Suppression of COUP-TFII upregulates angiogenin and promotes angiogenesis in endometriosis.

Abstract

How does hypoxia-mediated downregulation of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) promote angiogenesis in endometriosis? Suppression of COUP-TFII by hypoxia stimulates angiogenesis through induction of angiogenin (ANG). The level of COUP-TFII is downregulated in endometriotic tissues, and downregulation of COUP-TFII contributes to the development of endometriosis. Twenty-seven patients of reproductive age with endometriosis were recruited in this study. Eutopic endometrial and ectopic endometriotic stromal cells were isolated, cultured and subjected to various treatments. Microarray hybridization, quantitative RT-PCR, and Western blot were used to detect gene expression in normal and endometriotic samples. A luciferase reporter assay and chromatin immunoprecipitation in normoxia- or hypoxia-treated primary cultures of human endometrial stromal cells were performed. Tube formation analysis was performed using primary human umbilical vein endothelial cells (HUVECs). Protein level of COUP-TFII was downregulated by hypoxia (P < 0.05, normoxia versus hypoxia). Loss of COUP-TFII increased the angiogenic capacity of endometrial stromal cells (P < 0.05, COUP-TFII knockdown versus knockdown control). A novel COUP-TFII target gene, ANG, was identified through microarray analysis. Chromatin immunoprecipitation and promoter activity assays demonstrated that the ANG promoter was bound and suppressed by COUP-TFII (P < 0.05, COUP-TFII overexpression versus empty vector). The levels of ANG mRNA and protein were elevated in ectopic endometriotic stromal cells and negatively correlated with COUP-TFII (P < 0.05, endometrial versus endometriotic tissues/stromal cells). Both knockdown and forced-expression of COUP-TFII further demonstrated that ANG expression and ANG-mediated angiogenic activity were negatively regulated by COUP-TFII (P < 0.05, COUP-TFII knockdown versus knockdown control, and COUP-TFII overexpression versus empty vector). This study was conducted in primary human endometrial stromal cell cultures and HUVECs, therefore, may not fully reflect the situation in vivo. The raw data were submitted to Gene Expression Omnibus (GSE107469). This is the first study to highlight that the aberrant expression of ANG in endometriotic lesions is mediated by hypoxia-suppressed COUP-TFII expression, which reveals an as yet unidentified molecular pathogenesis of endometriosis. This work was supported by research grants (MOST 105-2314-B-006-059-MY3 to M.H.W. and MOST 104-2320-B-006-036-MY3 to S.J.T.) from the Ministry of Science and Technology, Taiwan. The authors declare that there is no conflict of interest.