TGF-β1-induced connective tissue growth factor (CCN2) expression in human renal proximal tubule epithelial cells requires Ras/MEK/ERK and Smad signalling

Background: Connective tissue growth factor ( CTGF, CCN2) plays a fundamental role in the development of tissue fibrosis by stimulating matrix deposition and mediating many of the pro-fibrotic effects of transforming growth factor (TGF)-beta. CCN2 induction by TGF-beta in renal proximal tubule epithelial cells (PTECs) is likely to play an important role in the development of tubulointerstitial fibrosis. In this study, we investigated the induction of CCN2 by TGF-beta 1 and the possible mechanisms of this induction in human PTECs. Methods: Experiments were performed on primary and transformed (human kidney cell (HKC)-clone 8) human PTECs. Induction of CCN2 in response to TGF-beta 1 was studied at the gene promoter level by reporter gene assay, mRNA by semi-quantitative RT-PCR and protein by immunoblotting. While chemical inhibitors were used to assess the role of Ras/MEK/ERK1,2 signalling, an HKC cell line over-expressing Smad7 was used to assess the role of Smad signalling in induction of CCN2 by TGF-beta 1. Results: TGF-beta 1 induced CCN2 promoter activity, mRNA and protein in human PTECs. TGF-beta 1-dependent CCN2 promoter activity was reduced by inhibiting Ras and MEK activation. MEK inhibition also resulted in inhibition of the TGF-beta 1-induced secreted CCN2 protein. There was no significant increase in CCN2 gene promoter activity or protein by TGF-beta 1 in Smad7 over-expressing HKCs. Conclusions: TGF-beta 1 induces the expression of CCN2 in human PTECs. This induction is dependent on Ras/MEK/ERK and Smad signalling. Inhibiting TGF-beta induced CCN2 by targeting Smad and/or Ras/MEK/ERK1,2 signalling pathways could be of therapeutic value in renal fibrosis. Copyright (C) 2005 S. Karger AG, Basel.