Nitro-Fatty Acids Activate Nrf2 by a Keap1 Cysteine 151-Independent Mechanism

EMILIA KANSANEN, GUSTAVO BONACCI, HENNA-KAISA JYRKKÄNEN, FRANCISCO J. SCHOPFER, STEVEN R. WOODCOCK, SEPPO YLÄ-HERTTUALA, BRUCE A. FREEMAN, ANNA-LIISA LEVONEN.

SAN FRANCISCO (CA)

Congreso; Arteriosclerosis, Thrombosis and Vascular Biology (ATVB); 2010

American Heart Association

Objective. Nitro-fatty acids (nitro-FAs) are electrophilic signaling mediators formed in vivo via NO-dependent reactions. Nitro-FAs signal via mechanisms that inhibit platelet and neutrophil activation, NF-_B-mediated proinflammatory reactions and vascular smooth muscle cell proliferation, as well as potently inducing anti-inflammatory heme oxygenase-1 gene expression. Nitro-FAs mediate beneficial cardiovascular actions in vivo, inhibiting neointimal hyperplasia after endoluminal vessel injury and myocardial ischemia-reperfusion injury in murine models. Transcriptional profiling of human endothelial cells responses to 9- and 10-nitro-octadecenoic acid (nitro-OA) was performed by microarray analysis. The endothelial cell transcriptome revealed that nitro-OA activates two distinct transcriptional response pathways, one being activation of the transcription factor Nrf2 and the other involving HSF-mediated gene transcription. We now report the molecular mechanisms by which nitro-FAs activate Nrf2 signaling responses, focusing on the hypothesis that post-translational modification of Keap1 cysteines occurs by Michael addition of nitro-FAs to Keap1, and that this event is a significant component of Nrf2 activation and cardiovascular signaling by nitro-FAs. Results. We identified the most nitro-OA-reactive Keap1 cysteine residues by mass spectrometry (MS) and found that nitro-OA covalently adducts C38, C226, C257, C273, C288 and C489. In addition, we show by MS-based “capture” of adducted nitro-FAs, via an exchange reaction with added _ME, that nitro-OA binds to Keap1 at nanomolar concentrations in a cellular milieu. Notably, the mutation of the highly-reactive C151 of Keap1 did not affect nitro-OA binding. We also utilized reporter gene expression analysis to reveal the functional role of Keap1 C151 and found that, in contrast to a “classical” Nrf2-reactive electrophile (the cruciferous vegetable constituent sulforaphane), nitro-OA activated Nrf2-dependent responses independent of adduction with Keap1 C151. Conclusions. Keap1 is a critical molecular target for nitro-FAs in Nrf2 activation. Moreover, Keap1 C151-independent activation of Nrf2 contributes to the protective effects of nitro-FAs in the vasculature.