Role of Src tyrosine kinase in the atherogenic effects of the 12/15-lipoxygenase pathway in vascular smooth muscle cells.
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.


RESULTS
Rat VSMCs (RVSMCs) were stimulated with 12(S)-HETE (0.1 micromol/L) in the presence or absence of the Src inhibitor PP2 (10 micromol/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.
Reddy
Marpadga A
MA
Division of Diabetes, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Sahar
Saurabh
S
Villeneuve
Louisa M
LM
Lanting
Linda
L
Natarajan
Rama
R
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000378:metabolism*; D000107:Acetylation; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D001093:Arachidonate 15-Lipoxygenase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D050197:Atherosclerosis / Q000201:enzymology* / Q000235:genetics; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004789:Enzyme Activation; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D006657:Histones / Q000378:metabolism; D007249:Inflammation / Q000201:enzymology* / Q000235:genetics; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011743:Pyrimidines; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
C475064:12-15-lipoxygenase; C412373:AG 1879; C491768:Ccl2 protein, mouse; C491766:Ccl2 protein, rat; D018932:Chemokine CCL2; D017362:Cyclic AMP Response Element-Binding Protein; D006657:Histones; D015850:Interleukin-6; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt; D048051:p38 Mitogen-Activated Protein Kinases