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

Background. Transforming growth factor-beta (TGF beta)-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 alpha-smooth muscle cell actin (alpha SMA) expression, a hallmark of myofibroblast formation, induced by TGF beta 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 alpha SMA synthesis was assessed by western blotting or immunofluorescent staining of cellular alpha SMA. To assess the regulation of the alpha SMA promoter, tubular cells were transiently transfected with a 785 bp alpha 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 TGF beta-induced alpha SMA synthesis in renal tubular cells. Adenoviral expression of dominant negative (DN) p38 beta but not of p38 alpha potently inhibited alpha SMA expression. Furthermore, adenoviral expression of DN MKK6b but not of DN MKK3b caused a substantial inhibition of the TGF beta effect, confirming the role of p38 beta in the regulation of TGF beta-induced alpha SMA expression. Finally, inhibiting the Smad pathway with adenovirally delivered Smad7 and DN Smad3 also blocked TGF beta-induced alpha SMA synthesis. Conclusion. TGF beta-induced alpha 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.