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. 2013 Aug;27(8):3100-12.
doi: 10.1096/fj.12-222620. Epub 2013 May 2.

Tissue transglutaminase regulates β-catenin signaling through a c-Src-dependent mechanism

Salvatore Condello  1 , Liyun Cao, Daniela Matei
Affiliations

Tissue transglutaminase regulates β-catenin signaling through a c-Src-dependent mechanism

Salvatore Condello et al. FASEB J. 2013 Aug.

Abstract

Tissue transglutaminase (TG2) is a multifunctional enzyme involved in protein cross-linking and cell adhesion to fibronectin (FN). In cancer, TG2 induces an epithelial to mesenchymal transition, contributing to metastasis. Because cadherins bind β-catenin at cell-cell junctions, disruption of adherens junctions destabilizes cadherin-catenin complexes. The goal of the present study was to analyze whether and how TG2 interacts with and regulates β-catenin signaling in ovarian cancer (OC) cells. We observed a significant correlation between TG2 and β-catenin expression levels in OC cells and tumors. TG2 augmented Wnt/β-catenin signaling, as evidenced by enhanced β-catenin transcriptional activity, inducing transcription of target genes cyclin D1 and c-Myc. By promoting integrin-mediated cell adhesion to FN, TG2 physically associates with and recruits c-Src, which in turn phosphorylates β-catenin at Tyr(654), releasing it from E-cadherin and rendering it available for transcriptional regulation. By interacting with FN and enhancing β-catenin signaling, complexed TG2 stimulates OC cell proliferation. In summary, our data demonstrate that TG2 regulates β-catenin expression and function in OC cells and define the c-Src-dependent mechanism through which this occurs.

Keywords: EMT; Wnt signaling; fibronectin; β-integrin.

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Figures

Figure 1.
Figure 1.
TG2 and β-catenin expression levels correlate in OC cells and tumors. A) Western blotting for TG2, β-catenin, and GAPDH protein levels in OC cells (SKOV3, OV90, and IGROV-1) stably transduced with AS-TG2, TG2, or siRNA targeting TG2, respectively (see Materials and Methods). B) Semiquantitative RT-PCR quantified β-catenin and GAPDH in SKOV3 (pcDNA3.1 and AS-TG2), OV90 (pQXCIP and TG2), and IGROV-1 cells (shRNA control and targeting TG2). Densitometry quantified β-catenin expression levels relative to those of GAPDH. C) IF staining for β-catenin in pcDNA3.1- and AS-TG2-transfected SKOV3 cells. D) SKOV3 OC cells were stained with polyclonal anti-TG2 antibody (Alexa Fluor 488, green) and anti-β-catenin (Cy5, red; ×600). Protein colocalization is identified by yellow spectra on merged images. Nuclei are visualized by DAPI. E) Representative IHC staining for β-catenin and TG2 in OC (×200).
Figure 2.
Figure 2.
TG2 regulates β-catenin transcription function in OC cells. A) Western blotting for β-catenin and poly(ADP-ribose) polymerase (PARP; control) in nuclear extracts from pcDNA3.1- and AS-TG2-transfected SKOV3 cells. B, C) TCF/LEF1 promoter activity measured by a reporter assay using SKOV3 cells stably transfected with pcDNA3.1 and AS-TG2 (B) and OV90 cells transduced with pQXCIP and TG2 (C), grown in complete (+FBS) or serum-free medium (−FBS). Values for luminescence were normalized to Renilla activity. Data are means ± sd of duplicate measurements. *P < 0.05; **P < 0.01. D, E) Quantitative RT-PCR for cyclin D1 (D) and c-Myc (E) in SKOV3/pcDNA3.1 and AS-TG2 cells treated with Wnt-3a (150 nM) for 4 h. Data are means ± sd of duplicate measurements. *P < 0.05; **P < 0.01. ctr, control. F) Western blotting for β-catenin, TG2, phospho-GSK-3α/β, and GAPDH in SKOV3, OV90, and IGROV-1 cells expressing TG2 or not and treated with Wnt-3a (150 nM) for 4 h.
Figure 3.
Figure 3.
TG2, β-catenin, and activated c-Src form a complex in OC cells. A, B) Equal amounts of lysates of SKOV3/pcDNA3.1 and SKOV3/AS-TG2 cells were immunoprecipitated (IP) with β-catenin antibody. Western blotting was performed using E-cadherin, TG2, and phospho (p)-β-catenin (Y654) antibodies (A) or phospho-Src (Y416) antibodies (B). C) Total β-catenin was immunoprecipitated from lysates of pcDNA3.1- and AS-TG2-transfected SKOV3 cells treated with dasatinib (100 nM) or dimethyl sulfoxide (DMSO; control) for 24 h. Western blotting was performed using phospho-β-catenin (Y654), phospho-Src (Y416), and total c-Src antibodies. D, E) Conversely, c-Src was immunoprecipitated from lysates of pcDNA3.1- and AS-TG2-transfected SKOV3 cells treated with dasatinib (100 nM) or DMSO (control) for 24 h. Western blotting was performed using phospho-β-catenin (Y654), phospho-Src (Y416), and total β-catenin (D) or TG2 and β1-integrin antibodies (E). Nonspecific IgG was used as control.
Figure 4.
Figure 4.
TG2, β-catenin, and β-integrin form a complex with c-Src in OC cells. A−C) Immunofluorescence staining for β1-integrin (Cy5, red) and c-Src (Alexa Fluor 488, green; A), β-catenin (Cy5, red) and c-Src (B), and TG2 (Cy5, red) and c-Src (C) in SKOV3 cells transfected with AS-TG2 and pcDNA3.1. D) IF with polyclonal anti-TG2 antibody (Alexa Fluor 488, green) and anti-β1-integrin (Cy5, red; ×600). Protein colocalization is identified by emergence of yellow spectra on merged images.
Figure 5.
Figure 5.
TG2 binding to FN activates c-Src and stabilizes β-catenin. A) Western blotting for β-catenin and phospho (p)-Src (Y416) in pcDNA3.1- and AS-TG2-transfected SKOV3 cells plated for 30 min onto uncoated or FN-coated (10 μg/ml) plates. B) Western blotting for p-FAK (Y576/577) in SKOV3/pcDNA3.1 and SKOV3/AS-TG2 cells plated on FN for 2 and 6 h. C) Western blotting for p-Src (Y416), β-catenin, and p-FAK (Y576/577) in SKOV3 cells plated on uncoated plastic (ctr, control), FN, collagen-1 (Col; 10 μg/ml), VN (5 μg/ml), laminin (LM; 5 μg/ml), and hyaluronan (HA; 200 μg/ml) for 30 min. D, E) Western blotting for p-Src (Y416; D) or β-catenin (E) in SKOV3 cells plated for 24 h onto uncoated or FN-coated (10 μg/ml) plates and treated with control neutralizing antibody against TG2-FN binding domain (4G3; 10 μg/ml) or α5β1-integrins (IIA1; 5 μg/ml). F) Western blotting for β-catenin and E-cadherin of OV90 cells stably transduced with TG2, pQCXIP, and δFN. G) Western blotting for p-Src (Y416) of OC cells expressing TG2 or not (SKOV3+/−AS-TG2; OV90+/−TG2, and IGROV-1+/−Sh-TG2) and treated with Wnt-3a (150 nM) or control cells for 4 h. H) Proposed mechanism by which TG2 regulates the transfer of β-catenin from its complex with E-cadherin at intercellular junctions to the cytoplasmic and nuclear pools.
Figure 6.
Figure 6.
TG2 increases OC cell proliferation in the presence of FN through a β-catenin-dependent mechanism. A) MTT assay measuring proliferation of pcDNA3.1 and AS-TG2-transfected SKOV3 cells grown on uncoated or FN-coated (10 μg/ml) plates for 48 h. Proliferation is expressed as fold increase compared with that for control cells. B) TCF/LEF promoter activity measured by a reporter assay using SKOV3/pcDNA3.1 and SKOV3/AS-TG2 cells plated onto uncoated or FN-coated plates for 6 h. Luciferase activity normalized to Renilla is expressed as fold increase compared with that of control cells. C) MTT assay measuring proliferation of SKOV3/pcDNA3.1 and SKOV3/AS-TG2 cells transfected with scrambled or β-catenin-targeting siRNA and grown on uncoated or FN-coated (10 μg/ml) plates for 48 h. Proliferation is expressed as fold increase compared with that for control cells. D) Quantitative RT-PCR measuring cyclin D1 mRNA expression levels in SKOV3/pcDNA3.1 and SKOV3/AS-TG2 cells transfected with scrambled or β-catenin-targeting siRNA and plated onto uncoated or FN-coated plates for 48 h. Data are means ± sd of duplicate measurements. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
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