Abstract 2091: Nitrooleic acids induce localization of heat shock protein 70 in PAM212 keratinocyte caveolae

Abstract

Abstract Keratinocytes can be stimulated to express inducible nitric oxide synthase and generate nitric oxide by proinflammatory mediators including γ-interferon and lipopolysaccharide. Once formed, nitric oxide and its oxidation products form signaling molecules via redox mediated nitration reactions. Important targets for nitration in mammalian cells are unsaturated fatty acids. This can result in the production of reactive electrophilic species including 9-nitrooleate (9-NO) and 10-nitrooleate (10-NO). Oxidative stress is known to upregulate expression of heat shock proteins which are important chaperones functioning in protein-protein interactions including folding and trafficking. In the present studies we characterized nitro-fatty acid-induced expression of heat shock protein 70 (hsp70) in PAM212 mouse keratinocytes. Western blot analysis showed low constitutive levels of hsp70. Treatment of keratinocytes with 5-25 μM 9-NO or 10-NO for 6 hr markedly increased hsp70 expression. 10-NO was more active than 9-NO. Constitutive hsp70 was found to be localized in cytosolic fractions of keratinocytes, 9- or 10-NO did not alter constitutive cytosolic hsp70. In contrast, following treatment of keratinocytes with the nitro-fatty acids, hsp70 was selectively upregulated in lipid raft membrane fractions. Both hsp70 and caveolin-1 were co-localized in the lipid raft fractions of the membranes demonstrating that the lipid rafts are caveolae. In mouse keratinocytes, nitro-fatty acids readily stimulate MAP kinase signaling including JNK and p38 MAP kinases. Inhibitors of JNK (SP600125, 20 μM) or p38 (SB203580, 10 μM) had no effect on induction of hsp70 or its localization in caveolae indicating that nitro-fatty acid-induced expression of this protein was MAP kinase-independent. Taken together these data demonstrate that nitro-fatty acids are important in the control of heat shock protein expression. Moreover, selective localization of hsp70 in caveolae suggests that it functions in protecting caveolar proteins from cellular stress. Supported by CA132624, ES004738, ES005022, GM034310 and AR055073. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2091. doi:10.1158/1538-7445.AM2011-2091