Polarity of Response to Transforming Growth Factor-β1 in Proximal Tubular Epithelial Cells Is Regulated by β-Catenin

Abstract

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.It is accepted that the rate of progression of renal disease is closely correlated with the degree of interstitial fibrosis. Much of our work has focused on the possible mechanisms of the pathogenesis of interstitial fibrosis and in particular the role of PTC.3 Recent studies have highlighted the role of PTC epithelial-mesenchymal transformation in the genesis of renal interstitial fibrosis (1, 2). Transforming growth factor-␤ (TGF-␤) is an important mediator of the pathogenesis of numerous fibrotic diseases and is known to be an important stimulus driving epithelial-mesenchymal transformation (3). One important step in this process is the loss of cell-cell contact (4). Epithelial cells have discrete specialized regions of cell-cell adhesion comprising the tight junction, which forms the main barrier to paracellular traffic and adherens junctions. We have previously demonstrated that alterations in PTC morphology upon TGF-␤1 stimulation are associated with disassembly of tight and adherens junction complexes (5, 6). Evidence now suggests that the adherens junction is an important target for the regulation of intracellular signaling pathways (reviewed in Ref. Our studies sugge...

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“…Our current data demonstrated that inhibition of ß-catenin expression induced the expression of Smad3 protein, but reduced TERT protein expression. Another previous study presents similarity to our data such that the inhibition of ß-catenin augmented Smad3 signaling in proximal tubular epithelial cells (13). Smad3 has been know to block telomerase activation as shown in recent studies (38).…”

“…Even so, recently, STAT3 is reported to be involved in the nuclear accumulation of ß-catenin in colorectal cancer (11), and Wnt/ ß-catenin pathway upregulated levels of STAT3 mRNA and protein in esophageal cancer cells (12). Smad3 signaling in proximal tubular epithelial cells is augmented by the inhibition of ß-catenin expression (13). ß-catenin expression is related to the upregulation of hTERT in gallbladder carcinoma.…”

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“…Several recent reports have highlighted the complex, intertwined nature of the two signaling pathways [42]. Zhang and collaborators supported the hypothesis that b-catenin in renal epithelial cells, rather than in mesenchymal cells, may suppress TGF-b1-dependent signaling [43]. Furthermore, analysis of TGF-b1 transgenic mice showed reduced GS expression resulting from apoptosis of GS-positive hepatocytes [44].…”

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