ReviewVolume 7 | 2009
Cite this Article: Nuclear Receptor Signaling (2009) 7, e003.
Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism
Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
The last few years have witnessed a rapid increase in our knowledge of the retinoid-related orphan receptors RORα, -β, and -γ (NR1F1-3), their mechanism of action, physiological functions, and their potential role in several pathologies. The characterization of ROR-deficient mice and gene expression profiling in particular have provided great insights into the critical functions of RORs in the regulation of a variety of physiological processes. These studies revealed that RORα plays a critical role in the development of the cerebellum, that both RORα and RORβ are required for the maturation of photoreceptors in the retina, and that RORγ is essential for the development of several secondary lymphoid tissues, including lymph nodes. RORs have been further implicated in the regulation of various metabolic pathways, energy homeostasis, and thymopoiesis. Recent studies identified a critical role for RORγ in lineage specification of uncommitted CD4+ T helper cells into Th17 cells. In addition, RORs regulate the expression of several components of the circadian clock and may play a role in integrating the circadian clock and the rhythmic pattern of expression of downstream (metabolic) genes. Study of ROR target genes has provided insights into the mechanisms by which RORs control these processes. Moreover, several reports have presented evidence for a potential role of RORs in several pathologies, including osteoporosis, several autoimmune diseases, asthma, cancer, and obesity, and raised the possibility that RORs may serve as potential targets for chemotherapeutic intervention. This prospect was strengthened by recent evidence showing that RORs can function as ligand-dependent transcription factors.
AF2: activation function 2; ApoA: apolipoprotein; ATRA: all-trans retinoic acid; ATXN1: ataxin 1; BMAL1: brain and muscle ARNT-like 1; CaMKIV: calmodulin-dependent kinase IV; ChIP: Chromatin immunoprecipitation; CRX: cone-rod homeobox factor; CRY: cryptochrome; CT: circadian time; Cyp7b1: oxysterol 7alpha-hydroxylase; DBD: DNA-binding domain; DBP: D site-binding protein; DHR3: Drosophila hormone receptor 3; DKO: double knockout; DP: double positive; EAE: experimental autoimmune encephalomyelitis; EGR: early growth response gene; FOXP3: forkhead box transcription factor p3; HDAC: histone deacetylase; HDL: high density lipoprotein; HIF1α: hypoxia-inducible factor α; IL: interleukin; IRF4: interferon regulatory factor 4; ISP: immature single positive; LBD: ligand binding domain; LPS: lipopolysaccharide; LTi: lymphoid tissue inducer cells; LXR: liver X receptor; NCOA: nuclear receptor coactivator; NCOR: nuclear receptor corepressor; NK: natural killer; Obfc2a: oligonucleotide/oligosaccharide-binding fold-containing 2a; Opn: opsin; OVA: ovalbumin; PER: period protein; PGC-1: peroxisome proliferator-activated receptors coactivator; PND: postnatal day; PPAR: peroxisome proliferators-activated receptor; PPs: Peyer’s patches; RAR: retinoic acid receptor; RIP140: receptor-interacting protein 140; ROR: RAR- or retinoid-related orphan receptor; RORE: ROR response element; RUNX1: runt-related transcription factor 1; RXR: retinoid X receptor; sg: staggerer; SCA1: spinocerebellar ataxia type 1; SCN: suprachiasmatic nucleus; Shh: Sonic hedgehog; SIRT1: sirtuin 1; SP: single positive; SRC: steroid receptor coactivator; SREBP1c: sterol regulatory element-binding protein 1, isoform c; STAT: signal transducer and activator of transcription; SULT: sulfotranferase; TGFβ: transforming growth factor β; Th: T helper; Thp: uncommitted (naïve) CD4+ T helper cells; TIP60: Tat-interactive protein, 60 kD; Treg: T regulatory; UBE2I: ubiquitin-conjugating enzyme I; WT: wild type
The cloning of several steroid hormone receptors in the 1980s led to an intense search by many laboratories for additional, novel members of the steroid hormone superfamily [Aranda and Pascual, 2001; Desvergne and Wahli, 1999; Escriva et al., 2000; Evans, 1988; Giguere, 1999; Kumar and Thompson, 1999; Novac and Heinzel, 2004; Willy and Mangelsdorf, 1998]. This resulted in the identification of a number of orphan receptors, including members of the retinoid-related orphan receptor (ROR) subfamily, which consists of RORα (NR1F1, RORA or RZRα)[Becker-Andre et al., 1993; Giguere et al., 1995; Giguere et al., 1994], RORβ (NR1F2, RORB or RZRβ) [Andre et al., 1998b; Carlberg et al., 1994; Schaeren-Wiemers et al., 1997], and RORγ (NR1F3, RORC or TOR) [He et al., 1998; Hirose et al., 1994; Medvedev et al., 1996; Ortiz et al., 1995; Sun et al., 2000]. ROR genes have been cloned from several mammalian species [Becker-Andre et al., 1993; Carlberg et al., 1994; Giguere et al., 1994; He et al., 1998; Hirose et al., 1994; Jetten and Joo, 2006; Koibuchi and Chin, 1998; Medvedev et al., 1996; Ortiz et al., 1995] and zebrafish [Flores et al., 2007]. ROR orthologues have also been identified in several lower species, including Drosophila hormone receptor 3 (DHR3) in Drosophila melanogaster [Carney et al., 1997; Gates et al., 2004; Horner et al., 1995; Koelle et al., 1992; Sullivan and Thummel, 2003; Thummel, 1995], CHR3 in Caenorhabditis elegans [Kostrouch et al., 1995; Palli et al., 1997; Palli et al., 1996], and MHR3 in Manduca sexta [Hiruma and Riddiford, 2004; Lan et al., 1999; Lan et al., 1997; Langelan et al., 2000; Palli et al., 1992; Riddiford et al., 2003]. This article will review the current status of our knowledge of RORs, with emphasis on recent developments in their physiological functions, roles in pathophysiological processes, and their potential as pharmatherapeutical targets.
The RORα gene maps to human chromosome 15q22.2 and spans a relatively large 730 kb genomic region comprised of 15 exons. The RORβ and RORγ genes map to 9q21.13, and 1q21.3 and cover approximately 188 and 24 kb, respectively. As a result of alternative promoter usage and exon splicing, each ROR gene generates several isoforms that differ only in their amino-terminus [Andre et al., 1998b; Giguere et al., 1994; Hamilton et al., 1996; He et al., 1998; Hirose et al., 1994; Matysiak-Scholze and Nehls, 1997; Medvedev et al., 1996; Sun et al., 2000; Villey et al., 1999] (Figure 1). Four human RORα isoforms, referred to as RORα1-4, have been identified, while only two isoforms, α1 and α4, have been reported for mouse. The mouse RORβ gene generates two isoforms, β1 and β2, while humans appear to express only the RORβ1 isoform [Andre et al., 1998b]. Both the mouse and human RORγ gene generate two isoforms, γ1 and γ2 [He et al., 1998; Hirose et al., 1994; Medvedev et al., 1996; Villey et al., 1999]. Most isoforms exhibit a distinct pattern of tissue-specific expression and are involved in the regulation of different physiological processes and target genes. For example, human RORα3 is only found in human testis [Steinmayr et al., 1998]. RORα1 and RORα4 are both prominently expressed in mouse cerebellum, while other mouse tissues express predominantly RORα4 [Chauvet et al., 2002; Hamilton et al., 1996; Matysiak-Scholze and Nehls, 1997]. RORγ2, most commonly referred to as RORγt, is exclusively detected in a few distinct cell types of the immune system, while RORγ1 expression is restricted to several other tissues [Eberl et al., 2004; He et al., 1998; Hirose et al., 1994; Kang et al., 2007]. In the mouse, expression of RORβ2 is restricted to the pineal gland and the retina, while RORβ1 is the predominant isoform in cerebral cortex, thalamus, and hypothalamus [Andre et al., 1998b]. Although most ROR isoforms are under the control of different promoters, little is known about the transcriptional regulation of their tissue-specific expression.
The ROR genes encode proteins of 459 to 556 amino acids (Figure 1). RORs exhibit a typical nuclear receptor domain structure consisting of four major functional domains: an N-terminal (A/B) domain followed by a highly conserved DNA-binding domain (DBD), a hinge domain, and a C-terminal ligand-binding domain (LBD) [Evans, 1988; Giguere, 1999; Jetten et al., 2001; Moras and Gronemeyer, 1998; Pike et al., 2000; Steinmetz et al., 2001; Willy and Mangelsdorf, 1998]. RORs regulate gene transcription by binding to specific DNA response elements (ROREs), consisting of the consensus RGGTCA core motif preceded by a 6-bp A/T-rich sequence, in the regulatory region of target genes [Andre et al., 1998a; Carlberg et al., 1994; Giguere et al., 1994; Greiner et al., 1996; Jetten et al., 2001; Medvedev et al., 1996; Moraitis and Giguere, 1999; Ortiz et al., 1995; Schrader et al., 1996]. RORs bind ROREs as a monomer and do not form heterodimers with retinoid-X receptors (RXRs) [Andre et al., 1998b; Carlberg et al., 1994; Giguere et al., 1994; Greiner et al., 1996; Medvedev et al., 1996; Moraitis and Giguere, 1999; Ortiz et al., 1995; Schrader et al., 1996] (Figure 2). The interaction of RORs with ROREs is mediated by the P-box, the loop between the last two cysteines within the first zinc finger, which recognizes the core motif in the major groove, and the C-terminal extension, a 30 residue region just downstream from the two zinc fingers, which interacts with the 5’-AT-rich segment of the RORE in the adjacent minor groove [Andre et al., 1998b; Giguere et al., 1995; Giguere et al., 1994; Jetten et al., 2001; McBroom et al., 1995; Sundvold and Lien, 2001; Vu-Dac et al., 1997]. Although RORα-γ and their different isoforms recognize closely-related ROREs, they exhibit distinct affinities for different ROREs. The amino-terminus (A/B domain) has been shown to play a critical role in conferring DNA binding specificity to the various ROR isoforms [Andre et al., 1998b; Giguere et al., 1995; Giguere et al., 1994; Sundvold and Lien, 2001; Vu-Dac et al., 1997]. In addition to the RORE sequence and the amino terminus, the promoter context may play an important factor in determining which ROR is recruited to a particular RORE.
In several instances, crosstalk between nuclear receptor pathways involves competition between receptors for binding to the same response element. Such an interplay has been demonstrated between RORs and the nuclear receptors REV-ERBAα and REV-ERBβ (NR1D1 and D2, respectively), which recognize a subset of ROREs [Burris, 2008; Giguere et al., 1995]. Because REV-ERB receptors act as transcriptional repressors, they are able to inhibit ROR-mediated transcriptional activation by competing with RORs for RORE binding [Austin et al., 1998; Bois-Joyeux et al., 2000; Downes et al., 1996; Forman et al., 1994; Liu et al., 2007b; Medvedev et al., 1997; Retnakaran et al., 1994] (Figure 2). Positive and negative regulation of RORE-mediated gene transcription by RORs and REV-ERBs have been reported to play a role in the control of brain and muscle ARNT-like 1 (BMAL1 or ARNTL) expression and may be part of several other regulatory feedback loops [Akashi and Takumi, 2005; Albrecht, 2002; Gachon et al., 2004; Guillaumond et al., 2005; Nakajima et al., 2004; Preitner et al., 2002; Triqueneaux et al., 2004].
The LBDs of nuclear receptors are multifunctional and play a role in ligand binding, nuclear localization, receptor dimerization, and provide an interface for the interaction with coactivators and corepressors. X-ray structural analysis demonstrated that RORs have a secondary domain structure that is characteristic of that of nuclear receptors [Kallen et al., 2002; Stehlin-Gaon et al., 2003; Stehlin et al., 2001]. The LBDs of RORs contain, in addition to the typical 12 canonical α-helices (H1-12), two additional helices, H2’ and H11’. The activation function 2 (AF2) in H12 consists of PLYKELF, which is 100% conserved among RORs (Figure 1). Deletion of the H12 or point mutations within H12 causes loss of the ROR transactivation activity and results in a dominant-negative ROR [Kurebayashi et al., 2004; Lau et al., 1999]. It is believed that H10 plays a critical role in the homo- and heterodimerization of nuclear receptors. Structure analyses revealed the presence of a kink in H10 of the LBD of RORα and RORβ that would greatly affect the dimerization capability of RORs [Kallen et al., 2002; Stehlin-Gaon et al., 2003; Stehlin et al., 2001]. This is consistent with the conclusion that RORs do not form homodimers or heterodimers with other RORs or RXRs.
Crystallography provided not only insights into the structure of the LBD, but also into size of the ligand binding pockets of RORs [Kallen et al., 2002; Stehlin et al., 2001]. The ligand-binding pocket of RORβ was calculated to be 766 Å3, similar to that of RORα (722 Å3). Homology modeling predicted that the ligand binding pocket of RORγ is similar in size (705 Å3), but different in shape. Cholesterol, 7-dehydrocholesterol, and cholesterol sulfate were identified as RORα agonists [Kallen et al., 2004; Kallen et al., 2002]. They were found to bind RORα in a reversible manner and to enhance RORE-dependent transcriptional activation by RORα in cells maintained in cholesterol-depleted medium. On this basis, it has been suggested that RORα might function as a lipid sensor and be implicated in the regulation of lipid metabolism. The latter would be consistent with reports indicating that RORα regulates the expression of several genes involved in lipid metabolism [Boukhtouche et al., 2004; Jakel et al., 2006; Kallen et al., 2002; Kang et al., 2007; Lau et al., 2008; Lau et al., 2004; Lind et al., 2005; Mamontova et al., 1998; Wada et al., 2008a]. However, whether cholesterol and cholesterol sulfate metabolites function as genuine physiological agonists of RORα, and whether other structurally-related lipids serve as endogenous ligands of RORα, needs to be established.
X-ray structure analysis of the RORβ(LBD) identified stearic acid as a fortuitously-captured ligand that appeared to act as a stabilizer by filling the ligand-binding pocket, rather than as a functional ligand [Stehlin et al., 2001]. Subsequently, several retinoids, including all-trans retinoic acid (ATRA) and the synthetic retinoid ALRT 1550 (ALRT), were identified as functional ligands for RORβ [Stehlin-Gaon et al., 2003]. ATRA and ALRT 1550 were able to bind RORβ(LBD) reversibly and with high affinity and reduced RORβ-mediated transcriptional activation, suggesting that they act as partial antagonists. These retinoids were also able to bind RORγ and inhibit RORγ-mediated transactivation, but did not bind RORα or affect RORα-induced transactivation [Stehlin-Gaon et al., 2003]. Interestingly, repression of RORβ-mediated transcription was observed only in neuronal cells and not in other cell types tested, indicating that this antagonism may be rather cell type-dependent and involve an interaction with (a) neuronal cell-specific RORβ repressor(s) [Stehlin-Gaon et al., 2003]. Future studies have to determine whether ATRA acts as a genuine physiological ligand of RORβ and RORγ and establish the physiological significance of such an interaction in neuronal and non-neuronal cells.
Although future research needs to determine whether in vivo ROR activity is regulated by endogenous ligands, these crystallographic and structural studies do support the concept that ROR activity can be modulated by specific endogenous and/or synthetic (ant)agonists [Kallen et al., 2004; Kallen et al., 2002; Stehlin-Gaon et al., 2003]. This conclusion is highly relevant to the emerging roles of RORs in several pathologies, including inflammation, various autoimmune diseases, obesity, and asthma, and the promise that these receptors might serve as potential targets for pharmacological intervention in these diseases (Figure 2).
For many receptors, binding of a ligand functions as a switch that induces a conformational change in the receptor that involves a repositioning of H12 (AF2) [Darimont et al., 1998; Glass and Rosenfeld, 2000; Harding et al., 1997; Heery et al., 2001; Heery et al., 1997; McInerney et al., 1998; Nagy et al., 1999; Nolte et al., 1998; Xu et al., 1999]. When RORs are in a transcriptionally-active conformation, H12 with H3 and H4 form a hydrophobic cleft and a charge clamp that involves the participation of a conserved Lys in H3 and a conserved Glu