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 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.
Lin
C C
CC
Department of Physiology and Pharmacology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
Shyr
M H
MH
Chien
C S
CS
Wang
C C
CC
Chiu
C T
CT
Hsiao
L D
LD
Yang
C M
CM
D000730:Androstadienes / Q000494:pharmacology; D016227:Benzoquinones; D015153:Blotting, Western; D002455:Cell Division; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D047029:Lactams, Macrocyclic; D020935:MAP Kinase Signaling System; D008301:Maleimides / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology* / Q000201:enzymology*; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology; D013997:Time Factors; D014132:Trachea / Q000166:cytology*; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000077191:Wortmannin; D018631:ras Proteins / Q000378:metabolism
D000730:Androstadienes; D016227:Benzoquinones; D002614:Chelating Agents; D002867:Chromones; D004791:Enzyme Inhibitors; D004963:Estrenes; D005419:Flavonoids; D007211:Indoles; D047029:Lactams, Macrocyclic; D008301:Maleimides; D009025:Morpholines; D020033:Protein Isoforms; D011760:Pyrrolidinones; D011809:Quinones; D010566:Virulence Factors, Bordetella; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D017828:Rifabutin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004533:Egtazic Acid; C020754:herbimycin; D019833:Genistein; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin; D018631:ras Proteins; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C070515:bisindolylmaleimide I; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D000077191:Wortmannin