Endothelin-1 Stimulates PAI-1 Protein Expression via Dual Transactivation Pathway Dependent ROCK and Phosphorylation of Smad2L
"In addition to the carboxy region, Smad2 transcription factor can be phosphorylated in the linker region as<br />well. Phosphorylation of Smad2 linker region (Smad2L) promotes the expression of plasminogen activator inhibitor type<br />1 (PAI-1) which leads to cardiovascular disorders such as atherosclerosis. The purpose of this study was to evaluate the role of dual transactivation of EGF and TGF-β receptors in phosphorylation of Smad2L and protein expression of PAI-1 induced by endothelin-1 (ET-1) in bovine aortic endothelial cells (BAECs). In addition, as an intermediary of G protein-coupled receptor (GPCR) signaling, the functions of ROCK and PLC were investigated in dual transactivation pathways.<br /><strong>Materials and Methods:</strong> The experimental study is an in vitro study performed on BAECs. Proteins were investigated<br />by western blotting using protein-specific antibodies against phospho-Smad2 linker region residues (Ser245/250/255),<br />phospho-Smad2 carboxy residues (465/467), ERK1/(Thr202/Thr204), and PAI-1.<br /><strong>Results:</strong> TGF (2 ng/ml), EGF (100 ng/ml) and ET-1 (100 nM) induced the phosphorylation of Smad2L. This response was<br />blocked in the presence of AG1478 (EGFR antagonists), SB431542 (TGFR inhibitor), and Y27632 (Rho-associated protein kinase (ROCK antagonist). Moreover, ET-1-increased protein expression of PAI-1 was decreased in the presence of bosentan (ET receptor inhibitor), AG1478, SB431542, and Y27632.<br /><strong>Conclusion:</strong> The results indicated that ET-1 increases the phosphorylation of Smad2L and protein expression of PAI-1<br />via induced the transactivation pathways of EGFR and TGFR. This study is the first attempt to scrutinize the significant role of ROCK in the protein expression of PAI-1."