Linda B. Moore

A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis.

Bile acids repress the transcription of cytochrome P450 7A1 (CYP7A1), which catalyzes the rate-limiting step in bile acid biosynthesis. Although bile acids activate the farnesoid X receptor (FXR), the mechanism underlying bile acid-mediated repression of CYP7A1 remained unclear. We have used a potent, nonsteroidal FXR ligand to show that FXR induces expression of small heterodimer partner 1 (SHP-1), an atypical member of the nuclear receptor family that lacks a DNA-binding domain. SHP-1 represses expression of CYP7A1 by inhibiting the activity of liver receptor homolog 1 (LRH-1), an orphan nuclear receptor that is known to regulate CYP7A1 expression positively. This bile acid-activated regulatory cascade provides a molecular basis for the coordinate suppression of CYP7A1 and other genes involved in bile acid biosynthesis.

Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway

Accumulation of cholesterol causes both repression of genes controlling cholesterol biosynthesis and cellular uptake and induction of cholesterol 7α-hydroxylase, which leads to the removal of cholesterol by increased metabolism to bile acids. Here, we report that LXRα and LXRβ, two orphan members of the nuclear receptor superfamily, are activated by 24(S),25-epoxycholesterol and 24(S)-hydroxycholesterol at physiologic concentrations. In addition, we have identified an LXR response element in the promoter region of the rat cholesterol 7α-hydroxylase gene. Our data provide evidence for a new hormonal signaling pathway that activates transcription in response to oxysterols and suggest that LXRs play a critical role in the regulation of cholesterol homeostasis.

The Pregnane X Receptor: A Promiscuous Xenobiotic Receptor That Has Diverged during Evolution

Transcription of genes encoding cytochrome P450 3A (CYP3A) monooxygenases is induced by a variety of xenobiotics and natural steroids. There are marked differences in the compounds that induce CYP3A gene expression between species. Recently, the mouse and human pregnane X receptor (PXR) were shown to be activated by compounds that induce CYP3A expression. However, most studies of CYP3A regulation have been performed using rabbit and rat hepatocytes. Here, we report the cloning and characterization of PXR from these two species. PXR is remarkably divergent between species, with the rabbit, rat, and human receptors sharing only approximately 80% amino acid identity in their ligand-binding domains. This sequence divergence is reflected by marked pharmacological differences in PXR activation profiles. For example, the macrolide antibiotic rifampicin, the antidiabetic drug troglitazone, and the hypocholesterolemic drug SR12813 are efficacious activators of the human and rabbit PXR but have little activity on the rat and mouse PXR. Conversely, pregnane 16alpha-carbonitrile is a more potent activator of the rat and mouse PXR than the human and rabbit receptor. The activities of xenobiotics in PXR activation assays correlate well with their ability to induce CYP3A expression in primary hepatocytes. Through the use of a novel scintillation proximity binding assay, we demonstrate that many of the compounds that induce CYP3A expression bind directly to human PXR. These data establish PXR as a promiscuous xenobiotic receptor that has diverged during evolution.

Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of glucose, lipid, and cholesterol metabolism. We report the x-ray crystal structure of the ligand binding domain of PPARα (NR1C1) as a complex with the agonist ligand GW409544 and a coactivator motif from the steroid receptor coactivator 1. Through comparison of the crystal structures of the ligand binding domains of the three human PPARs, we have identified molecular determinants of subtype selectivity. A single amino acid, which is tyrosine in PPARα and histidine in PPARγ, imparts subtype selectivity for both thiazolidinedione and nonthiazolidinedione ligands. The availability of high-resolution cocrystal structures of the three PPAR subtypes will aid the design of drugs for the treatments of metabolic and cardiovascular diseases.

Functional and structural comparison of PXR and CAR.

The nuclear receptors pregnane X receptor (PXR, NR1I2) and constitutive active receptor (CAR, NR1I3) have both been proposed to function as xenosensors, but the details of their respective physiological roles are still being elucidated. We have contrasted these two receptors in a variety of experiments including gene expression assays, cell-based ligand profiling assays, and crystallographic/structural modeling analyses. These data highlight key differences between PXR and CAR.

Recombinant Baculoviruses Used to Study Estrogen Receptor Function in Human Osteosarcoma Cells

We report that modified baculoviruses, termed BacMam viruses, can efficiently deliver multiple genes into mammalian cells to generate a heterologous transcription factor/reporter gene system. Using human estrogen receptor (ER) as a model nuclear receptor, we demonstrate how this approach can be successfully applied to assay development in Saos-2 human osteosarcoma cells. BacMam viruses containing full-length cDNAs were constructed for both human ER subtypes, ERalpha and ERbeta, and a third BacMam virus containing an ER-responsive reporter gene cassette. Using these viruses, we found that BacMam-ER expression/reporter constructs could be used to profile the effects of the agonist 17beta-estradiol and the partial agonist raloxifene in human Saos-2 cells. A comparison of assay data obtained with the BacMam-based system with that using standard DNA transfections demonstrates that the two systems are functionally equivalent, giving comparable EC(50) and IC(50) values for estrogen and estrogen plus raloxifene treatments, respectively. Our results indicate that BacMam-mediated gene transfer offers a novel and efficient method for delivery of nuclear receptors and associated genes for mammalian cell-based assay development.

Bone targeted drugs: 2. Synthesis of estrogens with hydroxyapatite affinity

Abstract The utility of the bone targeting 4-carboxy-3-hydroxy-1,2-pyrazole heterocycle was tested by the synthesis of hybrids with the non-steroidal estrogen hexestrol. Compounds 1 and 2 demonstrated significant hydroxyapatite affinity, while maintaining weak estrogenic activity in whole cell assays.

Use of in vitro pregnane X receptor assays to assess CYP3A4 induction potential of drug candidates.

Publisher Summary This chapter describes two such assays: a cell-based reporter assay and an in vitro competition binding assay. The two assays discussed in the chapter provide researchers with the tools needed to profile the potency and efficacy of large numbers of compounds on pregnane X receptor (PXR). The availability of these assays permits the removal of compounds with CYP3A4 induction potential at the very earliest stages of the drug discovery process, thus reducing costs and expediting the development of safer medicines. The new receptor named the “pregnane X receptor” (PXR) based on its activation by natural and synthetic C21 steroids. PXR binds as a heterodimer with the 9-cis-retinoic acid receptor (RXR) to DNA response elements composed of two copies of the consensus sequence AGTFCA organized as either a direct repeat with a three nucleotide spacer (DR3) or an everted repeat with a six nucleotide spacer (ER6). The discovery of PXR and its role in the regulation of CYP3A has provided the insights necessary to develop reliable, cost-effective assays for predicting the induction potential of drug candidates. During the course of drug development, it is critical to assess the potential for a compound to induce levels of liver enzymes, including CYP3A4. With the discovery of the nuclear receptor PXR and the elucidation of its role in regulating CYP3A expression, it is now possible to predict the induction potential of a drug candidate before it is tested in vivo.

Production and characterization of an estrogen receptor β subtype-specific mouse monoclonal antibody

An important step in differentiating the unique physiological roles of the alpha and beta forms of estrogen receptor is to determine the precise expression pattern of each of these receptors. We report the generation and characterization of a murine IgG1 monoclonal antibody (MAb), ER15.64A that is ERbeta subtype-specific and capable of recognizing full-length human ERbeta as well as all of its known protein isoforms. ER15.64A, raised against a ERbeta peptide (aa2-18)-keyhole limpet hemocyanine conjugate, reacted to the immunizing peptide and the full-length E. coli expressed ERbeta in ELISA and BIAcore assays. It also immunostained nuclei of Sf9 insect cells that were infected with an ERbeta-baculovirus. In Western analysis, ER15.64A recognized ERbeta1 and ERbeta2 proteins from a reticulocyte in vitro transcription/translation preparation. This antibody did not cross-react with recombinant ERalpha in ELISA, BIAcore, immunocytochemistry, or Western blot analysis. The specificity of ER15.64A should make this antibody a useful tool for monitoring expression of ERbeta and its isoforms at the protein level and should aid in distinguishing the pattern of ERbeta receptor expression from that of ERalpha.