Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H2O2. The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H2O2-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H2O2 treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H2O2-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H2O2-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H2O2 activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.

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