Intestinal Resection-Induced Enterocyte Apoptosis Is Mediated by p38α MAPK-Directed Activation of BAX
Derek Wakeman*1, Jun Guo1, Jethrina a. Santos2, John Schneider3, Jennifer a. Leinicke1, Kathryn J. Rowland1, Christopher R. Erwin1, Brad W. Warner1
1Surgery, Washington University School of Medicine, Saint Louis, MO; 2Texas A&M University, College Station, TX; 3Washington University, Saint Louis, MO
Introduction: Increased crypt apoptosis is an important aspect of intestinal adaptation after massive small bowel resection (SBR). We have previously shown expression of the pro-apoptotic factor Bax is required for resection-induced apoptosis. In addition, in vitro experiments have demonstrated p38α MAPK is required for activation of Bax and apoptosis. The present study was designed to test the hypothesis that p38α MAPK is a key regulator of Bax activation during adaptation after SBR in vivo.Methods: p38α MAPK was deleted in mice specifically in the intestinal mucosa by tamoxifen injection via a Cre/LoxP recombination system. p38α-deficient and wild type (WT) mice were sacrificed seven days after tamoxifen administration. The small intestine was harvested and the intestinal crypt cells were isolated and fractionated. Cytochrome c was quantified by Western blotting in cytoplasmic fractions. Activated Bax was immunoprecipitated using an activated Bax isoform-specific antibody (6A7) followed by Western blot analysis. To investigate Bax activation during adaptation, mice underwent either proximal SBR or sham operation. Mice were sacrificed 3 days after the operation and activated Bax was quantified as described above. Results: Rates of apoptosis were significantly lower in the p38α-deficient mice compared to their WT littermates both at baseline and after SBR (baseline: 4.9 +/- 0.6 vs 8.9 +/- 1.3 apoptotic bodies/100 crypts, p<.05; after SBR 6.9 +/- 0.6 vs. 10.9 +/- 0.8 apoptotic bodies/100 crypts, p<.05). Consistent with lower apoptotic rates, there were significantly lower levels of cytoplasmic cytochrome c in the p38α-deficient mice compared to WT littermates (57% reduction, p<.05). In addition, the amount of activated Bax present in intestinal crypts was reduced by 67% in mice after p38α MAPK deletion (p<.05). Finally, activated Bax levels were two-fold higher in mice after SBR (n=3) relative to sham levels (p=.08)Conclusions: Deleting p38α MAPK in an inducible, intestine-specific manner leads to reduced levels of enterocyte apoptosis, attenuated cytoplasmic cytochrome c, and diminished levels of the activated isoform of Bax. These results support our hypothesis that p38α MAPK regulates Bax activation in small intestine enterocytes during resection-induced enterocyte apoptosis.
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