Thrombin induces increased expression and secretion of VEGF from human FS4fibroblasts, DU145 prostate cells and CHRF megakaryocytes

Angiogenesis is required for tumor growth and metastasis. It has recently b een suggested that thrombin is a potent promoter of angiogenesis. Ve theref ore examined the possibility that thrombin could be inducing the expression of vascular endothelial growth factor (VEGF), which promotes endothelial g rowth. Primary human FS4 fibroblasts as well as tumor cell lines: prostate DU145 and megakaryocyte CHRF were incubated with thrombin (0.25-1 unit/ml) for 1-8 hrs and then examined for mRNA by Northern Analysis. Enhanced mRNA (similar to3-4 fold over base line) was noted at 2-4 hrs, with 0.5 u/ml. th rombin, The effect was specific for thrombin activity on its PAR-I receptor , since equal units of hirudin completely inhibited the response and the th rombin effect could be mimicked with the 14 mer thrombin receptor activatio n peptide (TRAP). Upregulation of mRNA was associated with enhanced VEGF pr otein synthesis and secretion as assayed by immunoblot. Enhanced expression of VEGF mRNA was not secondary to enhanced transcription (nuclear run on e xperiments), but due to an >3 fold stabilization of mRNA (Actinomycin D cha se experiment). Enhanced VEGF mRNA stabilization is promoted by the PI3Kina se and serine/threonine kinase pathways, since thrombin-induced mRNA expres sion is inhibited by Wortmanin and HT No effect was noted with the MAPKinas e inhibitor, PD98059. Thus, thrombin-induced tumorigenesis and metastasis i s associated with enhanced VEGF protein synthesis and secretion via the sta bilization of VEGF mRNA promoted by the PI3Kinase and serine/threonine kina se pathways. This could help explain how thrombin promotes angiogenesis.