PKC-delta/c-Src-mediated EGF receptor transactivation regulates thrombin-induced COX-2 expression and PGE(2) production in rat vascular smooth muscle cells.
The thrombin/proteinase-activated receptors (PARs) have been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. Thrombin up-regulates expression of several proteins including cyclooxygenase (COX)-2 in vascular smooth muscle cells (VSMCs) and contributes to vascular diseases. However, the mechanisms underlying thrombin-regulated COX-2 expression in VSMCs remain unclear. Western blotting, RT-PCR, and EIA kit analyses showed that thrombin induced the expression of COX-2 mRNA and protein and PGE(2) release in a time-dependent manner, which was attenuated by inhibitors of PKC (GF109203X and rottlerin), c-Src (PP1), EGF receptor (EGFR; AG1478) and MEK1/2 (U0126), or transfection with dominant negative mutants of PKC-delta, c-Src or extracellular regulated kinase (ERK) and ERK1 short hairpin RNA interference (shRNA). These results suggest that transactivation of EGFR participates in COX-2 expression induced by thrombin in VSMCs. Accordingly, thrombin stimulated phosphorylation of ERK1/2 which was attenuated by GF109203X, rottlerin, PP1, GM6001, CRM197, AG1478, or U0126, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by selective inhibitors of AP-1 and NF-kappaB, curcumin and helenalin, respectively. Moreover, thrombin-stimulated activation of NF-kappaB, AP-1, and COX-2 promoter activity was blocked by the inhibitors of c-Src, PKC, EGFR, MEK1/2, AP-1 and NF-kappaB, suggesting that thrombin induces COX-2 promoter activity mediated through PKC(delta)/c-Src-dependent EGFR transactivation, MEK-ERK1/2, AP-1, and NF-kappaB. These results demonstrate that in VSMCs, activation of ERK1/2, AP-1 and NF-kappaB pathways was essential for thrombin-induced COX-2 gene expression. Understanding the regulation of COX-2 expression and PGE(2) release by thrombin/PARs system on VSMCs may provide potential therapeutic targets of vascular inflammatory disorders including arteriosclerosis.
Hsieh
Hsi-Lung
HL
Department of Physiology and Pharmacology, Chang Gung University, Tao-Yuan, Taiwan.
Sun
Chi-Chin
CC
Wang
Tze-Shyuan
TS
Yang
Chuen-Mao
CM
D000818:Animals; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000096:biosynthesis / Q000235:genetics*; D015232:Dinoprostone / Q000096:biosynthesis*; D066246:ErbB Receptors / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D020782:Matrix Metalloproteinases / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013917:Thrombin / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism
D016328:NF-kappa B; D018808:Transcription Factor AP-1; D051546:Cyclooxygenase 2; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src); D051745:Protein Kinase C-delta; D048049:Extracellular Signal-Regulated MAP Kinases; D013917:Thrombin; D020782:Matrix Metalloproteinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D015232:Dinoprostone