Aim: To investigate whether Smad6 and Smad7 can regulate TGF-beta-induced epithelial-mesenchymal transition in human renal proximal tubule epithelial cells.
Methods: Two recombinant adeno-associated viruses (AAV) expressing Smad6 and Smad7 genes were produced without helper virus and then they were delivered into human renal proximal tubule epithelial cells (HKCs). The cells were randomly divided into normal controls, TGF-beta1-treated group, Smad7-infected control, LacZ-infected control, TGF-beta1+Smad7 group or TGF-beta1+Smad6 group, and TGF-beta1 + LacZ group. 10 microg/L of TGF-beta1 was added into the cell culture at the time of 15 min, 30 min, 60 min, and 120 min and the third day. The levels of phospho-Smad2, alpha-smooth muscle actin (alpha-SMA) and E-cadherin proteins were measured by Western blot. The concentration of hydroxyproline excreted into the culture supernatant was determinated by ELISA. The morphological changes of the cells detected by inverted microscope.
Results: Compared with the cells in absence of TGF-beta1, the expression level of P-Smad2 in HKCs increased at 15 min, 30 min, 60 min, and 120 min with the TGF-beta1 stimulation. It reached peak at 30 min. TGF-beta1 treatment for 72 hours resulted in significant up-regulation of alpha-SMA protein expression and hydroxyproline secretion, but a marked decrease in E-cadherin expression in the culture of HKCs. 10 microg/L of TGF-beta1 treatment for 72 hours also resulted in marked alteration in cell morphology. The cells lost their regular cuboidal appearance, and become elongated and spindle shaped. In the Smad7-infected cells, the overexpression of Smad7 resulted in a marked decrease of alpha-SMA protein and hydroxyproline synthesis, but a increase of E-cadherin protein, as well as the retainment of epithelial phenotypic appearance. These effects were associated with the inhibition of TGF-induced Smad2 activation, whereas the overexpression of Smad6 had no effect on the TGF-beta-induced changes in HKCs.
Conclusion: The overexpression of Smad7 instead of Smad6 can efficiently inhibit TGF-beta-induced epithelial-mesenchymal transition by blocking Smad2 activation in human renal proximal tubule epithelial cells.