J. D. Lyons1, N. Klingensmith1, Z. Liang1, C. Coopersmith1 1Emory University,Surgery,Atlanta, GA, USA
Introduction: Changes in intestinal epithelial cell apoptosis and proliferation have been associated with mortality in animal models of sepsis, and preventing gut cell death by local overexpression of Bcl-2 has repeatedly been shown to increase survival. However, our unpublished observations indicate that when anti-apoptotic Bcl-2 is overexpressed in conjunction with the pro-proliferative transgene SV40 T-antigen, the epithelium is not protected from sepsis-induced apoptosis, but instead displays a paradoxical increase in cell death. We here attempt to shed light on this phenomenon by examining mechanisms of cell death in the gut of septic Bcl2xTAg animals.
Methods: Fabpi-Bcl2 mice were crossed to fabpl-TAg mice to produce bi-transgenic Bcl2xTAg mice with small intestinal enterocyte-specific overexpression of Bcl-2 and SV40 T-antigen. Mice aged 8-10 weeks old were subjected to cecal ligation and puncture (CLP) and sacrificed at 24 hours. Jejunal sections were collected at time of sacrifice for western blot analysis. Septic Bcl2xTAg mice were compared to septic TAg mice to determine how the additional expression of Bcl-2 might lead to the increased cell death seen in Bcl2xTAg enterocytes.
Results: Septic Bcl2xTAg mice expressed the Bcl-2 protein at a concentration 4.1-fold greater than septic TAg controls (p=0.001, n=9-10). Expression of the apoptosis effector caspase-3 was significantly increased in these animals (3.1-fold increase, p=0.017, n=5-6). Bcl2xTAg mice also displayed increased expression of caspase-9 (24.5-fold increase, p=0.005, n=7-9) and caspase-8 (1.8-fold increase, p=0.013, n=9-10), suggesting both intrinsic and extrinsic signaling upstream of caspase-3 activation. These changes were associated with trends in increased concentrations of the mitochondrial apoptosis mediators Bax (8.9-fold increase, p=0.051, n=9-11) and cytochrome C (2.0-fold increase, p=0.052, n-5-6), but not apoptosis inducing factor (1.06-fold increase, p=0.65, n=5-6).
Conclusion: The overexpression of Bcl-2 in the gut of Bcl2xTAg mice paradoxically results in increased sensitivity to sepsis-induced enterocyte apoptosis by both intrinsic and extrinsic mechanisms. The greatly increased caspase-9 expression suggests the intrinsic pathway may predominate. Overexpression of an anti-apoptotic factor, when coupled with overexpression of a pro-proliferative factor, may trigger a counter compensatory response that increases the likelihood of cell death rather than survival.