Regulation of keratinocyte expression of stress proteins and antioxidants by the electrophilic nitrofatty acids 9- and 10-nitrooleic acid

Nitric oxide and various by-products including nitrite contribute to tissue injury by forming novel intermediates via redox-mediated nitration reactions. Nitration of unsaturated fatty acids generates electrophilic nitrofatty acids such as 9-nitrooleic acid (9-NO) and 10-nitrooleic acid (10-NO), which are known to initiate intracellular signaling pathways. In these studies, we characterized nitrofatty acid-induced signaling and stress protein expression in mouse keratinocytes. Treatment of keratinocytes with 5-25μM 9-NO or 10-NO for 6h upregulated mRNA expression of heat shock proteins (hsp's) 27 and 70; primary antioxidants heme oxygenase-1 (HO-1) and catalase; secondary antioxidants glutathione S-transferase (GST) A1/2, GSTA3, and GSTA4; and COX-2, a key Enzyme in prostaglandin biosynthesis. The greatest responses were evident with HO-1, hsp27, and HSP70. In keratinocytes, 9-NO activated JNK and p38 MAP kinases. JNK inhibition suppressed 9-NO-induced HO-1, hsp27, and HSP70 mRNA and protein expression, whereas p38 MAP kinase inhibition suppressed HO-1. In contrast, inhibition of constitutive expression of ERK1/2 suppressed only HSP70, indicating that 9-NO modulates expression of stress proteins by distinct mechanisms. 9-NO and 10-NO also upregulated expression of caveolin-1, the major structural component of caveolae. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation revealed that HO-1, hsp27, and HSP70 were localized within caveolae after nitrofatty acid treatment of keratinocytes, suggesting a link between induction of stress response proteins and caveolin-1 expression. These data indicate that nitrofatty acids are effective signaling molecules in keratinocytes. Moreover, caveolae seem to be important in the localization of stress proteins in response to nitrofatty acids.