Glutathione Supplementation Potentiates Hypoxic Apoptosis by S-Glutathionylation of p65-NFκB

• Published in: The Journal of biological chemistry 2007-06, Vol.282 (25), p.18427-18436
• Main Authors: Qanungo, Suparna, Starke, David W., Pai, Harish V., Mieyal, John J., Nieminen, Anna-Liisa
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M610934200

In murine embryonic fibroblasts, N-acetyl-l-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFκB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFκB that are responsible for NFκB inactivation. In MIA PaCa-2 pancreatic cancer cells, hypoxia increased p65-NFκB DNA binding and NFκB transactivation by 2.6- and 2.8-fold, respectively. NAC blocked these events without having an effect on p65-NFκB protein levels and p65-NFκB nuclear translocation during hypoxia. Pharmacological inhibition of the NFκB pathway also induced hypoxic apoptosis, indicating that the NFκB signaling pathway is a major protective mechanism against hypoxic apoptosis. In cell lysates after hypoxia and treatment with N-ethylmaleimide (thiol alkylating agent), dithiothreitol (disulfide reducing agent) was not able to increase binding of p65-NFκB to DNA, suggesting that most sulfhydryls in p65-NFκB protein were in reduced and activated forms after hypoxia, thereby being blocked by N-ethylmaleimide. In contrast, with hypoxic cells that were also treated with NAC, dithiothreitol increased p65-NFκB DNA binding. Glutaredoxin (GRx), which specifically catalyzes reduction of protein-SSG mixed disulfides, reversed inhibition of p65-NFκB DNA binding in extracts from cells treated with hypoxia plus NAC and restored NFκB activity. This finding indicated that p65-NFκB-SSG was formed in situ under hypoxia plus NAC conditions. In cells, knock-down of endogenous GRx1, which also promotes protein glutathionylation under hypoxic radical generating conditions, prevented NAC-induced NFκB inactivation and hypoxic apoptosis. The results indicate that GRx-dependent S-glutathionylation of p65-NFκB is most likely responsible for NAC-mediated NFκB inactivation and enhanced hypoxic apoptosis.