Nitroalkene fatty acids mediate activation of Nrf2/ARE-dependent and PPARγ-dependent transcription by distinct signaling pathways and with significantly different potencies.

Nitroalkene fatty acids (NAs)1 are naturally occurring electrophilic derivatives of unsaturated fatty acids formed via •NO-dependent oxidative reactions (1). Due to the natural abundance of oleic and linoleic acids in the diet, circulation, and membranes, their nitrated derivatives, NO2-OA and NO2-LA, are quantitatively important species that mediate a variety of cellular responses including •NO-dependent vasodilation (2), anti-inflammatory processes (3, 4), heat shock responses (5), and others (1). Additionally, among the more striking specific properties of NAs are their abilities to activate PPARγ- and Nrf2-dependent transcription of genes containing, respectively, PPRE and ARE promoter elements (6–8). PPARγ, by governing the expression of a large network of genes, is important in the control of lipid and carbohydrate homeostasis, adipocyte differentiation, inflammation/anti-inflammation, and other functions (9–12). Nrf2, on the other hand, is crucial for mediating the expression of a family of genes associated with cellular defense and protection in response to oxidative and electrophilic stress (13, 14). NO2-LA and NO2-OA are among the most potent naturally occurring ligand agonists of PPARγ yet identified (6, 7, 15) and, more recently, have been shown to activate Nrf2/ARE-dependent transcription (8). What is not known is the relative potency of NAs and their physiological relevance, at NA levels likely to be achieved in vivo, towards activation of these alternative transcriptional programs. It is well established that NO2-LA and NO2-OA activate PPARγ via direct interaction with the ligand-binding pocket of the transcription factor (15–18). Relatively less is known about the mechanism of NA-activation of Nrf2-dependent transcription. Under basal conditions, Nrf2 associates with its inhibitory partner, Keap1, which serves as an adaptor protein to bridge Nrf2 with a Cul3-based E3 ubiquitin ligase thereby targeting Nrf2 for ubiquitinylation and proteosomal degradation (19, 20). It is widely accepted that a major mechanism by which electrophiles activate Nrf2-dependent transcription is through disrupting Nrf2/Keap1 interactions resulting in Nrf2 stabilization, accumulation, and translocation to the nucleus where it can facilitate transcription of its target genes. This electrophile-mediated disruption of Nrf2/Keap1 interactions often occurs as a consequence of redox modification or direct adduction of key cysteine residues within Keap1 (21–24). Indeed, Kansanen et al. recently demonstrated formation of such adducts between Keap1 and the 9- and 10-E-nitro isomers of NO2-OA (25). While formation of these adducts is likely to contribute significantly to Nrf2 stabilization, other pathways are also implicated in the control of electrophile-stimulated Nrf2/ARE-dependent transcription—pathways that include PI3K/AKT, MAPK, and PKC signaling (26–34). Accordingly, the studies described herein were designed to: 1) evaluate the roles of these kinase signaling pathways in NA-mediated activation of Nrf2/ARE-dependent transcription, 2) determine whether NA-mediated activation of the two alternative transcription programs (PPARγ- and Nrf2-driven) are independent of each other or if they share common signaling pathways or exhibit significant activation pathway cross-talk, and 3) quantitatively compare Nrf2/ARE versus PPARγ/PPRE transcriptional responses to NA over a range of NA concentrations in order to ascertain which transcription program will dominate at NA levels likely to be encountered in vivo.