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J. Biol. Chem., Vol. 282, Issue 39, 28639-28647, September 28, 2007

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Polarity of Response to Transforming Growth Factor-1 in Proximal Tubular Epithelial Cells Is Regulated by -Catenin^*

From the Institute of Nephrology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom

Transforming growth factor-1 (TGF-1)-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-1 response and to determine the significance of R-Smad--catenin association previously demonstrated to accompany adherens junction disassembly. Smad3 signaling response to TGF-1 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-1 led to increased Smad3 signaling compared with basolateral stimulation. Association of Smad proteins with -catenin was greater following basolateral TGF-1 stimulation, as was the expression of cytoplasmic Triton-soluble -catenin. Inhibition of -catenin expression by small interfering RNA augmented Smad3 signaling. Lithium chloride, a GSK-3 inhibitor, increased expression of -catenin and attenuated TGF-1-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 TGF-1 stimulation, suggesting that this is a generally applicable mechanism for the regulation of TGF-1-dependent R-Smads. Caco-2 cells are a colonic carcinoma cell line, with known resistance to the anti-proliferative effects of TGF-1 and increased expression of -catenin. We used this cell line to address the general applicability of our observations. Inhibition of -catenin in this cell line by small interfering RNA resulted in increased TGF-1-dependent Smad3 phosphorylation and restoration of TGF-1 anti-proliferative effects.

Received for publication, January 22, 2007 , and in revised form, July 2, 2007.

^* This work was supported in part by a grant from Diabetes UK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Welsh Office of Research Development/Medical Research Council clinician scientist fellowship funds.

² To whom correspondence should be addressed. Tel.: 44-2920-748411; Fax: 44-2920-748470; E-mail: PhillipsAO{at}cf.ac.uk.

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