ERß-dependent effects on uterine endothelial cells are cell specific and mediated via Sp1.

Greaves E, Collins F, Critchley HO, Saunders PT.
Source: Human Reproduction
Publication Date: (2013)
Issue: 28(9): 2490-501
Research Area:
Basic Research
STUDY QUESTION: What are the in vitro effects of estrogen receptor ß (ERß) 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 ERß 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 ERß-dependent 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 ERß), 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 ERß. STUDY DESIGN, SIZE, DURATION: Cells were treated with a vehicle control or the ERß agonist, DPN, for 2 h or 24 h (n = 5) followed by gene expression analysis. Functional assays were analyzed after a 16 h incubation with ligand (n = 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 ERß-dependent changes in gene expression, with validation using qPCR and immunohistochemistry. Molecular mechanisms involved in ERß 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 ERß+/ERa-. 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 ERß 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 ERß 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 ERß-dependent changes in EC gene expression in hormone-dependent disorders.