Endothelin-1 Stimulates PAI-1 Protein Expression via Dual Transactivation Pathway Dependent ROCK and Phosphorylation of Smad2L

Document Type : Original Article


1 Department of Biochemistry, Cellular & Molecular Research Center, Medical School , Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

2 Department of Biochemistry, Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

3 Hyperlipidemia Research Center, Department of Clinical Biochemistry, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

4 Department of Basic Sciences, Shoushtar Faculty of Medical Sciences, Shoushtar, Iran


Objective: In addition to the carboxy region, Smad2 transcription factor can be phosphorylated in the linker region as
well. Phosphorylation of Smad2 linker region (Smad2L) promotes the expression of plasminogen activator inhibitor type
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.
Materials and Methods: The experimental study is an in vitro study performed on BAECs. Proteins were investigated
by western blotting using protein-specific antibodies against phospho-Smad2 linker region residues (Ser245/250/255),
phospho-Smad2 carboxy residues (465/467), ERK1/(Thr202/Thr204), and PAI-1.
Results: TGF (2 ng/ml), EGF (100 ng/ml) and ET-1 (100 nM) induced the phosphorylation of Smad2L. This response was
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.
Conclusion: The results indicated that ET-1 increases the phosphorylation of Smad2L and protein expression of PAI-1
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.


1. Babaahmadi-Rezaei H, Kheirollah A, Rashidi M, Seif F, Niknam Z, Zamanpour M. EGF receptor transactivation by endothelin-1 increased CHSY-1 mediated by NADPH oxidase and phosphorylation of ERK1/2. Cell J. 2021; 23(5): 510-515.
2. Donato AJ, Gano LB, Eskurza I, Silver AE, Gates PE, Jablonski K, et al. Vascular endothelial dysfunction with aging: endothelin-1 and endothelial nitric oxide synthase. Am J Physiol Heart Circ Physiol. 2009; 297(1): H425-H432.
3. Li MW, Mian MOR, Barhoumi T, Rehman A, Mann K, Paradis P, et al. Endothelin-1 overexpression exacerbates atherosclerosis and induces aortic aneurysms in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol. 2013; 33(10): 2306-2315.
4. Seif F, Kheirollah A, Babaahmadi-Rezaei H. Efficient isolation and identification of primary endothelial cells from bovine aorta by collagenase P. Immunopathol Persa. 2020; 6(2): e15.
5. Zhang C, Wang X, Zhang H, Yao C, Pan H, Guo Y, et al. Therapeutic monoclonal antibody antagonizing endothelin receptor A for Pulmonary arterial hypertension. J Pharmacol Exp Ther. 2019; 370(1): 54-61.
6. Bassilana F, Nash M, Ludwig MG. Adhesion G protein-coupled receptors: opportunities for drug discovery. Nat Rev Drug Discov. 2019; 18(11): 869-884.
7. Burch ML, Ballinger ML, Yang SNY, Getachew R, Itman C, Loveland K, et al. Thrombin stimulation of proteoglycan synthesis in vascular smooth muscle is mediated by protease-activated receptor-1 transactivation of the transforming growth factor beta type I receptor. J Biol Chem. 2010; 285(35): 26798-26805.
8. Duc NM, Kim HR,Chung KY. Recent progress in understanding the conformational mechanism of heterotrimeric G protein activation. Biomol Ther (Seoul). 2017; 25(1): 4-11.
9. Kamato D, Thach L, Bernard R, Chan V, Zheng W, Kaur H, et al. Structure, function, pharmacology, and therapeutic potential of the G protein, Gα/q,11. Front Cardiovasc Med. 2015; 2: 14.
10. Kamato D, Thach L, Getachew R, Burch M, Hollenberg MD, Zheng W, et al. Protease activated receptor-1 mediated dual kinase receptor transactivation stimulates the expression of glycosaminoglycan synthesizing genes. Cell Signal. 2016; 28(1): 110-119.
11. Little PJ, Burch ML, Getachew R, Al-aryahi S, Osman N. Endothelin-1 stimulation of proteoglycan synthesis in vascular smooth muscle is mediated by endothelin receptor transactivation of the transforming growth factor-[beta] type I receptor. J Cardiovasc Pharmacol. 2010; 56(4): 360-368.
12. Sharifat N, Mohammad Zadeh G, Ghaffari MA, Dayati P, Kamato D, Little PJ, et al. Endothelin-1 (ET-1) stimulates carboxy terminal Smad2 phosphorylation in vascular endothelial cells by a mechanism dependent on ET receptors and de novo protein synthesis. J Pharm Pharmacol. 2017; 69(1): 66-72.
13. Seif F, Little PJ, Niayesh-Mehr R, Zamanpour M, Babaahmadi-Rezaei H. Endothelin-1 increases CHSY-1 expression in aortic endothelial cells via transactivation of transforming growth factor β type I receptor induced by type B receptor endothelin-1. J Pharm Pharmacol. 2019; 71(6): 988-995.
14. Rezaei HB, Kamato D, Ansari G, Osman N, Little PJ. Cell biology of Smad2/3 linker region phosphorylation in vascular smooth muscle. Clin Exp Pharmacol Physiol. 2012; 39(8): 661-667.
15. Hough C, Radu M, Doré JJ. Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling. PLoS One. 2012; 7(8): e42513.
16. Kamato D, Rostam MA, Piva TJ, Babaahmadi Rezaei H, Getachew R, Thach L, et al. Transforming growth factor β-mediated site-specific Smad linker region phosphorylation in vascular endothelial
cells. J Pharm Pharmacol. 2014; 66(12): 1722-1733.                                                                                                                17. Kamato D, Burch ML, Piva TJ, Rezaei HB, Rostam MA, Xu S, et al. Transforming growth factor-β signalling: role and consequences of Smad linker region phosphorylation. Cell Signal. 2013; 25(10): 2017-2024. 18. Mehr RNM, Kheirollah A, Seif F, Dayati PM, Babaahmadi-Rezaei H. Reactive oxygen species and p38MAPK Have a role in the Smad2 linker region phosphorylation induced by TGF-β. Iran J Med Sci. 2018; 43(4): 401-408.
19. Rabieian R, Boshtam M, Zareei M, Kouhpayeh S, Masoudifar A, Mirzaei H. Plasminogen activator inhibitor type-1 as a regulator of fibrosis. J Cell Biochem. 2018; 119(1): 17-27.
20. Samarakoon R, Higgins SP, Higgins CE, Higgins PJ. The TGF-β1/p53/PAI-1 signaling axis in vascular senescence: role of caveolin-1.Biomolecules. 2019; 9(8).
21. Cesari M, Pahor M, Incalzi RA. Plasminogen activator inhibitor-1 (PAI-1): a key factor linking fibrinolysis and age-related subclinical and clinical conditions. Cardiovasc Ther. 2010; 28(5): e72-e91.
22. Mkaouar H, Akermi N, Kriaa A, Abraham AL, Jablaoui A, Soussou S, et al. Serine protease inhibitors and human wellbeing interplay: new insights for old friends. PeerJ. 2019; 7: e7224.
23. Eitzman DT, Westrick RJ, Xu Z, Tyson J, Ginsburg D. Plasminogen activator inhibitor-1 deficiency protects against atherosclerosis progression in the mouse carotid artery. Blood. 2000; 96(13): 4212-4215.
24. Kamato D, Ta H, Afroz R, Xu S, Osman N, Little PJ. Mechanisms of PAR-1 mediated kinase receptor transactivation: Smad linker region phosphorylation. Cell Commun Signal. 2019; 13(4): 539-548.
25. Shah BH, Baukal AJ, Chen HD, Shah AB, Catt KJ. Mechanisms of endothelin-1-induced MAP kinase activation in adrenal glomerulosa cells. J Steroid Biochem. Mol Biol. 2006; 102(1-5): 79-88.
26. Hu GF. Neomycin inhibits angiogenin-induced angiogenesis. Proc Natl Acad Sci USA. 1998; 95(17): 9791-9795.
27. Schafer AE, Blaxall BC. G protein coupled receptor-mediated transactivation of extracellular proteases. J Cardiovasc Pharmacol. 2017; 70(1): 10.
28. Burch ML, Getachew R, Osman N, Febbraio MA, Little PJ. Thrombin-mediated proteoglycan synthesis utilizes both protein-tyrosine kinase and serine/threonine kinase receptor transactivation in vascular smooth muscle cells. J Biol Chem. 2013; 288(10): 7410-7419.
29. Kamato D, Burch ML, Osman N, Zheng W, Little PJ. Therapeutic implications of endothelin and thrombin G-protein-coupled receptor transactivation of tyrosine and serine/threonine kinase cell surface receptors. J Pharm Pharmacol. 2013; 65(4): 465-473.
30. Murasawa S, Mori Y, Nozawa Y, Gotoh N, Shibuya M, Masaki H, et al. Angiotensin II type 1 receptor–induced extracellular signal–regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor. Circ Res. 1998; 82(12): 1338-1348.
31. Wang D, Yu X, Cohen RA, Brecher P. Distinct effects of N-acetylcysteine and nitric oxide on angiotensin II-induced epidermal growth factor receptor phosphorylation and intracellular Ca2+ levels. J Biol Chem. 2000; 275(16): 12223-12230.
32. Gieseler F, Ungefroren H, Settmacher U, Hollenberg MD, Kaufmann R. Proteinase-activated receptors (PARs)–focus on receptor-receptor-interactions and their physiological and pathophysiological impact. Cell Commun Signal. 2013; 11(1): 1-26.
33. Chaplin R, Thach L, Hollenberg MD, Cao Y, Little PJ, Kamato D. Insights into cellular signalling by G protein coupled receptor transactivation of cell surface protein kinase receptors. Cell Commun Signal. 2017; 11(2): 117-125.
34. Wang TJ, Gona P, Larson MG, Tofler GH, Levy D, Newton-Cheh C, et al. Multiple biomarkers for the prediction of first major cardiovascular events and death. N Engl J Med. 2006; 355(25): 2631-2639.
35. Samarakoon R, Higgins SP, Higgins CE, Higgins PJ. TGF-β1-induced plasminogen activator inhibitor-1 expression in vascular smooth muscle cells requires pp60c-src/EGFRY845 and Rho/ROCK signaling. J Mol Cell Cardiol. 2008; 44(3): 527-538.
36. Kutz SM, Higgins CE, Samarakoon R, Higgins SP, Allen RR, Qi L, et al. TGF-β1-induced PAI-1 expression is E box/USF-dependent and requires EGFR signaling. Exp Cell Res. 2006; 312(7): 1093-1105.
37. Kwist K, Bridges W, Burg K. The effect of cell passage number on osteogenic and adipogenic characteristics of D1 cells. Cytotechnology. 2016; 68(4): 1661-1667.
38. Cockell KA, Ren S, Sun J, Angel A, Shen GX. Effect of thrombin on release of plasminogen activator inhibitor-1 from cultured primate arterial smooth muscle cells. Thromb Res. 1995; 77(2): 119-131.
39. Kerins DM, Hao Q, Vaughan DE. Angiotensin induction of PAI-1 expression in endothelial cells is mediated by the hexapeptide angiotensin IV. J Clin Invest. 1995; 96(5): 2515-2520.
40. Talati N, Kamato D, Piva TJ, Little PJ, Osman N. Thrombin promotes PAI-1 expression and migration in keratinocytes via ERK dependent Smad linker region phosphorylation. Cell Signal. 2018; 47: 37-43.