Role of Src Tyrosine Kinase in the Atherogenic Effects of the 12/15-Lipoxygenase Pathway in Vascular Smooth Muscle Cells

Abstract

Objective— The 12/15-Lipoxygenase (12/15-LO) and its metabolite 12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] mediate proatherogenic responses in vascular smooth muscle cells (VSMCs). We examined the role of the nonreceptor tyrosine kinase Src in the signaling and epigenetic chromatin mechanisms involved in these processes. Methods and Results— Rat VSMCs (RVSMCs) were stimulated with 12(S)-HETE (0.1 μmol/L) in the presence or absence of the Src inhibitor PP2 (10 μmol/L). Src activation and downstream signaling events including inflammatory gene expression and chromatin histone H3-Lys-9/14 acetylation were examined by immunoblotting, RT-PCR, and chromatin immunoprecipitation assays, respectively. 12(S)-HETE significantly activated Src, focal adhesion kinase, Akt, p38MAPK, and CREB. Expression of monocyte chemoattractant protein-1 and interleukin-6 genes and histone H3-Lys-9/14 acetylation on their promoters were also increased by 12(S)-HETE. PP2 inhibited these responses as well as 12(S)-HETE-induced VSMC migration. Furthermore, dominant negative mutants of Src, CREB, and a histone acetyltransferase p300 significantly blocked 12(S)-HETE–induced inflammatory gene expression. In addition, growth factor induced Src signaling and downstream events including H3-Lys-9/14 acetylation and migration were significantly attenuated in VSMCs derived from 12/15-LO −/− mice relative to WT. Conclusions— Src kinase signaling plays a central role in the proatherogenic responses mediated by 12/15-LO and its oxidized lipid metabolite 12(S)-HETE in VSMCs.