Mechanisms of thrombin-induced MAPK activation associated with cell proliferation in human cultured tracheal smooth muscle cells

The elevated level of thrombin has been detected in the ail-way fluids of a sthmatic 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 trache al smooth muscle cells (TSMCs). Thrombin stimulated [H-3]thymidine incorpor ation and p32/p44 MAPK phosphorylation in a time- and concentration-depende nt manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) signif icantly inhibited [H-3]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inh ibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipa se C (PLC) inhibitor U73122. protein kinase C (PKC) inhibitor GF109203X, re moval of Ca2+ by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY293002. In addition, thrombin-induced [H-3]-thymidine inc orporation and p42/p34 MAPK phosphorylation was completely inhibited by PD9 8059 tan inhibitor of MEK1/2), indicating that activation of MEK1/2 was req uired for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activat ion induced by thrombin and PDGF-BB, indicating that Ras and Raf may be req uired for activation of these kinases. These results conclude that the mito genic effect of thrombin was mediated through the activation of Ras/Raf/MEK /MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, C a2+, PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferati on in cultured human TSMCs. (C) 2001 Elsevier Science Inc. All rights reser ved.