NDT Advance Access originally published online on January 11, 2008
Nephrology Dialysis Transplantation 2008 23(5):1537-1545; doi:10.1093/ndt/gfm789
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Transforming growth factor-β-induced alpha-smooth muscle cell actin expression in renal proximal tubular cells is regulated by p38β mitogen-activated protein kinase, extracellular signal-regulated protein kinase1,2 and the Smad signalling during epithelial–myofibroblast transdifferentiation
1 Department of Pathophysiology, Faculty of Medicine, Semmelweis University 2 Hungarian Academy of Sciences, Semmelweis University Research Group for Pediatrics and Nephrology, Budapest, Hungary 3 Department of Dermatology, Department of Medical Biochemistry and Molecular Biology, and MediCity Research Laboratory, University of Turku, Finland 4 1st Department of Medicine, Faculty of Medicine, Semmelweis University, Budapest, Hungary 5 Present address: VTT Medical Biotechnology, FI-20520 Turku, Finland
Correspondence and offprint requests to: Istvan Mucsi, 1st Department of Medicine, Faculty of Medicine, Semmelweis University, Budapest, 2/A Koranyi S. u, Budapest, H-1083, Hungary. Tel: + 36-20-825-8671; Fax: + 36-1-210-1220; E-mail: istvan{at}nefros.net.
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Abstract |
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Background. Transforming growth factor-β (TGFβ)-induced epithelial–myofibroblast transdifferentiation is a central mechanism contributing to the pathogenesis of progressive tubulo-interstitial fibrosis. We wanted to dissect the role of extracellular signal-regulated protein kinase (ERK1,2), p38 mitogen-activated protein kinase (p38 MAPK) and the receptor-regulated Smad proteins in the regulation of 
-smooth muscle cell actin (SMA) expression, a hallmark of myofibroblast formation, induced by TGFβ in renal proximal tubular cells.
Methods. Activation of signalling molecules was assessed by western blotting using phospho-specific antibodies. To specifically interfere with signalling cascades, porcine proximal tubular cells (LLC-PK/AT1) were infected with recombinant replication-deficient adenoviruses. In other experiments, specific kinase inhibitors were used. The SMA synthesis was assessed by western blotting or immunofluorescent staining of cellular SMA. To assess the regulation of the SMA promoter, tubular cells were transiently transfected with a 785 bp SMA promoter–luciferase reporter construct and vectors interfering with the Smad pathway.
Results. Blocking ERK1,2 activation with PD98059 or p38 MAPK with SB 203580 potently inhibited the TGFβ-induced SMA synthesis in renal tubular cells. Adenoviral expression of dominant negative (DN) p38β but not of p38 potently inhibited SMA expression. Furthermore, adenoviral expression of DN MKK6b but not of DN MKK3b caused a substantial inhibition of the TGFβ effect, confirming the role of p38β in the regulation of TGFβ-induced SMA expression. Finally, inhibiting the Smad pathway with adenovirally delivered Smad7 and DN Smad3 also blocked TGFβ-induced SMA synthesis.
Conclusion. TGFβ-induced SMA expression is regulated by the coordinated activation of a complex system of parallel MAPK and Smad signalling pathways in renal proximal tubular cells during epithelial–mesenchymal transdifferentiation.
Keywords: alpha-smooth muscle cell actin; epithelial–myofibroblast transdifferentiation; p38 nitrogen-activated protein kinase; renal proximal tubular cell; Smad proteins
Received for publication: 16. 2.07
Accepted in revised form: 10.10.07