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. 2012 Mar 2;287(10):7026-38.
doi: 10.1074/jbc.M111.276311. Epub 2012 Jan 12.

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  1 , 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
Affiliations

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 et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
TGF-β1 activates β-catenin-dependent signaling. A, representative Western blots (upper panel) and quantitative analysis of α-SMA (lower panel) following treatment of RLE-6TN cells with TGF-β1 (2.5 ng/ml) and/or LiCl (7.5 mm) for 6 days. Controls include media only, NaCl (7.5 mm) and TGF-β vehicle. Lamin A/C is used as a loading control (n = 4, *, p < 0.05 compared with vehicle). B, RLE-6TN cells were transfected with a LEF/TCF TOPFLASH reporter or its mutant, FOPFLASH, followed by treatment with TGF-β1 (2.5 ng/ml) for 24 h. Luciferase activity was normalized to Renilla luciferase activity (n = 3, * = p < 0.05 compared with vehicle). C, ICAT expression plasmid pCS2/ICAT and TOPFLASH reporter were co-transfected in RLE-6TN cells followed by TGF-β1 treatment. Reporter activity was determined 48 h after transfection and normalized to Renilla luciferase activity (n = 3, * = p < 0.05 (significantly different from pCS2 in the absence of TGF-β1)). D, representative Western blot (n = 3) for β-catenin in nuclear and cytoplasmic fractions harvested from RLE-6TN cells treated with TGF-β1 for indicated times. Lamin A/C and GAPDH are used to verify purity of nuclear and cytosolic fractions, respectively. Treatment with Wnt3a is used as a positive control. Cell lysates from RLE-6TN cells treated with TGF-β1 for 6 h were immunoprecipitated using anti-active-β-catenin (E) or anti-p-Tyr654-β-catenin (F) Abs. Immunoprecipitated active-β-catenin, dephosphorylated on Ser-37 and Thr-41 (E) or p-Tyr654-β-catenin (F), were detected by Western blot (n = 2). Representative Western blot (G) and quantitative analysis of total β-catenin (H) and α-SMA (I) in lysate from RLE-6TN cells transfected with β-catenin or control siRNA followed by TGF- β treatment for 48 h. GAPDH is used as a loading control (n = 3; *, p < 0.05 compared with control siRNA; **, p < 0.05 compared with control siRNA in the absence of TGF-β1). J, α-SMA reporter was transfected into RLE-6TN cells transduced with lentiviral vector expressing ICAT or control vector expressing GFP. Firefly luciferase activity was measured following treatment with TGF-β1 and normalized to Renilla luciferase activity. Expression of Myc-tagged ICAT was detected by Western blotting using anti-myc Ab (inset). β-Actin is used as loading control (n = 3, *, p < 0.05 compared with cells transduced with GFP control vector treated with TGF-β1).
FIGURE 2.
FIGURE 2.
ICG-001 inhibits TGF-β1-induced α-SMA induction and EMT. Representative Western blot (A) and quantitative analysis (B) of α-SMA protein in RLE-6TN cells treated with TGF-β1 (0.5 ng/ml) ± ICG-001 for 2 days. Lamin A/C is used as loading control. C, RLE-6TN cells were transfected with 764-bp α-SMA reporter, followed by treatment with TGF-β1 (2.5 ng/ml) for 24 h in the presence and absence of ICG-001 (5 μm). Luciferase activity was normalized to Renilla luciferase activity (n = 5, *, p < 0.05 compared with other conditions). D, co-immunoprecipitation of β-catenin with CBP (n = 3) in RLE-6TN cells treated with TGF-β1 ± ICG-001 for 24 h. NE is RLE-6TN nuclear extract which was used as positive control for β-catenin. E, representative immunofluorescence image (n = 3) of staining for α-SMA and phalloidin in RLE-6TN cells treated with TGF-β1 ± ICG-001 for 4 days. RLE-6TN cells treated with TGF-β vehicle are shown as control. Scale bar = 20 μm.
FIGURE 3.
FIGURE 3.
TGF-β-induced α-SMA induction and transcription in RLE-6TN cells is Smad-dependent. Representative Western blot (A) and quantitative analysis (B) of α-SMA protein in RLE-6TN cells treated with TGF-β1 (or vehicle DMSO) ± SIS3. M denotes medium. Lamin A/C is used as a loading control (n = 3, *, p < 0.05 (significantly different from DMSO). Western blot (C) and quantitative analysis of Smad3 (D) and α-SMA (E) protein using cell lysate from RLE-6TN cells transduced with lentivirus expressing shRNA for Smad3 or control pGIPZ nonsilencing shRNA followed by TGF-β1 treatment. Lamin A/C and GAPDH were used as loading controls (n = 3, *, p < 0.05, significantly different from control shRNA). F, RLE-6TN cells were transfected with 764-bp α-SMA reporter, followed by treatment with TGF-β1 (2.5 ng/ml) for 24 h in the presence and absence of SIS3 (6 μm). Luciferase activity was normalized to Renilla luciferase activity (n = 5, *, p < 0.05 compared with TGF-β1 vehicle and TGF-β1/SIS3 together). G, co-transfection of RLE-6TN cells with rat α-SMA promoter reporter, together with Smad3 expression plasmid pRK-5F/Smad3 or empty vector pRK-5F, followed by TGF-β1 treatment for 48 h. Firefly luciferase activity is normalized to Renilla luciferase activity (n = 3, *, p < 0.05, significantly different from the pRK-5F). H, co-transfection of RLE-6TN cells with wild type α-SMAp-Luc and SBE1 and SBE2 mutants α-SMAp-Luc-SBEm1 and α-SMAp-Luc-SBEm2, followed by TGF-β1 treatment for 15 h. Firefly luciferase activity is normalized to Renilla luciferase activity (n = 8, *, p < 0.05; NS, not significantly different).
FIGURE 4.
FIGURE 4.
TGF-β1 induces CBP-dependent interaction between Smad3 and β-catenin and occupancy of the SBE1 region of the α-SMA promoter. A, co-IP was performed with anti-Smad3 Ab using nuclear extracts (NE) harvested from RLE-6TN cells treated with TGF-β1 vehicle and DMSO (lane 1), TGF-β1, and DMSO (lane 2) and TGF-β1 together with SIS3 (3 μm) (lane 3). IgG is used as control (lane 4). Associated β-catenin was analyzed by Western blotting. NE was used as WB control. B, ChIP assay was performed with anti-β-catenin Ab for pull-down using chromatin harvested from RLE-6TN cells in the presence or absence of TGF-β1 (0.5 ng/ml) ± SIS3 (3 μm), followed by amplification of the SBE1-containing region at the α-SMA promoter by qPCR (n = 3). IgG pull-down is used as control. C, ChIP-re-ChIP assay was performed first with mouse IgG (lane 4) or anti-β-catenin Ab (lanes 5, 6, and 7) and then with anti-Smad3 (lane 6) or rabbit IgG (lane 7) for pull-down using chromatin harvested from RLE-6TN cells treated with TGF-β1. Enrichment of SBE1-containing region at the α-SMA promoter was identified by PCR (n = 3). M and NT denote marker and no template, respectively. D, ChIP assay was performed with anti-β-catenin Ab for pull-down using chromatin harvested from RLE-6TN cells in the presence or absence of TGF-β1 (0.5 ng/ml) ± ICG-001 (7.5 μm), followed by amplification of SBE1-containing region at the α-SMA promoter by qPCR (n = 3). ChIP efficiency was calculated relative to untreated cells precipitated with anti-β-catenin Ab, which was set as 1. IgG pull-down is used as control.
FIGURE 5.
FIGURE 5.
TGF-β1 induces β-catenin and CBP occupancy of SBE1 region of the α-SMA promoter. A, ChIP assay was performed with anti-CBP Ab for pull-down using chromatin harvested from RLE-6TN cells treated with TGF-β1 (0.5 ng/ml) ± ICG-001 (7.5 μm), followed by amplification of SBE1-containing region of the α-SMA promoter by qPCR. Data were processed from two pull-downs with qPCR performed in quadruplicate. ChIP efficiency was calculated relative to untreated cells precipitated with anti-CBP Ab, which was set as 1. IgG pull-down is used as control. B, ChIP-re-ChIP assay was performed first with mouse IgG (lane 4) or anti-β-catenin Abs (lanes 5, 6, and 7) and then with anti-CBP (lane 6) or rabbit IgG (lane 7) Abs for pull-down using chromatin harvested from RLE-6TN cells treated with TGF-β1. Enrichment of SBE1-containing region at the α-SMA promoter was identified by PCR (n = 3). M and NT denote marker and no template, respectively.
FIGURE 6.
FIGURE 6.
Co-localization of β-catenin with Smad3 in hyperplastic AT2 cells of IPF lung. A, representative immunofluorescence staining for β-catenin (red) and Smad3 (green) in hyperplastic AT2 cells of IPF lung tissue with anti-β-catenin and anti-Smad3 Abs. Nuclei (blue) are stained with DAPI. B, immunofluorescence staining for β-catenin (red) and pro-SP-C (green, cytoplasmic) in hyperplastic AT2 cells of IPF lung tissue. Nuclei (blue) are stained with DAPI. Scale bar = 20 μm. C, immunofluorescence staining for Smad3 (green) and Nkx2.1 (red, nuclear) in hyperplastic AT2 cells of IPF lung tissue. Nuclei (blue) are stained with DAPI. Scale bar = 20 μm.
FIGURE 7.
FIGURE 7.
Expression of CBP in IPF lung. A, representative immunohistochemistry for staining of CBP (pink) in hyperplastic AT2 cells of IPF lung tissue using anti-CBP Ab. Nuclei (blue) are stained with hematoxylin. Scale bar = 20 μm. B, magnified views of the rectangles shown in A. C, negative control using IgG.
FIGURE 8.
FIGURE 8.
Model for transcriptional regulation of α-SMA by TGF-β1. In addition to initiating phosphorylation of Smad3 and nuclear translocation of p-Smad3, TGF-β1 induces nuclear accumulation of active β-catenin through phosphorylation at Tyr-654 and dephosphorylation at Ser-37 and Thr-41. In the nucleus, a multi-protein complex among p-Smad3, β-catenin, and CBP is formed which interacts with the Smad binding element (SBE) at the α-SMA promoter to regulate α-SMA expression during EMT.
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