title: Thrombin stimulates VSMC proliferation through an EGFR-dependent pathway: involvement of MMP-2.
abstract: In this study, the role of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK1/2), heparin-binding EGF-like growth factor (HB-EGF), general metalloproteinases, matrix metalloproteinases-2 (MMP-2) in mediating the mitogenic action of thrombin in rat vascular smooth muscle cells (VSMC) was investigated. The incubation of rat VSMC with thrombin (1 U/ml) for 5 min resulted in significant (p < 0.001) increase of ERK1/2 phosphorylation by 8.7 ± 0.9-fold, EGFR phosphorylation by 8.5 ± 1.3-fold (p < 0.001) and DNA synthesis by 3.6 ± 0.4-fold (p < 0.001). Separate 30-min pretreatments with EGFR tyrosine kinase irreversible inhibitor, 10 µM PD169540 (PD), and 20 µM anti-HB-EGF antibody significantly reduced thrombin-stimulated EGFR and ERK1/2 phosphorylation by 81, 72 % and by 48 and 61 %, respectively. Furthermore, the same pretreatments with PD or anti-HB-EGF antibody reduced thrombin-induced VSMC proliferation by 44 and 45 %, respectively. In addition, 30-min pretreatments with 10 µM specific MMP-2 inhibitor significantly reduced thrombin-stimulated phosphorylation of both EGFR and ERK1/2 by 25 %. Moreover, the same pretreatment with MMP-2 inhibitor reduced thrombin-induced VSMC proliferation by 45 %. These results show that the thrombin-induced DNA synthesis correlates with the level of ERK1/2 activation rather than EGFR activation. These results further suggest that thrombin acts through EGFR and ERK 1/2 signaling pathways involving MMP-2 to upregulate proliferation of VSMC.
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000, Belgrade, Serbia, katicas71@gmail.com.
authors.forename: Katarina
authors.initials: K
authors.lastname: Smiljanic
authors.forename: Milan
authors.initials: M
authors.lastname: Obradovic
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Jovanovic
authors.forename: Jelena
authors.initials: J
authors.lastname: Djordjevic
authors.forename: Branislava
authors.initials: B
authors.lastname: Dobutovic
authors.forename: Danimir
authors.initials: D
authors.lastname: Jevremovic
authors.forename: Pierre
authors.initials: P
authors.lastname: Marche
authors.forename: Esma R
authors.initials: ER
authors.lastname: Isenovic
mesh_terms: D000178:Acrylamides / Q000494:pharmacology; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011799:Quinazolines / Q000494:pharmacology; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology*; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: D000178:Acrylamides; C000601814:N-(4-(3-bromophenylamino)-7-(3-(4-morpholinyl)propoxy)-6-quinazolinyl)-2-propenamide; D011799:Quinazolines; D020032:Tyrphostins; C101044:RTKI cpd; C483803:Egfr protein, rat; D066246:ErbB Receptors; D048052:Mitogen-Activated Protein Kinase 3; D013917:Thrombin; D020778:Matrix Metalloproteinase 2