TGF-beta1-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-beta1 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-beta1 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-beta1.


RESULTS
TGF-beta1 induced CCN2 promoter activity, mRNA and protein in human PTECs. TGF-beta1-dependent CCN2 promoter activity was reduced by inhibiting Ras and MEK activation. MEK inhibition also resulted in inhibition of the TGF-beta1-induced secreted CCN2 protein. There was no significant increase in CCN2 gene promoter activity or protein by TGF-beta1 in Smad7 over-expressing HKCs.


CONCLUSIONS
TGF-beta1 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.
Phanish
Mysore K
MK
South West Thames Institute for Renal Research, St. Helier Hospital, Carshalton, Surrey, UK. mysore.phanish@epsom-sthelier.nhs.uk
Wahab
Nadia A
NA
Hendry
Bruce M
BM
Dockrell
Mark E C
ME
D055513:Connective Tissue Growth Factor; D004847:Epithelial Cells; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005355:Fibrosis; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis*; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis*; D007668:Kidney / Q000473:pathology*; D007680:Kidney Neoplasms / Q000473:pathology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000502:physiology*; D020935:MAP Kinase Signaling System / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051785:Smad Proteins / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000502:physiology*; D053773:Transforming Growth Factor beta1; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000502:physiology
C526317:CCN2 protein, human; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D051785:Smad Proteins; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D048049:Extracellular Signal-Regulated MAP Kinases; D018631:ras Proteins