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β-catenin activates TGF-β-induced epithelial-mesenchymal transition in adenomyosis - PubMed

β-catenin activates TGF-β-induced epithelial-mesenchymal transition in adenomyosis

Jung-Yoon Yoo et al. Exp Mol Med. 2020 Oct.

Abstract

Adenomyosis is defined as the presence of ectopic nests of endometrial glands and stroma within the myometrium. Adenomyosis is a common cause of dysmenorrhea, menorrhagia, and chronic pelvic pain but is often underdiagnosed. Despite its prevalence and severity of symptoms, its pathogenesis and etiology are poorly understood. Our previous study showed that aberrant activation of β-catenin results in adenomyosis through epithelial-mesenchymal transition. Using transcriptomic and ChIP-seq analysis, we identified activation of TGF-β signaling in the uteri of mutant mice that expressed dominant stabilized β-catenin in the uterus. There was a strong positive correlation between β-catenin and TGF-β2 proteins in women with adenomyosis. Furthermore, treatment with pirfenidone, a TGF-β inhibitor, increased E-cadherin expression and reduced cell invasiveness in Ishikawa cells with nuclear β-catenin. Our results suggest that β-catenin activates TGF-β-induced epithelial-mesenchymal transition in adenomyosis. This finding describes the molecular pathogenesis of adenomyosis and the use of TGF-β as a potential therapeutic target for adenomyosis.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Identification of Tgf-β2 as a target gene of β-catenin in the murine uterus.

a Venn diagram illustrating the overlap between nonredundant genes bound by β-catenin as determined by ChIP-seq and those genes regulated by β-catenin by transcriptomic analysis (>1.5-fold) in control and mutant mouse uteri. b Quantitative real-time PCR analysis of Tgf-β2 in the uterus of control and mutant mice at 4 weeks of age (n = 3 per genotype). c Representative photomicrographs and histological score (H-score) of TGF-β2 immunohistochemical staining in uteri of control and mutant mice at 4 weeks of age (n = 5 per genotype). Scale bars represent 50 μm. d Map of the β-catenin-binding site (CBS) and negative control (NC) on the Tgf-β2 promoter. e ChIP assay using the anti-β-catenin antibody on the Tgf-β2 promoter in control and mutant mouse uteri at 4 weeks of age (n = 3). The results represent the mean ± SEM. **p < 0.01 and ***p < 0.001.

Fig. 2
Fig. 2. The expression of TGF-β2 and E-cadherin in the mutant mouse uterus.

Immunofluorescence analysis of TGF-β2 and E-cadherin (a) or vimentin (b) in the uteri of control and mutant mice at 4 weeks of age (n = 5 per genotype). Nuclei were counterstained with DAPI. Arrowheads indicate TGF-β2- and vimentin-positive epithelial cells. Scale bars represent 50 μm.

Fig. 3
Fig. 3. Overexpression of TGF-β2 in eutopic endometrium and adenomyotic lesions from women with adenomyosis.

a Semiquantitative analysis of TGF-β2 levels in control endometrium from the proliferative (n = 8) and secretory (n = 13) phases and eutopic endometrium (n = 4 per phase) and adenomyotic lesions (n = 15 per phase) from the proliferative and secretory phases with adenomyosis analyzed by immunohistochemical H-score. The results represent the mean ± SEM. ***p < 0.001. b Representative photomicrographs of immunohistochemical staining of TGF-β2 in women endometrium with (eutopic and adenomyostic lesions) and without adenomyosis during the proliferative and secretory phases. Controls represent the endometrium from women with no history or evidence of adenomyosis. Eutopic refers to the endometrium, and adenomyosis refers to lesions from women with adenomyosis. Scale bars represent 50 μm.

Fig. 4
Fig. 4. Positive correlation between TGF-β2 and β-catenin in the human endometrium with and without adenomyosis.

a Representative photomicrographs of immunohistochemical staining of TGF-β2 and β-catenin in the endometrium of women with and without adenomyosis. b Correlation analysis of TGF-β2 and β-catenin in control (n = 14) and endometrial (n = 8) and adenomyotic lesions (n = 30) of adenomyosis based on immunohistochemistry results (correlation coefficient = 0.9136, p < 0.0001). c Immunofluorescence analysis and intensity quantification of TGF-β2 and E-cadherin in women endometrium without adenomyosis (n = 14) and endometrium (n = 13) and adenomyotic lesions (n = 29) of adenomyosis. Nuclei were counterstained with DAPI. Scale bars represent 50 μm. The results represent the mean ± SEM. **p < 0.01 and ***p < 0.001.

Fig. 5
Fig. 5. Increased TGF-β2 by β-catenin activation leads to EMT in adenomyosis development.

a Western blot analysis of β-catenin, TGF-β2, E-cadherin, and vimentin in Ishikawa cells transfected with exon 3-deleted β-catenin vector for 0, 18, 36, and 48 h. Actin was used as a sample loading control. b Quantification of western blot results in Ishikawa cells transfected with exon 3-deleted β-catenin vector for 0, 18, 36, and 48 h (n = 3). c Quantification of invasion through the Matrigel and transwell membrane of Ishikawa cells transfected with control or exon 3-deleted β-catenin vector and treated with TGF-β1 or TGF-β2 (n = 5). d Representative results of transwell invasion assays of control or exon 3-deleted β-catenin vector-transfected and TGF-β1- or TGF-β2-treated Ishikawa cells. The results represent the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bars represent 100 μm.

Fig. 6
Fig. 6. Decrease in the β-catenin-induced EMT process in Ishikawa cells by inhibition of TGF-β.

a Western blot analysis of β-catenin and E-cadherin in control or exon 3-deleted β-catenin vector-transfected Ishikawa cells treated with or without pirfenidone for 18 and 48 h. Actin was used as a sample loading control. b Quantification of western blot results in the control or exon 3-deleted β-catenin vector-transfected Ishikawa cells treated with or without pirfenidone for 18 and 48 h (n = 3). c Quantification of invasion through the Matrigel and transwell membrane control or exon 3-deleted β-catenin vector-transfected Ishikawa cells treated with or without pirfenidone for 48 h (n = 5). d Representative results of transwell invasion assays of control or exon 3-deleted β-catenin vector-transfected Ishikawa cells treated with or without pirfenidone for 48 h. The results represent the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bars represent 100 μm.

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