The anti-apoptosis protein, survivin, promotes cell survival and mitosis. Recent studies have demonstrated that survivin is expressed in normal gastric mucosa. Using an in vitro model, we examined whether survivin plays a role in the cytoprotection produced in gastric mucosa by mild irritant ethanol (ETOH) against subsequent exposure to concentrated ETOH. Pre-treatment of rat gastric epithelial cells with 1% ETOH reduced cell death, in response to subsequent incubation with 5% ETOH, by 94% (P < 0.005). This pre-treatment also resulted in increased total and phosphorylated survivin protein levels by 180% (P < 0.0001) and 540% (P < 0.0002), respectively, which required the p34(cdc2) cell cycle-dependent kinase. The cytoprotective effect was abrogated upon siRNA knockdown of survivin protein levels. Further, overexpression of exogenous survivin resulted in significant cytoprotection by 62% (P < 0.02) in the absence of any pre-treatment. We further examined the in vivo relevance of these findings. In fasted rats, administration of 20% ETOH, which we found to be 93% (P < 0.0001) cytoprotective against 50% ETOH challenge, resulted in increased total and phosphorylated survivin protein levels by 234% (P < 0.001) and 214% (P < 0.02), respectively. Administration of 20% ETOH resulted in increased gastric p34(cdc2) activity by 146% (P < 0.01). Inhibition of p34(cdc2) by the potent inhibitor, roscovitine, abolished the increased survivin levels in response to pre-administration of 20% ETOH and reduced the cytoprotection against 50% ETOH challenge by 59% (P < 0.01). These results indicate that survivin is a key mediator of cytoprotection against ETOH-induced gastric injury, acting at the epithelial cell level, by a mechanism that is dependent, in part, on p34(cdc2).
Jones MK, Zhu E, Sarino EV, Padilla OR, Takahashi T, Shimizu T, Shirasawa T. Loss of parietal cell superoxide dismutase leads to gastric oxidative stress and increased injury susceptibility in mice. Am J Physiol Gastrointest Liver Physiol 301: G537-G546, 2011. First published June 30, 2011 doi:10.1152/ajpgi.00177.2011Mitochondrial superoxide dismutase (SOD2) prevents accumulation of the superoxide that arises as a consequence of oxidative phosphorylation. However, SOD2 is a target of oxidative/nitrosative inactivation, and reduced SOD2 activity has been demonstrated to contribute to portal hypertensive gastropathy. We investigated the consequences of gastric parietal cell-specific SOD2 deficiency on mitochondrial function and gastric injury susceptibility. Mice expressing Cre recombinase under control of the parietal cell Atpase4b gene promoter were crossed with mice harboring loxP sequences flanking the sod2 gene (SOD2 floxed mice). Cre-positive mice and Cre-negative littermates (controls) were used in studies of SOD2 expression, parietal cell function (ATP synthesis, acid secretion, and mitochondrial enzymatic activity), increased oxidative/nitrosative stress, and gastric susceptibility to acute injury. Parietal cell SOD2 deficiency was accompanied by a 20% (P Ͻ 0.05) reduction in total gastric SOD activity and a 93% (P Ͻ 0.001) reduction in gastric SOD2 activity. In SOD2-deficient mice, mitochondrial aconitase and ATP synthase activities were impaired by 36% (P Ͻ 0.0001) and 44% (P Ͻ 0.005), respectively. Gastric tissue ATP content was reduced by 34% (P Ͻ 0.002). Basal acid secretion and peak secretagogue (histamine)-induced acid secretion were reduced by 43% (P Ͻ 0.0001) and 40% (P Ͻ 0.0005), respectively. There was a fourfold (P Ͻ 0.02) increase in gastric mucosal apoptosis and 41% (P Ͻ 0.001) greater alcohol-induced gastric damage in the parietal cell SOD2-deficient mice. Our findings indicate that loss of parietal cell SOD2 leads to mitochondrial dysfunction, resulting in perturbed energy metabolism, impaired parietal cell function, and increased gastric mucosal oxidative stress. These alterations render the gastric mucosa significantly more susceptible to acute injury.
We previously demonstrated that the anti‐apoptosis protein, survivin, plays a protective role against alcohol‐induced gastric injury. Since the endothelia is a primary target of alcohol‐induced gastric damage, we investigated whether survivin expression is a key factor in the greater susceptibility of gastric endothelial vs. epithelial cells to alcohol‐induced injury. Here, we demonstrate that rat gastric epithelial cells expressed 8‐fold (P<0.01) greater survivin levels vs. rat gastric endothelial cells. Survivin expression correlated (r=0.84, P<0.05) with resistance of gastric epithelial vs. endothelial cells to both alcohol‐induced cell damage (20 ± 3% vs. 65 ± 7%, P<0.05) and alcohol‐induced apoptosis (14 ± 2% vs. 32 ± 4%, P<0.05). Forced overexpression of survivin in gastric endothelial cells significantly increased resistance to both alcohol‐induced damage and apoptosis. Moreover, overexpression of a threonine‐34 to glutamate mutant survivin construct rendered gastric endothelial cells significantly more resistant to alcohol‐induced damage and apoptosis vs. non‐transfected gastric epithelial cells (P<0.05). These findings indicate that disparate survivin expression levels can explain the discrepancy between gastric epithelial and endothelial cell susceptibility to alcohol‐induced injury; and, survivin phosphorylation is important in mediating gastric mucosal protection.NIH R01AA14946
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