell line from mouse hippocampal neurons). We found that Okadaic acid (OA) induced the hyperphosphorylation of tau protein at Ser199, Ser396, Thr205, and Thr231 and produced oxidative stress to the HT22 cells. The oxidative stress suppressed the cell viability and decreased the levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), mitochondria membrane potential (MMP) and Glutathione peroxidase (GSH-Px). It up-regulated malondialdehyde (MDA) production and intracellular reactive oxygen species (ROS). In addition, phosphoinositide 3 kinase/protein kinase B/Glycogen synthase kinase3β (PI3K/Akt/GSK3β) and mitogen activated protein kinase (MAPK) were also involved in this process. We found that pre-treatment with quercetin can inhibited OA-induced the hyperphosphorylation of tau protein and oxidative stress. Moreover, pre-treatment with quercetin not only inhibited OA-induced apoptosis via the reduction of Bax, and up-regulation of cleaved caspase 3, but also via the inhibition of PI3K/Akt/GSK3β, MAPKs and activation of NF-κB p65. Our findings suggest the therapeutic potential of quercetin to treat AD.

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic (DA) neurons in the substantial nigra pars compacta. Increasing evidence showed that Wnt/β-catenin pathway and the orphan nuclear receptor Nurr1 play crucial roles in the survival and functional maintenance of DA neurons in the midbrain and GSK-3β antagonists LiCl and SB216763 were used to activate Wnt/β-catenin pathway experimentally. However, the detail mechanism underlying the neuroprotection against apoptosis on DA neuron is still unclear and the interaction between Wnt/β-catenin and Nurr1 remains undisclosed. In this study, using cell biological assay we investigated the function of Wnt/β-catenin and its crosstalk with Nurr1 on the course of PC12 cell degeneration in vitro. Our data showed that PC12 cell viability was inhibited by rotenone, but attenuated by GSK-3β antagonists LiCl or SB216763. The activity of Wnt/β-catenin pathway was deregulated on exposure of rotenone in a concentration-dependent manner. After the interference of β-catenin with siRNA, LiCl or SB216763 failed to protect PC12 cells from apoptosis by the rotenone toxicity. Our data confirmed that Wnt/β-catenin signaling activated by LiCl or SB216763 enhanced Nurr1 expression to 2.75 ± 0.55 and 4.06 ± 0.41 folds respectively compared with control detected by real-time PCR and the interaction of β-catenin with Nurr1 was identified by co-immunoprecipitate analysis. In conclusion, the data suggested that Wnt/β-catenin and Nurr1 are crucial factors in the survival of DA neurons, and the activation of Wnt/β-catenin pathway exerts protective effects on DA neurons partly by mean of a co-active pattern with Nurr1. This finding may shed a light on the potential treatment of Parkinson disease.

Acquisition of resistance to docetaxel (Doc) is one of the most important problems in treatment of breast cancer patients, but the underlying mechanisms are still not fully understood. In present study, Doc-resistant MDA-MB-231 and MCF-7 breast cancer cell lines (MDA-MB-231/Doc and MCF-7/Doc) were successfully established in vitro by gradually increasing Doc concentration on the basis of parental MDA-MB-231 and MCF-7 cell lines (MDA-MB-231/S and MCF-7/S). The potential miRNAs relevant to the Doc resistance were screened by miRNA microarray. We selected 5 upregulated miRNAs (has-miR-3646, has-miR-3658, has-miR-4438, has-miR-1246, and has-miR-574-3p) from the results of microarray for RT-qPCR validation. The results showed that expression level of miR-3646 in MDA-MB-231/Doc cells was significantly higher than in MDA-MB-231/S cells. Compared to MCF-7/S cells, miR-3646 expression was up-regulated in MCF-7/Doc cells. Further studies revealed that transfection of miR-3646 mimics into MDA-MB-231/S or MCF-7/S cells remarkably increased their drug resistance, in contrast, transfection of miR-3646 inhibitors into MDA-MB-231/Doc or MCF-7/Doc cells resulted in significant reduction of the drug resistance. By the pathway enrichment analyses for miR-3646, we found that GSK-3β/β-catenin signaling pathway was a significant pathway, in which GSK-3β was an essential member. RT-qPCR and Western blot results demonstrated that miR-3646 could regulate GSK-3β mRNA and protein expressions. Furthermore, a marked increase of both nuclear and cytoplasmic β-catenin expressions (with phosphorylated-β-catenin decrease) was observed in MDA-MB-231/Doc cells compared with MDA-MB-231/S cells, and their expression were positively related to miR-3646 and negatively correlated with GSK-3β. Taken together, our results suggest that miR-3646-mediated Doc resistance of breast cancer cells maybe, at least in part, through suppressing expression of GSK-3β and resultantly activating GSK-3β/β-catenin signaling pathway.

Fetal nicotine exposure increased risk of developing cardiovascular disease later in life. The present study tested the hypothesis that perinatal nicotine-induced programming of heart ischemia-sensitive phenotype is mediated by enhanced reactive oxygen species (ROS) in offspring. Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps from day 4 of gestation to day 10 after birth, in the absence or presence of a ROS inhibitor, N-acetyl-cysteine (NAC) in drinking water. Experiments were conducted in 8 month old age male offspring. Isolated hearts were perfused in a Langendorff preparation. Perinatal nicotine treatment significantly increased ischemia and reperfusion-induced left ventricular injury, and decreased post-ischemic recovery of left ventricular function and coronary flow rate. In addition, nicotine enhanced cardiac ROS production and significantly attenuated protein kinase Cε (PKCε) protein abundance in the heart. Although nicotine had no effect on total cardiac glycogen synthase kinase-3β (GSK3β) protein expression, it significantly increased the phosphorylation of GSK3β at serine 9 residue in the heart. NAC inhibited nicotine-mediated increase in ROS production, recovered PKCε gene expression and abrogated increased phosphorylation of GSK3β. Of importance, NAC blocked perinatal nicotine-induced increase in ischemia and reperfusion injury in the heart. These findings provide novel evidence that increased oxidative stress plays a causal role in perinatal nicotine-induced developmental programming of ischemic sensitive phenotype in the heart, and suggest potential therapeutic targets of anti-oxidative stress in the treatment of ischemic heart disease.

The development of the mammalian skull is a complex process that requires multiple tissue interactions and a balance of growth and differentiation. Disrupting this balance can lead to changes in the shape and size of skull bones, which can have serious clinical implications. For example, insufficient ossification of the bony elements leads to enlarged anterior fontanelles and reduced mechanical protection of the brain. In this report, we find that loss of Gsk3β leads to a fully penetrant reduction of frontal bone size and subsequent enlarged frontal fontanelle. In the absence of Gsk3β the frontal bone primordium undergoes increased cell death and reduced proliferation with a concomitant increase in Fgfr2-IIIc and Twist1 expression. This leads to a smaller condensation and premature differentiation. This phenotype appears to be Wnt-independent and is not rescued by decreasing the genetic dose of β-catenin/Ctnnb1. Taken together, our work defines a novel role for Gsk3β in skull development.

Background and Purpose: Previous studies of ischemia-reperfusion injury (IRI) in hearts from mice with cardiac-restricted overexpression of CCN2 have shown that CCN2 increases tolerance towards IRI. The objectives of this study were to investigate to what extent post-ischemic administration of recombinant human CCN2 (rhCCN2) would limit infarct size and improve functional recovery and what signaling pathways are involved., Experimental Approach: Isolated mice hearts were perfused ad modum Langendorff, subjected to no-flow, global ischemia, and subsequently, exposed to mammalian cell derived, full-length (38-40kDa) rhCCN2 (250 nM) or vehicle during the first 15 min of a 60 min reperfusion period., Key Results: Post-ischemic administration of rhCCN2 resulted in attenuation of infarct size from 58 ± 4% to 34 ± 2% (p < 0.001) which was abrogated by concomitant administration of the PI3 kinase inhibitor LY294002 (45 ± 3% vs. 50 ± 3%, ns). In congruence with reduction of infarct size rhCCN2 also improved recovery of left ventricular developed pressure (p < 0.05). Western blot analyses of extracts of ex vivo-perfused murine hearts also revealed that rhCCN2 evoked concentration-dependent increase of cardiac phospho-GSK3β (serine-9) contents., Conclusions and Implications: We demonstrate that post-ischemic administration of rhCCN2 increases the tolerance of ex vivo-perfused murine hearts to IRI. Mechanistically, this postconditioning effect of rhCCN2 appeared to be mediated by activation of the reperfusion injury salvage kinase pathway as demonstrated by sensitivity to PI3 kinase inhibition and increased CCN2-induced phosphorylation of GSK3β (Ser-9). Thus, the rationale for testing rhCCN2-mediated post-ischemic conditioning of the heart in more complex models is established.

Myogenesis and muscle hypertrophy account for muscle growth and adaptation to work overload, respectively. In adults, insulin and insulin-like growth factor 1 stimulate muscle growth, although their links with cellular energy homeostasis are not fully explained. Insulin plays critical role in the control of mitochondrial activity in skeletal muscle cells, and mitochondria are essential for insulin action. The aim of this study was to elucidate molecular mechanism(s) involved in mitochondrial control of insulin-dependent myogenesis. The effects of several metabolic inhibitors (LY294002, PD98059, SB216763, LiCl, rotenone, oligomycin) on the differentiation of C2C12 myoblasts in culture were examined in the short-term (hours) and long-term (days) experiments. Muscle cell viability and mitogenicity were monitored and confronted with the activities of selected genes and proteins expression. These indices focus on the roles of insulin, glycogen synthase kinase 3 beta (GSK-3β) and forkhead box protein O1 (FOXO1) on myogenesis using a combination of treatments and inhibitors. Long-term insulin (10 nM) treatment in "normoglycemic" conditions led to increased myogenin expression and accelerated myogenesis in C2C12 cells. Insulin-dependent myogenesis was accompanied by the rise of mtTFA, MtSSB, Mfn2, and mitochondrially encoded Cox-1 gene expressions and elevated levels of proteins which control functions of mitochondria (kinase--PKB/AKT, mitofusin 2 protein--Mfn-2). Insulin, via the phosphatidylinositol 3-kinase (PI3-K)/AKT-dependent pathway reduced transcription factor FOXO1 activity and altered GSK-3β phosphorylation status. Once FOXO1 and GSK-3β activities were inhibited the rise in Cox-1 gene action and nuclear encoded cytochrome c oxidase subunit IV (COX IV) expressions were observed, even though some mRNA and protein results varied. In contrast to SB216763, LiCl markedly elevated Mfn2 and COX IV protein expression levels when given together with insulin. Thus, inhibition of GSK-3β activity by insulin alone or together with LiCl raised the expression of genes and some proteins central to the metabolic activity of mitochondria resulting in higher ATP synthesis and accelerated myogenesis. The results of this study indicate that there are at least two main targets in insulin-mediated myogenesis: notably FOXO1 and GSK-3β both playing apparent negative role in muscle fiber formation.

Glycogen synthase kinase-3β (GSK-3β) regulates the sequential activation of caspase-2 and caspase-8 before mitochondrial apoptosis. Here, we report the regulation of Mcl-1 destabilization and cathepsin D-regulated caspase-8 activation by GSK-3β and caspase-2. Treatment with either the ceramide analogue C2-ceramide or the topoisomerase II inhibitor etoposide sequentially induced lysosomal membrane permeabilization (LMP), the reduction of mitochondrial transmembrane potential, and apoptosis. Following LMP, cathepsin D translocated from lysosomes to the cytoplasm, whereas inhibiting cathepsin D blocked mitochondrial apoptosis. Furthermore, cathepsin D caused the activation of caspase-8 but not caspase-2. Inhibiting GSK-3β and caspase-2 blocked Mcl-1 destabilization, LMP, cathepsin D re-localization, caspase-8 activation, and mitochondrial apoptosis. Expression of Mcl-1 was localized to the lysosomes, and forced expression of Mcl-1 prevented apoptotic signaling via the lysosomal-mitochondrial pathway. These results demonstrate the importance of GSK-3β and caspase-2 in ceramide- and etoposide-induced apoptosis through mechanisms involving Mcl-1 destabilization and the lysosomal-mitochondrial axis.

Glycogen synthase kinase-3β (GSK-3β) acts as a negative regulator of NF-E2 related factor 2 (Nrf2) by inducing Nrf2 degradation and nuclear export. Our previous study demonstrated that the flavonoid hyperoside elicits cytoprotection against oxidative stress by activating the Keap1-Nrf2-ARE signaling pathway, thus increasing the expression of antioxidant enzymes, such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD) and catalase. However, the role of GSK-3β in hyperoside-mediated Nrf2 activation is unclear. Here, we demonstrate that in a normal human hepatocyte cell line, (L02), hyperoside is capable of inducing the phosphorylation of GSK-3β at Ser9 without affecting the protein levels of GSK-3β and its phosphorylation at Thr390. Lithium chloride (LiCl) and short interfering RNA (siRNA)-mediated inhibition of GSK-3β significantly enhanced the ability of hyperoside to protect L02 liver cells from H2O2-induced oxidative damage, leading to increased cell survival shown by the maintenance of cell membrane integrity and elevated levels of glutathione (GSH), one of the endogenous antioxidant biomarkers. Further study showed that LiCl and siRNA-mediated inhibition of GSK-3β increased hyperoside-induced HO-1 expression, and the effect was dependent upon enhanced Nrf2 nuclear translocation and gene expression. These activities were followed by ARE-mediated transcriptional activation in the presence of hyperoside, which was abolished by the transfection of the cells with Nrf2 siRNA. Furthermore, the siRNA-mediated inhibition of Keap1 also enhanced hyperoside-induced Nrf2 nuclear accumulation and HO-1 expression, which was relatively smaller than the effects obtained from GSK-3β siRNA administration. Moreover, Keap1 siRNA administration alone had no significant effect on the phosphorylation and protein expression of GSK-3β. Collectively, our data provide evidence that hyperoside attenuates H2O2 -induced L02 cell damage by activating the Nrf2-ARE signaling pathway through both an increase in GSK-3β inhibitory phosphorylation at Ser9 and an inhibition of Keap1 and that hyperoside-mediated GSK-3β inhibition exhibits more significant effects.

Introduction: Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy., Methods: In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2',7'-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining., Results: In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10., Conclusions: In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via induction of hypertrophy and senescence. Indeed, GSK3β inactivation is responsible for ROS production, triggering oxidative stress and DNA damage response.

Understanding the mechanisms in the generation of neural stem cells from pluripotent stem cells is a fundamental step towards successful management of neurodegenerative diseases in translational medicine. Albeit all-trans retinoic acid (RA) has been associated with axon outgrowth and nerve regeneration, the maintenance of differentiated neurons, the association with degenerative disease like Parkinson's disease, and its regulatory molecular mechanism from pluripotent stem cells to neural stem cells remain fragmented. We have previously reported that RA is capable of differentiation of human trophoblast stem cells to dopamine (DA) committed progenitor cells. Intracranial implantation of such neural progenitor cells into the 6-OHDA-lesioned substantia nigra pars compacta successfully regenerates dopaminergic neurons and integrity of the nigrostriatal pathway, ameliorating the behavioral deficits in the Parkinson's disease rat model. Here, we demonstrated a dynamic molecular network in systematic analysis by addressing spatiotemporal molecular expression, intracellular protein-protein interaction and inhibition, imaging study, and genetic expression to explore the regulatory mechanisms of RA induction in the differentiation of human trophoblast stem cells to DA committed progenitor cells. We focused on the tyrosine receptor kinase (Trk), G proteins, canonical Wnt2B/β-catenin, genomic and non-genomic RA signaling transductions with Tyrosine hydroxylase (TH) gene expression as the differentiation endpoint. We found that at the early stage, integration of TrkA and G protein signalings aims for axonogenesis and morphogenesis, involving the novel RXRα/Gαq/11 and RARβ/Gβ signaling pathways. While at the later stage, five distinct signaling pathways together with epigenetic histone modifications emerged to regulate expression of TH, a precursor of dopamine. RA induction generated DA committed progenitor cells in one day. Our results provided substantial mechanistic evidence that human trophoblast stem cell-derived neural stem cells can potentially be used for neurobiological study, drug discovery, and as an alternative source of cell-based therapy in neurodegenerative diseases like Parkinson's disease.

Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function.

Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.

Microtubules are regulated by a diverse set of proteins that localize to microtubule plus ends (+TIPs) where they regulate dynamic instability and mediate interactions with the cell cortex, actin filaments, and organelles. Although individual +TIPs have been studied in depth and we understand their basic contributions to microtubule dynamics, there is a growing body of evidence that these proteins exhibit cross-talk and likely function to collectively integrate microtubule behavior and upstream signaling pathways. In this study, we have identified a novel protein-protein interaction between the XMAP215 homologue in Drosophila, Mini spindles (Msps), and the CLASP homologue, Orbit. These proteins have been shown to promote and suppress microtubule dynamics, respectively. We show that microtubule dynamics are regionally controlled in cells by Rac acting to suppress GSK3β in the peripheral lamellae/lamellipodium. Phosphorylation of Orbit by GSK3β triggers a relocalization of Msps from the microtubule plus end to the lattice. Mutation of the Msps-Orbit binding site revealed that this interaction is required for regulating microtubule dynamic instability in the cell periphery. Based on our findings, we propose that Msps is a novel Rac effector that acts, in partnership with Orbit, to regionally regulate microtubule dynamics.

The fractalkine (CX3CL1-CX3CR1) chemokine system is associated with obesity-related inflammation and type 2 diabetes, but data on effects of Cx3cr1 deficiency on metabolic pathways is contradictory. We examined male C57BL/6 Cx3cr1-/- mice on chow and high-fat diet to determine the metabolic effects of Cx3cr1 deficiency. We found no difference in body weight and fat content or feeding and energy expenditure between Cx3cr1-/- and WT mice. Cx3cr1-/- mice had reduced glucose intolerance assessed by intraperitoneal glucose tolerance tests at chow and high-fat fed states, though there was no difference in glucose-stimulated insulin values. Cx3cr1-/- mice also had improved insulin sensitivity at hyperinsulinemic-euglycemic clamp, with higher glucose infusion rate, rate of disposal, and hepatic glucose production suppression compared to WT mice. Enhanced insulin signaling in response to acute intravenous insulin injection was demonstrated in Cx3cr1-/- by increased liver protein levels of phosphorylated AKT and GSK3β proteins. There were no differences in adipose tissue macrophage populations, circulating inflammatory monocytes, adipokines, lipids, or inflammatory markers. In conclusion, we demonstrate a moderate and reproducible protective effect of Cx3cr1 deficiency on glucose intolerance and insulin resistance.

Hedgehog (Hh) morphogen signalling plays an essential role in tissue development and homeostasis. While much is known about the Hh signal transduction pathway, far less is known about the molecules that regulate the expression of the hedgehog (hh) ligand itself. Here we reveal that Shaggy (Sgg), the Drosophila melanogaster orthologue of GSK3β, and the N-end Rule Ubiquitin-protein ligase Hyperplastic Discs (Hyd) act together to co-ordinate Hedgehog signalling through regulating hh ligand expression and Cubitus interruptus (Ci) expression. Increased hh and Ci expression within hyd mutant clones was effectively suppressed by sgg RNAi, placing sgg downstream of hyd. Functionally, sgg RNAi also rescued the adult hyd mutant head phenotype. Consistent with the genetic interactions, we found Hyd to physically interact with Sgg and Ci. Taken together we propose that Hyd and Sgg function to co-ordinate hh ligand and Ci expression, which in turn influences important developmental signalling pathways during imaginal disc development. These findings are important as tight temporal/spatial regulation of hh ligand expression underlies its important roles in animal development and tissue homeostasis. When deregulated, hh ligand family misexpression underlies numerous human diseases (e.g., colorectal, lung, pancreatic and haematological cancers) and developmental defects (e.g., cyclopia and polydactyly). In summary, our Drosophila-based findings highlight an apical role for Hyd and Sgg in initiating Hedgehog signalling, which could also be evolutionarily conserved in mammals.

Bone regeneration disorders are a significant problem in patients with type 2 diabetes mellitus. Bone marrow stromal cells (BMSCs) are recognized as ideal seed cells for tissue engineering because they can stimulate osteogenesis during bone regeneration. Therefore, the aim of this study was to investigate the osteogenic potential of BMSCs derived from type 2 diabetic rats and the pathogenic characteristics of dysfunctional BMSCs that affect osteogenesis. BMSCs were isolated from normal and high-fat diet+streptozotocin-induced type 2 diabetic rats. Cell metabolic activity, alkaline phosphatase (ALP) activity, mineralization and osteogenic gene expression were reduced in the type 2 diabetic rat BMSCs. The expression levels of Wnt signaling genes, such as β-catenin, cyclin D1 and c-myc, were also significantly decreased in the type 2 diabetic rat BMSCs, but the expression of GSK3β remained unchanged. The derived BMSCs were cultured on calcium phosphate cement (CPC) scaffolds and placed subcutaneously into nude mice for eight weeks; they were detected at a low level in newly formed bone. The osteogenic potential of the type 2 diabetic rat BMSCs was not impaired by the culture environment, but it was impaired by inhibition of the Wnt signaling pathway, likely due to an insufficient accumulation of β-catenin rather than because of GSK3β stimulation. Using BMSCs derived from diabetic subjects could offer an alternative method of regenerating bone together with the use of supplementary growth factors to stimulate the Wnt signaling pathway.

Increased stromal stiffness is associated with hepatocellular carcinoma (HCC) development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN) expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3β and the expression of nuclear β-catenin were also elevated, indicating that GSK3β/β-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin β1 showed that OPN expression and p-GSK3β level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3β led to accumulation of β-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin β1/GSK-3β/β-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin β1/GSK-3β/β-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells.

Trophoblast differentiation during early placental development is critical for successful pregnancy and aberrant differentiation causes preeclampsia and early pregnancy loss. During the first trimester, cytotrophoblasts are exposed to low oxygen tension (equivalent to~2%-3% O2) and differentiation proceeds along an extravillous pathway (giving rise to invasive extravillous cytotrophoblasts) and a villous pathway (giving rise to multinucleated syncytiotrophoblast). Interstitial extravillous cytotrophoblasts invade the decidua, while endovascular extravillous cytotrophoblasts are involved in re-modelling uterine spiral arteries. We tested the idea that sodium butyrate (an epigenetic modulator) induces trophoblast differentiation in early gestation rhesus monkey trophoblasts through activation of the Wnt/β-catenin pathway. The results show that syncytiotrophoblast formation was increased by butyrate, accompanied by nuclear accumulation of β-catenin, and increased expression of EnvV2 and galectin-1 (two factors thought to be involved in trophoblast fusion). Surprisingly, the expression of GCM1 and syncytin-2 was not affected by sodium butyrate. When trophoblasts were incubated with lithium chloride, a GSK3 inhibitor that mimics Wnt activation, nuclear accumulation of β-catenin also occurred but differentiation into syncytiotrophoblast was not observed. Instead the cells differentiated to mononucleated spindle-shaped cells and showed molecular and behavioral characteristics of endovascular trophoblasts. Another highly specific inhibitor of GSK3, CHIR99021, failed to induce endovascular trophoblast characteristics. These observations suggest that activation of the Wnt/β-catenin pathway correlates with both trophoblast differentiation pathways, but that additional factors determine specific cell fate decisions. Other experiments suggested that the differential effects of sodium butyrate and lithium chloride might be explained by their effects on TNFα production. The results provide valuable tools to manipulate trophoblast differentiation in vitro and to better understand the differentiation pathways that occur during early gestation.

Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1) and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.

There is increasing evidence that embelin, an active component of Embelia ribes, induces apoptosis in human cancer cells, but the detailed mechanisms are still unclear. Here, we have investigated the effect of embelin on the growth of human prostate cancer cells. Embelin strongly inhibited cell growth especially in human prostate cancer cell lines, including PC3, DU145, LNCaP-LN3 and normal prostate epithelial cell, RWPE-1 compared to breast cancer (MDA-MB-231, MCF-7, and T47D), hepatoma (HepG2, Hep3B, and HuH-7), or choriocarcinoma (JEG-3). We observed that embelin induced apoptosis of PC3 cells in a time-dependent manner correlated with decreased expression of Bcl-2, Bcl-xL, and Mcl-1, increased translocation of Bax into mitochondria, and a reduction in the mitochondrial membrane potential. Furthermore, embelin induced voltage-dependent anion channel (VDAC) 1 expression and oligomerization, which may promote cytochrome c and AIF release. Because embelin was able to inhibit Akt activation and cyclooxygenase-2 expression, the effects on Wnt/ β-catenin signaling were determined. Embelin activated glycogen synthase kinase (GSK)-3β by preventing phosphorylation and suppressed β-catenin expression. Attenuation of β-catenin-mediated TCF transcriptional activity and gene transcription, such as cyclin D1, c-myc, and matrix metalloproteinase (MMP)-7, were shown in embelin-treated cells. The changes in β-catenin levels in response to embelin were blocked by lithium chloride, a GSK-3 inhibitor, indicating that embelin may decrease β-catenin expression via GSK-3β activation. Furthermore, exposure of PC3 cells to embelin resulted in a significant decrease in cell migration and invasion. In conclusion, these findings suggest that inhibition of Akt signaling and activation of GSK-3β partially contributes to the pro-apoptotic effect of embelin in prostate cancer cells.

Although memories appear to be elusive phenomena, they are stored in the network of physical connections between neurons. Dendritic spines, which are actin-rich dendritic protrusions, serve as the contact points between networked neurons. The spines' shape contributes to the strength of signal transmission. To acquire and store information, dendritic spines must remain plastic, i.e., able to respond to signals, by changing their shape. We asked whether glycogen synthase kinase (GSK) 3α and GSK3β, which are implicated in diseases with neuropsychiatric symptoms, such as Alzheimer's disease, bipolar disease and schizophrenia, play a role in a spine structural plasticity. We used Latrunculin B, an actin polymerization inhibitor, and chemically induced Long-Term Depression to trigger fast spine shape remodeling in cultured hippocampal neurons. Spine shrinkage induced by either stimulus required GSK3α activity. GSK3β activity was only important for spine structural changes after treatment with Latrunculin B. Our results indicate that GSK3α is an essential component for short-term spine structural plasticity. This specific function should be considered in future studies of neurodegenerative diseases and neuropsychiatric conditions that originate from suboptimal levels of GSK3α/β activity.

Glycogen synthase kinase 3 (GSK3) is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3β is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN), one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3β activity in bovine endothelial cells (BEC) regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3β at Ser9, and NF-κB p65 subunit (p65) at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3β. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor). Inhibition of GSK3α and GSK3β with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3β. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3β gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus.

Although miR-150 is implicated in the regulation of immune cell differentiation and activation, it remains unknown whether miR-150 is involved in liver biology and disease. This study was performed to explore the potential role of miR-150 in LPS/D-GalN and Fas-induced liver injuries by using wild type and miR-150 knockout (KO) mice. Whereas knockout of miR-150 did not significantly alter LPS/D-GalN-induced animal death and liver injury, it protected against Fas-induced liver injury and mortality. The Jo2-induced increase in serum transaminases, apoptotic hepatocytes, PARP cleavage, as well as caspase-3/7, caspase-8, and caspase-9 activities were significantly attenuated in miR-150 KO mice. The liver tissues from Jo2-treated miR-150 KO mice expressed higher levels of Akt1, Akt2, total Akt, as well as p-Akt(Ser473) compared to the wild type livers. Pretreatment with the Akt inhibitor V reversed Jo2-induced liver injury in miR-150 KO mice. The primary hepatocytes isolated from miR-150 KO mice also showed protection against Fas-induced apoptosis in vitro (characterized by less prominent PARP cleavage, less nuclear fragmentation and less caspase activation) in comparison to hepatocytes from wild type mice. Luciferase reporter assays in hepatocytes transfected with the Akt1 or Akt2 3'-UTR reporter constructs (with or without mutation of miR-150 binding site) established Akt1 and Akt2 as direct targets of miR-150. Tail vein injection of lentiviral particles containing pre-miR-150 enhanced Jo2-induced liver injury in miR-150 KO mice. These findings demonstrate that miR-150 deficiency prevents Fas-induced hepatocyte apoptosis and liver injury through regulation of the Akt pathway.

Aripiprazole is a wide-used antipsychotic drug with therapeutic effects on both positive and negative symptoms of schizophrenia, and reduced side-effects. Although aripiprazole was developed as a dopamine D2 receptor (D2R) partial agonist, all other D2R partial agonists that aimed to mimic aripiprazole failed to exert therapeutic effects in clinic. The present in vivo study aimed to investigate the effects of aripiprazole on the D2R downstream cAMP-PKA and Akt-GSK3β signalling pathways in comparison with a D2R antagonist--haloperidol and a D2R partial agonist--bifeprunox. Rats were injected once with aripiprazole (0.75 mg/kg, i.p.), bifeprunox (0.8 mg/kg, i.p.), haloperidol (0.1 mg/kg, i.p.) or vehicle. Five brain regions--the prefrontal cortex (PFC), nucleus accumbens (NAc), caudate putamen (CPu), ventral tegmental area (VTA) and substantia nigra (SN) were collected. The protein levels of PKA, Akt and GSK3β were measured by Western Blotting; the cAMP levels were examined by ELISA tests. The results showed that aripiprazole presented similar acute effects on PKA expression to haloperidol, but not bifeprunox, in the CPU and VTA. Additionally, aripiprazole was able to increase the phosphorylation of GSK3β in the PFC, NAc, CPu and SN, respectively, which cannot be achieved by bifeprunox and haloperidol. These results suggested that acute treatment of aripiprazole had differential effects on the cAMP-PKA and Akt-GSK3β signalling pathways from haloperidol and bifeprunox in these brain areas. This study further indicated that, by comparison with bifeprunox, the unique pharmacological profile of aripiprazole may be attributed to the relatively lower intrinsic activity at D2R.

Objective: To understand the molecular pathways underlying the cardiac preconditioning effect of short-term caloric restriction (CR)., Background: Lifelong CR has been suggested to reduce the incidence of cardiovascular disease through a variety of mechanisms. However, prolonged adherence to a CR life-style is difficult. Here we reveal the pathways that are modulated by short-term CR, which are associated with protection of the mouse heart from ischemia., Methods: Male 10-12 wk old C57bl/6 mice were randomly assigned to an ad libitum (AL) diet with free access to regular chow, or CR, receiving 30% less food for 7 days (d), prior to myocardial infarction (MI) via permanent coronary ligation. At d8, the left ventricles (LV) of AL and CR mice were collected for Western blot, mRNA and microRNA (miR) analyses to identify cardioprotective gene expression signatures. In separate groups, infarct size, cardiac hemodynamics and protein abundance of caspase 3 was measured at d2 post-MI., Results: This short-term model of CR was associated with cardio-protection, as evidenced by decreased infarct size (18.5±2.4% vs. 26.6±1.7%, N=10/group; P=0.01). mRNA and miR profiles pre-MI (N=5/group) identified genes modulated by short-term CR to be associated with circadian clock, oxidative stress, immune function, apoptosis, metabolism, angiogenesis, cytoskeleton and extracellular matrix (ECM). Western blots pre-MI revealed CR-associated increases in phosphorylated Akt and GSK3ß, reduced levels of phosphorylated AMPK and mitochondrial related proteins PGC-1α, cytochrome C and cyclooxygenase (COX) IV, with no differences in the levels of phosphorylated eNOS or MAPK (ERK1/2; p38). CR regimen was also associated with reduced protein abundance of cleaved caspase 3 in the infarcted heart and improved cardiac function.

Apoptosis regulation is essential for neutrophil homeostasis. We previously demonstrated that a process involving glycogen synthase kinase (GSK)-3β determines neutrophil apoptosis. As for this apoptotic process, an overdose of propofol (2,6-Diisopropylphenol; 25 μg/ml or 140 μM) also causes GSK-3β-mediated macrophage apoptosis; however, the early deactivation of GSK-3β with low-dose propofol has been shown. Therefore, we hypothesize that low-dose propofol may induce neutrophil survival via GSK-3β inactivation. Following in vitro culture, the therapeutic concentration of propofol (10 μg/ml or 56 μM) treatment decreased constitutive apoptosis in isolated human primary neutrophils and in granulocyte-differentiated HL60 cells after all-trans retinoic acid (1 μM) treatment. The inactivation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT and the activation of GSK-3β results in myeloid cell leukemia 1 (Mcl-1) down-regulation, the loss of the mitochondrial transmembrane potential, and caspase-3 activation in these cells, which is accompanied by apoptosis. Notably, propofol treatment attenuates these effects in a PI3-kinase-regulated manner. We found that propofol initiates PI3-kinase/AKT-mediated GSK-3β inactivation and Mcl-1 stabilization, rescuing the constitutive apoptosis in primary neutrophils and granulocyte-differentiated acute promyelocytic leukemia HL60 cells.

Valproate (VPA) has recently been shown to influence the behavioral effects of psycho-stimulants. Although glycogen synthase kinase 3β (GSK3β) signaling in the nucleus accumbens (NAc) plays a key role in mediating dopamine (DA)-dependent behaviors, there is less direct evidence that how VPA acts on the GSK3β signaling in the functionally distinct sub-regions of the NAc, the NAc core (NAcC) and the NAc shell (NAcSh), during psycho-stimulant-induced hyperactivity. In the present study, we applied locomotion test after acute methamphetamine (MA) (2 mg/kg) injection to identify the locomotor activity of rats received repeated VPA (300 mg/kg) pretreatment. We next measured phosphor-GSK3β at serine 9 and total GSK3β levels in NAcC and NAcSh respectively to determine the relationship between the effect of VPA on MA-induced hyperlocomotor and changes in GSK3β activity. We further investigated whether microinjection of VPA (300 μg/0.5 μl/side, once daily for 7 consecutive days) into NAcC or NAcSh could affect hyperactivity induced by MA. Our data indicated that repeated VPA treatment attenuated MA-induced hyperlocomotor, and the effect was associated with decreased levels of phosphorylated GSK3β at Ser 9 in the NAcC. Moreover, repeated bilateral intra-NAcC, but not intra-NAcSh VPA treatment, significantly attenuated MA-induced hyperactivity. Our results suggested that GSK3β activity in NAcC contributes to the inhibitory effects of VPA on MA-induced hyperactivity.

In some esophageal cancer patients, radiotherapy may not prevent distant metastasis thus resulting in poor survival. The underlying mechanism of metastasis in these patients is not well established. In this study, we have demonstrated that ionizing radiation may induce epithelial-mesenchymal transition (EMT) accompanied with increased cell migration and invasion, through downregulation of phosphatase and tensin homolog (PTEN), and activation of Akt/GSK-3β/Snail signaling. We developed a radioresistant (RR) esophageal squamous cancer cell line, KYSE-150/RR, by fractionated ionizing radiation (IR) treatment, and confirmed its radioresistance using a clonogenic survival assay. We found that the KYSE-150/RR cell line displayed typical morphological and molecular characteristics of EMT. In comparison to the parental cells, KYSE-150/RR cells showed an increase in post-IR colony survival, migration, and invasiveness. Furthermore, a decrease in PTEN in KYSE-150/RR cells was observed. We postulated that over-expression of PTEN may induce mesenchymal-epithelial transition in KYSE-150/RR cells and restore IR-induced increase of cell migration. Mechanistically, fractionated IR inhibits expression of PTEN, which leads to activation of Akt/GSK-3β signaling and is associated with the elevated levels of Snail protein, a transcription factor involved in EMT. Correspondingly, treatment with LY294002, a phosphatidylinositol-3-kinase inhibitor, mimicked PTEN overexpression effect in KYSE-150/RR cells, further suggesting a role for the Akt/GSK-3β/Snail signaling in effects mediated through PTEN. Together, these results strongly suggest that fractionated IR-mediated EMT in KYSE-150/RR cells is through PTEN-dependent pathways, highlighting a direct proinvasive effect of radiation treatment on tumor cells.

We previously identified hypothetical protein Cpn1027 as a novel inclusion membrane protein that is unique to Chlamydia pneumoniae. In the current study, using a yeast-two hybrid screen assay, we identified host cell cytoplasmic activation/proliferation-associated protein 2 (Caprin2) as an interacting partner of Cpn1027. The interaction was confirmed and mapped to the C-termini of both Cpn1027 and Caprin2 using co-immunoprecipitation and GST pull-down assays. A RFP-Caprin2 fusion protein was recruited to the chlamydial inclusion and so was the endogenous GSK3β, a critical component of the β-catenin destruction complex in the Wnt signaling pathway. Cpn1027 also co-precipitated GSK3β. Caprin2 is a key regulator of the Wnt signaling pathway by promoting the recruitment of the β-catenin destruction complex to the cytoplasmic membrane in the presence of Wnt signaling while GSK3β is required for priming β-catenin for degradation in the absence of Wnt signaling. The Cpn1027 interactions with Caprin2 and GSK3β may allow C. pneumoniae to actively sequester the β-catenin destruction complex so that β-catenin is maintained even in the absence of extracellular Wnt activation signals. The maintained β-catenin can trans-activate Wnt target genes including Bcl-2, which may contribute to the chlamydial antiapoptotic activity. We found that the C. pneumoniae-infected cells were more resistant to apoptosis induction and the anti-apoptotic activity was dependent on β-catenin. Thus, the current study suggests that the chlamydial inclusion protein Cpn1027 may be able to manipulate host Wnt signaling pathway for enhancing the chlamydial anti-apoptotic activity.