Abstract 1528: Nitric Oxide-Induced Heme Oxygenase-1 Gene Transcription via the Nrf2 Pathway Promotes Vascular Smooth Muscle Cell Survival
Nitric oxide (NO) is a well established signaling molecule that regulates numerous vascular functions, including cell viability. We previously reported that NO stimulates the transcription of heme oxygenase-1 (HO-1) in vascular smooth muscle cells (SMC) in a cGMP-independent manner. In the present study, we determined the signaling pathway responsible for the induction of HO-1 and its functional significance. Cultured rat aortic SMC were transiently transfected with a promoter-luciferase construct that contained a previously identified enhancer region (E1) coupled to a minimum HO-1 promoter and exposed to various NO donors (10–3000 μM; 0–24 hour), including sodium nitroprusside, S-nitroso-N-acetyl-penicillamine, 3-morpholinosydnonimine, Angeli’s salt, and spermine NONOate. All the NO donors stimulated a concentration- and time-dependent increase (~3-fold) in HO-1 promoter activity. Mutation of the antioxidant response elements (AREs) in the HO-1 promoter or overexpression of a dominant-negative mutant of Nrf2 abolished the activation by NO. NO rapidly (1 hour) increased cellular Nrf2 protein levels and stimulated the translocation of Nrf2 from the cytosol to the nucleus. In addition, electromobility shift and chromatin immunoprecipitation assays demonstrated that NO promoted the binding of Nrf2 to the HO-1 ARE and E1 enhancer, respectively. Since MAPK and PI3K have been implicated in the activation of Nrf2, the role of these kinases was also investigated. Although NO rapidly (2 minutes) stimulated the activation of ERK, p38 MAPK, JNK, and PI3K, pharmacological inhibition of these kinases failed to prevent the activation of Nrf2. Finally, treatment of vascular SMC with the various NO donors (1 mM for 48 hour) stimulated apoptosis and this was dramatically increased by the HO inhibitors, zinc or tin protoporphyrin-IX (10 μM). In conclusion, this study demonstrates that different redox forms of NO stimulate HO-1 gene transcription by activating the Nrf2/ARE complex in a MAPK- and PI3K-independent manner to counteract NO-induced apoptosis of vascular SMC. The ability of NO to induce HO-1 may represent a critical adaptive response to maintain cell viability at sites of vascular inflammation and atherosclerosis.