Regulation of the estrogen receptor activity in human endometrium

Abstract

The human endometrium is a specialized tissue that undergoes sequential phases of proliferation and secretory changes in order to support the implantation and growth of an embryo. In particular, estradiol has a central role in the coordination of these events. All major actions of estradiol are mediated by the estrogen receptor (ER). The classical mechanism of ER action involves estradiol binding and subsequent interaction between the activated ER and estrogen responsive elements (ERE) located in the promoters of target genes. However, ER can also regulate gene expression through interaction with other DNA-binding transcription factors, e.g. AP-1, Sp1 and NF-kB. Whatever the mechanism of ER activation, transcriptional activity requires the interaction of the ER with coregulator proteins (coactivators and corepressors) that are recruited at the level the target gene promoters. The objective of the present study was to study the regulation of ER-alfa activity and the role of coregulators in human endometrium. Two model systems were used: 1) The first consisted of a human endometrial cancer cell line. Two genes, known to be differentially regulated by estradiol in the human endometrium, were selected for investigation: GW112 and GADD45B. The promoters of both genes showed numerous putative regulatory motifs, including ERE half sites, Sp1, AP-1 and NF-kB sites indicating that ER-alfa could bind directly or indirectly to the promoters of these genes. The human endometrial cancer cell line RL95.2 was used to study the regulation of both genes. First, the interaction between ER-alfa and distinct coregulators was investigated in solution using co-immunoprecipitation. This showed that after estradiol treatment both coactivators and corepressors could interact with ER-alfa in solution. Thus, it could be hypothesized that selective recruitment of coregulators by ER-alfa occurs at the level of target gene promoters. To examine this hypothesis in the RL95.2 cells, chromatin immunoprecipitation (ChIP) could be used. However, this method had never been performed on these cells, therefore the ChIP protocol needed to be optimized. 2) The second model consisted of human endometrium to study all genes that are targeted by the ER-alfa in vivo. To address this question, the ChIP protocol can be coupled to genomewide DNA micro-arrays. Also in this case, the protocol had to be optimized first. In conclusion, the regulation of a particular gene by estradiol is thought to be not only influenced by the ligand but also by the cellular and promoter context. Knowledge of the mechanisms of estradiol and ER-alfa signaling to regulate gene expression are necessary to get a better understanding in the normal and abnormal endometrial physiology.