One class of the nuclear receptor AF-2 coactivator complexes contains the SRC-1/TIF2 family, CBP/p300 and an RNA coactivator, SRA. We identi®ed a subfamily of RNA-binding DEAD-box proteins (p72/p68) as a human estrogen receptor a (hERa) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC-1/TIF2 family protein and the hERa A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC-1) and SRA were co-immunoprecipitated with estrogenbound hERa in MCF7 cells and in partially puri®ed complexes associated with hERa from HeLa nuclear extracts. Estrogen induced co-localization of p72 with hERa and TIF2 in the nucleus. The presence of p72/ p68 potentiated the estrogen-induced expression of the endogenous pS2 gene in MCF7 cells. In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the estrogen-induced transactivation of hERa. These ®nd-ings indicate that p72/p68 acts as an ER subtypeselective coactivator through ERa AF-1 by associating with the coactivator complex to bind its AF-2 through direct binding with SRA and the SRC-1/TIF2 family proteins.

The hormone estrogen is involved in both female and male reproduction, as well as numerous other biological systems including the neuroendocrine, vascular, skeletal, and immune systems. Therefore, it is also implicated in many different diseases and conditions such as infertility, obesity, osteoporosis, endometriosis, and a variety of cancers. Estrogen works through its two distinct nuclear receptors, Estrogen Receptor alpha (ERα) and Estrogen Receptor beta (ERβ). Various transcriptional regulation mechanisms have been identified as the mode of action for estrogen, mainly the classical mechanism with direct DNA binding but also a non-genomic mode of action and one using tethered or indirect binding. The expression profiles of ERα and ERβ are unique with the primary sites of ERα expression being the uterus and pituitary gland and the main site of ERβ expression being the granulosa cells of the ovary. Mouse models with knockout or mutation of Esr1 and Esr2 have furthered our understanding of the role each individual receptor plays in physiology. From these studies, it is known that the primary roles for ERα are in the uterus and neuroendocrine system, as female mice lacking ERα are infertile due to impaired ovarian and uterine function, whereas female mice lacking ERβ are subfertile due to ovarian defects. The development of effective therapies for estrogen-related diseases has relied on an understanding of the physiological roles and mechanistic functionalities of ERα and ERβ in various human health and disease.

The estrogen receptor (ER) is a ligand-dependent transcription factor containing two transcriptional activation domains. AF-1 is in the N terminus of the receptor protein and AF-2 activity is dependent on helix 12 of the C-terminal ligand-binding domain. Two point mutations of leucines 543 and 544 to alanines (L543A, L544A) in helix 12 minimized estrogen-dependent transcriptional activation and reversed the activity of the estrogen antagonists ICI182780 (ICI) and tamoxifen (TAM) into agonists in a similar manner that TAM activated WT ERα through AF-1 activation. To evaluate the physiological role of AF-1 and AF-2 for the tissueselective function of TAM, we generated an AF-2-mutated ERα knock-in (AF2ERKI) mouse model. AF2ERKI homozygote female mice have hypoplastic uterine tissue and rudimentary mammary glands similar to ERα-KO mice. Female mice were infertile as a result of anovulation from hemorrhagic cystic ovaries and elevated serum LH and E2 levels, although the mutant ERα protein is expressed in the AF2ERKI model. The AF2ERKI phenotype suggests that AF-1 is not activated independently, even with high serum E2 levels. ICI and TAM induced uterotropic and ER-mediated gene responses in ovariectomized AF2ERKI female mice in the same manner as in TAM-and E2-treated WT mice. In contrast, ICI and TAM did not act as agonists to regulate negative feedback of serum LH or stimulate pituitary prolactin gene expression in a different manner than TAM-or E2-treated WT mice. The functionality of the mutant ERα in the pituitary appears to be different from that in the uterus, indicating that ERα uses AF-1 differently in the uterus and the pituitary for TAM action.

Background: Endocrine-disrupting chemicals (EDCs) are widely found in the environment. Estrogen-like activity is attributed to EDCs, such as bisphenol A (BPA), bisphenol AF (BPAF), and zearalenone (Zea), but mechanisms of action and diversity of effects are poorly understood.Objectives: We used in vitro models to evaluate the mechanistic actions of BPA, BPAF, and Zea on estrogen receptor (ER) α and ERβ.Methods: We used three human cell lines (Ishikawa, HeLa, and HepG2) representing three cell types to evaluate the estrogen promoter activity of BPA, BPAF, and Zea on ERα and ERβ. Ishikawa/ERα stable cells were used to determine changes in estrogen response element (ERE)-mediated target gene expression or rapid action-mediated effects.Results: The three EDCs showed strong estrogenic activity as agonists for ERα in a dose-dependent manner. At lower concentrations, BPA acted as an antagonist for ERα in Ishikawa cells and BPAF acted as an antagonist for ERβ in HeLa cells, whereas Zea was only a partial antagonist for ERα. ERE-mediated activation by BPA and BPAF was via the AF-2 function of ERα, but Zea activated via both the AF-1 and AF-2 functions. Endogenous ERα target genes and rapid signaling via the p44/42 MAPK pathway were activated by BPA, BPAF, and Zea.Conclusion: BPA and BPAF can function as EDCs by acting as cell type–specific agonists (≥ 10 nM) or antagonists (≤ 10 nM) for ERα and ERβ. Zea had strong estrogenic activity and activated both the AF-1 and AF-2 functions of ERα. In addition, all three compounds induced the rapid action-mediated response for ERα.