Crucial role of membrane type 1 Matrix Metalloproteinase (MT1- MMP) in RhoA/Rac1-dependent signaling Pathways in Thrombin- stimulated endothelial cells

Aim: thrombin induces vascular responses including the promotion of tissue factor (tf) and plasminogen activator inhibitor-1 (pai-1) protein expression,which is modulated by small gtpases rhoa and rac1,ca 2+ signaling and reactive oxygen species (ros). recent studies have shown that membrane type 1 matrix metalloproteinase (mt1-mmp) functions not only as a protease but also as a signaling molecule. in this study,we hypothesized that mt1-mmp may mediate rhoa and rac1 activation and their downstream events in thrombin-stimulated endothelial cells. methods: we used cultured human aortic endothelial cells (haecs). mt1-mmp was silenced by small interfering rna (sirna). rhoa was inhibited by c3 exoenzyme,whereas adenovirus-mediated gene transfection of dominant negative rhoa and rac1 was used for the inhibition of rhoa and rac1. rhoa and rac1 activation was determined by pull-down assays. intracellular ca 2+ concentrations ([ca 2+]i) were fluorescently measured by fura-2 assay. nadph oxidase activity was determined by lucigenin-enhanced chemiluminescence. results: inhibition of rhoa attenuated thrombin-triggered [ca 2+]i increase and tf and pai-1 expression in haecs,whereas thrombin-triggered ros generation and tf and pai-1 expression were blocked by inhibition of rac1. silencing of mt1-mmp attenuated thrombin-triggered rhoa and rac1 activation,resulting in the attenuation of downstream events including ca 2+ signaling,nadph oxidase activity,ros generation,and tf and pai-1 expression. conclusions: the present study shows that mt1-mmp mediates the rhoa/ca 2+ and rac1/nadph oxidase-dependent signaling pathways in thrombin-induced vascular responses.