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, Ca2+ 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 Ca2+ concentrations ([Ca2+]i) were fluorescently measured by fura-2 assay. NADPH oxidase activity was determined by lucigenin-enhanced chemiluminescence. Results: Inhibition of RhoA attenuated thrombin-triggered [Ca2+]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 Ca2+ signaling, NADPH oxidase activity, ROS generation, and TF and PAI-1 expression. Conclusions: The present study shows that MT1-MMP mediates the RhoA/Ca2+ and Rac1/NADPH oxidase-dependent signaling pathways in thrombin-induced vascular responses.