Smad2 and Smad3 play differential roles in the regulation of matrix deposition-related enzymes in renal mesangial cells

Glomerulosclerosis, the very common feature of the end-stage renal disease is characterized by excessive extracellular matrix (ECM) deposition, especially by mesangial matrix deposition in glomeruli. Transforming growth factor-beta1-Smad (TGF-beta 1-Smad) signaling and connective tissue growth factor (CTGF) play important roles in the development of glomerulosclerosis. Together with membrane-type-1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2), matrix metalloproteinase-2 (MMP-2) is responsible for the mesangial matrix degradation. In the present study we aim to investigate the distinct contributions of Smad2 and Smad3 to the expressions of MMP-2, MT1-MMP, TIMP-2 and CTGF in response to TGF-beta 1 treatment in an in vitro culture model of HBZY-1 (rat glomerular mesangial cells). RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. Cellular MT1-MMP, MMP-2, TIMP-2 and CTGF were assessed by Western blot. Their corresponding mRNA expressions were also assessed by real time RT-PCR. TGF-beta 1 treatment induced a fibrotic response with decreased expressions of MMP-2 and MT1-MMP while increased expressions of TIMP-2 and CTGF. The down-regulation of MMP-2 and MT1-MMP induced by TGF-beta 1 were Smad2-dependent, whereas the upregulation of CTGF were Smad3-dependent. Increases in TIMP-2 expression were dependent on both Smad2 and Smad3 and were abolished by combined knockdown of both Smad2 and Smad3. In conclusion, we have demonstrated the distinct roles of Smad2 and Smad3 in TGF-beta 1-induced expression of CTGF and the key enzymes responsible for mesangial matrix deposition in glomerular mesangial cells. This can be of therapeutic value in designing targeted anti-fibrotic therapies for glomerulosclerosis.