The role of apoptosis repressor with a caspase recruitment domain (ARC) in oxidative stress induced apoptosis.

摘要

Oxidative stress has been implicated in several patho-physiological conditions including ischemia/reperfusion injury to the heart and brain and some neurodegenerative diseases. Prevention of oxidative stress-induced damage is considered an important approach to alleviate the severity of these conditions. In this study, we studied the function of ARC, a special type of caspase inhibitor, in oxidative stress response.; When ARC was reduced by the RNAi approach, H9c2 cells became more sensitive to oxidative stress, exhibiting typical apoptotic characteristics including fragmented nuclei and caspase-3 activation. Overexpression of wild type ARC in those cells restored their resistance.; When individual domains of ARC, the CARD or the P/E (p&barbelow;roline/g&barbelow;lutamic acid) domain were introduced into H9c2 cells, no protection was observed, suggesting the intact ARC protein is required to defend against oxidative stress.; When phosphorylation of ARC was blocked by either mutation or casein kinase II inhibitor, ARC failed to translocate to mitochondria, did not block the translocation of Bax, did not inhibit the activation of caspase-2 and -3, and could not prevent apoptosis induced by the overexpression of caspase-2. These data demonstrated the importance of phosphorylation for the antiapoptotic function of ARC.; This study identified two new proteins that might affect the function of ARC, phosphatase type 2C (PP2C) being a potential dephosphorylation enzyme for ARC and structural protein Kazrin providing structural support for the localization of ARC within the cell.; This study further demonstrated that ARC was significantly elevated and constitutively phosphorylated at a high level in multiple cancer cell lines, which was not affected by oxidative stress. HeLa cells with reduced ARC became more sensitive to oxidative stress. In contrast, ARC expression in H9c2 cells was downregulated by oxidative stress and its phosphorylation was minimal under normal conditions. The reduction of ARC protein in H9c2 cells by oxidative stress was mediated through proteasome degradation.; No changes in ARC expression were observed in multiple tissues from either MnSOD or caspase-2 knockout mice, suggesting no compensatory changes of ARC gene expression in these mice. However, overexpression of ARC did restore the sensitivity of MnSOD deficient cells to oxidative stress.; In summary, maintenance of sufficient level of intact ARC protein with proper phosphorylation is critical to the protection of cells against oxidative stress induced apoptosis.