Induction of KDR expression in bovine arterial endothelial cells by thrombin: involvement of nitric oxide.
Thrombin, a multifunctional serine protease, is generated at the site with vascular injuries. It not only participates in the coagulation cascade, but also can induce a lot of events related to cell mitogenesis and migration. In this study, we investigated the effect of thrombin on endothelial cell proliferation induced by vascular endothelial growth factor (VEGF). Thrombin promoted proliferation of cultured bovine carotid endothelial cells in a time- and dose-dependent manner. Moreover, it drastically enhanced the cell growth stimulated by VEGF. This stimulatory effect was reduced by inhibitors of either protein kinase C (PKC) or mitogen-activated protein kinase kinase (MAPKK). Thrombin induced a significant increase in the level of mRNA of the kinase domain-containing receptor (KDR), but not tms-like tyrosine kinase (Flt-1), in a time-dependent manner, which reached the maximum after 24 h of stimulation. This increase coincides well with the KDR protein expression. The luciferase assay showed that thrombin induced an about 7.5-fold increase in the KDR promoter activity compared with the control. This enhanced KDR promoter activity was also abolished by inhibitors of either PKC or MAPKK. The deletion analyses indicated that the region between -115 and -97 (containing Sp1 binding region) within the KDR promoter gene was required for the enhanced KDR expression induced by thrombin and VEGF. Moreover, the nitric oxide synthase (NOS) inhibitor abolished both the accelerated cell proliferation and the increased KDR expression induced by thrombin and VEGF. This inhibition was abrogated by DETA NONOate, a NO donor with long half-life. These findings suggest that thrombin might potentiate the VEGF-induced angiogenic activity through increasing the level of the VEGF receptor KDR, in which production of NO is involved.
Wang
Jie
J
Section of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.
Morita
Ikuo
I
Onodera
Mitsue
M
Murota
Sei-Itsu
SI
D000818:Animals; D001158:Arteries / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D002417:Cattle; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D015536:Down-Regulation / Q000502:physiology; D016228:Endothelial Growth Factors / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D017353:Gene Deletion; D008222:Lymphokines / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D013917:Thrombin / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000502:physiology; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D012333:RNA, Messenger; D017978:Receptors, Growth Factor; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin