Hepatic Expression of Phosphorylated Kinase Akt and Glycogen Synthase Kinase-3 Isoforms in Patients with Chronic Hepatitis C.

Some patients with chronic hepatitis C also demonstrate liver steatosis, but the mechanism remains elusive. To analyze the hepatic expression of phosphorylated kinase Akt at Thr 308 and phosphorylated GSK-3 (Glycogen synthase kinase-3) isoforms, GSK3α at Ser 21 and GSK3ß at Ser 9, in chronic hepatitis C patients with normal body weight, glucose, and lipid profiles depending on homeostasis model assessment of insulin resistance (HOMA-IR) levels and histological parameters. The study group consisted of 31 patients with chronic hepatitis C. The hepatic expression of kinase Akt (Thr308), GSK3ß (Ser9), and GSK3α (Ser21) was measured using Western blot assay. Liver steatosis was observed in 41.93% of patients with HCV infection, in those with increased HOMA-IR index (p = 0.02). However, the hepatic expression of Akt (Thr308), GSK3ß (Ser9), and GSK3α (Ser21) was not related to progression of liver steatosis, inflammation, and fibrosis. There was no significant difference in the hepatic expression of kinase Akt (Thr308), GSK3ß (Ser9), and GSK3α (Ser21) in relation to HOMA-IR. Liver steatosis was found to be positively associated with HOMA-IR levels in patients with chronic hepatitis C without metabolic disorders. However, the hepatic expression of Akt (Thr308), GSK3ß (Ser9), and GSK3α (Ser21) did not correspond to progression of liver disease.

Glycogen Synthase Kinase 3ß Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists.

The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3ß (GSK-3ß) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3ß interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3ß redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3ß at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3ß in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3ß. In vitro activity assays demonstrated that GSK-3ß phosphorylation mediated by PKCζ enhanced GSK-3ß activity. We mapped Ser147 of GSK-3ß as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3ß activity, resulting in ß-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3ß basal activity. Thus, we found that PKCζ phosphorylates GSK-3ß at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3ß activity in opposite manners in normal and malignant colon cells.

Clinicopathological significance of wnt/ß-catenin signaling pathway in esophageal squamous cell carcinoma.

BACKGROUND/AIM: Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors. It has been reported that Wnt signaling pathway plays an important role in Esophageal Cancer progression, metastasis and invasion. However the clinicopathological significance of Wnt2, GSK3ß, and ß-catenin in ESCC has been little reported. In the present study, the aim of this study was to investigate the clinicopathologic and prognosis roles of Wnt2, GSK3ß, and ß-catenin in ESCC tissue. METHODS: 265 ESCC samples were analyzed by immunohistochemistry using Wnt2, GSK3ß, and ß-catenin antibodies. Then, correlation of Wnt2, GSK3ß, and ß-catenin expression with clinicopathological features and prognosis of ESCC patients was statistically analyzed. RESULTS: Cytoplasmic Wnt2 overexpression was detected in 55.5% (147 of 265) ESCCs, which was significantly correlated with the degree of differentiation (P=0.031). Cytoplasmic GSK3ß overexpression was detected in 7.2% (19 of 265) ESCCs, and aberrant ß-catenin expression was identified in 54.3% (144 of 265) of ESCCs. The positive rate of Wnt2 significantly increased with the malignant degree of Kazak ESCC patients. The aberrant ß-catenin expression in GSK3ß-negative ESCC was significantly associated with the ethnic, tumor size, tumor location, degree of differentiation, AJCC stage, lymph node status. Furthermore, the expression of ß-catenin implicated the ethnic difference (P=0.019). In Kaplan-Meier curve analysis, no significant correlation was observed between the expression of Wnt2, GSK3ß, ß-catenin and the poor prognosis of ESCCs. CONCLUSION: The aberrant ß-catenin expression could be an adverse underlying factor in carcinogenesis and progression of ESCC. There was a different statistical signification for ß-catenin in Kazakhs to compare with Hans.

Differential expression of GSK3ß and pS9GSK3ß in normal human tissues: can pS9GSK3ß be an epithelial marker?

Glycogen synthase kinase 3ß (GSK3ß) and phosphorylated GSK3ß at Ser9 (pS9GSK3ß) are crucial in cellular proliferation and metabolism. GSK3ß and pS9GSK3ß are deregulated in many diseases including tumors. Data on altered expression of GSK3ß and pS9GSK3ß are mainly limited to tumor tissues, thus the expression of GSK3ß and pS9GSK3ß in normal human tissue has been largely unknown. Thus, we examined the immunohistochemical localization of GSK3ß and pS9GSK3ß in human fetal and adult tissues, and also compared the expression pattern of GSK3ß and pS9GSK3ß with that of the CK7 and CK20. We found GSK3ß expression in neurons of brain, myenteric plexus in gastrointestinal tract, squamous epithelium of skin, and mammary gland. The expression of pS9GSK3ß was restricted to the epithelial cells of breast and pancreaticobiliary duct, distal nephron of kidney, gastrointestinal tract, fallopian tube, epididymis, secretory cell of prostatic gland, and umbrella cell of urinary tract. The staining pattern of pS9GSK3ß and CK7 was overlapped in most organs except for gastrointestinal tract where CK7 was negative and CK20 was positive. Our results show that the expression of GSK3ß may be associated with differentiation of ectodermal derived tissues and pS9GSK3ß with that of epithelial cells of endodermal derived tissues in human. In addition, the expression of pS9GSK3ß in the selective epithelial cells may indicate its association with secretory or barrier function of specific cells and may serve as another immunohistochemical marker for epithelial cells.

Effect of atorvastatin on wound healing in rats.

Skin-wound healing is a complex and dynamic biological process involving inflammation, proliferation, and remodeling. Recent studies have shown that statins are new therapeutical options because of their actions, such as anti-inflammatory and antioxidant activity, on vasodilation, endothelial dysfunction and neoangiogenesis, which are independent of their lipid-lowering action. Our aim was to investigate the effect of atorvastatin on tissue repair after acute injury in healthy animals. Rats were divided into four groups: placebo-treated (P), topical atorvastatin-treated (AT), oral atorvastatin-treated (AO), topical and oral atorvastatin-treated (ATO). Under anesthesia, rats were wounded with an 8-mm punch in the dorsal region. Lesions were photographed on Days 0, 1, 3, 7, 10, 12, and 14 post-injury and samples taken on Days 1, 3, 7, and 14 for protein-expression analysis of insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), glycogen synthase kinase (GSK)-3, endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), extracellular signal-regulated kinase (ERK), interleukin (IL)-10, IL-1ß, IL-6, and tumor necrosis factor (TNF)-α. Upon macroscopic examination, we observed significant reductions of lesion areas in groups AT, AO, and ATO compared to the P group. Additionally, AT and AO groups showed increased expression of IRS-1, PI3K, Akt, GSK-3, and IL-10 on Days 1 and 3 when compared with the P group. All atorvastatin-treated groups showed higher expression of IRS-1, PI3K, Akt, GSK-3, IL-10, eNOS, VEGF, and ERK on Day 7. On Days 1, 3, and 7, all atorvastatin-treated groups showed lower expression of IL-6 and TNF-α when compared with the P group. We conclude that atorvastatin accelerated tissue repair of acute lesions in rats and modulated expressions of proteins and cytokines associated with cell-growth pathways.

Unremitting cell proliferation in the secretory phase of eutopic endometriosis: involvement of pAkt and pGSK3ß.

OBJECTIVE: Endometriosis is linked to altered cell proliferation and stem cell markers c-kit/stem cell factor (SCF) in ectopic endometrium. Our aim was to investigate whether c-kit/SCF also plays a role in eutopic endometrium. DESIGN: Eutopic endometrium obtained from 35 women with endometriosis and 25 fertile eumenorrheic women was analyzed for in situ expression of SCF/c-kit, Ki67, RAC-alpha serine/threonine-protein kinase (Akt), phosphorylated RAC-alpha serine/threonin-protein kinase (pAkt), Glycogen synthase kinase 3 beta (GSK3ß), and phosphorylated glycogen synthase kinase 3 beta (pGSK3ß), throughout the menstrual cycle. RESULTS: Expression of Ki67 and SCF was higher in endometriosis than in control tissue (P < .05) and greater in secretory rather than proliferative (P < .01) endometrium in endometriosis. Expression of c-kit was also higher in endometriosis although similar in both phases. Expression of Akt and GSK3ß was identical in all samples and cycle phases, whereas pAkt and pGSK3ß, opposed to control tissue, remained overexpressed in the secretory phase in endometriosis. CONCLUSION: Unceasing cell proliferation in the secretory phase of eutopic endometriosis is linked to deregulation of c-kit/SCF-associated signaling pathways.

Sensitization of cancer cells through reduction of total Akt and downregulation of salinomycin-induced pAkt, pGSk3ß, pTSC2, and p4EBP1 by cotreatment with MK-2206.

MK-2206 is an inhibitor of Akt activation. It has been investigated as an anticancer drug in clinical trials assessing the potential of pAkt targeting therapy. The purpose of this study was to identify conditions that increase the sensitivity of cancer cells to MK-2206. We found that the treatment of cancer cells with a high concentration of salinomycin (Sal) reduced total Akt protein levels but increased activated Akt levels. When cancer cells were cotreated with MK-2206 and Sal, both pAkt and total Akt levels were reduced. Using microscopic observation, an assessment of cleaved PARP, FACS analysis of pre-G1 region, and Hoechst staining, we found that Sal increased apoptosis of MK-2206-treated cancer cells. These results suggest that cotreatment with MK-2206 and Sal sensitizes cancer cells via reduction of both pAkt and total Akt. Furthermore, cotreatment of cancer cells with Sal and MK-2206 reduced pp70S6K, pmTOR, and pPDK1 levels. In addition, Sal-induced activation of GSK3ß, TSC2, and 4EBP1 was abolished by MK-2206 cotreatment. These results suggest that cotreatment using MK-2206 and Sal could be used as a therapeutic method to sensitize cancer cells through targeting of the PI3K/Akt/mTOR pathway. Our findings may contribute to the development of MK-2206-based sensitization therapies for cancer patients.

Glycogen synthase kinase-3ß is associated with the prognosis of hepatocellular carcinoma and may mediate the influence of type 2 diabetes mellitus on hepatocellular carcinoma.

BACKGROUND: Although many studies have shown glycogen synthase kinase-3ß (GSK-3ß) was associated with type 2 diabetes mellitus (T2DM) and implicated with a wide range of cancers, the role of GSK-3ß in hepatocellular carcinoma(HCC) and the correlation among GSK-3ß, T2DM and HCC remains unclear. Our objectives were to identify the effect of p-Ser9-GSK-3ß on the prognosis of patients with HCC and to learn more about the interaction among T2DM, GSK-3ß and the prognosis of HCC. METHODS: Firstly we used reverse transcriptase-PCR(RT-PCR) and western blotting to determine the expression levels of GSK-3ß and p-Ser9-GSK-3ß in human HCC samples. We then used immunohistochemical staining to evaluate the expression pattern of p-Ser9-GSK-3ß in 178 patients with HCC after curative partial hepatectomy. Finally we statistically analyzed the association of p-Ser9-GSK-3ß and T2DM with the prognosis of patients with HCC. RESULTS: P-Ser9-GSK-3ß was over-expressed in tumor tissues compared with their normal counterparts. Correlation and regression analysis indicated that the over-expression of p-Ser9-GSK-3ß was significantly associated with T2DM, and the correlation coefficient was 0.259 (P = 0.001). Multivariate analysis showed that the over-expression of p-Ser9-GSK-3ß(P<0.001) and T2DM(P = 0.008) were independently associated with poor prognosis of HCC, respectively. Further analysis demonstrated that these two variables are closely related with each other. CONCLUSION: The over-expression of p-Ser9-GSK-3ß and T2DM are strongly correlated with worse surgical outcome of HCC. P-Ser9-GSK-3ß may play a significant role in mediating the influence of T2DM on the prognosis of HCC.

Pioglitazone ameliorates intracerebral insulin resistance and tau-protein hyperphosphorylation in rats with type 2 diabetes.

OBJECTIVE: To investigate intracerebral insulin resistance and its relationship with tau-protein hyperphosphorylation. METHODS: A rat model of type 2 diabetes (T2D) was established with streptozotocin (STZ). Diabetic rats received intragastric administration of pioglitazone (PIO group) or normal saline (T2D group) for 4 weeks. As a control, non-diabetic rats received intragastric normal saline (CTL group). The insulin concentrations in cerebrospinal fluid (CSF) and blood were determined with radioimmunoassay, and blood glucose concentration was determined using a glucose oxidation technique. Total and phosphorylated levels of protein kinase B (AKT), glycogen synthase kinase-3ß (GSK-3ß) and tau-protein in the hippocampus were analyzed using western blotting. RESULTS: The plasma insulin level in the T2D group was higher, and the CSF insulin level in the T2D group lower than in the CTL group. Hippocampal phosphorylated AKT and phosphorylated GSK-3ß levels were significantly lower in the T2D group than in the CTL group. Hippocampal tau-protein in the T2D group was hyperphosphorylated at Ser199 and Ser396. Plasma insulin levels in the PIO group were lower than in the T2D group, with no differences in CSF insulin levels. Phosphorylated AKT and phosphorylated GSK-3ß levels in the PIO group were significantly higher than in the T2D group Hippocampal phosphorylated tau-protein (Ser199/Ser396) was lower in the PIO group than in the T2D group. CONCLUSION: Hyperphosphorylation of tau-protein in pioglitazone-treated rats with T2D was improved. Rats with T2D have both cerebral insulin resistance and cerebral hypoinsulinism. Pioglitazone can ameliorate intracerebral insulin resistance and decrease tau-protein hyperphosphorylation, but cannot increase intracerebral insulin levels.

Glycogen synthase kinase 3 in chronic rhinosinusitis: two faces of a single enzyme in one disease.

BACKGROUND: The origin and pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) remain unclear. Glycogen synthase kinase 3 (GSK-3) is a unique multitasking kinase involved in the regulation of inflammation and apoptosis and is an important messenger in the downstream signaling of interleukin 6. OBJECTIVE: To analyze the possible role of GSK-3 in the pathogenesis of CRSwNP. METHODS: We examined tissue samples of nasal polyps and the inferior turbinate of patients with CRSwNP and the inferior turbinate of individuals without chronic sinusitis (healthy mucosa). Expression levels of GSK-3 and its inactivated form phosphorylated GSK-3 (pGSK-3) were analyzed using DNA microarray, protein array, Western hybridization, and immunohistochemical analysis. RESULTS: We found increased expression of GSK-3 in both the nasal polyps and the inferior turbinate of patients with CRSwNP compared with those with healthy mucosa (P < .01). We did not observe a difference between nasal polyps and the inferior turbinate of patients with CRSwNP, but a highly significant increase in the phosphorylation rate of GSK-3 was detected in the tissue of nasal polyps compared with the turbinates of patients with CRSwNP (P < .01). CONCLUSION: GSK-3 may play a crucial role in the inflammatory process in CRSwNP. Nasal polyps originate mainly in the mucosa of the middle meatus of the nose and rarely occur in the region of the inferior turbinate. The inhibition of GSK-3 by phosphorylation in nasal polyps, in contrast to the inferior turbinate, is a possible explanation for the different behavior of the mucosa of the middle meatus and the inferior turbinate.

[Expression of RAGE in Helicobacter pylori infested gastric biopsies]. / Lesiones gástricas en pacientes infectados con Helicobacter pylori: expresión de RAGE (receptor de productos de glicosilización avanzada) y otros inmunomarcadores.

BACKGROUND: Inflammation is a common phenomenon present in gastric mucosa of patients infected with H. pylori. Activation of the RAGE/multiligand axis is thought to be a relevant factor in cancer-mediated inflammation. RAGE is a membrane receptor, belonging to the immunoglobulin family, and the over-expression of RAGE has been associated with increased invasiveness and metastasis generation in different types of cancer, including gastric cancer. Furthermore recent experiences show that the use of its soluble form (sRAGE) or silencing of the gene coding for this receptor could provide therapeutic benefits in cancer. AIM: To evaluate the immunohistochemical expression of RAGE, MUC-1, ß-Catenin free and phosphorylated, Cyclin-D1 and GSK3 in gastric biopsy specimens infected with H. pylori. MATERIAL AND METHODS: Immunohistochemical analysis was carried out in gastric biopsies from 138 patients: 55 with inflammatory injury (no atrophic gastritis), 42 with pre-cancerous conditions (atrophy or intestinal metaplasia) and 41 with dysplastic lesions or in situ adenocarcinoma. RESULTS: There was a high rate of positive RAGE expression in the three groups of biopsies. Biopsies with dysplasia or in situ carcinoma had a significantly higher percentage of RAGE expression than the other groups of biopsies. CONCLUSIONS: The increased RAGE expression reported in both dysplasia and incipient cancer support the role of the multiligand/RAGE axis in gastric carcinogenesis.

Noxious stimulation attenuates ketamine-induced neuroapoptosis in the developing rat brain.

BACKGROUND: Ketamine induces neuroapoptosis in neonatal rodents. However, these experimental paradigms were performed without concurrent noxious stimulation, a condition that does not reflect the interaction of anesthesia and surgical stimulation. Noxious stimulation with and without concurrent analgesic drugs has been shown to have divergent patterns of neuronal activation and cell death. We hypothesized that concurrent noxious stimulation would attenuate ketamine-induced caspase-3 activation. METHODS: Postnatal day 7 Sprague-Dawley rat pups were randomized to a 6-h exposure to ketamine with and without peripheral noxious stimulation by intraplantar injection of complete Freund's adjuvant. A cohort of naïve rat pups with and without complete Freund's adjuvant injections served as control subjects. Neuroapoptosis was measured by cleaved caspase-3 expression and terminal deoxynucleotidyl-transferase mediated 2'-deoxyuridine 5'-triphosphate nick end labeling staining. In order to determine if concurrent noxious simulation altered the expression of cell survival and cell cycle proteins, levels of protein kinase B and glycogen synthase kinase-3ß and cyclin D1 were measured. RESULTS: Ketamine induced a significant increase in cleaved caspase-3 expression and terminal deoxynucleotidyl-transferase mediated 2'-deoxyuridine 5'-triphosphate nick end labeling staining with increases in cyclin D1 levels. Concurrent noxious stimulation with ketamine attenuated caspase-3 activation and maintained cyclin D1 levels. Phosphorylation of protein kinase B and glycogen synthase kinase-3ß was not definitively altered under these conditions. CONCLUSION: The administration of ketamine with concurrent noxious stimulation results in the attenuation of the neuroapoptotic response. These findings suggest that concurrent surgery and procedural pain attenuates ketamine-induced neuroapoptosis.

Effects of herpes simplex virus type 1 infection on immune functions of human neutrophils.

BACKGROUND AND OBJECTIVE: Herpesviruses may play roles in the development of periodontal diseases. This study analyzed the effects of herpes simplex virus type 1 (HSV-1) infection on neutrophil function. The effects of lipopolysaccharide (LPS) from the periodontal pathogen, Porphyromonas gingivalis, during HSV-1 infection were also determined. MATERIAL AND METHODS: Purified HSV-1 was pretreated with buffer containing no serum, with HSV-1 immunoglobulin G (IgG)-positive serum (HSV-1 antiserum) or with control serum. Neutrophils were mock-infected or infected with the pretreated HSV-1. Viral binding and phagosome formation were detected using immunostaining. Intracellular reactive oxygen species (ROS) were determined using 2',7'-dichlorofluorescin diacetate and fluorometry. Leukotriene B(4) (LTB(4)) and interleukin-8 (IL-8) were detected using enzyme immunoassays. Release of matrix metalloproteinase-9 (MMP-9) was examined using gelatin zymography. Phosphorylation of Akt/glycogen synthase kinase-3 (GSK-3) was determined using western blotting. RESULTS: HSV-1 bound directly to neutrophils and enhanced the release of MMP-9. HSV-1 immune complexes, formed in the HSV-1 antiserum, bound neutrophils and induced the formation of early phagosome more effectively than did HSV-1 alone. The relative levels of ROS and phosphorylation of Akt/GSK-3 were increased significantly in neutrophils after infection with HSV-1 immune complexes. Infection with HSV-1 and HSV-1 immune complexes also stimulated the production of inflammatory mediators, LTB(4) and IL-8. Moreover, LPS enhanced the HSV-1-stimulatory production of IL-8. CONCLUSION: This study demonstrated differences in neutrophils infected with HSV-1 alone or with HSV-1 immune complexes, suggesting that opsonization of HSV-1 might enhance its effects on neutrophils. The in vitro findings suggest that HSV-1 infection may induce the inflammatory response and affect periodontal health.

Aberrant expression of ß-catenin and its association with ΔNp63, Notch-1, and clinicopathological factors in oral squamous cell carcinoma.

The present study focuses on the correlation between the expression pattern of ß-catenin (component of Wnt signaling), ΔNp63 (proliferation marker), and Notch 1 (transmembrane receptor) in oral squamous cell carcinoma. The study also aims to investigate the interaction between ß-catenin and ΔNp63 in oral cancer. Furthermore, we also analyzed the prognostic significance of ß-catenin, ΔNp63, and Notch 1 in oral squamous cell carcinoma. Immunohistochemical analysis of ß-catenin, ΔNp63, and Notch 1 were done in 62 cases of oral squamous cell carcinoma. Co-immunoprecipitation analysis was done to study the possible interaction between ß-catenin and ΔNp63 in oral cancer. Kaplan-Meier method was used to estimate overall and disease-free survival, and the Log-rank test was used to compare the resulting curves. Statistically significant positive correlation was found between the localization of ß-catenin and the expression of ΔNp63 (p = 0.001**, r (s) = 0.427), whereas, no significant association was found between the expression pattern of ß-catenin and Notch 1. Interestingly, interaction between ß-catenin and ΔNp63 was observed in oral carcinoma. Moreover, ß-catenin and ΔNp63 may be related to worst survival in oral carcinoma. Statistically significant positive association between localization of ß-catenin and expression of ΔNp63 suggests that they might have dependent roles in maintaining the proliferation of oral carcinoma cells. In addition, the downregulated expression of Notch 1 was related to invasion and differentiation status of oral carcinoma cells. Furthermore, ß-catenin and ΔNp63 may be used as independent prognostic markers of oral carcinoma. On the other hand, interaction of ß-catenin with ΔNp63 may be a key event in maintaining the proliferation of oral carcinoma cells. The present study indicates that ß-catenin and ΔNp63 may be used as independent prognostic markers of oral carcinoma and the interaction of ß-catenin with ΔNp63 may be a crucial event in regulating proliferation and differentiation of oral carcinoma cells, which may be used as a target for therapeutic implications.

Frat is a phosphatidylinositol 3-kinase/Akt-regulated determinant of glycogen synthase kinase 3ß subcellular localization in pluripotent cells.

Suppressing the activity of Gsk3ß is critical for maintenance of murine pluripotent stem cells. In murine embryonic stem cells (mESCs), Gsk3ß is inhibited by multiple mechanisms, including its inhibitory phosphorylation on serine 9 by protein kinase B (Akt), a major effector of the canonical phosphatidylinositol 3-kinase (PI3K) pathway. A second PI3K/Akt-regulated mechanism promotes the nuclear export of Gsk3ß, thereby restricting its access to nuclear substrates such as c-myc and ß-catenin. Although Gsk3ß shuttles between the nucleus and cytoplasm under self-renewing conditions, its localization is primarily cytoplasmic because its rate of nuclear export exceeds its rate of nuclear import. In this report, we show that Gsk3ß is exported from the nucleus in a complex with Frat. Loss of PI3K/Akt activity results in dissociation of this complex and retention of Gsk3ß in the nucleus. Frat continues to shuttle between the nucleus and cytoplasm under these conditions and remains predominantly in the cytoplasm. These results indicate that Frat carries Gsk3ß out of the nucleus under self-renewing conditions and that PI3K regulates this by promoting its association with Frat. These findings provide new links between PI3K/Akt signaling and regulation of Gsk3ß activity by Frat, an oncogene previously shown to cooperate with Myc in tumorigenesis.

STAT3 and beta-catenin signaling pathway may affect GSK-3beta expression in hepatocellular carcinoma.

BACKGROUND/AIMS: To study the correlation and significance of beta-catenin, STAT3 and GSK-3beta signaling pathway in hepatocellular carcinoma (HCC). METHODOLOGY: The HCC cell line HepG2 was transfected with small interfering RNA (siRNA) directed against 8-catenin or STAT3. After 72 and 96h, protein was extracted and the protein expression of beta-catenin, STAT3, and GSK-3beta was detected by Western blot analysis. RESULTS: After siRNA directed against beta-catenin was transfected into HepG2 cells, beta-catenin protein expression was decreased at 72 and 96h, GSK-3beta and p-GSK-3beta protein expression increased gradually at 72 and 96h, and STAT3 protein expression showed no change following transfection. After siRNA directed against STAT3 was transfected into HepG2 cells, STAT3 protein expression was decreased at 72 and 96h and beta-catenin, GSK-3beta and p-GSK-3beta protein expression all increased at 72h and decreased at 96 h after transfection. CONCLUSION: In HCC, the beta-catenin signaling pathway may regulate GSK-3beta protein expression and the STAT3 signaling pathway may regulate beta-catenin and GSK-3beta protein expression, thereby playing key roles during HCC genesis and development.

Development of a cellular tau enzyme-linked immunosorbent assay method for screening GSK-3ß inhibitors.

Glycogen synthase kinase-3ß (GSK-3ß), a serine/threonine kinase also known as tau protein kinase I, has been implicated in the pathogenic conditions of Alzheimer's disease. Many investigators have focused on GSK-3 inhibitor as a therapeutic drug. In this study, we established a cell-based assay for the screening of novel GSK-3ß inhibitors. For this purpose, four-repeat tau cDNAs were stably expressed in human embryonic kidney 293 (HEK293) cells (HEK293-Tau). The proliferation of HEK293-Tau cells was no different from that of HEK293 cells, as measured by the bromodeoxyuridine enzyme-linked immunosorbent assay (BrdU ELISA). The concentration-dependent reduction of tau phosphorylation by GSK-3 inhibitors, LiCl, Chir98023, and SB415286, was examined by immunoblot analysis and Tau ELISA (in situ ELISA). Highly consistent data were obtained, suggesting that this novel ELISA method is highly reproducible. Using this ELISA strategy, we isolated a few candidate compounds, including compounds 114 and 149, from several hundreds of synthetic agents and demonstrated that such candidates protect nerve growth factor-differentiated PC12 cells against amyloid-ß-induced cell death. These data indicate that this Tau ELISA method in HEK293-Tau cells may be a suitable cell-based assay system to screen for GSK-3ß inhibitors.

The effects of estradiol and catecholestrogens on uterine glycogen metabolism in mink (Neovison vison).

Glycogen is a uterine histotroph nutrient synthesized by endometrial glands in response to estradiol. The effects of estradiol may be mediated, in part, through the catecholestrogens, 2-hydroxycatecholestradiol (2-OHE2) and 4-hydroxycatecholestradiol (4-OHE2), produced by hydroxylation of estradiol within the endometrium. Using ovariectomized mink, our objectives were to determine the effects of estradiol, 4-OHE2, and 2-OHE2 on uterine: 1) glycogen concentrations and tissue localization; 2) gene expression levels for glycogen synthase, glycogen phosphorylase, and glycogen synthase kinase-3B; and 3) protein expression levels for glycogen synthase kinase-3B (total) and phospho-glycogen synthase kinase-3B (inactive). Whole uterine glycogen concentrations (mean ± SEM, mg/g dry wt) were increased by estradiol (43.79 ± 5.35), 4-OHE2 (48.64 ± 4.02), and 2-OHE2 (41.36 ± 3.23) compared to controls (4.58 ± 1.16; P ≤ 0.05). Percent glycogen content of the glandular epithelia was three-fold greater than the luminal epithelia in response to estradiol and 4-OHE2 (P ≤ 0.05). Expression of glycogen synthase mRNA, the rate limiting enzyme in glycogen synthesis, was increased by 4-OHE2 and 2-OHE2 (P ≤ 0.05), but interestingly, was unaffected by estradiol. Expression of glycogen phosphorylase and glycogen synthase kinase-3B mRNAs were reduced by estradiol, 2-OHE2, and 4-OHE2 (P ≤ 0.05). Uterine phospho-glycogen synthase kinase-3B protein was barely detectable in control mink, whereas all three steroids increased phosphorylation and inactivation of the enzyme (P ≤ 0.05). We concluded that the effects of estradiol on uterine glycogen metabolism were mediated in part through catecholestrogens; perhaps the combined actions of these hormones are required for optimal uterine glycogen synthesis in mink.

VHL mutations and dysregulation of pVHL- and PTEN-controlled pathways in multilocular cystic renal cell carcinoma.

Multilocular cystic renal cell carcinoma is a rare renal cell carcinoma with an excellent prognosis. To clarify the relationship with typical clear cell renal cell carcinoma, we evaluated 15 cases of multilocular cystic renal cell carcinomas diagnosed according to the 2004 WHO classification. Von Hippel Lindau (VHL) gene mutations were determined by whole genome amplification and direct sequencing. Carbonic anhydrase 9 (CAIX), a hypoxia-inducible factor (HIF) target, paired box gene 2 (PAX2), cyclin-dependent kinase inhibitor p27 and glycogen synthase kinase 3-ß (GSK3ß) were immunohistochemically evaluated as members of the VHL protein (pVHL)- and phosphatase and tensin homolog (PTEN)-controlled pathways. VHL mutations were identified in 3 of 12 (25%) tumors. Inactivated GSK3ß, decreased PTEN expression and PAX2 positivity were observed in the vast majority of the multilocular cystic renal cell carcinomas. Strong nuclear staining of p27 was seen in 14 of 15 cases. Compared with multilocular cystic renal cell carcinomas, expression frequencies of PAX2, p-GSK3ß, PTEN and CAIX were similar in a set of low-grade, early-stage clear cell renal cell carcinomas, whereas only 30% had strong p27 positivity. These results are consistent with the hypothesis that multilocular cystic renal cell carcinomas are related at the molecular level with clear cell renal cell carcinomas. Maintenance of a strong subcellular p27 expression in all multilocular cystic renal cell carcinomas analyzed may in part explain the excellent prognosis of these tumor patients.

The EDD E3 ubiquitin ligase ubiquitinates and up-regulates beta-catenin.

Wnt/ß-catenin signaling plays a central role in development and is also involved in a diverse array of diseases. ß-Catenin activity is tightly regulated via a multiprotein complex that includes the kinase glycogen synthase kinase-3ß (GSK-3ß). GSK-3ß phosphorylates ß-catenin, marking it for ubiquitination and degradation via the proteasome. Thus in regulation of the Wnt pathway, the ubiquitin system is known to be involved mostly in mediating the turnover of ß-catenin, resulting in reduced Wnt signaling levels. Here we report that an arm of the ubiquitin system increases ß-catenin protein levels. We show that GSK-3ß directly interacts with the E3 ubiquitin ligase identified by differential display (EDD) that also binds ß-catenin. Expression of EDD leads to enhanced nuclear accumulation of both GSK-3ß and ß-catenin and results in up-regulation of ß-catenin expression levels and activity. Importantly, EDD ubiquitinates ß-catenin through Lys29- or Lys11-linked ubiquitin chains, leading to enhanced stability of ß-catenin. Our results demonstrate a role for the ubiquitin system in up-regulation of the Wnt signaling pathway, suggesting that EDD could function as a colorectal oncogene.