Lopes Flavia L, Desmarais Joëlle, Ledoux Sandra, Gévry Nicolas Y, Lefevre Pavine, Murphy Bruce D
Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, Québec J2S 7C6, Canada.
J Biol Chem. 2006 Aug 25;281(34):24602-11. doi: 10.1074/jbc.M602146200. Epub 2006 Jun 21.
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.
血管内皮生长因子（VEGF）是一种重要的血管生成信号分子，它通过其两种酪氨酸激酶受体发挥作用，诱导内皮细胞增殖和血管通透性增加。鉴于血管生成和血管新生对早期妊娠的重要性，了解此阶段调节血管发育的机制具有重要意义。我们之前在一种独特的动物模型——水貂（一种表现出强制性胚胎滞育的物种）中证明，在胚胎植入期间VEGF及其受体表达上调。在此，我们研究了前列腺素E2（PGE₂）在妊娠早期作为VEGF调节剂的作用，并确定了这种调节的机制。我们证明，活化的胚胎分泌PGE₂，并且子宫中PGE合成酶蛋白的表达取决于植入期间与侵入的滋养层细胞的直接接触。使用转染了驱动荧光素酶报告基因的水貂VEGF启动子的水貂子宫基质细胞，我们表明PGE₂诱导启动子反式激活，并且这种反应可通过蛋白激酶A的阻断而消除。用PGE₂受体EP2和EP4拮抗剂处理可消除转染细胞中PGE₂诱导的反应。启动子的缺失研究表明，转录起始位点上游99 bp的区域是PGE₂诱导反式激活所必需的。在99 bp内发现的AP2/Sp1簇的突变完全消除了PGE₂反应。此外，染色质免疫沉淀试验证实了AP2和Sp1转录因子与子宫细胞中内源性水貂VEGF启动子的结合。PGE₂刺激了与含有AP2/Sp1簇的启动子区域相关的组蛋白H3的乙酰化。综上所述，这些结果表明PGE₂在调节子宫从而胎盘血管发育中起重要作用，通过其受体EP2和EP4发挥作用，激发蛋白激酶A激活AP2和Sp1以及组蛋白H3的乙酰化以反式激活VEGF启动子。
