Nitric oxide induces cell death by regulating anti-apoptotic BCL-2 family members.

Nitric oxide (NO) activates the intrinsic apoptotic pathway to induce cell death. However, the mechanism by which this pathway is activated in cells exposed to NO is not known. Here we report that BAX and BAK are activated by NO and that cytochrome c is released from the mitochondria. Cells deficient in Bax and Bak or Caspase-9 are completely protected from NO-induced cell death. The individual loss of the BH3-only proteins, Bim, Bid, Puma, Bad or Noxa, or Bid knockdown in Bim(-/-)/Puma(-/-) MEFs, does not prevent NO-induced cell death. Our data show that the anti-apoptotic protein MCL-1 undergoes ASK1-JNK1 mediated degradation upon exposure to NO, and that cells deficient in either Ask1 or Jnk1 are protected against NO-induced cell death. NO can inhibit the mitochondrial electron transport chain resulting in an increase in superoxide generation and peroxynitrite formation. However, scavengers of ROS or peroxynitrite do not prevent NO-induced cell death. Collectively, these data indicate that NO degrades MCL-1 through the ASK1-JNK1 axis to induce BAX/BAK-dependent cell death.

Mitochondrial regulation of cell survival and death during low-oxygen conditions.

Mitochondria can initiate cell death or activate genes that promote cell survival in response to low oxygen. The BCL-2 family of proteins regulate cell death in response to anoxia (0-0.5% O2). By contrast, under hypoxia (0.5-3% O2), mitochondrial oxidative stress activates hypoxia-inducible factors (HIFs) to promote cell survival. In this review, we discuss how mitochondria, BCL-2 proteins, and HIFs are crucial for cellular responses to low oxygen.

BH3 peptides induce mitochondrial fission and cell death independent of BAX/BAK.

BH3 only proteins trigger cell death by interacting with pro- and anti-apoptotic members of the BCL-2 family of proteins. Here we report that BH3 peptides corresponding to the death domain of BH3-only proteins, which bind all the pro-survival BCL-2 family proteins, induce cell death in the absence of BAX and BAK. The BH3 peptides did not cause the release of cytochrome c from isolated mitochondria or from mitochondria in cells. However, the BH3 peptides did cause a decrease in mitochondrial membrane potential but did not induce the opening of the mitochondrial permeability transition pore. Interestingly, the BH3 peptides induced mitochondria to undergo fission in the absence of BAX and BAK. The binding of BCL-X(L) with dynamin-related protein 1 (DRP1), a GTPase known to regulate mitochondrial fission, increased in the presence of BH3 peptides. These results suggest that pro-survival BCL-2 proteins regulate mitochondrial fission and cell death in the absence of BAX and BAK.

Hyperoxia-induced premature senescence requires p53 and pRb, but not mitochondrial matrix ROS.

Senescence is a potential tumor-suppressing mechanism and a commonly used model of cellular aging. One current hypothesis to explain senescence, based in part on the correlation of oxygen with senescence, postulates that it is caused by oxidative damage from reactive oxygen species (ROS). Here, we further test this theory by determining the mechanisms of hyperoxia-induced senescence. Exposure to 70% O(2) led to stress-induced, telomere-independent senescence. Although hyperoxia elevated mitochondrial ROS production, overexpression of antioxidant proteins was not sufficient to prevent hyperoxia-induced senescence. Hyperoxia activated AMPK; however, overexpression of a kinase-dead mutant of LKB1, which prevented AMPK activation, did not prevent hyperoxia-induced senescence. Knocking down p21 via shRNA, or suppression of the p16/pRb pathway by either BMI1 or HPV16-E7 overexpression, was also insufficient to prevent hyperoxia-induced senescence. However, suppressing p53 function resulted in partial rescue from senescence, suggesting that hyperoxia-induced senescence involves p53. Suppressing both the p53 and pRb pathways resulted in almost complete protection, indicating that both pathways cooperate in hyperoxia-induced senescence. Collectively, these results indicate a ROS-independent but p53/pRb-dependent senescence mechanism during hyperoxia.