Thrombin regulates insulin-like growth factor-1 receptor transcription in vascular smooth muscle: characterization of the signaling pathway.
We have previously demonstrated that thrombin upregulation of insulin-like growth factor-1 receptor (IGF-1R) is essential for thrombin-induced mitogenic signaling. To characterize the mechanisms involved, we studied transcription of the IGF-1R gene in rat aortic smooth muscle cells. Thrombin markedly increased IGF-1R mRNA levels, peaking at 3 hours (112+/-7% above control). This effect was mimicked by the hexapeptide SFFLRN (that functions as a tethered ligand) and was blocked by the thrombin inhibitor hirudin. Nuclear run-on assays indicated that thrombin stimulated IGF-1R gene transcription by 2.1-fold, and this was confirmed with the use of actinomycin D. Thrombin-mediated upregulation of IGF-1R mRNA and protein levels was protein kinase C independent but was completely inhibited by the protein tyrosine kinase inhibitor genistein and by the antioxidants N-acetyl-L-cysteine and pyrrolidinedithiocarbamate, suggesting the involvement of reactive oxygen species. The thrombin-induced increase in IGF-1R mRNA was inhibitable by diphenyleneiodonium chloride but not by other inhibitors of cellular oxidase systems, suggesting that NAD(P)H oxidase was necessary for the increase. Furthermore, inhibitors of the epidermal growth factor receptor kinase, Janus kinase-2 kinase, and Src kinase did not block the effect. Thus, thrombin transcriptionally regulates the IGF-1R gene via a redox-sensitive protein tyrosine kinase-dependent pathway that does not require protein kinase C activation. In view of our prior data indicating that IGF-1R density is a critical determinant of vascular smooth muscle cell growth, our findings have particular relevance to understanding mechanisms whereby growth factors such as thrombin regulate vascular proliferation in vivo.
Du
J
J
Division of Nephrology, Emory University, Atlanta, GA, USA.
Brink
M
M
Peng
T
T
Mottironi
B
B
Delafontaine
P
P
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001011:Aorta; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D019833:Genistein / Q000494:pharmacology; D006629:Hirudins / Q000494:pharmacology; D053614:Janus Kinase 2; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors; D019255:NADPH Oxidases; D009861:Onium Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000235:genetics / Q000378:metabolism*; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
D000975:Antioxidants; D004791:Enzyme Inhibitors; D006629:Hirudins; D009861:Onium Compounds; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; C007517:diphenyleneiodonium; D019833:Genistein; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D011505:Protein-Tyrosine Kinases; D017526:Receptor, IGF Type 1; C507927:Jak2 protein, rat; D053614:Janus Kinase 2; D019061:src-Family Kinases; D011493:Protein Kinase C; D013917:Thrombin