Bcl-2 attenuation of oxidative cell death is associated with up-regulation of gamma-glutamylcysteine ligase via constitutive NF-kappaB activation

Abstract

Oxidative stress induced by reactive oxygen intermediates often causes cell death via apoptosis, which is regulated by many functional genes and their protein products. The evolutionarily conserved protein Bcl-2 blocks apoptosis induced by a wide array of death signals. Despite extensive research, the molecular milieu that characterizes the anti-apoptotic function of Bcl-2 has not been fully clarified. In this work, we have investigated the role of bcl-2 in protecting against oxidative death induced by H(2)O(2) in cultured rat pheochromocytoma PC12 cells. Transfection with the bcl-2 gene rescued PC12 cells from apoptotic death caused by H(2)O(2). Addition of NF-kappaB inhibitors such as pyrrolidine dithiocarbamate and N-tosyl-l-phenylalanine chloromethyl ketone to the medium aggravated oxidative cell death. PC12 cells overexpressing bcl-2 exhibited relatively high constitutive DNA binding and transcriptional activities of NF-kappaB compared with vector-transfected control cells. Western blot analysis and immunocytochemistry revealed that bcl-2-transfected PC12 cells retained a higher level of p65 (the functionally active subunit of NF-kappaB) in the nucleus compared with vector-transfected controls. In addition, sustained activation of ERK1/2 (upstream of NF-kappaB) was observed in bcl-2-overexpressing cells. In contrast, the cytoplasmic inhibitor IkappaBalpha was present in lower amounts in cells overexpressing bcl-2. The ectopic expression of bcl-2 increased the cellular glutathione level and gamma-glutamylcysteine ligase expression, which were attenuated by NF-kappaB inhibitors. These results suggest that NF-kappaB plays a role in bcl-2-mediated protection against H(2)O(2)-induced apoptosis in PC12 cells through augmentation of antioxidant capacity.