Brandee L. Wagner

BISPHENOL A INTERACTS WITH THE ESTROGEN RECEPTOR ALPHA IN A DISTINCT MANNER FROM ESTRADIOL

We investigated the interaction of bisphenol A (BPA, an estrogenic environmental contaminant used in the manufacture of plastics) with the estrogen receptor alpha (ERalpha) transfected into the human HepG2 hepatoma cell line and expanded the study in vivo to examine the effect of BPA on the immature rat uterus. Bisphenol A was 26-fold less potent in activating ER-WT and was a partial agonist with the ERalpha compared to E2. The use of ERalpha mutants in which the AF1 or AF2 regions were inactivated has permitted the classification of ER ligands into mechanistically distinct groups. The pattern of activity of BPA with the ERalpha mutants differed from the activity observed with weak estrogens (estrone and estriol), partial ERalpha agonists (raloxifene or 4-OH-tamoxifen), or a pure antagonist (ICI 182, 780). Intact immature female Sprague-Dawley rats were exposed to BPA alone or with E2 for 3 days. Unlike E2, BPA had no effect on uterine weight; however, like E2, both peroxidase activity and PR levels were elevated, though not to the level induced by E2. Following simultaneous administration, BPA antagonized the E2 stimulatory effects on both peroxidase activity and PR levels but did not inhibit E2-induced increases of uterine weight. These results demonstrate that BPA is not merely a weak estrogen mimic but exhibits a distinct mechanism of action at the ERalpha.

Identification of macrophage liver X receptors as inhibitors of atherosclerosis

Recent studies have identified the liver X receptors (LXRα and LXRβ) as important regulators of cholesterol metabolism and transport. LXRs control transcription of genes critical to a range of biological functions including regulation of high density lipoprotein cholesterol metabolism, hepatic cholesterol catabolism, and intestinal sterol absorption. Although LXR activity has been proposed to be critical for physiologic lipid metabolism and transport, direct evidence linking LXR signaling pathways to the pathogenesis of cardiovascular disease has yet to be established. In this study bone marrow transplantations were used to selectively eliminate macrophage LXR expression in the context of murine models of atherosclerosis. Our results demonstrate that LXRs are endogenous inhibitors of atherogenesis. Additionally, elimination of LXR activity in bone marrow-derived cells mimics many aspects of Tangier disease, a human high density lipoprotein deficiency, including aberrant regulation of cholesterol transporter expression, lipid accumulation in macrophages, splenomegaly, and increased atherosclerosis. These results identify LXRs as targets for intervention in cardiovascular disease.

Reduction of atherosclerosis in apolipoprotein E knockout mice by activation of the retinoid X receptor

A common feature of many metabolic pathways is their control by retinoid X receptor (RXR) heterodimers. Dysregulation of such metabolic pathways can lead to the development of atherosclerosis, a disease influenced by both systemic and local factors. Here we analyzed the effects of activation of RXR and some of its heterodimers in apolipoprotein E −/− mice, a well established animal model of atherosclerosis. An RXR agonist drastically reduced the development of atherosclerosis. In addition, a ligand for the peroxisome proliferator-activated receptor (PPAR)γ and a dual agonist of both PPARα and PPARγ had moderate inhibitory effects. Both RXR and liver X receptor (LXR) agonists induced ATP-binding cassette protein 1 (ABC-1) expression and stimulated ABC-1-mediated cholesterol efflux from macrophages from wild-type, but not from LXRα and β double −/−, mice. Hence, activation of ABC-1-mediated cholesterol efflux by the RXR/LXR heterodimer might contribute to the beneficial effects of rexinoids on atherosclerosis and warrant further evaluation of RXR/LXR agonists in prevention and treatment of atherosclerosis.

The Nuclear Corepressors NCoR and SMRT Are Key Regulators of Both Ligand- and 8-Bromo-Cyclic AMP-Dependent Transcriptional Activity of the Human Progesterone Receptor

ABSTRACT Previously, we defined a novel class of ligands for the human progesterone receptor (PR) which function as mixed agonists. These compounds induce a conformational change upon binding the receptor that is different from those induced by agonists and antagonists. This establishes a correlation between the structure of a ligand-receptor complex and its transcriptional activity. In an attempt to define the cellular components which distinguish between different ligand-induced PR conformations, we have determined, by using a mammalian two-hybrid assay, that the nuclear receptor corepressor (NCoR) and the silencing mediator for retinoid and thyroid hormone receptor (SMRT) differentially associate with PR depending upon the class of ligand bound to the receptor. Specifically, we observed that the corepressors preferentially associate with antagonist-occupied PR and that overexpression of these corepressors suppresses the partial agonist activity of antagonist-occupied PR. Binding studies performed in vitro, however, reveal that recombinant SMRT can interact with PR in a manner which is not influenced by the nature of the bound ligand. Thus, the inability of SMRT or NCoR to interact with agonist-activated PR when assayed in vivo may relate more to the increased affinity of PR for coactivators, with a subsequent displacement of corepressors, than to an inherent low affinity for the corepressor proteins. Previous work from other groups has shown that 8-bromo-cyclic AMP (8-bromo-cAMP) can convert the PR antagonist RU486 into an agonist and, additionally, can potentiate the transcriptional activity of agonist-bound PR. In this study, we show that exogenous expression of NCoR or SMRT suppresses all 8-bromo-cAMP-mediated potentiation of PR transcriptional activity. Further analysis revealed that 8-bromo-cAMP addition decreases the association of NCoR and SMRT with PR. Thus, we propose that 8-bromo-cAMP-mediated potentiation of PR transcriptional activity is due, at least in part, to a disruption of the interaction between PR and the corepressors NCoR and SMRT. Cumulatively, these results suggest that NCoR and SMRT expression may play a pivotal role in PR pharmacology.

Promoter-Specific Roles for Liver X Receptor/Corepressor Complexes in the Regulation of ABCA1 and SREBP1 Gene Expression

ABSTRACT Liver X receptors (LXRs) regulate the expression of genes involved in cholesterol and fatty acid homeostasis, including the genes for ATP-binding cassette transporter A1 (ABCA1) and sterol response element binding protein 1 (SREBP1). Loss of LXR leads to derepression of the ABCA1 gene in macrophages and the intestine, while the SREBP1c gene remains transcriptionally silent. Here we report that high-density-lipoprotein (HDL) cholesterol levels are increased in LXR-deficient mice, suggesting that derepression of ABCA1 and possibly other LXR target genes in selected tissues is sufficient to result in enhanced HDL biogenesis at the whole-body level. We provide several independent lines of evidence indicating that the repressive actions of LXRs are dependent on interactions with the nuclear receptor corepressor (NCoR) and the silencing mediator of retinoic acid and thyroid hormone receptors (SMRT). While dissociation of NCoR and SMRT results in derepression of the ABCA1 gene in macrophages, it is not sufficient for derepression of the SREBP1c gene. These findings reveal differential requirements for corepressors in the regulation of genes involved in cholesterol and fatty acid homeostasis and raise the possibility that these interactions may be exploited to develop synthetic ligands that selectively modulate LXR actions in vivo.

Regulation of Cholesterol Homeostasis and Lipid Metabolism in Skeletal Muscle by Liver X Receptors

Recent studies have identified the liver X receptors (LXRα and LXRβ) as important regulators of cholesterol and lipid metabolism. Although originally identified as liver-enriched transcription factors, LXRs are also expressed in skeletal muscle, a tissue that accounts for ∼40% of human total body weight and is the major site of glucose utilization and fatty acid oxidation. Nevertheless, no studies have yet addressed the functional role of LXRs in muscle. In this work we utilize a combination of in vivoand in vitro analysis to demonstrate that LXRs can functionally regulate genes involved in cholesterol metabolism in skeletal muscle. Furthermore we show that treatment of muscle cellsin vitro with synthetic agonists of LXR increases the efflux of intracellular cholesterol to extracellular acceptors such as high density lipoprotein, thus identifying this tissue as a potential important regulator of reverse cholesterol transport and high density lipoprotein levels. Additionally we demonstrate that LXRα and a subset of LXR target genes are induced during myogenesis, suggesting a role for LXR-dependent signaling in the differentiation process.

Characterization of a CREB Gain-of-Function Mutant with Constitutive Transcriptional Activity In Vivo

ABSTRACT The cyclic AMP (cAMP)-responsive factor CREB promotes cellular gene expression, following its phosphorylation at Ser133, via recruitment of the coactivator paralogs CREB-binding protein (CBP) and p300. CBP and p300, in turn, appear to mediate target gene induction via their association with RNA polymerase II complexes and via intrinsic histone acetyltransferase activities that mobilize promoter-bound nucleosomes. In addition to cAMP, a wide variety of stimuli, including hypoxia, UV irradiation, and growth factor addition, induce Ser133 phosphorylation with stoichiometry and kinetics comparable to those induced by cAMP. Yet a number of these signals are incapable of promoting target gene activation via CREB phosphorylation per se, suggesting the presence of additional regulatory events either at the level of CREB-CBP complex formation or in the subsequent recruitment of the transcriptional apparatus. Here we characterize a Tyr134Phe CREB mutant that behaves as a constitutive activator in vivo. Like protein kinase A (PKA)-stimulated wild-type CREB, the Tyr134Phe polypeptide was found to stimulate target gene expression via the Ser133-dependent recruitment of CBP and p300. Biochemical studies reveal that mutation of Tyr134 to Phe lowers the Km for PKA phosphorylation and thereby induces high levels of constitutive Ser133 phosphorylation in vivo. Consistent with its constitutive activity, Tyr134Phe CREB strongly promoted differentiation of PC12 cells in concert with suboptimal doses of nerve growth factor. Taken together, these results demonstrate that Ser133 phosphorylation is sufficient for cellular gene activation and that additional signal-dependent modifications of CBP or p300 are not required for recruitment of the transcriptional apparatus to the promoter.

The novel progesterone receptor antagonists RTI 3021-012 and RTI 3021-022 exhibit complex glucocorticoid receptor antagonist activities: implications for the development of dissociated antiprogestins.

We have identified two novel compounds (RTI 3021–012 and RTI 3021–022) that demonstrate similar affinities for human progesterone receptor (PR) and display equivalent antiprogestenic activity. As with most antiprogestins, such as RU486, RTI 3021–012, and RTI 3021–022 also bind to the glucocorticoid receptor (GR) with high affinity. Unexpectedly, when compared with RU486, the RTI antagonists manifest significantly less GR antagonist activity. This finding indicates that, with respect to antiglucocorticoid function, receptor binding affinity is not a good predictor of biological activity. We have determined that the lack of a clear correlation between the GR binding affinity of the RTI compounds and their antagonist activity reflects the unique manner in which they modulate GR signaling. Previously, we proposed a two step “active inhibition” model to explain steroid receptor antagonism: 1) competitive inhibition of agonist binding; and 2) competition of the antagonist bound receptor with that activated by a...

Liver X receptor (LXR) partial agonists: biaryl pyrazoles and imidazoles displaying a preference for LXRβ.

A series of biaryl pyrazole and imidazole Liver X Receptor (LXR) partial agonists has been synthesized displaying LXRβ selectivity. The LXRβ selective partial agonist 18 was identified with potent induction of ATP binding transporters ABCA1 and ABCG1 in human whole blood (EC50=1.2μM, 55% efficacy). In mice 18 displayed peripheral induction of ABCA1 at 3 and 10mpk doses with no significant elevation of plasma or hepatic triglycerides at these doses, showing an improved profile compared to a full pan-agonist.

Saccharomyces cerevisiae As a Model System to Study Steroid Hormone Receptors

The human estrogen (ER) and progesterone receptor (PR) are members of the steroid receptor superfamily of ligand-dependent transcription factors (Evans, 1988). The cloning of the cDNAs for these receptors — ER in 1986 (Green et al, 1986) and hPR in 1987 (Misrahi et al, 1987) — and their subsequent expression in heterologous cell types permitted the reconstitution of hormone-responsive transcription systems that have been amenable to genetic analysis. The availability of these systems, coupled with existing biochemical techniques, has in the last ten years advanced our understanding of these transcription factors considerably. Currently, we believe that the steroid hormone receptor signal transduction pathway is comprised of multiple steps that can be genetically or pharmacologically separated The human estrogen (ER) and progesterone receptor (PR) are members of the steroid receptor superfamily of ligand-dependent transcription factors (Evans, 1988). The cloning of the cDNAs for these receptors — ER in 1986 (Green et al, 1986) and hPR in 1987 (Misrahi et al, 1987) — and their subsequent expression in heterologous cell types permitted the reconstitution of hormone-responsive transcription systems that have been amenable to genetic analysis. The availability of these systems, coupled with existing biochemical techniques, has in the last ten years advanced our understanding of these transcription factors considerably. Currently, we believe that the steroid hormone receptor signal transduction pathway is comprised of multiple steps that can be genetically or pharmacologically separated (McDonnell, 1995) A general outline of the proposed pathway is shown in Figure 1. In the absence of hormone, the sex steroid receptors (SRs) reside in a transcriptionally latent form in the nuclei of target cells (Beato et al, 1987). These inactive receptors have been shown biochemically to be sequestered in large macromolecular complex containing the receptor and a series of associated heat-shock proteins (Bagchi et al, 1991; Pratt, 1990; Smith and Toft, 1993). The role of these associated proteins in vivo is unknown. However, it has been postulated that they are involved in the folding of the receptor and in maintaining it in a transcriptionally inactive state within target cells (Smith and Toft, 1993). Upon interaction with their cognate hormone, the SRs undergo a conformational change (Allan et al, 1992; McDonnell et al, 1995). This event promotes the displacement of heat -shock proteins and other associated proteins, permitting dimerization and the subsequent association of the activated SRs with specific DNA response elements (SREs) located within the regulatory regions of target gene promoters (Beato et al, 1987; Kumar and Chambon, 1988). Coincident with this conformational change, and possibly as a consequence of it, the SRs undergo a series of phosphorylations (Bagchi et al, 1992; Denner et al, 1989; Takimoto et al, 1992). The resulting biological response to the ligand is determined by the cell and promoter context of the DNA-bound receptor (Tora et al, 1988; Tzukerman et al, 1994). The precise mechanism by which the DNA-bound receptors affect target gene transcription is unknown, although it has been shown that hormone-activated PR can stabilize the formation of the transcription pre-initiation complex when assayed in vitro (Klein-Hitpass et al, 1990)