ER b-dependent effects on uterine endothelial cells are cell specific and mediated via Sp 1

study question: What are the in vitro effects of estrogen receptor b (ERb) activation on the function of endothelial cells (ECs) from different vascular beds: human endometrial ECs (HEECs; endometrium), uterine myometrial microvascular ECs (UtMVECs; myometrium) and human umbilical vein ECs (HUVECs)? summary answer: Studies conducted in vitro demonstrate that the ERb agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) has EC type-specific effects on patterns of gene expression and network formation. Identification of a key role for the transcription factor Sp1 in ERbdependent signaling in uterine ECs offers new insights into cell-specific molecular mechanisms of estrogen action in the human uterus. what is known already: Estrogens, acting via ERs (ERa and ERb), have important, body-wide impacts on the vasculature. The human uterus is an estrogen target organ, the endometrial lining of which exhibits physiological, cyclical angiogenesis. In fixed tissue sections, human endometrial ECs are immunopositive for ERb. study design, size, duration: Cells were treated with a vehicle control or the ERb agonist, DPN, for 2 h or 24 h (n 1⁄4 5) followed by gene expression analysis. Functional assays were analyzed after a 16 h incubation with ligand (n 1⁄4 5). participant/materials, setting, methods: Analysis of DPN-treated ECs using Taqman gene array cards focused on genes involved in angiogenesis and inflammation identified cell type-specific ERb-dependent changes in gene expression, with validation using qPCR and immunohistochemistry. Molecular mechanisms involved in ERb signaling were investigated using bioinformatics, reporter assays, immunoprecipitation, siRNA and a specific inhibitor blocking Sp1-binding sites. The endometrium and myometrium from women with regular menses were used to validate the protein expression of candidate genes. main results and the role of chance: HEECs and UtMVECs were ERb+/ERa2. Treatment of ECs with DPN had opposite effects on network formation: a decrease in network formation in HEECs (P ≤ 0.001) but an increase in UtMVECs (P ≤ 0.05). Genomic analysis identified opposite changes in ERb target gene expression with only three common transcripts (HEY1, ICAM1, CASP1) in all three ECs; a unique profile was observed for each. An important role for Sp1 was identified, consistent with the regulation of ERb target genes via association with the transcription factor (‘tethered’ mechanism). limitations, reasons for caution: The study was mainly carried out in vitro using ECs of which one type was immortalized. Although the analysis of the protein expression of candidate genes was carried out using intact tissue samples from patients, investigations into in vivo angiogenesis were not carried out. wider implications of the findings: These results have implications for our understanding of the mechanisms responsible for ERb-dependent changes in EC gene expression in hormone-dependent disorders. study funding/competeing interest(s): The study was funded by a Medical Research Council Programme Grant. E.G. is the recipient of an MRC Career Development Fellowship. The authors have nothing to disclose.