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

Abstract

STUDY QUESTIONWhat are the in vitro effects of estrogen receptor β (ERβ) 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 ANSWERStudies conducted in vitro demonstrate that the ERβ 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 ERβ-dependent signaling in uterine ECs offers new insights into cell-specific molecular mechanisms of estrogen action in the human uterus.WHAT IS KNOWN ALREADYEstrogens, acting via ERs (ERα and ERβ), 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 ERβ.STUDY DESIGN, SIZE, DURATIONCells were treated with a vehicle control or the ERβ 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, METHODSAnalysis of DPN-treated ECs using Taqman gene array cards focused on genes involved in angiogenesis and inflammation identified cell type-specific ERβ-dependent changes in gene expression, with validation using qPCR and immunohistochemistry. Molecular mechanisms involved in ERβ 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 CHANCEHEECs and UtMVECs were ERβ+/ERα−. 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 ERβ 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 ERβ target genes via association with the transcription factor (‘tethered’ mechanism).LIMITATIONS, REASONS FOR CAUTIONThe 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 FINDINGSThese results have implications for our understanding of the mechanisms responsible for ERβ-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 Ca...

Endocrine disruption of oestrogen action and female reproductive tract cancers

“…Although the uterine weight in ERbKO mice is normal, they are hyper-responsive to E 2 -stimulated uterine proliferation (Dupont et al 2000), a finding consistent with studies using cell lines that have demonstrated co-expression of ERb with ERa can restrain the activities of the latter (Hall & McDonnell 1999). A study exploring the effects of oestrogens on endothelial cells derived from the endometrium and myometrium has recently reported that signalling via ERb has opposite effects on network formation in the two cell types (Greaves et al 2013). Thus, oestrogen action is dependent on ER isoform expression, which is cell-context-dependent.…”

“…In the cervix, expression of ERa and ERb has been documented in both stromal and epithelial cells (Taylor & Al-Azzawi 2000), whereas endothelial cells and leukocytes appear to express ERb alone as is the case in the endometrium (Stygar et al 2001). Both receptor subtypes are expressed in the myometrium with evidence for altered ratios between ERa and ERb in tissue recovered from pre-and postmenopausal women (Sakaguchi et al 2003), a reported increase in ERb in myometrium at the end of pregnancy (Wu et al 2000) and evidence that myometrial endothelial cells are ERb-positive/ERa-negative (Greaves et al 2013).…”

“…A truncated variant (ERb2) that can form heterodimers with full-length ERs but does not contain a functional ligand binding pocket is also expressed (Critchley et al 2002, Sierens et al 2004 as are orphan receptors implicated in regulation of oestrogen responsiveness (Bombail et al 2008(Bombail et al , 2010. Notably, endothelial and immune cells within the human endometrium are immunopositive for ERb but do not contain ERa protein (Henderson et al 2003, Greaves et al 2013. In the human fallopian tube, ER mRNAs are constitutively expressed during the menstrual cycle and have been immunolocalised to epithelial, stromal and smooth muscle cells (Horne et al 2009).…”

“…For example, co-expression of ERb with ERa and consequent formation of ERa-ERb heterodimers reduced ERa-driven transcription of some genes by competing for shared response elements (Charn et al 2010). Furthermore, EDC such as phytoestrogens that demonstrate high affinity binding to ERb may have a disproportionate effect on the function of endothelial and immune cells (Greaves et al 2013), providing a potential link between EDC and the inflammatory processes known to contribute to the aetiology of endometrial cancers (Wallace et al 2010).…”

Estradiol Is a Critical Mediator of Macrophage-Nerve Cross Talk in Peritoneal Endometriosis

“…We have previously demonstrated that endothelial cells within both normal endometrium 14,24 and peritoneal lesions 21 are ERb þ /ERa À . Notably, E2 treatment of immortalized human endometrial endothelial cells resulted in changes in gene expression of the axonal guidance factor SLIT3, consistent with the suggestion that neuroangiogenesis can be modulated by estrogen.…”