Abstract
Vascular endothelial growth factor (VEGF) is an essential angiogenic signaling element that acts through its two tyrosine kinase receptors, inducing both proliferation of endothelial cells and vascular permeability. Given the importance of vasculogenesis and angiogenesis to early pregnancy, it is of interest to understand the mechanisms regulating vascular development at this stage. We previously demonstrated that VEGF and receptors are up-regulated during embryo implantation in an unique animal model, the mink, a species displaying obligate embryonic diapause. Herein we examined the role of prostaglandin E2 (PGE(2)) as a regulator of VEGF during early pregnancy and established the mechanisms of this regulation. We demonstrate that activated embryos secrete PGE(2) and that expression of PGE synthase protein in the uterus is dependent upon direct contact with invading trophoblast cells during implantation. Using mink uterine stromal cells transfected with mink VEGF promoter driving the luciferase reporter gene, we show that PGE(2) induces promoter transactivation and that this response can be eliminated by blockade of protein kinase A. Treatment with antagonists to PGE(2) receptors EP2 and EP4 eliminated the PGE(2)-induced response in transfected cells. Deletional studies of the promoter revealed that a region of 99 bp upstream of the transcription start site is required for PGE(2)-induced transactivation. Mutation of an AP2/Sp1 cluster, found within the 99 bp, completely eliminated the PGE(2) response. Furthermore, chromatin immunoprecipitation assays confirmed binding of the AP2 and Sp1 transcription factors to the endogenous mink VEGF promoter in uterine cells. PGE(2) stimulated acetylation of histone H3 associated with the promoter region containing the AP2/Sp1 cluster. Taken together, these results demonstrate that PGE(2) plays an important role in regulating uterine and thus placental vascular development, acting through its receptors EP2 and EP4, provoking protein kinase A activation of AP2 and Sp1 as well as acetylation of histone H3 to transactivate the VEGF promoter.
Highlights
The role of prostaglandins in reproductive processes has been extensively investigated
Wang et al [10] identified prostaglandin E2 (PGE2) as the major prostaglandin at implantation sites in hamsters, and expression of microsomal PGE synthase was correlated with expression of COX-2
Expression of PGE Synthase and EP Receptor mRNA in the Uterus—Uterine tissues collected from implantation and interimplantation sites as well as uteri collected from pseudopregnant females contained mRNA for PGE synthase
Summary
Animals and Sample Collection—All treatment protocols involving the use of animals were approved by the Comitede Deontologie, Facultede Medecine Veterinaire, Universitede Montreal in accordance with regulations of the Canadian Council of Animal Care. We and others have shown that prolactin injections terminate obligate embryonic diapause and induce embryo activation and implantation in the mink [32,33,34], and a standard protocol consisting of daily intramuscular injections of 1 mg/kg prolactin (Sigma) was employed beginning 1 week following last mating and continuing for 12 days. Pseudopregnancy was induced in nonmated females by two injections of GnRH (10 g/kg Factrel; Ayerst, Guelph, Canada) 7 days apart. Uterine tissues were collected at the early stages of implantation from randomly selected females at days 13 and 15 following the first prolactin injections as well as from pseudopregnant animals. As we have previously shown [35], the natural increases in prolactin and consequent progesterone synthesis take place in pseudopregnant animals, and samples were collected and collected at 30 days after induction of the first ovulation.
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