Chronic ethanol consumption-induced pancreatic {beta}-cell dysfunction and apoptosis through glucokinase nitration and its down-regulation.

Chronic ethanol consumption is known as an independent risk factor for type 2 diabetes, which is characterized by impaired glucose homeostasis and insulin resistance; however, there is a great deal of controversy concerning the relationships between alcohol consumption and the development of type 2 diabetes. We investigated the effects of chronic ethanol consumption on pancreatic ß-cell dysfunction and whether generated peroxynitrite participates in the impaired glucose homeostasis. Here we show that chronic ethanol feeding decreases the ability of pancreatic ß-cells to mediate insulin secretion and ATP production in coordination with the decrease of glucokinase, Glut2, and insulin expression. Specific blockade of ATF3 using siRNA or C-terminally deleted ATF3(ΔC) attenuated ethanol-induced pancreatic ß-cell apoptosis or dysfunction and restored the down-regulation of glucokinase (GCK), insulin, and pancreatic duodenal homeobox-1 induced by ethanol. GCK inactivation and down-regulation were predominantly mediated by ethanol metabolism-generated peroxynitrite, which were suppressed by the peroxynitrite scavengers N(γ)-monomethyl-L-arginine, uric acid, and deferoxamine but not by the S-nitrosylation inhibitor DTT, indicating that tyrosine nitration is the predominant modification associated with GCK down-regulation and inactivation rather than S-nitrosylation of cysteine. Tyrosine nitration of GCK prevented its association with pBad, and GCK translocation into the mitochondria results in subsequent proteasomal degradation of GCK following ubiquitination. This study identified a novel and efficient pathway by which chronic ethanol consumption may induce GCK down-regulation and inactivation by inducing tyrosine nitration of GCK, resulting in pancreatic ß-cell apoptosis and dysfunction. Peroxynitrite-induced ATF3 may also serve as a potent upstream regulator of GCK down-regulation and ß-cell apoptosis.

HBx-induced hepatic steatosis and apoptosis are regulated by TNFR1- and NF-kappaB-dependent pathways.

Hepatitis B virus X (HBx) protein is an important regulator of hepatic steatosis observed in patients with hepatitis B virus; however, its underlying molecular mechanism remains unclear. TNF receptor 1 (TNFR1) is an essential pathway for the HBx-mediated nuclear factor kappaB (NF-kappaB) activation involved in hepatic liver injury. Here, we show that HBx-mediated steatosis and apoptosis are regulated by TNFR1- and NF-kappaB-dependent pathways. HBx-mediated tumor necrosis factor alpha (TNF-alpha) production and NF-kappaB activation were completely diminished in anti-TNF-alpha-treated cells and TNF-alpha(-)(/-) or TNFR1(-/-) mice. HBx and TNFR1, which are potentiated by TNF-alpha, are physically associated and colocalize in the plasma membrane. Similarly, TNFR1 depletion inhibits lipid droplets, and lipogenic genes such as sterol regulatory element binding protein (SREBP) 1 and peroxisome proliferator-activated receptor (PPAR) gamma increased in HBx-Tg mice and HepG2-GFPHBx stable cells. Furthermore, lipid accumulation and expression of SREBP1c and PPAR gamma are significantly increased in AdHBx-GFP-injected (intravenous) wild-type mice, but not in TNFR1(-/-) mice. HBx-enhanced transcriptional activities of SREBP1 and PPAR gamma are significantly attenuated by the NF-kappaB inhibitor Bay 11-7082, as well as by TNFR1 depletion. Also, AdHBx-GFP potentiates TNF-alpha-induced apoptosis, which is completely inhibited in TNFR1-depleted cells. Our results suggest that HBx-induced NF-kappaB activation was mediated by direct interaction with TNFR1 and thereby induced TNF-alpha production. HBx-induced NF-kappaB activation is also associated with the induction of hepatic steatosis and apoptosis, which is determined by a TNFR1-dependent pathway.

Role of activating transcription factor 3 on TAp73 stability and apoptosis in paclitaxel-treated cervical cancer cells.

Taxol (paclitaxel) is a potent anticancer drug that has been found to be effective against several tumor types, including cervical cancer. However, the exact mechanism underlying the antitumor effects of paclitaxel is poorly understood. Here, paclitaxel induced the apoptosis of cervical cancer HeLa cells and correlated with the enhanced activation of caspase-3 and TAp73, which was strongly inhibited by TAp73beta small interfering RNA (siRNA). In wild-type activating transcription factor 3 (ATF3)-overexpressed cells, paclitaxel enhanced apoptosis through increased alpha and beta isoform expression of TAp73; however, these events were attenuated in cells containing inactive COOH-terminal-deleted ATF3 [ATF3(DeltaC)] or ATF3 siRNA. In contrast, paclitaxel-induced ATF3 expression did not change in TAp73beta-overexpressed or TAp73beta siRNA-cotransfected cells. Furthermore, paclitaxel-induced ATF3 translocated into the nucleus where TAp73beta is expressed, but not in ATF3(DeltaC) or TAp73beta siRNA-transfected cells. As confirmed by the GST pull-down assay, ATF3 bound to the DNA-binding domain of p73, resulting in the activation of p21 or Bax transcription, a downstream target of p73. Overexpression of ATF3 prolonged the half-life of TAp73beta by inhibiting its ubiquitination and thereby enhancing its transactivation and proapoptotic activities. Additionally, ATF3 induced by paclitaxel potentiated the stability of TAp73beta, not its transcriptional level. Chromatin immunoprecipitation analyses show that TAp73beta and ATF3 are recruited directly to the p21 and Bax promoter. Collectively, these results reveal that overexpression of ATF3 potentiates paclitaxel-induced apoptosis of HeLa cells, at least in part, by enhancing TAp73beta's stability and its transcriptional activity. The investigation shows that ATF3 may function as a tumor-inhibiting factor through direct regulatory effects on TAp73beta, suggesting a functional link between ATF3 and TAp73beta.

Superoxide dismutase 1 mutants related to amyotrophic lateral sclerosis induce endoplasmic stress in neuro2a cells.

One of the common features of damaged neurons in many neurodegenerative diseases is the presence of abnormal aggregates of the disease-related proteins. In amyotrophic lateral sclerosis (ALS) of both sporadic and familial forms, protein aggregates are found in the affected spinal cords. In familial ALS with mutations in copper-zinc superoxide dismutase 1 (SOD1), the propensity of SOD1 for aggregation is known to increase with the mutation. In the present study, we examined whether the aggregate-prone SOD1 mutants induce endoplasmic reticulum (ER) stress and the inhibition of the ER stress protects the cells. The ALS-related mutant G85R SOD1 and G93A SOD1 formed visible aggregates and caused cell death possibly by apoptosis when over-expressed in neuro2a cells. Interestingly, the rate of the mutant SOD1-induced cell death was greater than that of the visible aggregate formation. Expression of the mutant SOD1 caused signs of both early and late ER stress responses, namely, RNA-dependent protein kinase-like ER kinase and eukaryotic initiation factor alpha phosphorylation, Jun amino-terminal kinase activation, activating transcription factor 6-translocation, X-box binding protein 1 mRNA splicing, and caspase 12 activation. The X-box binding protein 1 mRNA splicing activation was also detected in the mutant SOD1-expressing cells even without the visible aggregates. The cell death induced by the mutant SOD1 over-expression looked like apoptosis as evidenced by nuclear morphology and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling. Importantly, an ER stress inhibitor, salubrinal delayed the formation of insoluble aggregates of the mutant SOD1 and suppressed the mutant-induced cell death. In addition, over-expression of the ER-targeted Bcl-xL protected the cells from the mutant SOD1-induced cytotoxicity. These results suggest that the misfolding of ALS-related mutant SOD1 induces ER stress possibly prior to the formation of visible aggregates, which may contribute to the motor neuron degeneration in ALS pathogenesis.

The role of STAT1/IRF-1 on synergistic ROS production and loss of mitochondrial transmembrane potential during hepatic cell death induced by LPS/d-GalN.

Previously, we demonstrated that signal transducer and activator of transcription factor 1 (STAT1) plays an essential role in liver injury induced by lipopolysaccharide (LPS)/D-galactosamine (D-GalN); however, the underlying mechanism involved remains unclear. Here, we showed that LPS/D-GalN administration induced secretion of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma), which mediated apoptosis synergistically. Moreover, LPS/D-GalN-induced apoptosis was associated with increased inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production, as well as elevated reactive oxygen species (ROS) production, which were all strongly inhibited by treatment with the antioxidant N-acetyl-L-cysteine (NAC) and an iNOS/NO inhibitor, L-NMMA. Although STAT1 activation and expression did not change significantly in TNF-alpha/IFN-gamma-cotreated cells compared with cells treated with IFN-gamma alone, the absence of STAT1 or interferon regulatory factor 1 (IRF-1) in genetic knockout mice strongly abrogated the observed effects of TNF-alpha/IFN-gamma on iNOS/NO induction, ROS production, loss of mitochondrial transmembrane potential (DeltaPsim), and apoptosis compared with STAT1(+/+) and IRF-1(+/+) mice. Additionally, the synergistic effects of TNF-alpha/IFN-gamma on iNOS/NO induction, ROS production, and apoptosis were significantly inhibited by overexpression of dominant negative STAT1 in contrast to overexpression of wild-type STAT1. In STAT1-deficient mice, nuclear factor kappaB (NF-kappaB) activation by TNF-alpha/IFN-gamma was attenuated and strongly inhibited by both NAC and L-NMMA. Moreover, the proteasome inhibitor, MG132, inhibited NF-kappaB activation and strongly inhibited iNOS/NO induction, ROS production, and loss of DeltaPsim induced by TNF-alpha/IFN-gamma, thereby inhibiting apoptosis. Interestingly, it appears peroxynitrite, which is produced by TNF-alpha/IFN-gamma, may interfere with STAT1 phosphorylation by inducing STAT1 nitration. Collectively, these findings demonstrate that TNF-alpha/IFN-gamma synergistically potentiates iNOS/NO induction, ROS production, and loss of DeltaPsim via STAT1 overexpression, playing an important role in promoting apoptosis and liver injury induced by LPS/D-GalN.

AICAR potentiates ROS production induced by chronic high glucose: roles of AMPK in pancreatic beta-cell apoptosis.

We previously demonstrated that chronic high glucose (33.3 mM) induced beta-cell dysfunction and apoptosis through glucokinase (GCK) downregulation, but the exact mechanisms involved remain unclear. Here, we show that prolonged exposure of 5-aminoimidazole-4-carboxamide (AICA)-riboside potentiated apoptosis induced by high glucose in MIN6N8 pancreatic beta-cells, correlating with enhanced GCK downregulation and decreased production of ATP and insulin. These events are potentiated in AMPK-overexpressing cells, but are prevented in cells transfected with mutant dominant-negative AMPK (AMPK-K45R). Furthermore, AMPK activation increases production of reactive oxygen species (ROS) and loss of mitochondria membrane potential induced by high glucose, which is significantly inhibited by treatment with compound C or by AMPK-K45R overexpression. Overexpression of GCK prevents apoptosis; decreased cellular ATP and insulin secretion, and ROS production enhanced by AICAR, but does not affect AMPK activation. Similar results are obtained using isolated primary islet cells. Collectively, these data demonstrate that AMPK activation potentiates beta-cell apoptosis induced by chronic high glucose through augmented GCK downregulation mediated by enhanced ROS production.

AIF translocates to the nucleus in the spinal motor neurons in a mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of motor neurons in the brain stem and the spinal cords. One of the causes for the familial ALS has been attributed to the mutations in copper-zinc superoxide dismutase (SOD1). Although the toxic function of the mutant enzyme has not been fully understood, the final cell death pathway has been suggested as caspase-dependent. In the present study, we present evidence that the activation of apoptosis inducing factor (AIF) may play a role to induce motor neuron death during ALS pathogenesis. In the spinal cord of SOD1 G93A transgenic mice, expression of AIF was detected in the motor neurons and astrocytes. The level of AIF expression increased as the disease progressed. In the symptomatic SOD1 G93A transgenic mice, AIF released from the mitochondria and translocated into the nucleus in the motor neurons as evidenced by confocal microscopy and biochemical analysis. These results suggest that AIF may play a role to induce motor neuron death in a mouse model of ALS.

Scutellariae radix extracts suppress ethanol-induced caspase-11 expression and cell death in N(2)a cells.

Scutellariae radix is a Chinese herbal medicine that has been used to treat disease conditions accompanying inflammation and oxidative stress. In the present study, we examined the effect of Scutellariae radix extracts during acute ethanol exposure in N(2)a neuroblastoma. The Scutellariae radix extracts effectively inhibited ethanol-induced apoptosis and caspase-3/-7 activation. Ethanol induced the expression of caspase-11 that has been known as a dual regulator of pathological apoptosis and inflammatory response. The ethanol-induced caspase-11 expression was suppressed by pretreatment of the Scutellariae radix extracts. Furthermore, the activation of caspase-3/-7 and apoptosis were significantly inhibited in caspase-11-/- mouse embryonic fibroblasts following ethanol treatment. These results suggest that caspase-11 has a regulatory role in ethanol-induced apoptosis, and the suppression of caspase-11 may be a mechanism by which Scutellariae radix exerts its cytoprotective effect.