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)

Interactions between transforming growth factor-beta (TGF-beta) 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 beta-catenin-dependent and transforming growth factor-beta 1 (TGF-beta 1) 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 beta-catenin/CBP (but not beta-catenin/p300) interaction, ameliorates and reverses pulmonary fibrosis and inhibits TGF-beta 1-mediated alpha-smooth muscle actin (alpha-SMA) and collagen induction in AEC. We now demonstrate that TGF-beta 1 induces LEF/TCF TOPFLASH reporter activation and nuclear beta-catenin accumulation, while LiCl augments TGF-beta-induced alpha-SMA expression, further confirming co-operation between beta-catenin- and TGF-beta-dependent signaling pathways. Inhibition and knockdown of Smad3, knockdown of beta-catenin and overexpression of ICAT abrogated effects of TGF-beta 1 on alpha-SMA transcription/expression, indicating a requirement for beta-catenin in these Smad3-dependent effects. Following TGF-beta treatment, co-immunoprecipitation demonstrated direct interaction between endogenous Smad3 and beta-catenin, while chromatin immunoprecipitation (ChIP)re-ChIP identified spatial and temporal regulation of alpha-SMA via complex formation among Smad3, beta-catenin, and CBP. ICG-001 inhibited alpha-SMA expression/transcription in response to TGF-beta as well as alpha-SMA promoter occupancy by beta-catenin and CBP, demonstrating a previously unknown requisite TGF-beta 1/beta-catenin/CBP-mediated pro-EMT signaling pathway. Clinical relevance was shown by beta-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-beta.