Polarity of response to transforming growth factor-beta1 in proximal tubular epithelial cells is regulated by beta-catenin.
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.
Zhang
Mei
M
Institute of Nephrology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom.
Lee
Chien-Hung
CH
Institute of Nephrology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom.
Luo
Dong Dong
DD
Institute of Nephrology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom.
Krupa
Aleksandra
A
Institute of Nephrology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom.
Fraser
Donald
D
Institute of Nephrology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom.
Phillips
Aled
A
Institute of Nephrology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom. Electronic address: PhillipsAO@cf.ac.uk.
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*
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