The elevated level of thrombin has been detected in the airway fluids of asthmatic
patients and shown to stimulate cell proliferation in tracheal smooth muscle cells
(TSMCs). However, the implication of thrombin in the cell proliferation was not completely
understood. In this study, thrombin stimulated [3H]thymidine incorporation and p42/p44
mitogen-activated protein kinase (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 inhibitor GF109203X, removal of Ca2+ by addition of BAPTA/AM plus EGTA, PI 3-kinase
inhibitors wortmannin and LY294002, and inhibitor of MEK1/2 PD98059. Furthermore,
overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, 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, Ca2+, PKC, tyrosine kinase,
and PI 3-kinase associated with cell proliferation in canine cultured TSMCs.