title: Polarity of response to transforming growth factor-beta1 in proximal tubular epithelial cells is regulated by beta-catenin.
abstract: Transforming growth factor-beta1 (TGF-beta1)-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-beta1 response and to determine the significance of R-Smad-beta-catenin association previously demonstrated to accompany adherens junction disassembly. Smad3 signaling response to TGF-beta1 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-beta1 led to increased Smad3 signaling compared with basolateral stimulation. Association of Smad proteins with beta-catenin was greater following basolateral TGFbeta-1 stimulation, as was the expression of cytoplasmic Triton-soluble beta-catenin. Inhibition of beta-catenin expression by small interfering RNA augmented Smad3 signaling. Lithium chloride, a GSK-3 inhibitor, increased expression of beta-catenin and attenuated TGF-beta1-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 TGFbeta-1 stimulation, suggesting that this is a generally applicable mechanism for the regulation of TGF-beta1-dependent R-Smads. Caco-2 cells are a colonic carcinoma cell line, with known resistance to the anti-proliferative effects of TGF-beta1 and increased expression of beta-catenin. We used this cell line to address the general applicability of our observations. Inhibition of beta-catenin in this cell line by small interfering RNA resulted in increased TGF-beta1-dependent Smad3 phosphorylation and restoration of TGF-beta1 anti-proliferative effects.
authors.affiliation: Institute of Nephrology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom.
authors.forename: Mei
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Institute of Nephrology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom.
authors.forename: Chien-Hung
authors.initials: CH
authors.lastname: Lee
authors.affiliation: Institute of Nephrology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom.
authors.forename: Dong Dong
authors.initials: DD
authors.lastname: Luo
authors.affiliation: Institute of Nephrology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Krupa
authors.affiliation: Institute of Nephrology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom.
authors.forename: Donald
authors.initials: D
authors.lastname: Fraser
authors.affiliation: Institute of Nephrology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom. Electronic address: PhillipsAO@cf.ac.uk.
authors.forename: Aled
authors.initials: A
authors.lastname: Phillips
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000276:Adjuvants, Immunologic / Q000494:pharmacology; D018938:Caco-2 Cells; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism*; D016764:Cell Polarity / Q000187:drug effects* / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D018021:Lithium Chloride / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism / Q000494:pharmacology*; D051176:beta Catenin / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000276:Adjuvants, Immunologic; D034741:RNA, Small Interfering; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; D051176:beta Catenin; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride