title: Crucial role of membrane type 1 matrix metalloproteinase (MT1- MMP) in RhoA/Rac1-dependent signaling pathways in thrombin- stimulated endothelial cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Cardiology and Hematology, Fukushima Medical University, Fukushima, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Ando
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Ishibashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ohkawara
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Inoue
authors.forename: Koichi
authors.initials: K
authors.lastname: Sugimoto
authors.forename: Hironori
authors.initials: H
authors.lastname: Uekita
authors.forename: Chengjun
authors.initials: C
authors.lastname: Hu
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Okamoto
authors.forename: Yoh
authors.initials: Y
authors.lastname: Takuwa
authors.forename: Yasuchika
authors.initials: Y
authors.lastname: Takeishi
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D049449:Luminescence; D053511:Matrix Metalloproteinase 14 / Q000235:genetics / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology*; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; D013917:Thrombin; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D002118:Calcium