Polarity of response to transforming growth factor-β1 in proximal tubular epithelial cells is regulated by β-catenin

Abstract

Transforming growth factor-β1 (TGF-β1)-mediated loss of proximal tubular epithelial cell-cell interaction is regulated in a polarized fashion. The aim of this study was to further explore the polarity of the TGF-β1 response and to determine the significance of R-Smad-β-catenin association previously demonstrated to accompany adherens junction disassembly. Smad3 signaling response to TGF-β1 was assessed by activity of the Smad3-responsive reporter gene construct (SBE)4-Lux and by immunoblotting for phospho-Smad proteins. Similar results were obtained with both methods. Apical application of TGF-β1 led to increased Smad3 signaling compared with basolateral stimulation. Association of Smad proteins with β-catenin was greater following basolateral TGFβ-1 stimulation, as was the expression of cytoplasmic Triton-soluble β-catenin. Inhibition of β-catenin expression by small interfering RNA augmented Smad3 signaling. Lithium chloride, a GSK-3 inhibitor, increased expression of β-catenin and attenuated TGF-β1-dependent Smad3 signaling. Lithium chloride did not influence degradation of Smad3 but resulted in decreased nuclear translocation. Smad2 activation as assessed by Western blot analysis and activity of the Smad2-responsive reporter constructs ARE/MF1 was also greater following apical as compared with basolateral TGFβ-1 stimulation, suggesting that this is a generally applicable mechanism for the regulation of TGF-β1-dependent R-Smads. Caco-2 cells are a colonic carcinoma cell line, with known resistance to the anti-proliferative effects of TGF-β1 and increased expression of β-catenin. We used this cell line to address the general applicability of our observations. Inhibition of β-catenin in this cell line by small interfering RNA resulted in increased TGF-β1-dependent Smad3 phosphorylation and restoration of TGF-β1 anti-proliferative effects. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.