title: ERβ-dependent effects on uterine endothelial cells are cell specific and mediated via Sp1.
abstract: OBJECTIVE
  What 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)?
  
  
  CONCLUSIONS
  Studies 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.
  
  
  BACKGROUND
  Estrogens, 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.
  
  
  METHODS
  Cells 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).
  
  
  METHODS
  Analysis 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.
  
  
  RESULTS
  HEECs 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).
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  These results have implications for our understanding of the mechanisms responsible for ERβ-dependent changes in EC gene expression in hormone-dependent disorders.
authors.affiliation: The University of Edinburgh, Edinburgh, UK. egreaves@ed.ac.uk
authors.forename: Erin
authors.initials: E
authors.lastname: Greaves
authors.forename: Frances
authors.initials: F
authors.lastname: Collins
authors.forename: Hilary O D
authors.initials: HO
authors.lastname: Critchley
authors.forename: Philippa T K
authors.initials: PT
authors.lastname: Saunders
mesh_terms: D002460:Cell Line; D002478:Cells, Cultured; D004717:Endometrium / Q000098:blood supply* / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D047629:Estrogen Receptor beta / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D055806:Microvessels / Q000187:drug effects / Q000378:metabolism; D009215:Myometrium / Q000098:blood supply* / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D009928:Organ Specificity; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D015398:Signal Transduction / Q000187:drug effects; D016329:Sp1 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011422:Propionates
chemical_list: D047629:Estrogen Receptor beta; D004967:Estrogens; D009570:Nitriles; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; C486182:2,3-bis(4-hydroxyphenyl)-propionitrile; D011422:Propionates
keywords: Sp1; angiogenesis; endothelial; estrogen; uterus