Autocrine production of basic fibroblast growth factor translated from novel synthesized mRNA mediates thrombin-induced mitogenesis in smooth muscle cells.
Thrombin is known to stimulate smooth muscle cell (SMC) growth in culture but the mechanisms underlying growth stimulation remain unclear. Previous works have observed a significant increase in platelet-derived growth factor AA and basic fibroblast growth factor (bFGF) release by bovine aortic SMC after addition of thrombin. The aim of this study was to clarify the link between thrombin, bFGF and SMC proliferation by examining the kinetics of autocrine production of bFGF by thrombin-stimulated SMC and its contribution to thrombin-induced mitogenesis. Experiments were performed to assess the dynamics of thrombin-induced bFGF mRNA transcription and to distinguish, following thrombin stimulus, between the activation of 'old' bFGF protein and/or bFGF mRNA, or novel mRNA synthesis and subsequent translation. Bovine aortic SMCs were stimulated with thrombin in serum-free culture. bFGF mRNA expression was determined by RT-PCR. Mitogenic activity of thrombin was determined by 3H-thymidine uptake. Our results demonstrate that the peak of bFGF mRNA expression occurred 24 h after thrombin stimulation. Experiments performed with cycloheximide, a translation inhibitor, revealed a translation peak later than 24 h after thrombin stimulation. Thrombin-induced mitogenic activity in SMCs was partially inhibited by the addition of anti-bFGF antibody (p<0.001) and of hirudin (p<0.001). When hirudin was added 24 h after stimulation, thrombin-induced mitogenic activity was not inhibited. In conclusion, thrombin-induced mitogenesis was partially mediated by the autocrine production of bFGF, mainly due to protein synthesis by novel mRNA with a transcription peak at 24 h and a later translation peak.
Cucina
Alessandra
A
First Department of Surgery, University of Rome La Sapienza, Rome, Italy. acucina@uniroma1.it
Borrelli
Valeria
V
Lucarelli
Marco
M
Sterpetti
Antonio V
AV
Cavallaro
Antonino
A
Strom
Roberto
R
Santoro-D'Angelo
Luciana
L
Scarpa
Sigfrido
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D019898:Autocrine Communication; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D003513:Cycloheximide / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D006629:Hirudins / Q000378:metabolism; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D016895:Culture Media, Serum-Free; D006629:Hirudins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D016222:Fibroblast Growth Factor 2; D003513:Cycloheximide; D013917:Thrombin