Estrogen receptors ER␣ and ER␤ are members of the family of nuclear hormone receptors and act as ligandinducible transcriptional factors, which regulate the expression of target genes on binding to cognate response elements. We report here the characterization of steroid receptor coactivator-3 (SRC-3), a coactivator of nuclear receptor transcription that is a member of a family of steroid receptor coactivators that includes SRC-1 and transcription intermediate factor-2. SRC-3 enhanced ER␣ and progesterone receptor-stimulated gene transcription in a ligand-dependent manner, but stimulation of ER␤-mediated transcription was not observed. Protein-protein interaction assays, including real-time interaction analyses with BIAcore, demonstrated that the affinity of the ER␣ interaction with SRC-3 was much higher than that observed for the ER␤ interaction with SRC-3. Mutational analysis suggests a potential interplay between the transactivation function-1 and -2 domains of ER␣ and SRC-3. Furthermore, an intrinsic transactivation function was observed in the C-terminal half of SRC-3. Finally, SRC-3 was differentially expressed in various tissues and, among several tumor cells examined, was most abundant in the nuclear fraction of MCF-7 breast cancer cells. Therefore, SRC-3, a third member of a family of steroid receptor coactivators, has a distinct tissue distribution and intriguing selectivity between ER␣ and ER␤. Estradiol (E 2 )1 exerts numerous biological effects in different tissues through an interaction with the estrogen receptor (ER), a member of the steroid/nuclear hormone receptor superfamily (1, 2). Amino acid sequence analyses, transient transfection studies, and mutational dissections of ER indicate that ER can be subdivided into several functional domains (3). The N-terminal A/B domain contains a transactivation function, referred to as TAF-1. The DNA binding domain, the C region, contains two zinc fingers and is responsible for DNA recognition. The ligand binding domain (LBD) and a second transactivation function, referred to as TAF-2, is located at the C-terminal of ER. On binding to hormone, the receptor undergoes an activation and transformation step. The activated ER interacts with specific estrogen response elements that are located in the promoter region of estrogen-regulated genes and influences the rate of gene transcription. Over the past decade, numerous studies have provided a basic understanding of both the effects of ligand (agonist and antagonist) on the ER and the relationship between the structure and function of the ER (4). Nevertheless, little is known regarding the mechanisms involved in the gene-specific and tissue-selective effects mediated by either estrogens or antiestrogens. Furthermore, the molecular mechanisms by which ligand-activated ER influences the basal transcriptional machinery and regulates target gene transcription are mostly unknown.Recently, a new estrogen receptor, named ER␤, has been isolated from rat prostate and human testis (5, 6). The DNA binding domain of ER␤ is 9...

Binding of the thyroid hormone receptor (TR) to thyroid hormone-responsive elements (TREs) is crucial for regulation of gene expression by thyroid hormone. The TR binds to each half-site of a palindromic TRE separately, as a monomer, or simultaneously, as a homodimer. In addition, the TR monomer interacts with a 42-kDa protein that may be responsible for an increase in the apparent size and stability of the TR-TRE complex after incubation with liver nuclear extract. The multiple DNA-binding forms of the TR contact the TRE differently but compete for binding in a dynamic equilibrium which is highly dependent on the relative concentrations of TR and nuclear protein. Thus, protein-protein interactions are likely to determine the context in which the TR binds to target genes and regulates the transcriptional response to thyroid hormone.Thyroid hormone (T3) is required for normal growth, development, and adult function in a wide range of species, from Xenopus laevis to humans (44). T3 regulates gene transcription by binding to nuclear thyroid hormone receptors (TRs) (47). In mammals, three TRs (axl, ,13, and ,B2) with different tissue-specific, hormonal, and pharmacological regulation have been described (34). The TRs are cellular homologs of the viral oncoprotein v-ErbA (48,54) and are members of a superfamily of ligand-activated transcription factors (15, 24). The most highly conserved region of these receptors, located near their amino termini, is the DNAbinding domain, which contains basic amino acids arranged into two zinc fingers. The carboxyl termini, which are less well conserved, mediate binding of specific ligands. Although T3 is not required for interactions between TRs and T3-responsive elements (TREs) to occur in vitro (33,35,40), its presence can determine whether transfected TRs have repressive or stimulatory effects on gene transcription in cultured cells (6,11,23).The palindromic sequence AGGTCATGACCT (TREp) has been shown to be a strong TRE (21), although some T3-responsive sequences contain direct repeats (7) while others consist of only a single half-site (28). It has been suggested that TRs bind as dimers to the two half-sites of TREp. This view has been supported by the observation of functional repression by non-T3-binding TR mutants which retain heptad repeats of hydrophobic amino acids in their carboxyl termini (18), which have been referred to as interaction domains (22). Such mutant TRs are similarly able to inhibit the action of retinoic acid receptors (RARs), also perhaps through formation of inactive heterodimers. TRs by themselves form multiple TRE-containing complexes, as revealed by the gel electrophoretic mobility shift assay (EMSA) (33), but biochemical identification of monomeric or dimeric TRs in these complexes has not previously been achieved. TR binding to TREs is also affected by nuclear proteins which enhance binding (8,40) and retard the migration of the TR-TRE complexes in the EMSA (33).Since TR-TRE interaction is a key step in T3 action, the existence of a nuclear protein ...

This article is available online at http://www.jlr.org Supplementary key words cynomolgus monkey • dyslipidemia • fi broblast growth factor 19 • hypertriglyceridemiaAtherosclerosis is the major cause of cardiovascular disease and its incidence is on the rise due to its tight relationship to obesity and diabetes. Therapeutic interventions targeted at reducing elevated plasma low-density lipoprotein cholesterol (LDLc), the primary risk factor for development of atherosclerosis, do not eliminate cardiovascular risk particularly in several high-risk subpopulations. The statin class of drugs achieve dramatic reductions in LDLc yet reduce heart attack risk only 33% per 1.5 mmol/L reduction in LDL ( 1 ). As statins primarily limit disease progression through the inhibition of endogenous cholesterol synthesis, newer treatment modalities directed at reversing established atherosclerotic plaque are likely to provide additional benefi t and can have important clinical implications for disease management. This is exemplifi ed by the exploratory clinical studies targeting the enhancement of high-density lipoprotein ( 2 ). In this study, intravenous

The use of estrogen-based therapies and the selective estrogen receptor (ER) modulator (SERM), raloxifene, which are approved for postmenopausal osteoporosis, is associated with side effects such as uterine/breast hyperproliferation, thromboembolism, and hot flashes. A combination of a new SERM, bazedoxifene (BZA), and Premarin (conjugated estrogens; CE) is under investigation to mitigate the estrogen/SERM side effects with promising results in Phase III clinical trials. To explore the mechanism of BZA/CE action, we investigated the recruitment of cofactor peptides to ERalpha by components of CE and a mixture containing the 10 major components of CE with or without three different SERMs. Here, we demonstrate differential recruitment of cofactor peptides to ERalpha by the individual CE components using a multiplex nuclear receptor-cofactor peptide interaction assay. We show that estrone and equilin are partial agonists in comparison with 17beta-estradiol in recruiting cofactor peptides to ERalpha. Further, CE was more potent than 17beta-estradiol in mediating ERalpha interaction with cofactor peptides. Interestingly, BZA was less potent than other SERMs in antagonizing the CE-mediated cofactor peptide recruitment to ERalpha. Finally, in accordance with these biochemical findings, 17beta-estradiol and CE, as well as SERM/CE combinations, showed differential gene regulation patterns in MCF-7 cells. In addition, BZA showed antagonism of a unique set of CE-regulated genes and did not down-regulate the expression of a number of CE-regulated genes, the expression of which was effectively antagonized by the other two SERMs. These results indicate that SERMs in combination with CE exhibit differential pharmacology, and therefore, combinations of other SERMs and estrogen preparations may not yield the same beneficial effects that are observed in clinic by pairing BZA with CE.

Thyroid hormone receptor (TR) binds to DNA as a monomer, homodimer, and heterodimer with nuclear proteins. We have confirmed that the TR can heterodimerize with retinoid X receptors (RXRs)-alpha and -beta, and have found that another member of the nuclear receptor superfamily, chicken ovalbumin upstream promoter transcription factor (COUP-TF), also formed heterodimers with the TR in the context of binding to a palindromic thyroid hormone-responsive element (TREp). The interaction between COUP-TF and the TR was confirmed using specific antibodies which supershifted the COUP-TF/TR DNA complexes. The complex between the TR and the major TR heterodimerization partner in liver was unaffected by antibodies to COUP-TF and RXR beta, but was supershifted by an anti-RXR alpha antibody, indicating that the liver protein is highly related to RXR alpha. Indeed, the TR/RXR and TR/liver protein heterodimers contact the same guanidine residues in TREp. The retinoic acid receptor (RAR) also heterodimerized with COUP-TF as well as with RXR alpha, RXR beta, and the TR heterodimerization partner in liver. In contrast to its ability to heterodimerize with the TR and RAR, we did not detect heterodimers between COUP-TF and either RXR alpha, RXR beta, or the liver nuclear protein in the context of binding to the TREp. These results show that the major TR heterodimerization partner in liver is highly related to RXR alpha, but that other nuclear receptors such as COUP-TF can heterodimerize with the TR and RAR, suggesting that selective protein-protein interactions may be involved in the tissue and target gene specificities of hormone action.