GSK3B Research Articles

Abstract 2934: GSK3β modulates chemoresistance in epithelial ovarian cancer

Abstract Introduction: Epithelial ovarian cancer is one of the most common gynecological malignancies and the fifth most frequent cause of cancer death in women, affecting over 22,000 women annually. Nearly 15,500 affected women die from this disease annually, and chemoresistance from the commonly prescribed platinum-based drug, carboplatin, is a major contributor to this mortality. Previous studies have identified genes with CpG islands that are methylated and transcriptionally silenced in resistant epithelial ovarian cancer patients. One of these genes is GSK3β, an important regulator of apoptosis and cell growth in the Wnt pathway. Thus, understanding the role of GSK3β suppression in chemoresistance of epithelial ovarian cancer can help contribute to more effective treatments for this disease. By performing different assays our study examined the functional role that GSK3β plays in carboplatin chemoresistance. Procedure: Human ovarian surface epithelium (HOSE) 6-3 cell line was utilized, which is characterized as sensitive to carboplatin therapy. The cells were studied in six groups: 1) untreated; 2) treated with Lithium Chloride (LiCl); 3) treated with carboplatin; 4) treated with carboplatin and LiCl; 5) treated with doxorubicin as control; 6) treated with doxorubicin and LiCl as another control. LiCl is known to suppress GSK3β gene expression. We took images of cells using a fluorescence microscope. We also performed the Neutral Red Dye assay that determines cell viability, Vybrant® MTT Cell Assay which measures amount of non-viable cells, Caspase 3 Assay which measures cell apoptosis, and we did cell counting using a hemocytometer and a light microscope. Data analysis was done by T-tests using Microsoft Excel®, with p < .05 for significance. Summary of Data: More growth was observed in the carboplatin and LiCl group compared to the carboplatin group alone on microscopy. Neutral Red Dye assay: Compared to the cells exposed to carboplatin alone, those exposed to carboplatin and LiCl were more viable (p <0.01). Vybrant® MTT Cell Assay: the cells treated with carboplatin and LiCl showed lesser amounts of nonviable cells as compared to the carboplatin alone group (p<0.01). Caspase 3 Assay: the cells treated with carboplatin and LiCl were less apoptotic, compared to cells treated with carboplatin alone (p <0.01). Cell Counting: cells treated with carboplatin and LiCl had significantly more growth compared to the cells treated with carboplatin alone (p <0.01). Conclusion: Our results show that cells with suppressed GSK3β had increased proliferation and reduced apoptosis, strongly suggesting that silenced GSK3β expression contributes to carboplatin resistance and GSK3β expression is vital to carboplatin chemosensitivity. Future in vivo studies could further investigate the role of GSK3β methylation to facilitate the design of potential genome-guided treatments for patients with chemoresistant epithelial ovarian cancer. Citation Format: Naeha Pathak, Noelle L. Cutter. GSK3β modulates chemoresistance in epithelial ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2934.

Local anesthetic bupivacaine induced ovarian and prostate cancer apoptotic cell death and underlying mechanisms in vitro.

Retrospective studies indicate that the use of regional anesthesia can reduce cancer recurrence after surgery which could be due to ranging from immune function preservation to direct molecular mechanisms. This study was to investigate the effects of bupivacaine on ovarian and prostate cancer cell biology and the underlying molecular mechanisms. Cell viability, proliferation and migration of ovarian carcinoma (SKOV-3) and prostate carcinoma (PC-3) were examined following treatment with bupivacaine. Cleaved caspase 3, 8 and 9, and GSK-3β, pGSK-3βtyr216 and pGSK-3βser9 expression were assessed by immunofluorescence. FAS ligand neutralization, caspase and GSK-3 inhibitors and GSK-3β siRNA were applied to further explore underlying mechanisms. Clinically relevant concentrations of bupivacaine reduced cell viability and inhibited cellular proliferation and migration in both cell lines. Caspase 8 and 9 inhibition generated partial cell death reversal in SKOV-3, whilst only caspase 9 was effective in PC-3. Bupivacaine increased the phosphorylation of GSK-3βTyr216 in SKOV-3 but without measurable effect in PC3. GSK-3β inhibition and siRNA gene knockdown decreased bupivacaine induced cell death in SKOV-3 but not in PC3. Our data suggests that bupivacaine has direct ‘anti-cancer’ properties through the activation of intrinsic and extrinsic apoptotic pathways in ovarian cancer but only the intrinsic pathway in prostate cancer.

EZH2-mediated repression of GSK-3β and TP53 promotes Wnt/β-catenin signaling-dependent cell expansion in cervical carcinoma.

Enhancer of zeste homolog 2 (EZH2), a catalytic core component of the Polycomb repressive complex 2 (PRC2), stimulates the silencing of target genes through histone H3 lysine 27 trimethylation (H3K27me3). Recent findings have indicated EZH2 is involved in the development and progression of various human cancers. However, the exact mechanism of EZH2 in the promotion of cervical cancer is largely unknown. Here, we show that EZH2 expression gradually increases during the progression of cervical cancer. We identified a significant positive correlation between EZH2 expression and cell proliferation in vitro and tumor formation in vivo by the up-regulation or down-regulation of EZH2 using CRISPR-Cas9-mediated gene editing technology and shRNA in HeLa and SiHa cells. Further investigation indicated that EZH2 protein significantly accelerated the cell cycle transition from the G0/G1 to S phase. TOP/FOP-Flash reporter assay revealed that EZH2 significantly activated Wnt/β-catenin signaling and the target genes of Wnt/β-catenin pathway were up-regulated, including β-catenin, cyclin D1, and c-myc. Moreover, dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays confirmed that EZH2 inhibited the expression of glycogen synthase kinase-3β (GSK-3β) and TP53 through physically interacting with motifs in the promoters of the GSK-3β and TP53 genes. Additionally, blockage of the Wnt/β-catenin pathway resulted in significant inhibition of cell proliferation, and activation of the Wnt/β-catenin pathway resulted in significant enhancement of cell proliferation, as induced by EZH2. Taken together, our data demonstrate that EZH2 promotes cell proliferation and tumor formation in cervical cancer through activating the Wnt/β-catenin pathway by epigenetic silencing via GSK-3β and TP53.

3, 3', 5-triiodo-L-thyronine Increases In Vitro Chondrogenesis of Mesenchymal Stem Cells From Human Umbilical Cord Stroma Through SRC2.

Our group focuses on the study of mesenchymal stem cells (MSCs) from human umbilical cord stroma or Warthońs jelly and their directed differentiation toward chondrocyte-like cells capable of regenerating damaged cartilage when transplanted into an injured joint. This study aimed to determine whether lactogenic hormone prolactin (PRL) or 3, 3', 5-triiodo-L-thyronine (T3), the active thyroid hormone, modulates chondrogenesis in our in vitro model of directed chondrogenic differentiation, and whether Wnt signalling is involved in this modulation. MSCs from human umbilical cord stroma underwent directed differentiation toward chondrocyte-like cells by spheroid formation. The addition of T3 to the chondrogenic medium increased the expression of genes linked to chondrogenesis like collagen type 2, integrin alpha 10 beta 1, and Sox9 measured by quantitative real time polymerase chain reaction (qRT-PCR) analysis. Levels of collagen type 2 and aggrecane analyzed by immunohistochemistry, and staining by Safranin O were increased after 14 days in spheroid culture with T3 compared to those without T3 or only with PRL. B-catenin, Frizzled, and GSK-3β gene expressions were significantly higher in spheroids cultured with chondrogenic medium (CM) plus T3 compared to CM alone after 14 days in culture. The increase of chondrogenic differentiation was inhibited when the cells were treated with T3 plus ML151, an inhibitor of the T3 steroid receptor. This work demonstrates, for first time, that T3 promotes differentiation towards chondrocytes-like cells in our in vitro model, that this differentiation is mediated by steroid receptor co-activator 2 (SRC2) and does not induce hypertrophy. J. Cell. Biochem. 117: 2097-2108, 2016. © 2016 Wiley Periodicals, Inc.

Cardioprotective effects of lipoic acid, quercetin and resveratrol on oxidative stress related to thyroid hormone alterations in long-term obesity

This study investigated possible mechanisms for cardioprotective effects of lipoic acid (LA), quercetin (Q) and resveratrol (R) on oxidative stress related to thyroid hormone alterations in long-term obesity. Female C57BL/6 mice were fed on high-fat diet (HFD), HFD+LA, HFD+R, HFD+Q and normal diet for 26weeks. Body weight, blood pressure, thyroid hormones, oxidative stress markers, angiotensin converting enzyme (ACE), nitric oxide synthase (NOS) and ion pump activities were measured, and expression of cardiac genes was analyzed by real-time polymerase chain reaction. HFD induced marked increase (P<.05) in body weight, blood pressure and oxidative stress, while plasma triidothyronine levels reduced. ACE activity increased (P<.05) in HFD mice (0.69±0.225U/mg protein) compared with controls (0.28±0.114U/mg protein), HFD+LA (0.231±0.02U/mg protein) and HFD+Q (0.182±0.096U/mg protein) at 26weeks. Moreover, Na+/K+-ATPase and Ca2+-ATPase activities increased in HFD mice whereas NOS reduced. A 1.5-fold increase in TRα1 and reduction in expression of the deiodinase iodothyronine DIO1, threonine protein kinase and NOS3 as well as up-regulation of AT1α, ACE, ATP1B1, GSK3β and Cja1 genes also occurred in HFD mice. Conversely, LA, Q and R inhibited weight gain; reduced TRα1 expression as well as increased DIO1; reduced ACE activity and AT1α, ATP1B1 and Cja1 gene expression as well as inhibited GSK3β; increased total antioxidant capacity, GSH and catalase activity; and reduced blood pressure. In conclusion, LA, resveratrol and quercetin supplementation reduces obesity thereby restoring plasma thyroid hormone levels and attenuating oxidative stress in the heart and thus may have therapeutic potential in heart diseases.

MicroRNA-224 sustains Wnt/β-catenin signaling and promotes aggressive phenotype of colorectal cancer.

BackgroundGrowing evidence suggests that Wnt/β-catenin pathway plays an important role in CRC development, progression and metastasis. Aberrant miR-224 expression has been reported in CRC. However, the mechanism of miR-224 promotes both proliferation and metastatic ability largely remains unclear.MethodsReal-time PCR was used to quantify miR-224 expression. Luciferase reporter assays were conducted to confirm the activity of Wnt/β-catenin pathway and target gene associations, and immunofluorescence staining assay was performed to observe the nuclear translocation of β-catenin. Bioinformatics analysis combined with in vivo and vitro functional assays showed the potential target genes, GSK3β and SFRP2, of miR-224. Specimens from forty patients with CRC were analyzed for the expression of miR-224 and the relationship with GSK3β/SFRP2 by real-time PCR and western blot.ResultsBioinformatics and cell luciferase function studies verified the direct regulation of miR-224 on the 3’-UTR of the GSK3β and SFRP2 genes, which leads to the activation of Wnt/β-catenin signaling and the nuclear translocation of β-catenin. In addition, knockdown of miR-224 significantly recovered the expression of GSK3β and SFRP2 and attenuated Wnt/β-catenin-mediated cell metastasis and proliferation. The ectopic upregulation of miR-224 dramatically inhibited the expression of GSK3β/SFRP2 and enhanced CRC proliferation and invasion.ConclusionOur research showed mechanistic links between miR-224 and Wnt/β-catenin in the pathogenesis of CRC through modulation of GSK3β and SFRP2.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0287-1) contains supplementary material, which is available to authorized users.

Prolonged hyperinsulinemia affects metabolic signal transduction markers in a tissue specific manner

Insulin dysregulation is common in horses although the mechanisms of metabolic dysfunction are poorly understood. We hypothesized that insulin signaling in striated (cardiac and skeletal) muscle and lamellae may be mediated through different receptors as a result of receptor content, and that transcriptional regulation of downstream signal transduction and glucose transport may also differ between tissues sites during hyperinsulinemia. Archived samples from horses treated with a prolonged insulin infusion or a balanced electrolyte solution were used. All treated horses developed marked hyperinsulinemia and clinical laminitis. Protein expression was compared across tissues for the insulin receptor and insulin-like growth factor 1 receptor (IGF-1R) by immunoblotting. Gene expression of metabolic insulin-signaling markers (insulin receptor substrate 1, Akt2, and glycogen synthase kinase 3 beta [GSK-3β]) and glucose transport (basal glucose transporter 1 and insulin-sensitive glucose transporter 4) was evaluated using real-time reverse transcription polymerase chain reaction. Lamellar tissue contained significantly more IGF-1R protein than skeletal muscle, indicating the potential significance of IGF-1R signaling for this tissue. Gene expression of the selected markers of insulin signaling and glucose transport in skeletal muscle and lamellar tissues was unaffected by prolonged hyperinsulinemia. In contrast, the significant upregulation of Akt2, GSK-3β, GLUT1, and GLUT4 gene expression in cardiac tissue suggested that the prolonged hyperinsulinemia induced an increase in insulin sensitivity and a transcriptional activation of glucose transport. Responses to insulin are tissue-specific, and extrapolation of data across tissue sites is inappropriate.

WNT-associated gene expression in human mesenchymal stromal cells under hypoxic stress.

The effect of short-term stress on the expression of 84 Wnt-related genes in mesenchymal stromal cells (MSCs) was analyzed. In MSCs permanently expanded at 5% O2, Dkk1, RHOA, CSNK1A1, CSNK2A1, GSK3β, and PORCN genes were most highly expressed. FZD1, FZD3, SFRP1, and SFRP4 genes were up-regulated after short-term hypoxic stress (0.1% O2), whereas the expression of LEF-1 and RUVBL1 genes was significantly inhibited (down-regulated). The data suggest that Wnt signal transduction was suppressed under hypoxic stress, whereas the noncanonical Wnt signaling prevailed under the "physiological hypoxia" (5% O2).

Impairment of PI3K/AKT and WNT/β-catenin pathways in bone marrow mesenchymal stem cells isolated from patients with myelodysplastic syndromes

Bone marrow mesenchymal stem cells (BM-MSCs) exhibit multiple abnormalities in myelodysplastic syndromes (MDS), including impaired proliferative and clonogenic capacity, altered morphology, increased senescence, impaired immunoregulatory properties, and reduced hematopoietic support capacity. Common signaling pathways, such as PI3K/AKT and WNT/β-catenin, regulate multiple MSC properties, including proliferation, differentiation, and cell-cell interaction. Here, with polymerase chain reaction arrays, we investigated the expression of 84 genes belonging to the PI3K/AKT signaling pathways in BM-MSCs isolated from patients with MDS, acute myeloid leukemia, and therapy-related myeloid neoplasms, using as a control BM-MSCs isolated from patients with untreated early-stage lymphomas without BM involvement. Statistically significant downregulation of GSK3β, SOS1, RASA1, and MTCP1 gene expression was observed in BM-MSCs isolated from patients with de novo MDS, as compared with controls. Moreover, expression of the GSK3β protein was reduced in MDS-derived MSCs, and was associated with concomitant reduction of phosphorylation at Ser-9. The role of GSK3β in the downstream WNT/β-catenin signaling pathway was assessed. We investigated β-catenin protein levels and expression of 84 genes belonging to the WNT target gene pathway using PCR arrays in MDS BM-MSCs, as compared with control BM-MSCs. GSK3β impairment translated into decreased β-catenin protein levels and downregulation of several WNT/β-catenin target genes (SOX9, EGR1, WISP1). These findings suggest that deregulation of genes involved in the PI3K/AKT and WNT signaling pathways may contribute to the phenotypical abnormalities of MDS BM-MSCs.

Violacein induces apoptosis in human breast cancer cells through up regulation of BAX, p53 and down regulation of MDM2

We aimed to explore the anticancer potential of violacein and its time, dose dependent mechanism of action in human MCF-7 breast cancer cells. We observed, violacein inhibit MCF-7 cells viability in a time and dose-dependent manner, IC50 value was 4.5μM in 24h, 1.7μM in 48h and 0.51μM in 72h. Violacein triggered generation of intra cellular ROS even from the lower doses, significant ROS production was observed from 0.25, 0.45μM dose range and it is relative to higher doses. Further we fixed 0.45μM and 4.5μM as an experimental dose for relative dose dependent analysis. In nuclear staining, after 48h 0.45μM dose showed characteristic apoptotic morphological changes such as, 59% of cells in apoptosis and 11% of cells in necrotic stage, also in 72h we found 68% in apoptosis and 12% in necrotic stage. However, 4.5μM (IC50) dose of violacein, 78% of cells became apoptotic and 21% in necrotic after 48h; but in 72h only 61% cells are in apoptotic, necrosis was increased to 38%. Violacein increased both mitochondrial and extra mitochondrial apoptotic pathway related gene expressions; it was confirmed by increased CYP1A, GPX, GSK3β and TNF-α gene. Further, 0.45 and 4.5μM of violacein increased apoptotic genes, such as Bax, p53, caspase 3, Fas, FADD and markedly reduced Bcl-2 and MDM2 expression levels to two fold when compared to control. In addition violacein upregulated poly ADP-ribose polymerase (PARP), CDKN1A and caspase-9 significantly (p≤0.05) when compared to control. Relative quantification of caspase-8 was differently expressed; there were no changes in 0.45μM, but in 4.5μM we found two fold increased caspase-8 expression. In conclusion, lower dose of violacein treatment induced apoptosis in human breast cancer MCF-7 cells through TNF-α and p53 dependent mitochondrial pathways.

Exploring Genetic Variability at PI, GSK3, HPA, and Glutamatergic Pathways in Lithium Response: Association With IMPA2, INPP1, and GSK3B Genes.

Lithium is considered the first-line treatment in bipolar disorder, although response could range from an excellent response to a complete lack of response. Response to lithium is a complex phenotype in which different factors, part of them genetics, are involved. In this sense, the aim of this study was to investigate the potential association of genetic variability at genes related to phosphoinositide, glycogen synthetase kinase-3 (GSK3), hypothalamic-pituitary-adrenal, and glutamatergic pathways with lithium response. A sample of 131 bipolar patients (99 type I, 32 type II) were grouped and compared according to their level of response: excellent responders (ER), partial responders (PR), and nonresponders (NR). Genotype and allele distributions of the rs669838 (IMPA2), rs909270 (INNP1), rs11921360 (GSK3B), and rs28522620 (GRIK2) polymorphisms significantly differed between ER, PR, and NR. When we compared the ER versus PR+NR, the logistic regression showed significant association for rs669838-C (IMPA2; P = 0.021), rs909270-G (INPP1; P = 0.009), and rs11921360-A (GSK3B; P = 0.004) with lithium nonresponse. Haplotype analysis showed significant association for the haplotypes rs3791809-rs4853694-rs909270 (INPP1) and rs1732170-rs11921360-rs334558 (GSK3B) and lithium response. Our study is in line with previous studies reporting association between genetic variability at these genes and lithium response, pointing to an effect of IMPA2, INPP1, and GSK3B genes to lithium response in bipolar disorder patients. Further studies with larger samples are warranted to assess the strength of the reported associations.

FXR1P is a GSK3β substrate regulating mood and emotion processing.

Inhibition of glycogen synthase kinase 3β (GSK3β) is a shared action believed to be involved in the regulation of behavior by psychoactive drugs such as antipsychotics and mood stabilizers. However, little is known about the identity of the substrates through which GSK3β affects behavior. We identified fragile X mental retardation-related protein 1 (FXR1P), a RNA binding protein associated to genetic risk for schizophrenia, as a substrate for GSK3β. Phosphorylation of FXR1P by GSK3β is facilitated by prior phosphorylation by ERK2 and leads to its down-regulation. In contrast, behaviorally effective chronic mood stabilizer treatments in mice inhibit GSK3β and increase FXR1P levels. In line with this, overexpression of FXR1P in the mouse prefrontal cortex also leads to comparable mood-related responses. Furthermore, functional genetic polymorphisms affecting either FXR1P or GSK3β gene expression interact to regulate emotional brain responsiveness and stability in humans. These observations uncovered a GSK3β/FXR1P signaling pathway that contributes to regulating mood and emotion processing. Regulation of FXR1P by GSK3β also provides a mechanistic framework that may explain how inhibition of GSK3β can contribute to the regulation of mood by psychoactive drugs in mental illnesses such as bipolar disorder. Moreover, this pathway could potentially be implicated in other biological functions, such as inflammation and cell proliferation, in which FXR1P and GSK3 are known to play a role.

The Glycogen Synthase Kinase 3α and β Isoforms Differentially Regulates Interleukin-12p40 Expression in Endothelial Cells Stimulated with Peptidoglycan from Staphylococcus aureus.

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.

Molecular Mechanism on Inhibition of MB Angiogenesis by Curcumin Blocking the Wnt/β-Catenin and NF-κB Signaling Pathway and Inhibiting the Expression of VEGFs/VEGFRs

Medulloblastoma (MB) is one of the most common malignant tumors in pediatric brain. The genesis and development of MB are related to abnormal and persistent activation of the Wnt/β-catenin abnormal and NF-κB signaling pathway. The VEGF, as the main target gene regulated by the crosstalk between the two signaling pathways, is closely related to MB. In previous studies, We found that the genesis of MB is associated with the abnormal expression of key molecular GSK-3β,β-catenin and downstream target gene PPAR-gamma in Wnt /β-catenin signaling pathway.It also found that, there have abnormal expression of VEGF-A, C and its receptor VEGFR-2 in MB, and positively correlate with β-catenin. curcumin has anti-angiogenic function, but the exact mechanism is unclear. Combining with the early research findings and the latest literatures, we hypothesized that curcumin can inhibit the expression of VEGFs/VEGFRs by blocking the Wnt/β-catenin and NF-κB signaling pathway.

The GSK3B gene confers risk for both major depressive disorder and schizophrenia in the Han Chinese population

Background: Glycogen synthease kinase-3B is a key gene encoding a protein kinase which is abundant in brain, and is involved in signal transduction cascades of neuronal cell development and energy metabolism. Previous researches proposed GSK3B as a potential region for schizophrenia.Method: To validate the susceptibility of GSK3B to major depressive disorder, and to investigate the overlapping risk conferred by GSK3B for mental disorders, we performed a large-scale case-control study, analyzed 6 tag single nucleotide polymorphisms using TaqMan® technology in 1,045 major depressive disorder patients, 1,235 schizophrenia patients and 1,235 normal controls of Han Chinese origin.Results: We found rs334535 (Pallele=2.79E−03, Pgenotype=5.00E−03, OR=1.429) and rs2199503 (Pallele=0.020, Pgenotype= 0.040, OR=1.157) showed association with major depressive disorder before Bonferroni correction. rs6771023 (adjusted Pallele=1.64E−03, adjusted Pgenotype=6.00E−03, OR=0.701) and rs2199503 (adjusted Pallele=0.001, adjusted Pgenotype=0.002, OR=1.251) showed significant association with schizophrenia after Bonferroni correction. rs2199503 (adjusted Pallele=1.70E−03, adjusted Pgenotype=0.006, OR=1.208) remained to be significant in the combined cases of major depressive disorder and schizophrenia after Bonferroni correction.Limitations: Further validations of our findings in samples with larger scale are suggested, and functional genomic study is needed to elucidate the role of GSK3B in signal pathway and psychiatric disorders.Conclusions: Our results provide evidence that the GSK3B gene could be a promising region which contains genetic risk for both major depressive disorder and schizophrenia in the Han Chinese population. The study on variants conferring overlapping risk for multiple psychiatric disorders could be tangible pathogenesis support and clinical or diagnostic references.

Decreased Activity in Neuropathic Pain Form and Gene Expression of Cyclin-Dependent Kinase5 and Glycogen Synthase Kinase-3 Beta in Soleus Muscle of Wistar Male Rats.

Background:The relationship between decreased activity/neuropathic pain and gene expression alterations in soleus muscle has remained elusive.Objectives:In this experimental study, we investigated the effects of decreased activity in neuropathic pain form on Cyclin-Dependent Kinase 5 (CDK5) and Glycogen Synthase Kinase-3 β (GSK-3β) gene expression in soleus muscle of rats.Materials and Methods:Twelve male Wistar rats were randomly divided into three groups: (1) tight ligation of the L5 spinal nerve (SNL: n = 4); (2) sham surgery (Sham: n = 4), and (3) control (C: n = 4). The threshold to produce a withdrawal response to a mechanical and thermal stimulus was measured using von Frey filaments and radiation heat apparatus, respectively. Following 4 weeks after surgery, the left soleus muscle was removed and mRNA levels were determined by real-time Polymerase Chain Reaction (PCR).Results:Compared to control animals, L5 ligated animals developed mechanical and heat hypersensitivity during total period of study. Soleus muscle weight as well as CDK5 mRNA levels (less than ~ 0.4 fold) was decreased and GSK-3β mRNA levels (up to ~ 7 folds) increased in L5 ligated animals.Conclusions:These results showed enhanced muscle atrophy processes following peripheral nerve damage and might provide a useful approach to study underlying muscle mechanisms associated with clinical neuropathic pain syndromes.

Sirtuin 3 inhibits hepatocellular carcinoma growth through the glycogen synthase kinase-3β/BCL2-associated X protein-dependent apoptotic pathway

SIRT3 is a class III histone deacetylase that has been implicated in a variety of cancers. The role of SIRT3 in hepatocellular carcinoma (HCC) remains elusive. In this study, we found that SIRT3 expression was frequently repressed in HCC and its downregulation was closely associated with tumor grade and size. Ectopic expression of SIRT3 inhibited cell growth and induced apoptosis in HCC cells, whereas depletion of SIRT3 in immortalized hepatocyte promoted cell growth and decreased epirubicin-induced apoptosis. Mechanistic studies revealed that SIRT3 deacetylated and activated glycogen synthase kinase-3β (GSK-3β), which subsequently induced expression and mitochondrial translocation of the pro-apoptotic protein BCL2-associated X protein (Bax) to promote apoptosis. GSK-3β inhibitor or gene silencing of BAX reversed SIRT3-induced growth inhibition and apoptosis. Furthermore, SIRT3 overexpression also suppressed tumor growth in vivo. Together, this study reveals a role of SIRT3/GSK-3β/Bax signaling pathway in the suppression of HCC growth, and also suggests that targeting this pathway may represent a potential therapeutic approach for HCC treatment.

31 DEREGULATION OF PI3K/AKT SIGNALING AND WNT TARGET GENES IN BONE MARROW MESENCHYMAL STROMAL CELLS FROM PATIENTS WITH MDS

Bone marrow mesenchymal stem cells (BM-MSCs) exhibit multiple abnormalities in myelodysplastic syndromes (MDS), including impaired proliferative and clonogenic capacity, altered morphology, increased senescence, impaired immunoregulatory properties, and reduced hematopoietic support capacity. Common signaling pathways, such as PI3K/AKT and WNT/β-catenin, regulate multiple MSC properties, including proliferation, differentiation, and cell–cell interaction. Here, with polymerase chain reaction arrays, we investigated the expression of 84 genes belonging to the PI3K/AKT signaling pathways in BM-MSCs isolated from patients with MDS, acute myeloid leukemia, and therapy-related myeloid neoplasms, using as a control BM-MSCs isolated from patients with untreated early-stage lymphomas without BM involvement. Statistically significant downregulation of GSK3β, SOS1, RASA1, and MTCP1 gene expression was observed in BM-MSCs isolated from patients with de novo MDS, as compared with controls. Moreover, expression of the GSK3β protein was reduced in MDS-derived MSCs, and was associated with concomitant reduction of phosphorylation at Ser-9. The role of GSK3β in the downstream WNT/β-catenin signaling pathway was assessed. We investigated β-catenin protein levels and expression of 84 genes belonging to the WNT target gene pathway using PCR arrays in MDS BM-MSCs, as compared with control BM-MSCs. GSK3β impairment translated into decreased β-catenin protein levels and downregulation of several WNT/β-catenin target genes (SOX9, EGR1, WISP1). These findings suggest that deregulation of genes involved in the PI3K/AKT and WNT signaling pathways may contribute to the phenotypical abnormalities of MDS BM-MSCs.

Paclitaxel-induced neuropathy: potential association of MAPT and GSK3B genotypes.

BackgroundPaclitaxel treatment produces dose-limiting peripheral neurotoxicity, which adversely affects treatment and long-term outcomes. In the present study, the contribution of genetic polymorphisms to paclitaxel-induced neurotoxicity were assessed in 21 patients, focusing on polymorphisms involved in the tau-microtubule pathway, an important target of paclitaxel involved in neurotoxicity development.MethodsPolymorphisms in the microtubule-associated protein tau (MAPT) gene (haplotype 1 and rs242557 polymorphism) and the glycogen synthase kinase-3β (GSK3β) gene (rs6438552 polymorphism) were investigated. Neurotoxicity was assessed using neuropathy grading scales, neurophysiological studies and patient questionnaires.ResultsA significant relationship between the GSK-3B rs6438552 polymorphism and paclitaxel-induced neurotoxicity was evident.ConclusionsPolymorphisms in tau-associated genes may contribute to the development of paclitaxel-induced neurotoxicity, although larger series will be necessary to confirm these findings.

Multiple alternative splicing and differential expression patterns of the glycogen synthase kinase-3β (GSK3β) gene in Schizothorax prenanti.

Glycogen synthase kinase-3 (GSK-3) is a key protein kinase involved in numerous cellular processes, including embryonic development, protein synthesis, glycogen metabolism, mitosis and apoptosis. However, our knowledge of Schizothorax prenanti GSK-3 is very limited. In this study, we cloned and characterized the S. prenanti GSK3β gene. Using qPCR, we found that the GSK3β gene was widely expressed in eleven tissues of S. prenanti and had especially high expression levels in the liver and brain. Moreover, we screened and sequenced more than 100 positive clones to identify the alternative transcripts of GSK3β. Five novel isoforms of GSK3β were identified in different S. prenanti tissues; these were different from the GSK3β isoforms previously reported in the other species. We named the five transcripts as GSK3β1, GSK3β2, GSK3β3, GSK3β4 and GSK3β5. These consisted of 1266, 1153, 902, 836 and 654 base pairs, respectively. Our studies provide useful information for further research on the S. prenanti GSK3β gene.