Mechanisms of ER stress‐induced apoptosis in endothelial cells

Abstract

Intrinsic apoptotic mechanisms arising from endoplasmic reticulum (ER) stress have been recently described. Short-term disruption of ER homeostasis by the N-glycosylation inhibitor tunicamycin (TM) has been shown to induce the unfolded protein response (UPR) in proliferating endothelial cells. But prolonged dysfunction of the ER machinery results in the induction of apoptosis. Quantitative immunoblot, flow cytometry and enzymatic analyses were used to propose a signal transduction pathway that describes apoptotic induction by ER stress in endothelial cells. Results showed that short-term exposure to TM (<24 hrs) induced the UPR and an early activation of caspase-12, caspase-7 and a biphasic activation of caspase-9. After prolonged ER-stress (>24 hrs), activation of the executioner caspase-3 and irreversible apoptotic induction were evidenced by Annexin-V binding and DNA “laddering” assay. Early activation of caspase-7 resulted from caspase-9 cleavage. Early Ca2+ release from ER into the cytoplasm was detected after 16 hours by FURA-2 binding assay. Increased expression of AIF, SMAC/Diablo and Bcl-2 was detected after 24 hours. These results suggest that activation of caspase-12 and Ca2+ release from ER initiate the ER-stress signaling events. Caspase-12 activation results in early (cytochrome-c independent) caspase-9 activation and subsequent caspase-7 activation. ER-stress signals to mitochondria via Ca2+ activates the mitochondria-mediated apoptosis machinery after 24 hours. If TM was removed before 24 hours, cells would show ER stress but no apoptosis. These results suggest that the ER stress-induced apoptosis signaling mechanisms depend on the activation of the mitochondria-mediated apoptosis pathway. Supported by MBRS-RISE, NIH (USA): 1 R25 GM066250-01A1.