The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis

Abstract

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.

Proapoptotic Activities of Protein Disulfide Isomerase (PDI) and PDIA3 Protein, a Role of the Bcl-2 Protein Bak

“…The membrane filters were incubated with appropriate primary and secondary antibodies and 10% (w/v) nonfat dried skimmed milk (Bio-Rad) in blotting buffer (1ϫ PBS, 0.2% Tween 20). Proteins were detected using the enhanced chemiluminescence detection system (Thermo) as described previously (27).…”

The role of endogenous Bcl-xL in regulation of apoptotic signaling pathways.

“…Our studies indicate that during H202-induced apoptosis LMP induced Noxa expression increases in both MEFs and HCT116 cells (Figure 4.6). This induction of Noxa is unlikely caused by p53 activation, since MEFs examined in our studies have been immortalized by SV40 T antigen, a known p53 deactivator [151]. In contrast, the increase in Noxa expression in cells exposed to cisplatin or cisplatin-related compounds could depend on p53 [158,161].…”

“…Oxidative stress has been known to induce both necrosis and apoptosis dependent on the severity of the oxidative stress to cells. Mild to moderate oxidative stress causes apoptosis whereas severe oxidative stress triggers necrosis [93,151]. The exact mechanism through which oxidative stress inducers, including H 2 0 2 , promote apoptosis is unclear but recent evidence suggests the involvement of the Bcl-2 proteins.…”

“…Our previous studies have shown that the elevated expression of the pro-apoptotic BH3-only Bcl-2 protein Noxa mediates apoptosis triggered by H202 [151]. ... …”

“…Murine Noxa cDNA was subcloned into the retroviral expression vector pBABE-IRES-EGFP with the marker protein enhanced green fluorescence protein (EFGP) expressed from an internal ribosomal entry site (IRES) as described previously [151]. For retrovirus production, the human embryonic kidney (HEK)-293T cells were transfected with the plasmid pBABE-mNoxa-IRES-EGFP or the empty vector and the helper plasm ids pUVMC and pMDG2.0 using the jetPRIME® transfection reagent.…”