Notch activity mediates oestrogen-induced stromal cell invasion in endometriosis

in Reproduction
Correspondence should be addressed to Y Liu; Email: liqun94@163.com or to W Xiong: 176051948@qq.com
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Oestrogen has been reported to control the invasiveness of endometrial stromal cells in endometriosis. Notch signalling, a master regulator of cell invasion in tumours, is regulated by oestrogen in other diseases and hyperactivated in endometriotic stromal cells. Therefore, we hypothesized that an interaction between Notch signalling and oestrogen may exist in the regulation of endometrial stromal cell invasion, which is essential for the development of endometriosis. Western blot analysis of tissues showed that the expression levels of Notch components (JAG1 and NOTCH1) and Notch activity were markedly higher in ectopic endometria than in their eutopic and normal counterparts. Primary stromal cells obtained from normal endometria cultured with oestrogen presented significant increases in the expression of Notch components and Notch activity, the cytoplasmic and nuclear accumulation of NOTCH1 intracellular domain, the expression of matrix metallopeptidase 9 and vascular endothelial growth factor and cell invasiveness. Knockdown of NOTCH1 markedly alleviated oestrogen-induced matrix metallopeptidase 9 and vascular endothelial growth factor expression and cell invasion. ICI (an oestrogen receptor α antagonist) also blocked these oestrogenic effects. Oestrogen-responsive elements were found in the promoters of NOTCH1 and JAG1. A luciferase reporter analysis revealed that oestrogen regulated the expression of Notch components via oestrogen receptor alpha, which is bound to oestrogen-responsive elements in the JAG1 and NOTCH1 promoters. Collectively, our findings indicate that oestrogen engages in crosstalk with Notch signalling to regulate cell invasion in endometriosis via the activation of oestrogen receptor alpha and the enhancement of Notch activity. Notch signalling blockade may therefore be a novel therapeutic target for endometriosis.

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  • Supplementary Table 1. Primers used in real-time PCR
  • Supplementary Figure 1

 

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    JAG1, NOTCH1 and NOTCH1 intracellular domain (N1ICD) levels are significantly higher in eutopic and ectopic endometria in endometriosis than in normal endometria. (A) Left: Western blot analysis of JAG1, NOTCH1 and N1ICD protein levels in normal endometria obtained from patients (n = 10) with tubal infertility and paired eutopic and ectopic endometria obtained from patients (n = 10) with endometriosis. GAPDH was used as the internal control. Right: Bars indicate relative expression levels after normalization to GAPDH. Mean values were obtained from n = 3 independent experiments; *P < 0.05 by one-way ANOVA. Normal, normal endometria; Eu, eutopic endometria; Ec, ectopic endometria; N1ICD, NOTCH1 intracellular domain. (B) Representative photomicrograph of immunohistochemical staining for the JAG1 (left), NOTCH1 (middle) and N1ICD (right) proteins in human endometriotic endometrium.

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    Oestrogen increases Notch activity (assessed as the N1ICD level), the protein levels of JAG1, NOTCH1, MMP9 and VEGF and the intracellular distribution of N1ICD. (A) The protein levels of JAG1, NOTCH1, N1ICD, MMP9 and VEGF by time course analysis after treatment with 17β-oestradiol (E2) as determined by Western blot. The bars indicate relative expression levels after normalization to GAPDH. Mean values were obtained from n = 3 independent experiments; *P < 0.05 by one-way ANOVA. n.s., not significant. (B) Immunofluorescence showing the subcellular localization of N1ICD in the control (PBS + DMSO-treated) and E2-treated groups. (C) Immunohistochemistry showing the subcellular localization of N1ICD in the control and E2-treated groups. The scale bars represent 10, 5 and 2.5 µm, as indicated.

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    E2 enhances the migratory capacity and invasiveness of endometrial stromal cells (ESCs). Left: Representative photographs of ESC migration and invasion in the control (PBS + DMSO-treated) and E2-treated groups. Original magnification, 200×. Right: Quantified results of the migration and invasion assays. Mean values were obtained from n = 3 independent experiments; *P < 0.05 by unpaired Student’s t-test.

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    Knockdown of NOTCH1 by shRNA transfection alleviates oestrogen-mediated MMP9, VEGF expression and ThESC migration and invasion. (A) ThESCs were pre-transfected with scramble shRNA or NOTCH1 shRNA. NOTCH1 knockdown inhibited E2-induced MMP9 and VEGF expression determined by Western blot analysis. Bars indicate relative expression levels after normalization to GAPDH. Mean values were obtained from n = 3 independent experiments; *P < 0.05 by one-way ANOVA. (B) Representative photographs of ThESC migration and invasion after transfection with scrambled shRNA or NOTCH1 shRNA in the presence or absence of E2. (C) Bars correspond to the quantified results of the migration and invasion assays. Mean values were obtained from n = 3 independent experiments; *P < 0.05 by one-way ANOVA.

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    ERα mediates the oestrogen-induced activation of Notch signalling, the upregulation of JAG1, NOTCH1, MMP9 and VEGF, and the enhancement of migration and invasion of ESCs. (A and B) JAG1, NOTCH1, MMP9 and VEGF mRNA (A) and protein (B) levels together with N1ICD protein levels (B) were analysed after E2 and ICI treatment by real time-PCR (A) and Western blot (B) analysis. Bars indicate relative expression levels after normalization to GAPDH. Mean values were obtained from n = 3 independent experiments; *P < 0.05 by one-way ANOVA. (C) Representative photographs of migration and invasion of ESCs treated with or without ICI (left). Bars show the quantified results of migration and invasion assays (right). Mean values were obtained from n = 3 independent experiments; *P < 0.05 by one-way ANOVA.

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    The role of oestrogen in the activation of EREs in the promoters of JAG1 and NOTCH1 was assessed by luciferase assays. (A) Constructs: Full-length JAG1 promoter containing the putative ERE (FLJ1) and mutated ERE (MuJ1), full-length NOTCH1 promoter (FLN1) containing three putative EREs, and FLN1 with EREs deletion (N1a, N1b and N1c, respectively). (B and C) Luciferase activity after transfection of different constructs. Mean values were obtained from n = 3 independent experiments; *P < 0.05 by one-way ANOVA.

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