Interactions Between β-Catenin and Transforming Growth Factor-β Signaling Pathways Mediate Epithelial-Mesenchymal Transition and Are Dependent on the Transcriptional Co-activator cAMP-response Element-binding Protein (CREB)-binding Protein (CBP)

Beiyun Zhou,Yixin Liu,Michael Kahn,David K Ann,Arum Han,Hongjun Wang,Cu Nguyen,Per Flodby,Qian Zhong,Manda S Krishnaveni,Janice M Liebler,Parviz Minoo,Edward D Crandall,Zea Borok

doi:10.1074/jbc.m111.276311

Abstract

Interactions between transforming growth factor-β (TGF-β) and Wnt are crucial to many biological processes, although specific targets, rationale for divergent outcomes (differentiation versus block of epithelial proliferation versus epithelial-mesenchymal transition (EMT)) and precise mechanisms in many cases remain unknown. We investigated β-catenin-dependent and transforming growth factor-β1 (TGF-β1) interactions in pulmonary alveolar epithelial cells (AEC) in the context of EMT and pulmonary fibrosis. We previously demonstrated that ICG-001, a small molecule specific inhibitor of the β-catenin/CBP (but not β-catenin/p300) interaction, ameliorates and reverses pulmonary fibrosis and inhibits TGF-β1-mediated α-smooth muscle actin (α-SMA) and collagen induction in AEC. We now demonstrate that TGF-β1 induces LEF/TCF TOPFLASH reporter activation and nuclear β-catenin accumulation, while LiCl augments TGF-β-induced α-SMA expression, further confirming co-operation between β-catenin- and TGF-β-dependent signaling pathways. Inhibition and knockdown of Smad3, knockdown of β-catenin and overexpression of ICAT abrogated effects of TGF-β1 on α-SMA transcription/expression, indicating a requirement for β-catenin in these Smad3-dependent effects. Following TGF-β treatment, co-immunoprecipitation demonstrated direct interaction between endogenous Smad3 and β-catenin, while chromatin immunoprecipitation (ChIP)-re-ChIP identified spatial and temporal regulation of α-SMA via complex formation among Smad3, β-catenin, and CBP. ICG-001 inhibited α-SMA expression/transcription in response to TGF-β as well as α-SMA promoter occupancy by β-catenin and CBP, demonstrating a previously unknown requisite TGF-β1/β-catenin/CBP-mediated pro-EMT signaling pathway. Clinical relevance was shown by β-catenin/Smad3 co-localization and CBP expression in AEC of IPF patients. These findings suggest a new therapeutic approach to pulmonary fibrosis by specifically uncoupling CBP/catenin-dependent signaling downstream of TGF-β.

Highlights

Direct evidence for molecular interdependence between transforming growth factor-␤ (TGF-␤) and Wnt pathways in mesenchymal gene regulation during epithelial-mesenchymal transition (EMT) is limited
We demonstrate that ␤-catenin is necessary for induction of ␣-SMA transcription by Transforming growth factor (TGF)-␤ and that ␣-SMA induction by TGF-␤ is CBP-dependent suggesting that modulation of Smad/␤-catenin/CBP-dependent signaling via specific small molecule inhibitors such as ICG-001 may provide a novel approach to therapeutic intervention in fibrosis and EMT
To further investigate the mechanism(s) whereby these two signaling pathways may interact in the context of EMT, we used small-interfering RNA (siRNA) to knock down ␤-catenin expression in TGF-␤1treated RLE-6TN cells and examined effects on ␣-SMA expression (Fig. 1, G–I)

Summary

Background

Direct evidence for molecular interdependence between transforming growth factor-␤ (TGF-␤) and Wnt pathways in mesenchymal gene regulation during epithelial-mesenchymal transition (EMT) is limited. Evidence of nuclear accumulation of ␤-catenin, along with increased ␤-catenin target genes, Wnt ligands, Dickkopf proteins and phosphorylation of glycogen synthase kinase-3 (GSK-3␤) in hyperplastic type II (AT2) cells in IPF lung tissue [31,32,33,34,35,36] suggest a role for aberrant Wnt/␤-catenin pathway activation in the pathogenesis of IPF, perhaps through EMT Both TGF-␤ and Wnt signaling pathways are known to participate in regulation of EMT, evidence for interactions between these pathways in the context of EMT is limited and mechanistic studies have largely focused on regulation of components of one pathway by the other (i.e. reciprocal transregulation). We demonstrate that ␤-catenin is necessary for induction of ␣-SMA transcription by TGF-␤ and that ␣-SMA induction by TGF-␤ is CBP-dependent suggesting that modulation of Smad/␤-catenin/CBP-dependent signaling via specific small molecule inhibitors such as ICG-001 may provide a novel approach to therapeutic intervention in fibrosis and EMT

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION