Department of Cardiology and Hematology, Fukushima Medical University, Fukushima, Japan.
J Atheroscler Thromb. 2011;18(9):762-73. doi: 10.5551/jat.6783. Epub 2011 Jun 1.
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.
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+)) were fluorescently measured by fura-2 assay. NADPH oxidase activity was determined by lucigenin-enhanced chemiluminescence.
Inhibition of RhoA attenuated thrombin-triggered Ca(2+) 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.
The present study shows that MT1-MMP mediates the RhoA/Ca(2+) and Rac1/NADPH oxidase-dependent signaling pathways in thrombin-induced vascular responses.
凝血酶可诱导血管反应，包括促进组织因子（TF）和纤溶酶原激活物抑制剂-1（PAI-1）蛋白表达，该过程受小 G 蛋白 RhoA 和 Rac1、Ca2+信号和活性氧（ROS）调节。最近的研究表明，膜型 1 基质金属蛋白酶（MT1-MMP）不仅具有蛋白水解酶的功能，还具有信号分子的功能。在本研究中，我们假设 MT1-MMP 可能介导凝血酶刺激的内皮细胞中 RhoA 和 Rac1 的激活及其下游事件。
我们使用培养的人主动脉内皮细胞（HAEC）。通过小干扰 RNA（siRNA）沉默 MT1-MMP。用 C3 外切酶抑制 RhoA，而用腺病毒介导的显性负性 RhoA 和 Rac1 基因转染抑制 RhoA 和 Rac1。通过下拉实验测定 RhoA 和 Rac1 的激活。通过 fura-2 测定法荧光测定细胞内 Ca2+浓度(Ca2+)。通过荧光素增强化学发光测定 NADPH 氧化酶活性。
抑制 RhoA 可减弱凝血酶触发的 HAEC 中 Ca2+增加和 TF 和 PAI-1 的表达，而 Rac1 的抑制则阻断了凝血酶触发的 ROS 生成和 TF 和 PAI-1 的表达。沉默 MT1-MMP 可减弱凝血酶触发的 RhoA 和 Rac1 的激活，导致下游事件（包括 Ca2+信号、NADPH 氧化酶活性、ROS 生成和 TF 和 PAI-1 的表达）减弱。
本研究表明，MT1-MMP 介导了凝血酶诱导的血管反应中 RhoA/Ca2+和 Rac1/NADPH 氧化酶依赖的信号通路。
