Analyses of steroid receptors are important for understanding molecular details of transcriptional control, as well as providing insight as to how an individual transacting factor contributes to cell identity and function. These studies have led to the identification of a superfamily of regulatory proteins that include receptors for thyroid hormone and the vertebrate morphogen retinoic acid. Although animals employ complex and often distinct ways to control their physiology and development, the discovery of receptor-related molecules in a wide range of species suggests that mechanisms underlying morphogenesis and homeostasis may be more ubiquitous than previously expected.

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The steroid and thyroid hormone receptor superfamily

We have cloned a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. The receptor homologue is activated by a diverse class of rodent hepatocarcinogens that causes proliferation of peroxisomes. Identification of a peroxisome proliferator-activated receptor should help elucidate the mechanism of the hypolipidaemic effect of these hepatocarcinogens and aid evaluation of their potential carcinogenic risk to man.

Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators

Gene regulation by steroid hormones.

The Structural Basis of Estrogen Receptor/Coactivator Recognition and the Antagonism of This Interaction by Tamoxifen

All scientific investigations begin with distinct objectives: first is the hypothesis upon which studies are undertaken to disprove, and second is the overall aim of obtaining further information, from which future and more precise hypotheses may be drawn Studies focusing on the generation and use of gene-targeted animal models also apply these goals and may be loosely categorized into sequential phases that become apparent as the use of the model progresses Initial studies of knockout models often focus on the plausibility of the model based on prior knowledge and whether the generation of an animal lacking the particular gene will prove lethal or not Upon the successful generation of a knockout, confirmatory studies are undertaken to corroborate previously established hypotheses of the function of the disrupted gene product As these studies continue, observations of unpredicted phenotypes or, more likely, the lack of a phenotype that was expected based on models put forth from past investigations are noted Often the surprising phenotype is due to the loss of a gene product that is downstream from the functions of the disrupted gene, whereas the lack of an expected phenotype may be due to compensatory roles filled by alternate mechanisms As the descriptive studies of the knockout continue, use of the model is often shifted to the role as a unique research reagent, to be used in studies that 1) were not previously possible in a wild-type model; 2) aimed at finding related proteins or pathways whose existence or functions were previously masked; or 3) the subsequent effects of the gene disruption on related physiological and biochemical systems The alpha ERKO mice continue to satisfy the confirmatory role of a knockout quite well As summarized in Table 4, the phenotypes observed in the alpha ERKO due to estrogen insensitivity have definitively illustrated several roles that were previously believed to be dependent on functional ER alpha, including 1) the proliferative and differentiative actions critical to the function of the adult female reproductive tract and mammary gland; 2) as an obligatory component in growth factor signaling in the uterus and mammary gland; 3) as the principal steroid involved in negative regulation of gonadotropin gene transcription and LH levels in the hypothalamic-pituitary axis; 4) as a positive regulator of PR expression in several tissues; 5) in the positive regulation of PRL synthesis and secretion from the pituitary; 6) as a promotional factor in oncogene-induced mammary neoplasia; and 7) as a crucial component in the differentiation and activation of several behaviors in both the female and male The list of unpredictable phenotypes in the alpha ERKO must begin with the observation that generation of an animal lacking a functional ER alpha gene was successful and produced animals of both sexes that exhibit a life span comparable to wild-type The successful generation of beta ERKO mice suggests that this receptor is also not essential to survival and was most likely not a compensatory factor in the survival of the alpha ERKO In support of this is our recent successful generation of double knockout, or alpha beta ERKO mice of both sexes The precise defects in certain components of male reproduction, including the production of abnormal sperm and the loss of intromission and ejaculatory responses that were observed in the alpha ERKO, were quite surprising In turn, certain estrogen pathways in the alpha ERKO female appear intact or unaffected, such as the ability of the uterus to successfully exhibit a progesterone-induced decidualization response, and the possible maintenance of an LH surge system in the hypothalamus [ABSTRACT TRUNCATED]

Estrogen receptor null mice: what have we learned and where will they lead us?

https://www.cell.com/cell/pdf/0092-8674(87)90581-2.pdf

Functional domains of the human estrogen receptor.

Using chimeric recombinants transfected into HeLa cells and a transient expression assay, we demonstrate that the 5'-flanking region of the pS2 gene from position -428 to position -324 exhibits both constitutive and estrogen-inducible enhancer activity. The estrogen-inducible activity, but not the constitutive activity, was inhibited by antiestrogens. ICI 164,384 behaved as a pure antagonist, whereas hydroxytamoxifen was a partial agonist-antagonist. The estrogen-responsive element of the pS2 gene has been narrowed down by site-directed deletion mutagenesis to a 13-base-pair (position -405 to position -393) imperfectly palindromic sequence, which in isolation can confer estrogen inducibility to the heterologous rabbit beta-globin gene promoter. On the other hand, the sequences responsible for the constitutive enhancer activity are spread over the entire region.

https://www.pnas.org/content/pnas/86/4/1218.full.pdf

Estrogen-responsive element of the human pS2 gene is an imperfectly palindromic sequence

Functional analyses, performed with the estrogen receptor (ER) isolated from different sources or produced with various expression systems, led to contradictory results concerning the role of estrogen (E2) and antiestrogens in ER DNA binding. Here we report the DNA-binding properties of the human ER and show that the wild type ER (HEGO) binds in vitro to an estrogen response element (ERE) as a dimer, irrespective of the presence or absence of estrogen. We also show that the two antihormones, 4-hydroxytamoxifen (OHT, a partial ER agonist) and ICI 164,364 (a pure antagonist) do not impair HEGO dimerization and DNA binding in vitro. Exposure of HEGO to elevated temperature (37 C) in vitro results in a much faster reduction of its binding capacity to an ERE in the absence of ligand or in the presence of ICI 164,364 than in the presence of either E2 or OHT. The Gly to Val mutation at amino acid 400 present in the human ER that we initially cloned (HEO), is responsible for an even faster heat inactivation of unliganded receptor compared with HEGO and largely accounts for the previously observed in vitro ligand-dependent DNA binding of ER. We also show that, as previously observed for OHT, ICI 164,364 does not prevent ER binding to an ERE in viva, even though ICI 164,364 acts as a pure antagonist for transcriptional activation by ER. We discuss these results in the context of a ligand-dependent interaction between the C-terminal region E, which contains the ligand-binding domain, and the N-terminal A/B region, which contains the activation function AF-1. (Molecular Endocrinology 9: 579-591, 1995)

Effect of antagonists on DNA binding properties of the human estrogen receptor in vitro and in vivo.

The 5' flanking region of the human pS2 gene mediates its transcriptional activation by estrogen in MCF-7 cells.

The mechanisms by which oestrogens promote and stimulate the growth of a significant percentage of breast cancers are still poorly understood (1–4 and refs. therein), although the basis for the hormonal dependence is clearly the presence of the oestrogen receptor. The MCF-7 cell line, which is derived from a pleural effusion of a breast cancer, contains both oestrogen and progesterone receptors (5–8 and refs therein). The levels of a variety of mRNAs, proteins and secreted growth factors are under oestrogen control in MCF-7 cells (3, 4, 9 and refs therein). As such, these cells represent a good in vitro model to study the molecular mechanisms underlying oestrogen action in breast cancers. To elucidate these mechanisms, we have undertaken several years ago a study aimed at characterizing genes whose expression is controlled by oestrogen in these cells and at cloning the human oestrogen receptor (hER) cDNA. We will summarize here our analysis of one such induced gene, named pS2, and the characterization of the protein for which it codes. We will also describe how the cloning and analysis of the hER cDNA has provided information concerning the mechanism by which the receptor activates the transcription of this and other oestrogen-responsive genes.

Meera Berry

Papers

Functional domains of the human estrogen receptor.

Estrogen-responsive element of the human pS2 gene is an imperfectly palindromic sequence

Effect of antagonists on DNA binding properties of the human estrogen receptor in vitro and in vivo.

The 5' flanking region of the human pS2 gene mediates its transcriptional activation by estrogen in MCF-7 cells.

The Oestrogen-Induced pS2 Gene and the Oestrogen Receptor in Breast Cancer