Nuclear receptor biology in the Blumberg laboratory

Introduction
The endocrine system is responsible for coordinating cellular growth and differentiation, many aspects of reproduction and embryological development, the maintenance of homeostasis and cyclical phenomena. These processes require coordinated gene expression and are regulated by a large and diverse group of inter- and intracellular signaling pathways mediated by an equally diverse group of hormones and hormone receptors. An important set of hormonal signaling pathways is mediated by the steroid receptor superfamily. These "nuclear receptors" are ligand-modulated transcription factors that directly regulate the expression of target genes. The hormones are primarily lipophilic molecules such as steroids, retinoids, and vitamin D3 - all potent regulators of cell differentiation and development. They can diffuse from a source and penetrate to a target within tissues making them ideal candidates for developmental signals and an important model system to study the role of regulated transcription in fundamental biological processes.

In recent years, many receptor-like molecules were identified by molecular cloning. Although related to the known receptors, no physiological ligands or activators were known for these "orphan receptors" which are more numerous (19 families) than receptors with known ligands (11 families). Orphan nuclear receptors provide a unique, largely untapped, resource to illuminate novel and fundamental regulatory systems that impact health and human disease. Cloning new vertebrate receptors offers the opportunity to use molecular genetic techniques, biochemistry and genomic technology to identify new hormones important in embryonic development and adult physiology. We call the process of using the receptors to identify the endogenous hormones "reverse endocrinology".

My research goals are to understand the role of hormonal signaling in establishing positional information in the early embryo and physiologic function in the adult. One class of information transfer is mediated by morphogens, diffusible chemicals responsible for causing morphogenesis. An interest in identifying novel morphogens led me to design a strategy where candidate nuclear hormone receptor homologs are first isolated from a developmental system and then used to identify the corresponding ligand. I chose Xenopus because it affords an ideal combination of embryological and biochemical approaches to study embryonic signaling while remaining an appropriate model for higher vertebrates.

Experimental Approaches
Chemistry, robotics, signal transduction, gene regulation, reverse genetics and basic cell, developmental and molecular biology are key to identifying new hormonal signaling pathways. We have taken an integrated approach toward understanding the role of orphan steroid receptors in important biological processes.

One goal is to identify endogenous activators or antagonists for these orphan receptors and then to determine which are bona fide ligands. We designed sensitive, in vivo, cell-based assays to measure receptor activation and in vitro biochemical assays to measure the ability of candidate hormones to bind directly to the receptors, or to modulate important features of receptor biology such as interaction with coactivator and corepressor proteins. These assays have been miniaturized and optimized for a high-throughput robotic screening system.

We employ techniques of natural products chemistry with sophisticated purification and analytical procedures to identify potentially novel compounds with receptor agonist or antagonist activity. Our approach is inherently unbiased toward or against any particular class of compounds and does not presume any prior knowledge about the chemical nature of the activities. This is because chemical purification is guided simply by biological activity as measured by transcriptional regulation. This strategy allows us to identify compounds with potentially unusual fractionation and absorption properties.

BXR - a new embryonic signaling pathway
It remains to be determined how many orphan receptors regulate unidentified endocrine systems. To address this key issue for the five Xenopus orphan receptors previously identified, we first determined which could be transcriptionally activated by embryonic extracts and cocktails of candidate synthetic compounds. One receptor, now termed BXR for benzoate X receptor, was activated by embryonic extracts. We employed a bioassay-directed fractionation of embryonic extracts and identified alkyl esters of amino and hydroxy benzoic acids potent stereoselective activators and endogenous BXR ligands. These compounds are all related to the B-complex vitamin, p-amino benzoic acid, which is itself a component of the essential B-vitamin, folic acid. Benzoates comprise a new molecular class of nuclear receptor ligand and their activity suggests that BXR may control a previously unsuspected vertebrate signaling pathway. Immediate goals are to finish the purification and chemical characterization of endogenous BXR activators which occur in Xenopus embryos and bovine sera. That the agonist occurs in sera suggests both that it behaves like a true endocrine hormone in adults and that a mammalian BXR ortholog exists. Next, mutant receptors engineered to be either constitutively active or dominant transcriptional repressors will be employed to ascertain the effects of locally increasing or decreasing receptor function. Subsequent experiments will identify target genes. The elucidation of components in the gene regulatory network under the control of BXR in Xenopus provides an entry point for future study in other systems. More long-term goals for this project are to identify homologs in other species, to knock out BXR function in transgenic mice, and to identify potential human diseases associated with defects in this signaling pathway.

SXR and the steroid sensor hypothesis
An important requirement for physiologic homeostasis is the removal and detoxification of various endogenous hormones and xenobiotic compounds. Much of the detoxification is performed in the liver and gut by cytochrome P450 enzymes, which have broad substrate specificity and are inducible by numerous different compounds, including steroids. The ingestion of dietary steroids and lipids induces the same enzymes and thus, they would appear to be integrated into a coordinated metabolic pathway. Instead of possessing many receptors, one for each inducing compound, we proposed the existence of a few broad-specificity sensing receptors that would monitor aggregate levels of inducers to trigger production of metabolizing enzymes. Rather than responding to nanomolar levels of specific ligand the "sensor" should possess exactly the opposite properties - binding ligands with low affinity and broad specificity such that it could recognize and respond to a variety of compounds.

In support of this model, we isolated a novel nuclear receptor, SXR (steroid and xenobiotic receptor) that responds to an diversity of natural and synthetic steroid agonists and antagonists, xenobiotic drugs and bioactive dietary compounds such as phytoestrogens. SXR can activate transcription through response elements present in steroid and xenobiotic-inducible cytochrome P450 genes and is expressed in tissues where these catabolic enzymes are expressed. The ability of SXR to regulate expression of catabolic enzymes in response to this diversity of compounds is unprecedented for a nuclear receptor and provides a novel mechanism for direct regulation of metabolism.

We are pursuing several aspects of SXR biology. We are knocking out the mouse SXR-related gene, PXR, in collaboration with Wen Xie in Ron Evans's laboratory. It is reasonable to expect that SXR knockout mice will exhibit defects in steroid and xenobiotic elimination and/or a decreased tolerance to such compounds in the diet. One very interesting class of SXR activators is the phytoestrogens. These compounds are abundant in beans, whole grains, seeds and berries. Numerous epidemiological studies have linked intake of phytoestrogen-containing foods with lower incidence of cancers, heart disease and osteoporosis. For example, phytoestrogens, raloxifene (an estrogen antagonist) and vitamin K2 are all effective against osteoporosis yet it has been impossible to place these compounds in a common molecular pathway. However, each of these compounds is a potent activator of SXR suggesting a possible mechanism of action. These studies are being pursued in collaboration with Prof. Satoshi Inoue of Tokyo University. Similarly, phytoestrogens, fatty amides (e.g. anandamide), estrogen antagonists and RXR activators are all effective against breast cancers in vitro and in vivo. While it is obvious that estrogen antagonists may prevent the development of estrogen-dependent tumors, it is less obvious how fatty amides, phytoestrogens or RXR activators could be involved. Once again, all four classes of compounds activate SXR:RXR heterodimers which are known to be expressed in several breast cancer cell lines. We are working intensively on following up these promising studies in collaboration with a specialist in breast cancer, Dr. Powel Brown at the University of Texas, San Antonio Cancer Center.

Retinoids are required for patterning the central nervous system
Vitamin A and its derivatives, the retinoids, are required for many processes including growth, vision, reproduction, morphogenesis, hematopoiesis, immune function, and differentiation of normal and malignant tissues. A major focus of my research has been the role of retinoid signaling during development, particularly how retinoids influence anteroposterior (A/P) neural patterning and neuronal differentiation. Our approaches have been to identify endogenous retinoid receptor activators and to manipulate receptor function in embryos using reverse genetics and receptor-selective agonists and antagonists.

A voluminous literature suggested that retinoids could mimic factors involved in diverse developmental processes, including A/P patterning. However, the question of their necessity in these processes remained open since very few loss-of-function experiments had ever been performed. We investigated the developmental effects of locally altering RAR signaling with mutant receptors that resulted in dominant gain or loss of receptor function. Increased receptor activity suppressed anterior neural structures and led to an anterior shift of posterior marker genes (posteriorization). Decreased receptor activity led to anterior enhancement with attendant posterior coordinate shifts in marker gene expression and loss of posterior marker genes (anteriorization). These results showed unequivocally that retinoids are both necessary and sufficient to modulate A/P patterning, in vivo. Interference with retinoid signaling also led to ablation of primary neurons whereas increased retinoid signaling increased the number of primary neurons. These results established a critical role for retinoids in neuronal differentiation, in vivo.

My long term goal for the retinoid project is to understand how retinoids and their receptors pattern the developing nervous system. Despite recent progress, many questions remain to be answered. Do the six retinoid receptors regulate the same, different or overlapping sets of target genes? Do retinoids act as gradient morphogens to specify position along the A/P axis? Do retinoids diffuse from a discrete source in the embryo or are there multiple sources at different times of development? How does interfering with retinoid signaling lead to underproduction of neurons while excess signaling gives ectopic neurons?

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