Vascular endothelial growth factor (VEGF) plays a pivotal role in the regulation of microvascular permeability and angiogenesis, processes essential for normal endometrial growth and implantation. Estrogen [17beta-estradiol (E2)], via its receptor (ER alpha), rapidly stimulates VEGF expression in the uterus at the transcriptional level. The VEGF gene promoter, however, lacks a consensus estrogen response element (ERE), and attempts to identify the site through which E2 induces VEGF expression have yielded contradictory results. To address this question, we modified the chromatin immunoprecipitation method to identify transcription factors that interact with the VEGF promoter in the rat uterus in response to E2. Chromatin immunoprecipitation showed that both Sp1 and Sp3 were associated with a proximal, GC-rich region of the promoter before E2 treatment. E2 induced an increase in Sp1 binding and the recruitment of ER alpha, and the coactivator p300 to this region. The association of ER alpha persisted, however, after VEGF mRNA levels had declined again (at 4 h), indicating that other factors might be involved in that expression. Western analysis showed that both the alpha- and beta-subunits of the transcription factor hypoxia-inducible factor 1 (HIF-1), which regulates VEGF expression in response to hypoxia and several hormones and growth factors, were present in the uterus. Furthermore, E2 rapidly induced their recruitment to the -944 to -611 bp region of the VEGF promoter, which contains the hypoxia response element to which HIF-1 binds. This binding was transient, matching the pattern of E2-induced VEGF expression. These results indicate that HIF-1 is an important mediator of E2-induced VEGF expression in the uterus. In addition, E2 also induced a later increase in HIF-1alpha mRNA and protein expression in the uterus, suggesting that it may be required for longer term effects of E2 on the uterus as well. In contrast to the uterus, HEC1A cells cultured in 95% air-5% CO2 (and therefore 20% O2) contained no HIF-1alpha, consistent with the inability of E2 to stimulate the expression of VEGF by these and other cell types in vitro.

Increased uterine capillary permeability, which can be induced by both estrogen and relaxin, is required for endometrial growth and implantation. This effect is mediated in both cases by estrogen receptors (ERs), via stimulation of vascular endothelial growth factor (VEGF) expression. The sites on the VEGF promoter through which induction occurs, however, are completely unclear. We have used the technique of chromatin immunoprecipitation in vivo to localize the site of ER action and identify other transcription factors that are involved. We have found that ERa associates with Sp1/Sp3 at a GC-rich region of the promoter. More interesting, however, is the observation that estrogen also induces rapid, transient binding of hypoxia-inducible factor 1 (HIF-1), which mediates VEGF transcription in response to hypoxia, to the promoter. The estrogen-induced HIF-1 binding closely matches the estrogen-induced pattern of VEGF expression in the uterus, suggesting that HIF-1 is involved in that induction, and probably that of many other genes as well (HIF-1 is now known to regulate the expression of more than 40 genes). It is likely that studies now under way will also link relaxin-induced VEGF expression to HIF-1. This is based on the similarities in the effects of the two hormones on VEGF expression and on their shared ability to activate the PI3K and MAPK pathways, both of which can activate HIF-1.

Estrogen regulates the growth and differentiation of the uterus via binding to estrogen receptors (ERs), members of the nuclear receptor family of transcription factors. Two forms of ER exist: ERalpha and ERbeta. The former is a well-characterized mediator of estrogen-induced transcription, but the function of the latter is unclear. Recent in vitro studies suggest that both splicing forms of ERbeta expressed in rat tissues, beta1 and beta2, may function as inhibitors of ERalpha transcriptional activity. To gain insight into the role of ERbeta in estrogen action, we examined the effects of estrogen and relaxin, a ligand-independent activator of ERs, on the expression of ERbeta1 and ERbeta2 mRNA in the uterus in vivo. Eighteen-day-old female rats were ovariectomized and, after recovery, treated with 17beta-estradiol, relaxin, or vehicle. Quantitative reverse transcription-polymerase chain reaction analyses of uterine RNA from estrogen-treated animals revealed marked decreases in the steady-state levels of the mRNAs for both ERbeta1 and ERbeta2 at 3, 6, and 24 h after treatment. Relaxin induced a similar effect. Neither hormone had any significant effect on ERalpha mRNA levels. To determine if endogenous estrogen exerts this effect, we examined the expression of ERbetas in the uterus during the estrous cycle. Levels of both isoforms were highest at diestrus (low estrogen), were significantly lower at early proestrus (rising estrogen), reached a nadir during late proestrus (peak estrogen), and rebounded at estrus (declining estrogen). These data suggest that down-regulation of ERbeta expression may be required for estrogen to exert its full trophic effects on the uterus.

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