Regulation of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR by estradiol through VEGF in uterus.

The induction of vascular endothelial growth factor (VEGF) expression by 17beta-estradiol (E(2)) in many target cells, including epithelial cells, fibroblasts and smooth muscle cells, suggests a role for this hormone in the modulation of angiogenesis and vascular permeability. We have already described a cyclic increase in Flk-1/KDR-expressing capillaries in the human endometrium during the proliferative and mid-secretory phases, strongly suggestive of an E(2) effect on Flk-1/KDR expression in the endometrial capillaries. However, it is unclear whether these processes are due to a direct effect of E(2) on endothelial cells. Using immunohistochemistry, we report an increase in Flk-1/KDR expression in endometrial capillaries of ovariectomized mice treated with E(2), or both E(2) and progesterone. This process is mediated through estrogen receptor (ER) activation. In vitro experiments using quantitative RT-PCR analysis demonstrate that Flk-1/KDR expression was not regulated by E(2) in human endothelial cells from the microcirculation (HMEC-1) or macrocirculation (HUVEC), even in endothelial cells overexpressing ERalpha or ERbeta after ER-mediated adenovirus infection. In contrast, Flk-1/KDR expression was up-regulated by VEGF itself, in a time- and dose-dependent manner, with the maximal response at 10 ng/ml. Thus, we suggest that E(2) up-regulates Flk-1/KDR expression in vivo in endothelial cells mainly through the modulation of VEGF by a paracrine mechanism. It is currently unknown whether or not the endothelial origin might account for differences in the E(2)-modulation of VEGF receptor expression, particularly in relation to the vascular bed of sex steroid-responsive tissues.

The analysis of chimeric human/rainbow trout estrogen receptors reveals amino acid residues outside of P- and D-boxes important for the transactivation function.

The amino acid sequence of rainbow trout estrogen receptor (rtER) is highly conserved in the C domain but presents few similarities in the A/B and E domains with human estrogen receptor alpha (hER) [NR3A1]. A previous study has shown that rtER and hER have differential functional activities in yeast Saccharomyces cerevisiae. To determine the domain(s) responsible for these differences, chimeric human/rainbow trout estrogen receptors were constructed. The A/B, C/D or E/F regions of rtER were replaced by corresponding regions of hER and expressed in yeast cells. Ligand-binding and transcription activation abilities of these hybrid receptors were compared with those of wild-type rtER or hER. Surprisingly, our data revealed that the human C/D domains play an important role in the magnitude of transactivation of ER. Two other chimeric ERs carrying either a C or D domain of hER showed that the C domain was responsible for this effect whereas the D domain did not affect hybrid receptor activities. Moreover, a chimeric hER carrying the C domain of rtER showed maximal transcriptional activity similar to that observed with rtER. Gel shift assays showed that, whereas rtER and hER present a similar binding affinity to an estrogen response element (ERE) element, the rtER C domain is responsible for a weaker DNA binding stability compared to those of hER. In addition, the human C domain allows approximately 2 times faster association of ER to an ERE. Utilization of reporter genes containing one or three EREs confirms that rtER requires protein-protein interactions for its stabilization on DNA and that the C domain is involved in this stabilization. Moreover, AF-1 may be implicated in this synergistic effect of EREs. Interestingly, although E domains of these two receptors are much less conserved, replacement of this domain in rtER by its human counterpart resulted in higher estradiol sensitivity but no increase in the magnitude of transactivation. Data from the chimeric receptors, rtER(hC) and hER(rtC), demonstrated that rtER AF-1 and AF-2 activation domains activated transcription in the presence of estradiol similar to both AF-1 and AF-2 hER. This implies that these domains, which show poor sequence homology, may interact with similar basal transcription factors.

Function of N-terminal transactivation domain of the estrogen receptor requires a potential alpha-helical structure and is negatively regulated by the A domain.

Transcriptional activation by the estrogen receptor (NR3A1, or ER) requires specific ligand-inducible activation functions located in the amino (AF-1) and the carboxyl (AF-2 and AF-2a) regions of the protein. Although several detailed reports of ER structure and function describe mechanisms whereby AF-2 activates transcription, less precise data exist for AF-1. We recently reported that the rainbow trout and human estrogen receptors (rtERs and hERs, respectively), two evolutionary distant proteins, exhibit comparable AF-1 activities while sharing only 20% homology in their N-terminal region. These data suggested that the basic mechanisms whereby AF-1 and the ER N-terminal region activate transactivation might be evolutionary conserved. Therefore, a comparative approach between rtER and hER could provide more detailed information on AF-1 function. Transactivation analysis of truncated receptors and Gal4DBD (DNA binding domain of the Gal4 factor) fusion proteins in Saccharomyces cerevisiae defined a minimal region of 11 amino acids, located at the beginning of the B domain, necessary for AF-1 activity in rtER. Hydrophobic cluster analysis (HCA) indicated the presence of a potential alpha-helix within this minimal region that is conserved during evolution. Both rtER and hER sequences corresponding to this potential alpha-helical structure were able to induce transcription when fused to the Gal4DBD, indicating that this region can transactivate in an autonomous manner. Furthermore, point mutations in this 11-amino acid region of the receptors markedly reduced their transcriptional activity either within the context of a whole ER or a Gal4DBD fusion protein. Data were confirmed in mammalian cells and, interestingly, ERs with an inverted alpha-helix were as active as their corresponding wild-type proteins, indicating a conserved role in AF-1 for these structures. Moreover, using two naturally occurring rtER N-terminal variants possessing or not the A domain (rtER(L) and rtER(S), respectively), together with A domain-truncated hER and chimeric rtER/hER receptors, we demonstrated that the A domain of the ER plays an inhibitory role in ligand-independent activity of the receptor. In vitro and in vivo protein-protein interaction assays using both rtER and hER demonstrated that this repression is likely to be mediated by a ligand-sensitive direct interaction between the A domain and the C-terminal region of the ER.

Synergism between a half-site and an imperfect estrogen-responsive element, and cooperation with COUP-TFI are required for estrogen receptor (ER) to achieve a maximal estrogen-stimulation of rainbow trout ER gene.

In all oviparous, liver represents one of the main E2-target tissues where estrogen receptor (ER) constitutes the key mediator of estrogen action. The rainbow trout estrogen receptor (rtER) gene expression is markedly up-regulated by estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect estrogen-responsive element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-elements and/or trans-acting factors, such as ER itself or COUP-TF.

Inhibition of rainbow trout (Oncorhynchus mykiss) estrogen receptor activity by cadmium.

This study was conducted to determine if the cadmium-mediated inhibition of vitellogenesis observed in fish collected from contaminated areas or undergoing experimental exposure to cadmium correlated with modification in the transcriptional activity of the estrogen receptor. A recombinant yeast system expressing rainbow trout (Oncorhynchus mykiss) estradiol receptor or human estradiol receptor was used to evaluate the direct effect of cadmium exposure on estradiol receptor transcriptional activity. In recombinant yeast, cadmium reduced the estradiol-stimulated transcription of an estrogen-responsive reporter gene. In vitro-binding assays indicated that cadmium did not affect ligand binding to the receptor. Yeast one- and two-hybrid assays showed that estradiol-induced conformational changes and receptor dimerization were not affected by cadmium; conversely, DNA binding of the estradiol receptor to its cognate element was dramatically reduced in gel retardation assay. This study provides mechanistic data supporting the idea that cadmium is an important endocrine disrupter through a direct effect on estradiol receptor transcriptional activity and may affect a number of estrogen signaling pathways.