We have recently shown that silencing of the brain/islet specific c-Jun N-terminal Kinase3 (JNK3) isoform enhances both basal and cytokine-induced beta-cell apoptosis, whereas silencing of JNK1 or JNK2 has opposite effects. While it is known that JNK1 or JNK2 may promote apoptosis by inhibiting the activity of the pro-survival Akt pathway, the effect of JNK3 on Akt has not been documented. This study aims to determine the involvement of individual JNKs and specifically JNK3 in the regulation of the Akt signaling pathway in insulin-secreting cells. JNK3 silencing strongly decreases Insulin Receptor Substrate 2 (IRS2) protein expression, and blocks Akt2 but not Akt1 activation by insulin, while the silencing of JNK1 or JNK2 activates both Akt1 and Akt2. Concomitantly, the silencing of JNK1 or JNK2, but not of JNK3, potently phosphorylates the glycogen synthase kinase3 (GSK3β). JNK3 silencing also decreases the activity of the transcription factor Forkhead BoxO3A (FoxO3A) that is known to control IRS2 expression, in addition to increasing c-Jun levels that are known to inhibit insulin gene expression. In conclusion, we propose that JNK1/2 on one hand and JNK3 on the other hand, have opposite effects on insulin-signaling in insulin-secreting cells; JNK3 protects beta-cells from apoptosis and dysfunction mainly through maintenance of a normal IRS2 to Akt2 signaling pathway. It seems that JNK3 mediates its effects mainly at the transcriptional level, while JNK1 or JNK2 appear to mediate their pro-apoptotic effect in the cytoplasm.

Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) can differentiate into multiple lineages including osteogenic and adipogenic cells. Wnt signalling has been implicated in controlling BMSC fate, but the mechanisms are unclear and apparently conflicting data exist. Here we show that a novel glycogen synthase kinase 3β inhibitor, AR28, is a potent activator of canonical Wnt signalling using in vitro β-catenin translocation studies and TCF-reporter assays. In vivo, AR28 induced characteristic axis duplication and secondary regions of chordin expression in Xenopus laevis embryos. Using human BMSCs grown in adipogenic medium, we confirmed that AR28-mediated Wnt signalling caused a significant (p<0.05) dose-dependent reduction of adipogenic markers. In osteogenic media, including dexamethasone, AR28 caused significant (p<0.05) decreases in alkaline phosphatase (ALP) activity compared to vehicle controls, indicative of a reduced osteogenic response. However, when excluding dexamethasone from the osteogenic media, increases in both ALP and mineralisation were identified following AR28 treatment, which was blocked by mitomycin C. Pre-treatment of BMSCs with AR28 for 7 days before osteogenic induction also increased ALP activity and mineralisation. Furthermore, BMP2-induced osteogenic differentiation was strongly enhanced by AR28 addition within 3 days, but without concomitant changes in cell number, therefore revealing BMP-dependent and independent mechanisms for Wnt-induced osteogenesis.

Earlier reports suggest a link between mitochondrial dysfunction and development of hepatic insulin resistance. Here we used a murine model heterozygous (HET) for a mitochondrial trifunctional protein (MTP) gene defect to determine if a primary defect in mitochondrial long-chain fatty acid oxidation disrupts hepatic insulin action. Hyperinsulinemic-euglycemic clamps and signaling studies were performed for assessment of whole-body and hepatic insulin resistance/signaling. In addition, hepatic fatty acid oxidation and hepatic insulin action were assessed in vitro using primary hepatocytes isolated from HET and wildtype (WT) mice. In both hepatic mitochondria and isolated primary hepatocytes, heterozygosity of MTP caused an ∼50% reduction in mitochondrial fatty acid oxidation, a significantly impaired glucose disposal during the insulin clamp, and a markedly lower insulin-stimulated suppression of hepatic glucose production. HET mice also exhibited impaired insulin signaling, with increased hepatic phosphorylation of IRS2 (ser731) and reduced Akt phosphorylation (ser473) in both hepatic tissue and isolated primary hepatocytes. Assessment of insulin-stimulated FOXO1/phospho-FOXO1 protein content and PEPCK/G6Pase messenger RNA (mRNA) expression did not reveal differences between HET and WT mice. However, insulin-induced phosphorylation of GSK3β was significantly blunted in HET mice. Hepatic insulin resistance was associated with an increased methylation status of the catalytic subunit of protein phosphatase 2A (PP2A-C), but was not associated with differences in hepatic diacylglycerol content, activated protein kinase C-ϵ (PKC-ϵ), inhibitor κB kinase β (IKK-β), c-Jun N-terminal kinase (JNK), or phospho-JNK protein contents. Surprisingly, hepatic ceramides were significantly lower in the HET mice compared with WT.

CONCLUSIONS

A primary defect in mitochondrial fatty acid β-oxidation causes hepatic insulin resistance selective to hepatic glycogen metabolism that is associated with elevated methylated PP2A-C, but independent of other mechanisms commonly considered responsible for insulin resistance. (HEPATOLOGY 2013;).

Compared with the rodent, the domestic pig is a much better animal model for studying adipogenesis and obesity-related diseases. Currently, the role of Akt2 and Sirt1 in porcine adipogenesis remains elusive. In this study, we defined the effect of Akt2 and Sirt1 on porcine preadipocyte lipogenesis and the regulatory mechanism. First, we found that Akt2 was widely expressed in porcine various tissues and at high level in adipose tissue. Further analysis showed that the expression level of Akt2 was much higher in adipose tissue and adipocytes of the Bamei pig breed (a Chinese indigenous fatty pig) than in that of the Large White pig breed (a Lean type pig), whereas the level of Sirt1 expression was opposite. The expression levels of Sirt1 and Akt2 gradually increased during adipogenic differentiation. Adipogenesis was robustly inhibited in Akt2 deficient fat cells, whereas it was promoted in Sirt1 deficient cells using the lentiviral-mediated shRNA approach. Interestingly, adipogenesis returned to normal in Akt2 and Sirt1 dual-deficient cells, showing that the pro- and anti-adipogenic effects were balanced. Sirt1 inhibited transcriptional activity of Akt2 in a dose-dependent way. Interaction of endogenous Akt2 and Sirt1 was gradually enhanced before day 6 of differentiation, and then attenuated. Akt2 and Sirt1 also interacted with C/EBPα in adipocytes. Moreover, knockdown of Akt2 or/and Sirt1 affected pro-lipogenesis of insulin-stimulated by PI3K/Akt pathway. We further found that Sirt1 respectively interacted with PI3K and GSK3β which were key upstream and downstream components of PI3K/Akt pathway. Based on the above findings, we concluded that the crosstalk between C/EBPα and PI3K/Akt signaling pathways is implicated in Akt2 and Sirt1 regulation of adipogenesis.

Glycogen synthase kinase-3 (GSK3) is a constitutively active protein kinase that is involved in neuronal regulation and is a potential pharmacological target of neurological disorders. We found previously that GSK3β selectively interacts with 5-hydroxytryptamine-1B receptors (5-HT1BR) that have important functions in serotonin neurotransmission and behavior. In this study, we provide new information supporting the importance of GSK3β in 5-HT1BR-regulated signaling, physiological function, and behaviors. Using molecular, biochemical, pharmacological, and behavioral approaches, we tested 5-HT1BR's interaction with G(i)α(2) and β-arrestin2 and 5-HT1BR-regulated signaling in cells, serotonin release in mouse cerebral cortical slices, and behaviors in wild-type and β-arrestin2 knockout mice. Molecular ablation of GSK3β and GSK3 inhibitors abolished serotonin-induced change of 5-HT1BR coupling to G(i)α(2) and associated signaling but had no effect on serotonin-induced recruitment of β-arrestin2 to 5-HT1BR. This effect is specific for 5-HT1BR because GSK3 inhibitors did not change the interaction between serotonin 1A receptors and G(i)α(2). Two GSK3 inhibitors, N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418) and 3-(5-bromo-1-methyl-1H-indol-3-yl)-4-(benzofuran-3-yl)pyrrole-2,5-dione (BIP-135), efficiently abolished the inhibitory effect of the 5-HT1BR agonist anpirtoline on serotonin release in mouse cerebral cortical slices. GSK3 inhibitors also facilitated the 5-HT1BR agonist anpirtoline-induced behavioral effect in the tail suspension test but spared anpirtoline-induced locomotor activity. These results suggest that GSK3β is a functional selective modulator of 5-HT1BR-regulated signaling, and GSK3 inhibitors fine-tune the physiological and behavioral actions of 5-HT1BR. Future studies may elucidate the significant roles of GSK3 in serotonin neurotransmission and implications of GSK3 inhibitors as functional selective modulators of 5-HT1BR.

Haploinsufficiency for the p53 family member p73 causes behavioral and neuroanatomical correlates of neurodegeneration in aging mice, including the appearance of aberrant phospho-tau-positive aggregates. Here, we show that these aggregates and tau hyperphosphorylation, as well as a generalized dysregulation of the tau kinases GSK3β, c-Abl, and Cdk5, occur in the brains of aged p73+/- mice. To investigate whether p73 haploinsufficiency therefore represents a general risk factor for tau hyperphosphorylation during neurodegeneration, we crossed the p73+/- mice with 2 mouse models of neurodegeneration, TgCRND8+/Ø mice that express human mutant amyloid precursor protein, and Pin1-/- mice. We show that haploinsufficiency for p73 leads to the early appearance of phospho-tau-positive aggregates, tau hyperphosphorylation, and activation of GSK3β, c-Abl, and Cdk5 in the brains of both of these mouse models. Moreover, p73+/-;TgCRND8+/Ø mice display a shortened lifespan relative to TgCRND8+/Ø mice that are wild type for p73. Thus, p73 is required to protect the murine brain from tau hyperphosphorylation during aging and degeneration.

This study was conducted to test whether oxidative stress activates the intracellular protein kinase B (AKT1) signaling pathway, which culminates with cardiac hypertrophy in experimental hyperthyroidism. Male Wistar rats were divided into four groups: control, vitamin E, thyroxine (T(4)), and T(4)+vitamin E. Hyperthyroidism was induced by T(4) administration (12 mg/l in drinking water for 28 days). Vitamin E treatment was given during the same period via s.c. injections (20 mg/kg per day). Morphometric and hemodynamic parameters were evaluated at the end of the 4-week treatment period. Protein oxidation, redox state (reduced glutathione, GSH/glutathione dissulfide, GSSG), vitamin C, total radical-trapping antioxidant potential (TRAP), hydrogen peroxide (H2O2), and nitric oxide metabolites (NO(X)) were measured in heart homogenates. The p-AKT1/AKT1 ratio, p-glycogen-synthase kinase (GSK)3B/GSK3B ratio, FOS, and JUN myocardial protein expression were also quantified by western blot after 4 weeks. Increases in biochemical parameters, such as protein oxidation (41%), H2O2 (62%), and NO(X) (218%), and increase in the left ventricular end-diastolic pressure were observed in the T(4) group. T(4) treatment also caused a decrease in GSH/GSSG ratio (83%), vitamin C (34%), and TRAP (55%). These alterations were attenuated by vitamin E administration to the hyperthyroid rats. Expression of p-AKT1/AKT1, p-GSK3B/GSK3B, FOS, and JUN were elevated in the T(4) group (by 69, 37, 130, and 33% respectively), whereas vitamin E administration promoted a significant reduction in their expression. These results indicate that oxidative stress plays an important role in cardiac hypertrophy, and suggest redox activation of AKT1 and JUN/FOS signaling pathways with H2O2 acting as a possible intracellular mediator in this adaptive response to experimental hyperthyroidism.

Growth hormone releasing hormone (GHRH) antagonists have been developed for the treatment of various cancers. We investigated the effects of a novel GHRH antagonist, MIA-602, on nine breast cancer cell lines, differing in their expression for estrogen-, progesterone- and HER-2 receptors. We detected the presence of pituitary-type GHRH receptors (pGHRH-R) on 6 of the 9 breast cancer cell lines. The main splice variant of pGHRH-R, SV1, was found on all 9 cell lines. MTT assay showed that following treatment with MIA-602, cell viability decreased significantly in all 9 cell lines. The reduction in cell viability was greater in cells positive for both pGHRH-R and SV1, than in cells positive for only SV1, but the difference was not significant. Using Western blotting, we demonstrated that the levels of phospho-Akt, -GSK3β and -ERK1/2 decreased significantly following exposure to MIA-602 and the level of phospho-p38 increased after treatment. The reduction of the phosphorylated anti-apoptotic proteins was significantly greater in cells where both pGHRH-R and SV1 were present, than where only SV1 was expressed. In conclusion, our study shows that MIA-602 is effective against a wide range of breast cancer cells in vitro, independently of their receptor positivity, suggesting the potential use of GHRH antagonists also in the treatment of triple-negative breast cancer. The effect of MIA-602 was mediated nearly as well in tumors that expressed only the SV1 receptor compared to those in which both SV1 and pGHRH-R were present, although a difference could be detected at the level of cell signaling.

Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP(+)-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3β (GSK3β) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP(+)-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP(+)-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.

Ethanol exposure induces neurodegeneration in the developing central nervous system (CNS). Fetal alcohol spectrum disorders (FASD) are caused by ethanol exposure during pregnancy and are the most common nonhereditary cause of mental retardation. It is important to identify agents that provide neuroprotection against ethanol neurotoxicity. Multiple mechanisms have been proposed for ethanol-induced neurodegeneration, and oxidative stress is one of the most important mechanisms. Recent evidence indicates that glycogen synthase kinase 3β (GSK3β) is a potential mediator of ethanol-mediated neuronal death. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is a potent natural antioxidant. Our previous study suggested that C3G inhibited GSK3β activity in neurons. Using a third trimester equivalent mouse model of ethanol exposure, we tested the hypothesis that C3G can ameliorate ethanol-induced neuronal death in the developing brain. Intraperitoneal injection of C3G reduced ethanol-meditated caspase-3 activation, neurodegeneration, and microglial activation in the cerebral cortex of 7-day-old mice. C3G blocked ethanol-mediated GSK3β activation by inducing phosphorylation at serine 9 while reducing the phosphorylation at tyrosine 216. C3G also inhibited ethanol-stimulated expression of malondialdehyde (MDA) and p47phox, indicating that C3G alleviated ethanol-induced oxidative stress. These results provide important insight into the therapeutic potential of C3G.

Nasopharyngeal carcinoma (NPC) is endemic to Asia and over 40 % of NPC tissues harbor PIK3CA amplifications. This study characterized the preclinical activity of MK-2206, an oral allosteric inhibitor of AKT in 6 NPC cell lines: C666-1, HK1, HONE-1-EBV, HONE-1, CNE-2 and HNE-1. Exposure to increasing concentrations of MK-2206 resulted in over 95 % of growth inhibition in all NPC cell lines with IC50 values in the low micromolar range. Further experiments were performed in 3 representative NPC cell lines: CNE-2 (harbor PIK3CA mutation and most sensitive to MK-2206), C666-1 (carries PIK3CA amplification), and HONE-1-EBV (least sensitive to MK-2206). MK-2206 induced G0/G1 cycle arrest in all 3 cell lines, but could induce apoptosis only in CNE-2 cells. MK-2206 significantly abrogated AKT signaling in all 3 cell lines by inhibiting the activation of AKT and its downstream effectors (FKHR, GSK3β and BAD). MK-2206 also reduced mTOR signaling by reducing activation of mTOR and its downstream 4E-BP1 and p70S6 kinase. MAPK activation was observed in HONE-1 and C666-1 cells, but not in CNE-2 cells following exposure to MK-2206. The addition of MK-2206 to cisplatin (but not with paclitaxel) has a supra-additive inhibitory effect on growth in vitro. In summary, MK-2206 can inhibit growth and abrogate AKT and mTOR signaling in NPC cell lines. This agent is currently being evaluated in a phase II study in metastatic NPC.

β-Amyloid (Aβ) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2-coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac-tau) and tau phosphorylation (P-tau) by inhibiting activation of P300 and GSK3β. Aβ was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aβ accumulation was accompanied by increased Ac-tau and P-tau levels. Concomitantly, these cells showed increased P300 and GSK3β P-Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3-30 μM) and resveratrol (20 μM). Moreover, decreased expression of SIRT1 and its activity by Aβ were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 μM, PKA inhibitor), TBCA (20 μM, inhibitor of CK2), and sirtinol (20 μM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP-dependent protein kinase-linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau-related neurodegeneration in the AD brain.

This study was designed to investigate whether rosuvastatin could attenuate monocrotaline-induced pulmonary hypertension via regulation of Akt/eNOS signaling pathway and asymmetric dimethylarginine (ADMA) metabolism in rats. After a single-dose injection of monocrotaline (60 mg/kg), oral administration of rosuvastatin (5mg/kg) was started from day 1 to day 28 (preventive administration) or from day 15 to day 28 (therapeutic administration), or with vehicle as corresponding controls. 28 days after monocrotaline, significant pulmonary hypertension characterized by pulmonary arterial medial wall thickening, right ventricular hypertrophy and right heart failure was observed. Rosuvastatin (5mg/kg, for 14 days and 28 days) treatment significantly attenuated monocrotaline-induced pulmonary vascular remodeling, right ventricular hypertrophy and dysfunction, and normalized the down-regulated pulmonary Akt/p-Akt and eNOS/p-eNOS expressions, while increased DDAH2 expression accompanied by decreased serum level of ADMA. However expression of PRMT1 and GSK3β/p-GSK3β did not differ among all groups (all P>0.05). We concluded that rosuvastatin inhibits monocrotaline-induced pulmonary hypertension through normalization of Akt, eNOS and DDAH2 expressions, and decreasing the level of ADMA.

Skin cancer is the most common cancer in the United States. Ultraviolet B (UVB) radiation in sunlight is the major environmental factor causing skin cancer. p21, a p53-inducible protein, plays an important role in cell cycle, DNA repair, and apoptosis. Here we have investigated the effect of UVB radiation on p21 and its molecular mechanisms and function in human HaCaT keratinocytes, which we used as a premalignant cellular model because normal skin harbors numerous clones of p53-mutated keratinocytes. We found that in human HaCaT keratinocytes UVB induces rapid p21 down-regulation via a proteasomal degradation mechanism. In p53-defective HaCaT cells, the p21 protein levels remain decreased at a later time post-UVB, but in normal human and mouse epidermal keratinocytes with wild-type p53 the p21 levels are initially reduced but later increase post-UVB. These findings indicate that loss of p53 function leads to sustained p21 down-regulation in response to UVB damage. Degradation of p21 following UVB radiation does not require ATR, ATM, or both, because either the ATR/ATM inhibitor caffeine or siRNA knockdown of ATR, ATM, or both failed to reverse p21 degradation. However, inhibiting MDM2 or GSK3β partially reduced UVB-induced p21 degradation, while inhibiting both enzymes completely prevented it. Restoring the p21 protein levels in UVB-irradiated keratinocytes reduced apoptosis. Although at the molecular level increasing p21 expression has no effect on the protein levels of the Bcl-2 family members, it enhances the activation of AKT, a critical survival pathway to protect cells from apoptosis. Our results suggest a distinct mechanism of p21 degradation in keratinocytes by UVB, and this p21 degradation may significantly enhance UVB-induced apoptosis of premalignant keratinocytes with a p53 defect to eliminate damaged cells and therefore prevent skin cancer development.

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.

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.

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.

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.

We previously demonstrated that, in nasal epithelial cells (NECs) from smokers, methylation of an antiviral gene was associated with impaired antiviral defense responses. To expand these findings and better understand biological mechanisms underlying cigarette smoke (CS)-induced modifications of host defense responses, we aimed to compare DNA methylation of genes that may play a role in antiviral response. We used a two-tiered analytical approach, where we first implemented a genome-wide strategy. NECs from smokers differed in the methylation levels of 390 genes, the majority (84%) of which showed decreased methylation in smokers. Secondly, we generated an a priori set of 161 antiviral response-related genes, of which five were differentially methylated in NEC from smokers (CCL2, FDPS, GSK3B, SOCS3, and ULBP3). Assessing these genes at the systems biology level revealed a protein interaction network associated with CS-induced epigenetic modifications involving SOCS3 and ULBP3 signaling, among others. Subsequent confirmation studies focused on SOCS3 and ULBP3, which were hypomethylated and hypermethylated, respectively. Expression of SOCS3 was increased, whereas ULBP3 expression was decreased in NECs from smokers. Addition of the demethylating agent 5-Aza-2-deoxycytidine enhanced ULBP3 expression in NECs from smokers. Furthermore, infection of differentiated NECs with influenza virus resulted in significantly lower levels of ULBP3 in cells from smokers. Taken together, our findings show that genomic DNA methylation profiles are altered in NECs from smokers and that these changes are associated with decreased antiviral host defense responses, indicating that epigenenic dysregulation of genes such as SOCS3 and ULBP3 likely impacts immune responses in the epithelium.

Bcl-w a pro-survival member of the Bcl-2 protein family, is expressed in a variety of cancer types, including gastric and colorectal adenocarcinomas, as well as glioblastoma multiforme (GBM), the most common and lethal brain tumor type. Previously, we demonstrated that Bcl-w is upregulated in gastric cancer cells, particularly those displaying infiltrative morphology. These reports propose that Bcl-w is strongly associated with aggressive characteristic, such as invasive or mesenchymal phenotype of GBM. However, there is no information from studies of the role of Bcl-w in GBM. In the current study, we showed that Bcl-w is upregulated in human glioblastoma multiforme (WHO grade IV) tissues, compared with normal and glioma (WHO grade III) tissues. Bcl-w promotes the mesenchymal traits of glioblastoma cells by inducing vimentin expression via activation of transcription factors, β-catenin, Twist1 and Snail in glioblastoma U251 cells. Moreover, Bcl-w induces invasiveness by promoting MMP-2 and FAK activation via the PI3K-p-Akt-p-GSK3β-β-catenin pathway. We further confirmed that Bcl-w has the capacity to induce invasiveness in several human cancer cell lines. In particular, Bcl-w-stimulated β-catenin is translocated into the nucleus as a transcription factor and promotes the expression of target genes, such as mesenchymal markers or MMPs, thereby increasing mesenchymal traits and invasiveness. Our findings collectively indicate that Bcl-w functions as a positive regulator of invasiveness by inducing mesenchymal changes and that trigger their aggressiveness of glioblastoma cells.

Glycogen synthase kinase β (GSK3β) phosphorylates many substrates in mammalian cells, and functions in many physiological processes. We observed that GSK3β knockdown by siRNA perturbed both Golgi morphology in HeLa cells and the anterograde transport of cation-independent mannose 6-phosphate receptor (CI-M6PR) from the trans-Golgi network (TGN) to prelysosomal compartments (PLC), diverting it to the exocytic pathway. Moreover, we demonstrate that a portion of GSK3β was localized to the TGN through the Golgi peripheral protein p230 and that this localization regulated CLASP2 phosphorylation. Our results also show that GSK3β knockdown resulted in accumulation of CLASP2 at microtubule plus ends at the cell periphery. Our findings support the hypothesis that GSK3β at the TGN acts as a guide, activates exocytic transport, and redirects CI-M6PR from transport to the PLC into the exocytic pathway by regulating the affinity of CLASPs for microtubules.

BACKGROUND

Sulfatase 2 (SULF2), an extracellular heparan sulphate 6-O-endosulphatase, has an oncogenic effect in hepatocellular carcinoma (HCC) that is partially mediated through glypican 3, which promotes heparin-binding growth factor signalling and HCC cell growth. SULF2 also increases phosphorylation of the anti-apoptotic Akt kinase substrate GSK3β and SULF2 expression is associated with a decreased apoptotic index in human HCCs.

METHODS

We investigated the functional and mechanistic effects of SULF2 on drug-induced apoptosis of HCC cells using immunohistochemistry, Western immunoblotting, gene transfection, real-time quantitative polymerase chain reaction, MTT and apoptosis assays and immunocytochemistry.

RESULTS

The increased expression of SULF2 in human HCCs was confirmed by immunohistochemistry and immunoblotting. Treatment with inhibitors of MEK, JNK and PI3 kinases decreased the viability of SULF2-negative Hep3B HCC cells and induced apoptotic caspase 3 and 7 activity, which was most strongly induced by the PI3K inhibitor LY294002. Forced expression of SULF2 in Hep3B cells significantly decreased activity of the apoptotic caspases 3 and 7 and induced resistance to LY294002-induced apoptosis. As expected, LY294002 inhibited activation of Akt kinase by PI3K. Conversely, knockdown of SULF2 using an shRNA construct targeting the SULF2 mRNA induced profound cell growth arrest and sensitized the endogenously SULF2-expressing HCC cell lines Huh7 and SNU182 to drug-induced apoptosis. The effects of knockdown of SULF2 on HCC cells were mediated by decreased Akt phosphorylation, downregulation of cyclin D1 and the anti-apoptotic molecule Bcl-2, and upregulation of the pro-apoptotic molecule BAD.

CONCLUSIONS

The prosurvival, anti-apoptotic effect of SULF2 in HCC is mediated through activation of the PI3K/Akt pathway.

OBJECTIVE

The goal of this study was to examine the role of endoplasmic reticulum (ER) stress signaling and the contribution of glycogen synthase kinase (GSK)-3β activation in hyperglycemic, hyperhomocysteinemic, and high-fat-fed apolipoprotein E-deficient (apoE(-/-)) mouse models of accelerated atherosclerosis.

RESULTS

Female apoE(-/-) mice received multiple low-dose injections of streptozotocin (40 μg/kg) to induce hyperglycemia, methionine-supplemented drinking water (0.5% wt/vol) to induce hyperhomocysteinemia, or a high-fat (21% milk fat+0.2% cholesterol) diet to induce relative dyslipidemia. A subset of mice from each group was supplemented with sodium valproate (625 mg/kg), a compound with GSK3 inhibitory activity. At 15 and 24 weeks of age, markers of ER stress, lipid accumulation, GSK3β phosphorylation, and GSK3β activity were analyzed in liver and aorta. Atherosclerotic lesions were examined and quantified. Hyperglycemia, hyperhomocysteinemia, and high-fat diet significantly enhanced GSK3β activity and also increased hepatic steatosis and atherosclerotic lesion volume compared with controls. Valproate supplementation blocked GSK3β activation and attenuated the development of atherosclerosis and the accumulation of hepatic lipids in each of the models examined. The mechanism by which GSK3β activity is regulated in these models likely involves alterations in phosphorylation at serine 9 and tyrosine 216.

CONCLUSIONS

These findings support the existence of a common mechanism of accelerated atherosclerosis involving ER stress signaling through activation of GSK3β. Furthermore, our results suggest that atherosclerosis can be attenuated by modulating GSK3β phosphorylation.

Our in vitro experiments suggested that tetrahydroxystilbene glucoside (TSG) affords a significant neuroprotective effect against MPP⁺-induced damage and apoptosis in PC12 cells though activation of the PI3K/Akt pathway. This study was aimed to investigate the potential neuroprotective effect of TSG in 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP)-treated mouse model of Parkinson's disease (PD). We found that treatment of TSG protected dopaminergic neurons by preventing MPTP-induced decreases in substantia nigra tyrosine hydroxylase (TH)-positive cells and striatal dopaminergic transporter (DAT) protein levels. Furthermore, it was also associated with increasing striatal Akt and GSK3β phosphorylation, up-regulation of the Bcl-2/BAD ratio, and inhibition of the activation of caspase-9 and caspase-3. These results showed that TSG promoted dopamine neuron survival in vivo, the PI3K/Akt signaling pathway may have mediated the protection of TSG against MPTP, suggesting that TSG treatment might represent a neuroprotective treatment for PD.

BACKGROUND

c-Myb is expressed at high levels in immature progenitors of all the hematopoietic lineages. It is associated with the regulation of proliferation, differentiation and survival of erythroid, myeloid and lymphoid cells, but decreases during the terminal differentiation to mature blood cells. The cellular level of c-Myb is controlled by not only transcriptional regulation but also ubiquitin-dependent proteolysis. We recently reported that mouse c-Myb protein is controlled by ubiquitin-dependent degradation by SCF-Fbw7 E3 ligase via glycogen synthase kinase 3 (GSK3)-mediated phosphorylation of Thr-572 in a Cdc4 phosphodegron (CPD)-dependent manner. However, this critical threonine residue is not conserved in human c-Myb. In this study, we investigated whether GSK3 is involved in the regulatory mechanism for human c-Myb expression.

RESULTS

Human c-Myb was degraded by ubiquitin-dependent degradation via SCF-Fbw7. Human Fbw7 ubiquitylated not only human c-Myb but also mouse c-Myb, whereas mouse Fbw7 ubiquitylated mouse c-Myb but not human c-Myb. Human Fbw7 mutants with mutations of arginine residues important for recognition of the CPD still ubiquitylated human c-Myb. These data strongly suggest that human Fbw7 ubiquitylates human c-Myb in a CPD-independent manner. Mutations of the putative GSK3 phosphorylation sites in human c-Myb did not affect the Fbw7-dependent ubiquitylation of human c-Myb. Neither chemical inhibitors nor a siRNA for GSK3β affected the stability of human c-Myb. However, depletion of GSK3β upregulated the transcription of human c-Myb, resulting in transcriptional suppression of γ-globin, one of the c-Myb target genes.

CONCLUSIONS

The present observations suggest that human Fbw7 ubiquitylates human c-Myb in a CPD-independent manner, whereas mouse Fbw7 ubiquitylates human c-Myb in a CPD-dependent manner. Moreover, GSK3 negatively regulates the transcriptional expression of human c-Myb but does not promote Fbw7-dependent degradation of human c-Myb protein. Inactivation of GSK3 as well as mutations of Fbw7 may be causes of the enhanced c-Myb expression observed in leukemia cells. We conclude that expression levels of human and mouse c-Myb are regulated via different mechanisms.

Changes in cytoprotective signaling may influence cardiac aging, and underpin sensitization to ischemic insult and desensitization to 'anti-ischemic' therapies. We tested whether age-dependent shifts in ischemia-reperfusion (I-R) tolerance in murine and human myocardium are associated with reduced efficacies and coupling of membrane, cytoplasmic and mitochondrial survival-signaling. Hormesis (exemplified in ischemic preconditioning; IPC) and expression of proteins influencing signaling/stress-resistance were also assessed in mice. Mouse hearts (18 vs. 2-4 mo) and human atrial tissue (75±2 vs. 55±2 yrs) exhibited profound age-dependent reductions in I-R tolerance. In mice aging negated cardioprotection via IPC, G-protein coupled receptor (GPCR) agonism (opioid, A1 and A3 adenosine receptors) and distal protein kinase c (PKC) activation (4 nM phorbol 12-myristate 13-acetate; PMA). In contrast, p38-mitogen activated protein kinase (p38-MAPK) activation (1 μM anisomycin), mitochondrial ATP-sensitive K(+) channel (mKATP) opening (50 μM diazoxide) and permeability transition pore (mPTP) inhibition (0.2 μM cyclosporin A) retained protective efficacies in older hearts (though failed to eliminate I-R tolerance differences). A similar pattern of change in protective efficacies was observed in human tissue. Murine hearts exhibited molecular changes consistent with altered membrane control (reduced caveolin-3, cholesterol and caveolae), kinase signaling (reduced p70 ribosomal s6 kinase; p70s6K) and stress-resistance (increased G-protein receptor kinase 2, GRK2; glycogen synthase kinase 3β, GSK3β; and cytosolic cytochrome c). In summary, myocardial I-R tolerance declines with age in association with dysfunctional hormesis and transduction of survival signals from GPCRs/PKC to mitochondrial effectors. Differential changes in proteins governing caveolar and mitochondrial function may contribute to signal dysfunction and stress-intolerance.

Mitochondrial dysfunction and epithelial-to-mesenchymal transition (EMT) play important roles in cancer development and metastasis. However, very little is known about the connection between mitochondrial dysfunction and EMT. Tu translation elongation factor, mitochondrial (TUFM), a key factor in the translational expression of mitochondrial DNA, plays an important role in the control of mitochondrial function. Here, we show that TUFM is downregulated in human cancer tissues. TUFM expression level was positively correlated with that of E-cadherin and decreased significantly during the progression of human lung cancer. TUFM knockdown induced EMT, reduced mitochondrial respiratory chain activity, and increased glycolytic function and the production of reactive oxygen species (ROS). Mechanistically, TUFM knockdown activated AMPK and phosphorylated GSK3β and increased the nuclear accumulation of β-catenin, leading to the induction of EMT and increased migration and metastasis of A549 lung cancer cells. Although TUFM knockdown also induced EMT of MCF7 breast cancer cells, the underlying mechanism appeared somewhat different from that in lung cancer cells. Our work identifies TUFM as a novel regulator of EMT and suggests a molecular link between mitochondrial dysfunction and EMT induction.