The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFbeta1 responses in human proximal-tubule epithelial cells

Abstract

In chronic renal diseases, progressive loss of renal function correlates with advancing tubulo-interstitial fibrosis. TGFbeta1-Smad (transforming growth factor-beta1-Sma and Mad protein) signalling plays an important role in the development of renal tubulo-interstitial fibrosis. Secretion of CTGF (connective-tissue growth factor; CCN2) by PTECs (proximal-tubule epithelial cells) and EMT (epithelial-mesenchymal transdifferentiation) of PTECs to myofibroblasts in response to TGFbeta are critical Smad-dependent events in the development of tubulo-interstitial fibrosis. In the present study we have investigated the distinct contributions of Smad2 and Smad3 to expression of CTGF, E-cadherin, alpha-SMA (alpha-smooth-muscle actin) and MMP-2 (matrix-metalloproteinase-2) in response to TGFbeta1 treatment in an in vitro culture model of HKC-8 (transformed human PTECs). RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. Cellular E-cadherin, alpha-SMA as well as secreted CTGF and MMP-2 were assessed by Western immunoblotting. TGFbeta1 treatment induced a fibrotic phenotype with increased expression of CTGF, MMP-2 and alpha-SMA, and decreased expression of E-cadherin. TGFbeta1-induced increases in CTGF and decreases in E-cadherin expression were Smad3-dependent, whereas increases in MMP-2 expression were Smad2-dependent. Increases in alpha-SMA expression were dependent on both Smad2 and Smad3 and were abolished by combined knockdown of both Smad2 and Smad3. In conclusion, we have demonstrated distinct roles for Smad2 and Smad3 in TGFbeta1-induced CTGF expression and markers of EMT in human PTECs. This can be of therapeutic value in designing targeted anti-fibrotic therapies for tubulo-interstitial fibrosis.

Figure 1. Smad2 and Smad3 siRNA treatment results in knockdown of respective Smad proteins in human PTECs

HKCs were transfected with Smad2 and Smad3 siRNA as described in the Materials and methods section. At the end of the experimental period, cells were lysed and Smad knockdown was assessed by Western immunoblotting. Smad2 and Smad3 siRNA treatment resulted in about a 60% decrease in the band density of respective Smad proteins assessed 84 h after the start of transfection (A and B). This knockdown was comparable in the presence and absence of TGFβ1 treatment (A and B). Western immunoblots shown are representative blots for cellular Smad2 and Smad3 proteins. Results are means±S.E.M. and expressed as a percentage of the control value (n=6; **P<0.01).

Figure 2. Smad2 and Smad3 siRNA treatment results in selective and specific knockdown of respective Smad proteins

Effect of siRNAs targeting Smad2 and Smad3 was specific as non-targeting control siRNA had no effect on either Smad2 or Smad3 protein band densities (A and B). The knockdown was also selective, as Smad3 protein levels were reduced by Smad3 siRNA, but not by Smad2 siRNA (C). Smad2 protein levels were decreased by Smad2 siRNA and not by Smad3 siRNA (D). Neither of the transfections had any effect on the level of Smad4 protein (E). Western immunoblots shown are representative of at least four transfection experiments.

Figure 3. TGFβ1-induced secreted CTGF protein in human PTECs is Smad3-dependent and Smad2-independent

Following transfection with Smad2 and Smad3 siRNAs as described in the Materials and methods section, HKCs were treated with either vehicle (0.1% BSA) or TGFβ1 (5 ng/ml) for 24 h under serum-free conditions. Secreted CTGF was assessed by Western immunoblotting of the cell culture supernatants. TGFβ1 (5 ng/ml) treatment for 24 h resulted in significant induction of secreted CTGF protein. This induction was markedly attenuated by Smad3 knockdown, but not by Smad2 knockdown. The Western immunoblot shown is a representative blot for CTGF in the cell culture supernatants. Results are means±S.E.M. and expressed as a percentage of the control value (n=4; **P<0.01).

Figure 4. TGFβ1-induced secreted MMP-2 protein in human PTECs is Smad2-dependent and Smad3-independent

After transfection with Smad2 and Smad3 siRNAs as described in the Materials and methods section, HKCs were treated with either TGFβ1 (5 ng/ml) or vehicle (0.1% BSA) for 48 h under serum-free conditions. Secreted MMP-2 was assessed by Western immunoblotting of the cell culture supernatants. TGFβ1 treatment for 48 h resulted in significant induction of secreted MMP-2 protein. This induction was markedly attenuated by Smad2 knockdown, but not by Smad3 knockdown. Western immunoblot shown is a representative blot for MMP-2 in the cell culture supernatants. Results are means±S.E.M. and expressed as a percentage of the control value (n=4; **P<0.01, *P<0.05).

Figure 5. Decrease in E-cadherin expression in response to TGFβ1 in human PTECs is Smad3-dependent and Smad2-independent

After transfection with Smad2 and Smad3 siRNAs as described in the Materials and methods section, HKCs were treated with either TGFβ1 (5 ng/ml) or vehicle (0.1% BSA) for 24 h under serum-free conditions. Cells were then lysed and cellular E-cadherin was assessed by Western immunoblotting. TGFβ1 treatment for 24 h resulted in an approx. 50% decrease in cellular E-cadherin expression. This reduction was almost completely prevented by Smad3 knockdown (A), whereas Smad2 knockdown had a minimal, or no, effect (B). Results are means±S.E.M. and expressed as a percentage of the control value (n=6; **P<0.01, *P<0.05).

Figure 6. The TGFβ1-induced increase in α-SMA expression in human PTECs is Smad2- and Smad3-dependent

After transfection with Smad2 and Smad3 siRNAs as described in the Materials and methods section, HKCs were treated with either TGFβ1 (5 ng/ml) or vehicle (0.1% BSA) for 48 h under serum-free conditions. Cells were then lysed and cellular α-SMA was assessed by Western immunoblotting. TGFβ1 treatment for 48 h resulted in a 4-fold increase in α-SMA expression. Either Smad2 or Smad3 knockdown resulted in a 50% decrease in TGFβ1-induced α-SMA expression (A). Double knockdown of both Smad2 and Smad3 resulted in complete inhibition of TGFβ1-induced α-SMA expression (B). Results are means±S.E.M. and expressed as a percentage of the control value (n=4; *P<0.05, **P<0.01).