New blood vessel development in the ovary, uterus and placenta, which is critical to ultimate pregnancy outcome, is regulated by estradiol-17b (E2) acting through estrogen receptor a (ERa). The underlying processes include direct actions of E2 on endothelial cells to promote their growth and migration. These mechanisms also mediate cardiovascular protection by E2 remote from pregnancy. However, the basis for direct regulation of endothelial cell phenotype by E2 and ERa is poorly understood. We have shown that E2 stimulates endothelial NO synthase (eNOS) via the activation of plasma membrane-associated ERa, and that these receptors are G protein-coupled. In preliminary studies with a new estrogen-dendrimer conjugate (EDC), we have observed that G-protein-mediated membrane ERa activation of Src and eNOS underlies E2-induced endothelial cell growth and migration. The OBJECTIVE of the proposed research is to determine if estrogen regulates endothelial cell phenotype via direct coupling of membrane ERa to G proteins. In recent initial experiments we have identified dynamic protein-protein interactions between recombinant ERa and Gai and Gby.
Aim 1 is to determine the molecular basis of ERa-G protein interaction and Src activation. The domains of ERa mediating G protein interactions will be determined, and their roles in signaling to Src will be tested. The ability of peptide sequences derived from the ERa interaction domains to inhibit signaling to Src will also be assessed.
Aim 2 is to determine if membrane ERa coupling to G proteins regulates endothelial cell growth and migration in vitro. Using EDC as a membrane-directed ligand, the requirement for ERa will be determined by gain- and loss-of-function strategies, and additional interventions will test the requirements for Gai and Gby activation. Involvement of ERa-Gai and ERa-Gby interactions will be tested using interfering peptides identified in Aim 1.
Aim 3 is to determine if membrane ERa coupling to G proteins regulates endothelial cell migration in vivo. In a nonpregnant female mouse model of carotid artery, reendothelialization, responses to E2 versus EDC will be evaluated in wild-type versus ERa mice. The requirement for Ga and Gby activation will be tested by local blockade of their functions in the carotid artery, and the requirement for ER-Gai and ER-Gby interactions will be assessed by endothelial delivery of interfering peptides.
Aim 4 is to identify nuclear targets of membrane ERa-G protein activation in endothelium in vivo. Using focused microarrays and RT-PCR, changes in gene expression will be determined in endothelium sorted from Tie2-GFP;ERa+/+versus Tie2-GFP;Era mice treated with E2 versus EDC. Genes downstream of G protein activation will be identified by pertussis toxin treatment, and the requirement for ER-Gai and ER-Gby interactions will be tested by endothelial delivery of interfering peptides. The roles of identified target genes will be tested in gain- and loss-of-function studies of cultured endothelial cell phenotypes. By investigating the novel, most proximal processes by which E2 and ERa regulate endothelial cell phenotype, the proposed research will increase our fundamental understanding of how vascular actions of estrogen promote the reproductive and nonreproductive health of women. ? ? ?

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
2R01HD030276-14
Application #
7097608
Study Section
Pregnancy and Neonatology Study Section (PN)
Program Officer
Raju, Tonse N
Project Start
1993-04-01
Project End
2011-02-28
Budget Start
2006-04-01
Budget End
2007-02-28
Support Year
14
Fiscal Year
2006
Total Cost
$253,163
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Pediatrics
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
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Wu, Qian; Chambliss, Ken; Umetani, Michihisa et al. (2011) Non-nuclear estrogen receptor signaling in the endothelium. J Biol Chem 286:14737-43
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Chambliss, Ken L; Simon, Liliana; Yuhanna, Ivan S et al. (2005) Dissecting the basis of nongenomic activation of endothelial nitric oxide synthase by estradiol: role of ERalpha domains with known nuclear functions. Mol Endocrinol 19:277-89
McCurnin, Donald C; Pierce, Richard A; Chang, Ling Yi et al. (2005) Inhaled NO improves early pulmonary function and modifies lung growth and elastin deposition in a baboon model of neonatal chronic lung disease. Am J Physiol Lung Cell Mol Physiol 288:L450-9

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