CCN3, a key matricellular protein, distinctly inhibits TGFβ1-mediated Smad1/5/8 signalling in human podocyte culture

Growth factors like TGF beta and CTGF (CCN2) plays a vital role in various cellular functions. TGF beta and CTGF are overexpressed in renal fibrosis. CTGF act as profibrotic stimuli to TGF beta. CCN3 is a member of CCN family which also comprises CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2) and CCN6 (WISP-3). CCN3 has been shown to antagonise CTGF. In this study, we investigated the role of CCN3 in TGF beta 1-mediated signalling in human podocytes culture. This study describes the novel function of CCN3 in regulation of TGF beta 1 mediated non-canonical Smad signalling in human podocytes culture. Experiments were conducted on conditionally immortalised human podocytes incubated with TGF beta 1 (1.25ng/ml and 2.5ng/ml) and CCN3 (360ng/ml). Western blot study was performed to study signalling proteins. RT-PCR was performed to study alternative splicing of Fibronectin (Fn). Real time PCR was performed to look for gene expression of Fn and collagen IV and collagen I. TGF beta 1 induced the Smad1/5/8, Smad3 and p38 phosphorylation and CCN3 downregulated the TGF beta 1 induced Smad1/5/8 phosphorylation and did not affect Smad3 and p38 phosphorylation. In addition to this CCN3 induced alternative splicing of Extra domain A Fibronectin (EDA+ Fn). CCN3 also induced collagen IV, Collagen I and Fn gene expression. This is the first evidence of downregulation of TGF beta beta-mediated activation of a Smad1/5/8 signalling pathway by CCN3 in human podocytes and in any cell type. Targeting CCN3-mediated events could provide exciting outcomes in the understanding of molecular mechanism of fibrosis.