Thomas Perlmann

An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway.

Steroid hormones exert profound effects on differentiation, development, and homeostasis in higher eukaryotes through interactions with nuclear receptors. We describe a novel orphan nuclear receptor, termed the pregnane X receptor (PXR), that is activated by naturally occurring steroids such as pregnenolone and progesterone, and synthetic glucocorticoids and antiglucocorticoids. PXR exists as two isoforms, PXR.1 and PXR.2, that are differentially activated by steroids. Notably, PXR.1 is efficaciously activated by pregnenolone 16alpha-carbonitrile, a glucocorticoid receptor antagonist that induces the expression of the CYP3A family of steroid hydroxylases and modulates sterol and bile acid biosynthesis in vivo. Our results provide evidence for the existence of a novel steroid hormone signaling pathway with potential implications in the regulation of steroid hormone and sterol homeostasis.

Structure and Function of Nurr1 identifies a Class of Ligand-Independent Nuclear Receptors

Members of the nuclear receptor (NR) superfamily of transcription factors modulate gene transcription in response to small lipophilic molecules. Transcriptional activity is regulated by ligands binding to the carboxy-terminal ligand-binding domains (LBDs) of cognate NRs. A subgroup of NRs referred to as ‘orphan receptors’ lack identified ligands, however, raising issues about the function of their LBDs. Here we report the crystal structure of the LBD of the orphan receptor Nurr1 at 2.2 Å resolution. The Nurr1 LBD adopts a canonical protein fold resembling that of agonist-bound, transcriptionally active LBDs in NRs, but the structure has two distinctive features. First, the Nurr1 LBD contains no cavity as a result of the tight packing of side chains from several bulky hydrophobic residues in the region normally occupied by ligands. Second, Nurr1 lacks a ‘classical’ binding site for coactivators. Despite these differences, the Nurr1 LBD can be regulated in mammalian cells. Notably, transcriptional activity is correlated with the Nurr1 LBD adopting a more stable conformation. Our findings highlight a unique structural class of NRs and define a model for ligand-independent NR function.

Induction of a midbrain dopaminergic phenotype in Nurr1-overexpressing neural stem cells by type 1 astrocytes.

The implementation of neural stem cell lines as a source material for brain tissue transplants is currently limited by the ability to induce specific neurochemical phenotypes in these cells. Here, we show that coordinated induction of a ventral mesencephalic dopaminergic phenotype in an immortalized multipotent neural stem cell line can be achieved in vitro. This process requires both the overexpression of the nuclear receptor Nurr1 and factors derived from local type 1 astrocytes. Over 80% of cells obtained by this method demonstrate a phenotype indistinguishable from that of endogenous dopaminergic neurons. Moreover, this procedure yields an unlimited number of cells that can engraft in vivo and that may constitute a useful source material for neuronal replacement in Parkinsons disease.

Cellular expression of the immediate early transcription factors Nurr1 and NGFI-B suggests a gene regulatory role in several brain regions including the nigrostriatal dopamine system.

Nurr1 and NGFI-B are closely related orphan members of the steroid-thyroid hormone receptor family involved in immediate early responses to stimuli such as growth factors. In-situ hybridization in the developing and adult mouse and rat demonstrated Nurr1 mRNA in several regions during early central nervous system (CNS) development. Expression persisted through the pre- and postnatal periods and was also found in several areas in the adult CNS. Positive areas include the olfactory bulb, parts of the cortex, the hippocampal formation and substantia nigra where Nurr1 and tyrosine hydroxylase mRNAs were co-expressed. 6-Hydroxydopamine-induced degeneration of mesencephalic dopamine neurons led to a corresponding loss of Nurr1 mRNA, demonstrating a link between Nurr1 and dopaminergic neurons. NGFI-B mRNA was not found in the prenatal CNS but was highly expressed in the adult brain in many areas including the olfactory bulb, cortex, basal ganglia and hippocampus. The spatiotemporal distribution of Nurr1 and NGFI-B mRNAs suggests that these transcription factors are involved in the development and maturation of specific sets of CNS neurons. The experimental data imply that one of these functions may be to control gene regulatory events important for development and function of those neurons that degenerate in patients with Parkinsons disease.

Role of retinoids in the CNS: differential expression of retinoid binding proteins and receptors and evidence for presence of retinoic acid

Retinoic acid (RA), a retinoid metabolite, acts as a gene regulator via ligand‐activated transcription factors, known as retinoic acid receptors (RARs) and retinoid X receptors (RXRs), both existing in three different subtypes, α, β and γ. In the intracellular regulation of retinoids, four binding proteins have been implicated: cellular retinol binding protein (CRBP) types I and II and cellular retinoic acid binding protein (CRABP) types I and II. We have used in situ hybridization to localize mRNA species encoding CRBP‐ and CRABP I and II as well as all the different nuclear receptors in the developing and adult rat and mouse central nervous system (CNS), an assay to investigate the possible presence of RA, and immunohistochemistry to also analyse CRBP I‐ and CRABP Iimmunoreactivity (IR). RXRβ is found in most areas while RARα and ‐β and RXRα and ‐γ show much more restricted patterns of expression. RARα is found in cortex and hippocampus and RARβ and RXRγ are both highly expressed in the dopamine‐innervated areas caudate/putamen, nucleus accumbens and olfactory tubercle. RARγ could not be detected in any part of the CNS. Using an in vitro reporter assay, we found high levels of RA in the developing striatum. The caudate/putamen of the developing brain showed strong CRBP I‐IR in a compartmentalized manner, while at the same time containing many evenly distributed CRABP I‐IR neurons. The CRBP I‐ and CRABP I‐IR patterns were closely paralleled by the presence of the corresponding transcripts. The specific expression pattern of retinoid‐binding proteins and nuclear retinoid receptors as well as the presence of RA in striatum suggests that retinoids are important in many brain structures and emphasizes a role for retinoids in gene regulatory events in postnatal and adult striatum.

Polyunsaturated Fatty Acids Including Docosahexaenoic and Arachidonic Acid Bind to the Retinoid X Receptor α Ligand-binding Domain

Nuclear receptors (NRs) constitute a large and highly conserved family of ligand-activated transcription factors that regulate diverse biological processes such as development, metabolism, and reproduction. As such, NRs have become important drug targets, and the identification of novel NR ligands is a subject of much interest. The retinoid X receptor (RXR) belongs to a subfamily of NRs that bind vitamin A metabolites (i.e. retinoids), including 9-cis-retinoic acid (9-cis-RA). However, although 9-cis-RA has been described as the natural ligand for RXR, its endogenous occurrence has been difficult to confirm. Recently, evidence was provided for the existence of a different natural RXR ligand in mouse brain, the highly enriched polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) (Mata de Urquiza et al. (2000) Science 290, 2140–2144). However, the results suggested that supra-physiological levels of DHA were required for efficient RXR activation. Using a refined method for ligand addition to transfected cells, the current study shows that DHA is a more potent RXR ligand than previously observed, inducing robust RXR activation already at low micromolar concentrations. Furthermore, it is shown that other naturally occurring PUFAs can activate RXR with similar efficiency as DHA. In additional experiments, the binding of fatty acid ligands to RXRα is directly demonstrated by electrospray mass spectrometry of the noncovalent complex between the RXR ligand-binding domain (LBD) and its ligands. Data is presented that shows the noncovalent interaction between the RXR LBD and a number of PUFAs including DHA and arachidonic acid, corroborating the results in transfected cells. Taken together, these results show that RXR binds PUFAs in solution and that these compounds induce receptor activation, suggesting that RXR could function as a fatty acid receptor in vivo.

Vitamin A deprivation results in reversible loss of hippocampal long-term synaptic plasticity

Despite its long history, the central effects of progressive depletion of vitamin A in adult mice has not been previously described. An examination of vitamin-deprived animals revealed a progressive and ultimately profound impairment of hippocampal CA1 long-term potentiation and a virtual abolishment of long-term depression. Importantly, these losses are fully reversible by dietary vitamin A replenishment in vivo or direct application of all trans-retinoic acid to acute hippocampal slices. We find retinoid responsive transgenes to be highly active in the hippocampus, and by using dissected explants, we show the hippocampus to be a site of robust synthesis of bioactive retinoids. In aggregate, these results demonstrate that vitamin A and its active derivatives function as essential competence factors for long-term synaptic plasticity within the adult brain, and suggest that key genes required for long-term potentiation and long-term depression are retinoid dependent. These data suggest a major mental consequence for the hundreds of millions of adults and children who are vitamin A deficient.

Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells

Signaling factors involved in CNS development have been used to control the differentiation of embryonic stem cells (ESCs) into mesencephalic dopamine (mesDA) neurons, but tend to generate a limited yield of desired cell type. Here we show that forced expression of Lmx1a, a transcription factor functioning as a determinant of mesDA neurons during embryogenesis, effectively can promote the generation of mesDA neurons from mouse and human ESCs. Under permissive culture conditions, 75%–95% of mouse ESC-derived neurons express molecular and physiological properties characteristic of bona fide mesDA neurons. Similar to primary mesDA neurons, these cells integrate and innervate the striatum of 6-hydroxy dopamine lesioned neonatal rats. Thus, the enriched generation of functional mesDA neurons by forced expression of Lmx1a may be of future importance in cell replacement therapy of Parkinson disease.

Nuclear Receptors in Sicily: All in the Famiglia

NRs function as molecular machines to transduce a hormonal signal into a transcriptional response. As sequence-specific DNA binding proteins, the action of the receptor primarily occurs at the site of the target gene. Quiescent genes could be viewed as being wrapped in a protective chromatin shield to fend off centroviral advances from an errant RNA polymerase. Dramatic effects of hormones on chromatin structure have long been recognized; whether this was causal to the transcriptional response or merely its consequence was unclear. In addition, how such changes in chromatin structure were directed by receptors was simply unknown. One popular model is that mere binding by receptor was sufficient to initiate a change in chromatin by altering nucleosome position. In contrast, the effort to identify the biochemical basis for transcriptional regulation has led in recent years to the consideration that NR cofactors might serve as active mediators of the regulatory effect. From the view of an insider, it has often seemed that those labs studying chromatin remodeling and those studying NR cofactors appeared to be on opposite sides of the arena taking unrelated approaches to an otherwise common quest. However, in the last year, these two fields, like lost allies, have been reintroduced to each other and are enjoying a new-found synergy. M. Beato (University of Marburg) outlined the general pathway by which the MMTV LTR utilizes a chromatin-based structure to control its transcription. In this case, nucleosomes are phased in a fashion that allows glucocorticoid receptor but not other transcription factors to bind to target DNA. Accordingly, hormone treatment leads to a rapid alteration in chromatin structure and at the same time promotes cooperative binding of other transcription factors. Presumably, it is the packaging of DNA into a repressive chromatin structure that restricts the accessibility of the template to the basal transcription machinery. Thus, as suggested by Beato, and supported by experiments reported by O. Wrange (Karolinska Institute) using in vitro–reconstituted nucleosomes, the role of the glucocorticoid receptor is to function, at least in part, to counteract chromatin-mediated repression. As discussed by L. Krause (UCSD) and J. Kadonaga (UCSD), the “ground” state or default status of an endogenous target gene would be transcriptionally inactive. The role of a transcription factor would be to initially counteract the chromatin effect leading to a derepressed template. This, in turn, would be followed by a “true activation” event equivalent to robust transcriptional initiation. Kadonaga described dissection of this putative multistep process by creating chromatin templates to directly assess the action of NRs in an in vitro–reconstituted system. While previous in vitro transcription studies have been described, this is the first with chromatin-based templates. The effects with the estrogen receptor (ER) are clear and dramatic. Fifteen- to fifty-fold inductions were seen, all in a hormone-dependent fashion. The estrogen receptor can be added either before or after chromatin assembly, and the entire process was shown to be dependent on the known activation domains in the ER. The improved regulation appears to be a consequence of chromatin suppressing basal activity of the promoter thus increasing the fold of induction. Interestingly, estrogen antagonists failed to activate although these complexes can be bound to the template. Continuing in this vein, A. Wolffe (NICHD) described the use of replication-dependent chromatin assembly to identify three regulatory steps in the regulation of transcription by the thyroid hormone receptor (TR). These three steps include: (1) the establishment of a repressive chromatin structure; (2) disruption of the chromatin template; and (3) transcriptional activation. As is apparent, this is similar to the events described by Kadonaga and Beato although with a completely different regulatory system. In this approach, cloned templates are microinjected into the Xenopus oocyte nucleus, which are then assembled in a natural chromatin structure. In this way, Wolffe has been able to address one of the more vexing problems in NR transcription by demonstrating that nonliganded RXR:TR heterodimers can not only bind to chromatin but also may facilitate the formation of a repressive chromatin structure. Conclusions that arise out of this important model system include the demonstration that the nonliganded TR:RXR heterodimers bind to nucleosome DNA, make use of chromatin to repress transcription, control nucleosome position, and can influence both nucleosome modificat

International Union of Pharmacology. LXVI. Orphan nuclear receptors.

Half of the members of the nuclear receptors superfamily are so-called “orphan” receptors because the identity of their ligand, if any, is unknown. Because of their important biological roles, the study of orphan receptors has attracted much attention recently and has resulted in rapid advances that have helped in the discovery of novel signaling pathways. In this review we present the main features of orphan receptors, discuss the structure of their ligand-binding domains and their biological functions. The paradoxical existence of a pharmacology of orphan receptors, a rapidly growing and innovative field, is highlighted.