Lin C C, Shyr M H, Chien C S, Wang C C, Chiu C T, Hsiao L D, Yang C M
Department of Physiology and Pharmacology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
Cell Signal. 2001 Apr;13(4):257-67. doi: 10.1016/s0898-6568(01)00134-6.
The elevated level of thrombin has been detected in the airway fluids of asthmatic patients. However, the implication of thrombin in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study we investigated the effect of thrombin on cell proliferation and p42/p44 mitogen-activated protein kinase (MAPK) activation in human tracheal smooth muscle cells (TSMCs). Thrombin stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor GF109203X, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY294002. In addition, thrombin-induced [3H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca(2+), PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in cultured human TSMCs.
在哮喘患者的气道分泌物中检测到凝血酶水平升高。然而，凝血酶在支气管高反应性发病机制中的作用尚未完全明确。因此，在本研究中，我们调查了凝血酶对人气管平滑肌细胞（TSMCs）增殖及p42/p44丝裂原活化蛋白激酶（MAPK）激活的影响。凝血酶以时间和浓度依赖性方式刺激TSMCs中的[3H]胸腺嘧啶核苷掺入及p42/p44 MAPK磷酸化。用百日咳毒素（PTX）预处理TSMCs可显著抑制凝血酶诱导的[3H]胸腺嘧啶核苷掺入及MAPK磷酸化。这些反应可被酪氨酸激酶抑制剂染料木黄酮和赫曲霉素A、磷脂酰肌醇（PI）-磷脂酶C（PLC）抑制剂U73122、蛋白激酶C（PKC）抑制剂GF109203X、添加BAPTA/AM加EGTA去除Ca(2+)以及PI 3-激酶抑制剂渥曼青霉素和LY294002所减弱。此外，PD98059（MEK1/2抑制剂）可完全抑制凝血酶诱导的[3H]-胸腺嘧啶核苷掺入及p42/p44 MAPK磷酸化，表明这些反应需要MEK1/2的激活。此外，显性负性突变体RasN17和Raf-301的过表达可显著抑制凝血酶和血小板衍生生长因子-BB（PDGF-BB）诱导的p42/p44 MAPK激活，表明Ras和Raf可能是这些激酶激活所必需的。这些结果表明，凝血酶的促有丝分裂作用是通过Ras/Raf/MEK/MAPK途径的激活介导的。凝血酶介导的MAPK激活在培养的人TSMCs中受到与细胞增殖相关的PI-PLC、Ca(2+)、PKC、酪氨酸激酶和PI 3-激酶的调节。
