AgiAl Nuclear Receptor Research Publishing House Vol. 2 (2015), Article ID 101185, 24 pages doi:10.11131/2015/101185 http://www.agialpress.com/

Review Article

Retinoic Acid-Related Orphan Receptors (RORs): Regulatory Functions in Immunity, Development, Circadian Rhythm, and Metabolism

Donald N. Cook1, Hong Soon Kang2, and Anton M. Jetten2

1Immunogenetics Section, Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA 2Cell Biology Section, Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA Corresponding Author: Anton Jetten; email: [email protected]

Received 10 September 2015; Accepted 23 November 2015

Editor: Brian G. Rowan

Copyright © 2015 Donald N. Cook et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract. In this overview, we provide an update on recent progress made in understanding the mechanisms of action, physiological functions, and roles in disease of retinoic acid related orphan receptors (RORs). We are particularly focusing on their roles in the regulation of adaptive and innate immunity, brain function, retinal development, cancer, glucose and lipid metabolism, circadian rhythm, metabolic and inflammatory diseases and neuropsychiatric disorders. We also summarize the current status of ROR agonists and inverse agonists, including their regulation of ROR activity and their therapeutic potential for management of various diseases in which RORs have been implicated.

Keywords: retinoic acid-related orphan receptor; ROR훾; ROR훼; ROR훽; immunity; Th17 cells; innate lymphoid cells; autoimmune disease; retina; brain; cancer; metabolism; glucose homeostasis; lipid metabolism; insulin sensitivity; diabetes; circadian clock; agonists; cholesterol biosynthesis; autism; agonists; antagonist; transcription

1. Introduction generates four isoforms, ROR훼1-4, while RORb and RORc each generate two isoforms [4–10]. Most isoforms exhibit The retinoic acid-related orphan receptors alpha, beta, and a distinct tissue-specific pattern of expression and regulate gamma (ROR훼-훾, RORA-C or NR1F1-3) constitute a sub- different biological processes and target genes. For example, family of nuclear receptors that function as ligand-dependent the expression of ROR훾2, commonly referred to as ROR훾t, transcription factors [1–3]. By using different promoters is restricted to several immune cell types, while ROR훾1 is and/or alternative splicing, each ROR gene produces several only expressed in various peripheral tissues, including liver, isoforms that vary only at their N-terminus. The RORa gene adipose tissue, skeletal muscle, and kidney [1, 7, 8, 11–14]. 2 Nuclear Receptor Research

RORs are critical in the regulation of many physiological implicated, including various inflammatory and metabolic processes, including immunity, circadian rhythm, embryonic diseases and neuropsychiatric disorders. In this review, we development, and several metabolic pathways, and have summarize several areas of ROR research in which recently been implicated in several pathologies associated with those significant progress has been made. processes. RORs exhibit a domain structure that is typical of nuclear receptors and contain an N-terminal domain, a 2. RORs in Adaptive Immunity highly conserved DNA-binding domain (DBD) consisting The innate and adaptive immune systems are highly inte- of two C2-C2 zinc finger motifs, a ligand-binding domain grated and serve to protect the host from being overwhelmed (LBD), and a hinge domain spacing the DBD and LBD by pathogen invasion. Innate immune responses are imme- [1]. The RORs regulate gene transcription by binding as diate and utilize germline-encoded receptors to recognize monomers to ROR response elements (ROREs) consist- and respond to pathogens, whereas adaptive immunity is a ing of the RGGTCA consensus preceded by an A/T-rich delayed response that requires expansion of a small number sequence in the regulatory regions of target genes [6, 15]. of cells bearing antigen-specific receptors on the surface of The ability to bind ROREs is shared with several other lymphocytes. Genetically modified mice lacking ROR훼 or nuclear receptors, including the transcriptional repressors ROR훾t have revealed that each receptor plays a key role in Rev-Erb훼 and Rev-Erb훽 (NR1D1-2) [16]. By competing for the development of several immune cells and are critical for RORE binding, these receptors can antagonize each other’s some immune responses (Figure 1). effects on transcription. For example, crosstalk between RORs and Rev-Erbs plays a role in the transcriptional Lymphoid progenitor cells undergo several stages of regulation of a number of metabolic and clock genes [9, 16– differentiation in the thymus prior to becoming mature T 25]. cells. These stages can be identified in part by display of CD4 and CD8 on the cell surface. ROR훾t is selectively expressed Relatively little is known about posttranslational mod- in T cells that display both CD4 and CD8, typically called ifications and upstream signaling pathways that modulate double positive (DP) cells. ROR훾t is required in these cells ROR transcription activity. Protein kinase A (PKA) has been for expression of the anti-apoptotic gene Bcl-X퐿,[36–40]. reported to activate ROR훼4, and although PKA phospho- Bcl-X퐿 expression is repressed in DP thymocytes of ROR훾 rylates ROR훼4 at Ser99, mutation of this site had little null mice, resulting in accelerated apoptosis in vivo and in influence on ROR훼4 transcriptional activity [26], while vitro. Consequently, thymi of ROR훾 null mice have reduced phosphorylation of ROR훼4 at Thr128 by ERK2 enhances its numbers of DP cells and their descendants, including single transcriptional activity [27]. PGE2/PKC훼-dependent phos- positive (SP) mature CD4+ T helper cells (Th) and CD8+ phorylation of ROR훼 has been reported to attenuate Wnt cytotoxic cells. target gene expression in colon cancer cells [28], while Mature, but naïve CD4+T (Th0) cells can be differentially sumoylation of ROR훼 enhanced its transcriptional activity polarized to produce the cytokines characteristic of Th1, Th2 [29]. A recent study demonstrated that the deubiquitinase, and Th17 cells [1, 41]. ROR훾t is required for the development DUB, interacts with and stabilizes the ubiquitin ligase UBR5 of Th17 cells [12, 13, 42–45], whose name reflects their in response to TGF-훽 signaling [30]. This results in an ability to produce the cytokines IL-17A and IL-17F, as well increase in ROR훾t ubiquitination that leads to reduced as IL-21 and IL-22. Like ROR훾t, ROR훼 can also contribute ROR훾t stability and diminished transactivation of ROR훾t to Th17 development and acts synergistically with ROR훾t in target genes in T-helper type 17 (Th17) cells. Another study this regard [13, 44]. Th17 cells protect against extracellular reported that the protein deacetylase, Sirtuin 1 (SIRT1), pathogens, but are also associated with various diseases, deacetylates ROR훾t and increases its transcriptional activity, such as rheumatoid arthritis, inflammatory bowel disease, thereby enhancing Th17 generation [31]. multiple sclerosis and asthma [46, 47]. Forced expression Reports showing that cholesterol and cholesterol sulfate, of ROR훾t is sufficient for the induction of several Th17- as well as a series of other small molecules, were able to associated genes, including Il17, Il22, Ccr6, and the IL- bind the LBD of RORs and modulate its transcriptional 23 receptor (Il23r)[12, 48, 49]. Combinations of promoter activity indicated that RORs function as ligand-dependent and chromatin immunoprecipitation (ChIP) analysis, and transcription factors [2, 25, 32, 33]. Recently, several cistrome mapping showed that ROR훾t is recruited to ROREs intermediates of the cholesterol biosynthetic pathway were in several Th17 marker genes, including Il17a, Il17f, Irf4 and reported to act as endogenous agonists of ROR훾 [34, 35]. Il23r, and directly regulate their transcription [43, 44, 50]. These studies revealed that ROR훾 transcriptional activity and However, in addition to ROR훾t, several other transcriptional the physiological processes it regulates, can be controlled by factors are necessary to induce the full Th17 differentiation changes in the intracellular pool of these sterol intermediates. program, including BATF [51], IRF4 [52], and STAT3 [53]. In addition, these discoveries raised the possibility that Recent advances in ChIP and RNA-seq technologies have ROR ligands might be valuable in the development of shed light on the hierarchy and order of transcription factor new therapeutic strategies for diseases in which RORs are occupancy during Th17 differentiation [54, 55]. In Th0

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A. ROR t extracellular pathogen clearance, Th0 Th17 IL-17A IL-17F autoimmunity, asthma IL-22 ROR t + rapid producon of IL-17 after IL-17 17 TCR – infecon

LTi/ILC ROR t LTi LT Lymph node progenitor LT development TNF

ROR t ILC IL-17 ILC3 bacterial pathogen clearance, colis precursor IL-22

B. ROR IL-5 ILC helminth clearance, metabolism, ILC2 IL-13 allergic inflammaon precursor amphiregulin

Figure 1: Multiple functions of RORs in lymphocyte development. A. Roles of ROR훾t in the development of Th17 cells, 훾훿-17 T cells, lymphoid tissue inducer (LTi) cells and innate lymphoid cells 3 (ILC3) cells. (B) Role of ROR훼 in the development of ILC2 cells. ROR훼 has also a role in the regulation of Th17 cells. cells, the transcription factors, IRF4 and BATF, are co- The differentiation of Th0 cells into Treg and Th17 operatively bound to overlapping sites in chromatin near cells is dependent on the balance between the level of several genes, including Rorc and Il17, thereby increasing expression of Foxp3 and ROR훾t, respectively. Through its chromatin accessibility to other transcription factors (Figure interaction with ROR훾t, Foxp3 inhibits ROR훾t function and 2). In response to TGF훽 and IL-6, STAT3 becomes phos- promotes Treg differentiation [1, 57, 58]. This balance is phorylated (pSTAT3) and moves to the nucleus, where it controlled by the concentration of specific cytokines in the binds to chromatin and induces expression of Rorc. ROR훾t environmental milieu. Foxp3 expression and consequent Treg and pSTAT3 then cooperate with IRF4 or BATF and other development is favored in cultures containing high levels of factors to increase expression of Th17-associated genes, TGF-훽, IL-2 and retinoic acid, whereas Th17 development including Il17a, Il17f, Il23r, Ccl20 and Il1r1 [55]. Thus, is promoted by low amounts of TGF-훽 in combination IRF4 and BATF have broad and self-reinforcing effects on with the proinflammatory cytokines, IL-6 and IL-1 [58– chromatin remodeling, whereas ROR훾t specifically regulates 60]. IL-1 can repress the suppressor of cytokine signaling a relatively small number of key Th17-associated genes in 3 (SOCS3), an inhibitor of STAT3 phosphorylation [61], a manner potentially responsive to environmental cues. In thereby increasing Rorc expression. Th17 cells share an addition to transcriptional control by IRF4/BATF/STAT4, the overlapping developmental program with that of inducible PI3K-Akt-mTORC1-S6K1/1 cell signaling axis has also been regulatory T cells (iTregs) [62]. In the small intestine, a linked to the control of Th17 differentiation by ROR훾t [56]. number of ROR훾t+Foxp3+ T cells do not produce IL-17, but Activation of PI3K-Akt-mTORC1 induces ribosomal protein instead express IL-10. These ROR훾t+ Tregs develop outside S6 kinase 훽2 (RPS6KB2) expression that subsequently the thymus and require gut microbiota for their development. promotes the nuclear localization of ROR훾t and ROR훾t- In addition, dietary vitamin A favors the generation of these mediated Th17 differentiation. ROR훾t+ Tregs over that of Th17 cells [62]. ROR훾t+ Treg

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IL-6 IL-1 TGF- P STAT3 STAT3

ROR t ROR t P P BATF BATF BATF STAT3 STAT3 IRF4 IRF4 IRF4 Rorc Rorc Rorc P P BATF BATF BATF STAT3 STAT3 ROR t IRF4 IRF4 IRF4 ll-17a ll17 ll17

IL-17 Th0 Th17

Figure 2: Figure 2. ROR훾t-dependent induction of Th17 differentiation and Th17-associated genes. In Th0 cells, IRF4 and BATF are bound to chromatin near Th17-associated genes, but the loci are transcriptionally silent. Upon exposure to cytokines, such as IL-6, STAT-3 becomes phosphorylated and transfers to the nucleus, where it binds DNA near IRF4 and BATF and induces Rorc transcription. ROR훾t can then join the IRF4/BATF/STAT3 transcription factor complex and induce expression of Th17-associated genes, such as Il17 and Il23r.

cells regulate Th2 cells - but not Th1 or Th17 cells - many 훾훿-17 cells express IL-17 very early during their through a CTLA4-dependent regulation of CD80 and CD86 development in the thymus, even prior to TCR rearrangement on dendritic cells. [70, 71]. Thus, 훾훿-17 cells have the potential to be a major Th17 and IL17 have been implicated in several inflam- cell source of IL-17, especially during the early phases of matory and autoimmune diseases. Mice lacking ROR훾 are disease, prior to the development of antigen-specific Th17 partially protected against the development of diseases, cells [14, 72]. There are some commonalities, but also including autoimmune diseases such as experimental autoim- differences in the molecular pathways leading to 훾훿-17 and mune encephalomyelitis (EAE) and type II collagen-induced Th17 cells. For both cell types, ROR훾t is critically important. arthritis, as well as allergen-induced lung inflammation [12, Thus, mice deficient for ROR훾t lack 훾훿-17 cells in peripheral 44, 58, 63]. Mice lacking both ROR훼 and ROR훾 are greatly organs and lymphoid tissues [12]. However, the induction of protected from EAE [44]. Although IL-17A, IL-17F and IL- ROR훾t in Th17 cells requires the canonical c-Rel-dependent 22 are the signature cytokines of Th17 cells, they appear NF-kB pathway, whereas 훾훿-17 cells require RelB and the not to be sufficient for pathogenicity in EAE [64, 65]. In noncanonical NF-kB pathway [73]. In addition, IRF4 is this model, ROR훾t-dependent production of granulocyte required for the induction of Th17 cells, but this transcription macrophage colony stimulating factor (GM-CSF) is reported factor is dispensable for the development of 훾훿-17 T cells. to drive the effector phase of neuroinflammation [66, 67]. ROR훼 and ROR훾 also play a critical role in the generation However, the molecular requirements of pathogenicity might of innate lymphoid cells (ILCs). ILCs are a heterogeneous depend on the disease model because either IL-17A or IL- population of cells that possess the typical lymphoid cell 17F is required for Th17 cell-mediated intestinal inflamma- morphology, but lack some cell surface molecules typically tion [68]. Together, these studies raise the possibility that seen on lymphocytes [74]. In particular, ILCs lack TCRs and ROR훾 antagonists might be useful in the management of the associated CD3 complex found on conventional T cells. autoimmune disease. Consequently, ILCs cannot recognize specific antigens, and instead respond to cytokines produced during innate immune responses. ILCs have been classified into three groups, based 3. RORs in Innate Immunity on their cytokine production profiles and the transcription factors that regulate their development [75]. The cytokines Like conventional 훼훽 T cell receptor (TCR)+ cells, T cells produced by each of these groups mirrors those produced by expressing the 훾 and 훿 TCR chains (훾훿 T cells) develop specific T helper (Th) cell types: Group 1 ILCs and Th1 cells in the thymus, but they have a more limited repertoire than produce IFN-훾, Group 2 ILCs and Th2 cells produce IL-5 훼훽 TCR+ cells and lack major histocompatibility complex and IL-13, and Group 3 ILCs and Th17 cells produce IL-17A, (MHC) restriction [69]. Many 훾훿 T cells express IL-17 IL17F and IL-22. and are thus termed 훾훿-17 T cells [70, 71]. Unlike 훼훽 ROR훾t is required for the development of all ILC3s, TCR Th17 cells, which acquire effector functions only after a heterogeneous population of cells that also depends on encountering their cognate antigens in peripheral tissue, IL-7 for their development. The first discovered member

AgiAl Publishing House | http://www.agialpress.com/ Nuclear Receptor Research 5 of the Group 3 ILCs is the lymphoid tissue inducer (LTi) interleukin-1 receptor-associated kinase 1 (IRAK1), Toll- cell, a type of CD4+CD3− cell that displays transmembrane interleukin 1 receptor domain containing adaptor protein lymphotoxin 훼1훽2 [76, 77]. These cells are required for the (TIRAP), and the clock genes, Bmal1 and nuclear factor, development of secondary lymphoid organs, Peyer’s patches, interleukin 3 regulated (NFIL3 or E4BP4). The decreased and intestinal lymphoid follicles [78–81]. ROR훾t-deficient expression of ROR훼 in the intestine of microbiota-depleted mice lack these cells and therefore do not develop secondary mice further provides additional evidence of a relationship lymphoid organs [1, 36, 37, 82]. Recently, retinoic acid between ROR훼, commensal bacteria and diurnal regulation (RA) was found to control LTi cell maturation upstream of of immune-related genes in the gut [94]. ROR훾t by positively regulating ROR훾t expression directly through the recruitment of RA receptors (RARs) to the promoter region of ROR훾t [83]. Impairment of LTi mat- 4. ROR Functions in Brain and Retina uration in cells defective in RA signaling can be rescued by the exogenous expression of ROR훾t. More recently, ROR훼 is highly expressed and developmentally regulated in other ILC3 subpopulations have been identified that, like several regions of the brain, including cerebellar Purkinje Th17 cells, are abundant in the gut and can produce IL- cells and the thalamus [1, 95–97]. Genetically modified mice, 17A, IL-17F, IL-22, GM-CSF, and TNF, suggesting their in which ROR훼 is disrupted, and ROR훼-deficient staggerer importance for clearing extra-cellular pathogens. Gata-3 is (ROR훼푠푔/푠푔) mice display severe cerebellar ataxia due to critical for the development of gut ROR훾t+ ILC3s subsets cerebellar neurodegeneration [98–100]. Further characteriza- [84]. At least some ILC3s can transition to ILC1 cells [85], tion of these mice revealed that ROR훼 plays a critical role in reminiscent of Th17 cell conversion to a Th1-like phenotype. the regulation of the survival and differentiation of Purkinje This transition, which is accompanied by elevated levels cells from embryonic development throughout adulthood of TBX21, is driven by cytokine signals in the cellular [98, 100–103]. Deletion of ROR훼 in Purkinje cells between milieu. postnatal days 10-21 revealed that continued expression of Group 2 ILCs are the most homogeneous group of the ROR훼 is necessary after neuronal maturation to maintain ILCs and are dependent on ROR훼, but not ROR훾t, for mature morphological and innervation characteristics in the their development [86, 87]. They display the cell surface adult Purkinje cells [103]. Loss of Purkinje cells was also markers IL-7Ra (CD127), IL-2RA (CD25), Sca-1, KLRG1 observed in >6 months-old male ROR훼+/푠푔 mice and to and the IL-33 receptor, ST2. ILC2s are defined based a much smaller degree in ROR훼+/푠푔 females. The marked, on their ability to produce an array of type 2 cytokines, age-related Purkinje cell death in ROR훼+/푠푔 male mice including IL-4, IL-5, IL-9, and IL-13, as well the cytokine, has been linked to a premature decrease in circulating sex amphiregulin, and are implicated in helminth clearance steroids, which have been shown to be neuroprotective, and and allergic inflammation [88]. Several single nucleotide appeared not to be due to changes in cerebellar neurosteroids polymorphisms (SNPs) within the RORA gene are associated [104]. Genome-wide gene expression studies showed that with increased susceptibility to asthma [89–91], and ROR훼 ROR훼 regulates the expression of a number of genes linked null mice and ILC2-deficient mice generated by ROR훼- to Purkinje cell maturation, particularly dendritic differen- deficient bone marrow transplants have reduced type 2 tiation, and the glutamatergic pathway [98]. ChIP analysis cytokine production and partial protection from airway demonstrated that ROR훼 directly controls the transcription hyper-reactivity [87, 92]. ROR훼 expression was significantly of several of these genes, including the glutamate transporter upregulated in patients with therapy-resistant asthma [93]. Slc1a6, the calmodulin inhibitor Pcp4, and the IP3 receptor ROR훼 expression was also found to be significantly ele- (Itpr1). Several of the ROR훼 target genes were found to be vated in skin from patients with atopic dermatitis (AD), also down-regulated in Ski family transcriptional corepressor while in an experimental model of AD-like inflammation 2 (Skor2)-deficient mice; however, ROR훼 expression was not ROR훼-deficient mice exhibit a profound deficit in ILC2 altered in these mice [105]. It was proposed that ROR훼 and cells and significantly reduced allergic skin inflammation Skor2 cooperate in regulating Purkinje cell differentiation [88]. Together, these observations indicate that ROR훼 and gene expression. In Purkinje cells, ROR훼 also directly plays a critical role in ILC2 cell lineage determination regulates the expression of sonic hedgehog (Shh)[98], which and control of allergy-induced inflammation in multiple is required for the proliferation and survival of cerebellar tissues. granule precursor cells through its activation of Gli tran- ROR훼 also contributes to immune function in the intesti- scription factors. The degeneration of cerebellar granule cells nal epithelium by controlling the diurnal regulation of observed in ROR훼-deficient mice can at least in part be several pathogen recognition receptors, including Nod2 and attributed to this loss in Shh production. various Toll-like receptors [94]. The expression of these Accumulating evidence indicates a role for ROR훼 in genes in the intestine is reduced in ROR훼-deficient mice, several neuropsychiatric disorders, including autism spec- particularly when ROR훼 expression is at its highest and trum and bipolar disorder (ASD), schizophrenia, depres- bound to promoter regions of these genes. Other genes whose sion, and posttraumatic stress syndrome [106–117]. Studies diurnal expression is directly controlled by ROR훼 include demonstrating that the expression of RORA was reduced in

AgiAl Publishing House | http://www.agialpress.com/ 6 Nuclear Receptor Research sections of cerebellum and cortex of autistic subjects and somatosensory, auditory, and visual areas [125, 126]. This observations showing differential methylation of the RORA developmental pattern of expression was similar to that gene in lymphoblastoid cells from autistic and nonautistic reported for rat neocortex [127]. A possible connection was siblings supported a role of ROR훼 in the development of found between ROR훽 expression levels and the control of ASD [107]. This, together with reports showing reduced cytoarchitectural patterning of neocortical neurons during number and size of Purkinje cells (PC) in the majority of mouse development [128]. GWAS studies revealed an asso- cerebellar specimens from ASD patients, as was observed in ciation between a series of RORB genetic variants with ROR훼-deficient mice (Chugani 2014), is consistent with a schizophrenia, and bipolar I disorder [113, 116, 129, 130] and link between ROR훼, its regulation of Purkinje cell survival between ROR훽 expression in the temporal cortex and verbal and differentiation, and ASD. A connection between ROR훼 intelligence [131]. Similarly, a syndrome characterized by and ASD is further supported by genome-wide ChIP-Seq moderate facial dysmorphy, mental retardation, epilepsy, analysis showing that in human neuronal cells, ROR훼 was speech delay, and autistic behaviour in patients with a 9q21 associated with the promoter region of a number of ASD- deletion at the RORB locus identified ROR훽 as a strong associated genes, including ataxin binding protein (A2BP1), candidate for this neurological disorder [132–134]. Prenatal neuroligin 1 (NLGN1), and aromatase (CYP19A1)[118, ethanol exposure has been reported to lead to neurobiological 119]. Several of the genes down regulated in ROR훼- damage in early development. Newborns prenatally exposed deficient neuronal cells, including aromatase, were also to alcohol show neuroanatomical defects in the neocortex and found to be repressed in the frontal brain of individuals an abnormal neocortical expression pattern of ROR훽 that with ASD. The positive regulation by ROR훼 of aromatase, is associated with mental and intellectual dysfunction, and which converts testosterone to estrogen, is intriguing because behavioral and motor deficits [135]. estrogen has been reported to enhance ROR훼 expression Recent studies showed that the ROR훽1 and ROR훽2 and to exhibit a neuroprotective effect, as reported for isoforms exhibit distinct patterns of expression during reti- ROR훼. Thus, ROR훼, aromatase, and estrogen might be nal development [136, 137]. ROR훽 was shown to play a part of a positive regulatory pathway. Therefore, reduced critical role in regulating retinal progenitor proliferation and expression of aromatase and estrogen production in ROR훼- differentiation [123, 137–139]. ROR훽1-deficient mice lack deficiency might excerbate Purkinje cell death and enhance amacrine and horizontal interneurons, cells important for the the risk for ASD. Loss of Purkinje cells was also observed integration of visual information, suggesting that ROR훽1 +/푠푔 in >6 months-old male ROR훼 mice and to a much is critical for the differentiation of retinal progenitors into +/푠푔 lesser degree in ROR훼 females. The marked, age-related these interneurons [136]. This involves direct transcriptional +/푠푔 Purkinje cell death in ROR훼 male mice was linked regulation of Ptf1a, a key factor required for the generation to a premature decrease in circulating sex steroids, which of amacrine and horizontal cells, by ROR훽1. Re-expression have been shown to be neuroprotective, and not due to of ROR훽1 was able to rescue amacrine differentiation in changes in cerebellar neurosteroids [104]. Since both ROR훼 ROR훽 null mice. Retinal progenitors can also differentiate and estrogen exhibit neuroprotective effects, this pathway into two morphologically, developmentally, and functionally +/푠푔 might help to explain why ROR훼 female mice are distinct photoreceptors, rods and cones. Rods function in dim less susceptible to Purkinje cell loss during aging. Further light, while cones mediate daylight, and in most mammals, indications for a link between ROR훼 and ASD came from color vision. Mice lacking ROR훽 lose rods, but overproduce a recent study demonstrating that the microRNA MIR137, primitive S cones that lack outer segments [139]. ROR훽1 and which has been implicated in ASD and schizophrenia, ROR훽2 control rod cell differentiation through its transcrip- targets the 5’-UTR of ROR훼 [108]. ROR훼 has also been tional regulation of neural retina leucine zipper factor (NRL), reported to have neuroprotective functions in neurons and a transcription factor that promotes the differentiation of astrocytes during hypoxia [120, 121]. Protecting brain cells retinal progenitors into the rod cell lineage, while suppressing from the damaging effects of injury and stress might be an the cone cell lineage [137, 139, 140]. The lack of rod important function of ROR훼 and be relevant to several brain photoreceptors in ROR훽 null mice is in part due to the loss disorders. of NRL expression. This can be reversed by re-expression ROR훽 is highly expressed in the suprachiasmatic nucleus, of NRL in these mice [139]. ROR훽2 is expressed in rod the retina and pineal gland, and has been implicated in the photoreceptors and its transcription was shown to be directly regulation of circadian, motor, and visual functions [3, 122, regulated by NRL. Thus, ROR훽2 and NRL form two positive 123]. ROR훽 deficient mice displayed motor (“duck gait,” feedback loops that synergistically promote the commitment hind paw clasping reflex), olfactory deficits, reduced anxiety to a rod cell lineage [137]. In addition to these roles, ROR훽- and learned helplessness-related behaviors, and alterations in deficient mice fail to induce S opsin appropriately during circadian behavior [124]. Examination of ROR훽 expression postnatal cone development, suggesting a function for ROR훽 during embryonic and postnatal development of the mouse in morphological maturation of cone photoreceptors [123]. neocortex showed that after E16.5 ROR훽 transcripts increas- ROR훽 was found to activate the S opsin gene (Opn1sw) ingly localized to the primary sensory areas and reached peak expression through binding sites in the upstream promoter expression at P10 with strongest expression in the primary region.

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5. Role of RORs in Cancer by a recent study demonstrating that ROR훼 expression was decreased in tumor tissues compared to adjacent normal Very little is known about the role of ROR훽 and ROR훾 in tissues in human hepatocellular carcinoma patients and that cancer. Initial analysis of ROR훾 knockout mice revealed that this was associated with a change in glucose metabolism these mice develop T-cell lymphomas within the first months [157]. ROR훼 was shown to inhibit pyruvate dehydrogenase after birth that rapidly metastasize to liver and spleen [141]. kinase 2 (PDK2) expression and phosphorylation, thereby The mechanism underlying the development of thymic lym- promoting aerobic glycolysis rather than oxidative phospho- phomas in ROR훾 null mice has yet to be elucidated. A recent rylation, whereas reduced ROR훼 expression in tumor cells study reported that low levels of ROR훾 mRNA expression promotes oxidative phosphorylation and tumor cell growth. in somatotroph adenomas was associated with reduced E- Several studies revealed a role for a noncanonical ROR훼 cadherin expression and increased epithelial mesenchymal pathway in cancer that does not involve RORE binding, but in transition (EMT), and increased tumor size and invasiveness which ROR훼 functions as a transcriptional cofactor. In colon [142]. Similarly, higher expression of ROR훾 correlated with carcinoma cells, ROR훼 was shown to bind 훽-catenin directly longer metastasis-free survival in breast cancer [143]. With and inhibit 훽-catenin-mediated transcriptional activation of respect to ROR훽 and cancer, a recent study reported that the target genes, cyclin D1 (CCND1) and c-myc (MYC), RORB is overexpressed in primary leiomyosarcomas, the resulting in repression of cell proliferation and migration most common type of uterine sarcoma [144]. In contrast, [158]. ROR훼 was found to be recruited to the lymphoid RORB expression was found to be highly down-regulated in enhancer-binding factor 1 (LEF1)-binding sites of the LEF1- both serous and endometrioid types of endometrial cancer target gene, CCND1, together with LEF1 and 훽-catenin. [145]. ROR훼 interacted with residues within the armadillo repeat A number of studies reported that ROR훼 expression domains of 훽-catenin, which function as binding sites for is significantly down-regulated during tumor development a subset of coactivators. The interaction of ROR훼 with 훽- and progression, while expression of exogenous ROR훼 catenin required the N-terminus of ROR훼 and was dependent inhibited cell proliferation and tumor growth [146–151]. on the phosphorylation of Ser35 by protein kinase C훼 Reduced ROR훼 expression has been observed in colorectal (PKC훼) activated by the noncanonical Wnt pathway. Inter- and mammary carcinomas and found to be associated with estingly, in many colorectal carcinomas phosphorylation poorer prognosis in hepatocellular and breast carcinoma of ROR훼 was reduced. In another noncanonical pathway, patients [146, 147, 150, 152, 153]. In addition, silencing ROR훼 interacts with the heptad repeat and marked box ROR훼 in mammary epithelial cells significantly enhanced region of the transcription factor E2F1 and suppressed E2F1- cell proliferation in ductal epithelial cells and promoted side regulated transcription and cell cycle progression in epithe- branching of mammary ducts, suggesting that ROR훼 has an lial cells [159]. In mammary ducts, ROR훼 levels inversely important role in mammary gland branching morphogenesis correlated with the expression of E2F1 target genes and cell [150]. Conversely, restoring ROR훼 expression in cultured proliferation. Binding of ROR훼 was shown to inhibit E2F1 breast cancer cells was shown to inhibit cell migration and acetylation and its DNA-binding activity by enhancing its suppress tumor growth and metastasis in nude mice. This interaction with histone deacetylase 1 (HDAC1). Knockdown was accompanied by enhanced expression of semaphorin 3F of HDAC1 or inhibition of HDAC activity partially reversed (SEMA3F), a tumor suppressor that inhibits tumor growth the repression of E2F1 activity by ROR훼. In contrast to and invasiveness. ROR훼 was shown to regulate SEMA3F its growth inhibitory effects, ROR훼 was shown to enhance transcription directly through ROREs in its promoter region the proliferation in mammary carcinoma MCF7 cells and [150]. A different study indicated a role for SPARC, which is significantly induced the expression of aromatase mRNA by critical in the regulation of cell growth and adhesion, in the binding an RORE in the aromatase promoter region [160]. It anti-tumor and anti-proliferation effects of ROR훼 in human was proposed that the increase in aromatase expression by hepatoma cells [154]. SPARC was found to be a direct target ROR훼 accelerates the local production of estrogen, which gene of ROR훼. Treatment of colon carcinoma HCT116 cells then enhances the proliferation of breast cancer cells. with DNA-damage agents led to a p53-dependent increase in ROR훼 expression that is directly mediated through func- tional p53 response elements in the RORa promoter [155]. 6. RORs in the Regulation of Metabolism ROR훼 itself stabilized p53 by inhibiting its ubiquitination and enhanced p53 transcription in a HAUSP/Usp7-dependent Both ROR훼 and ROR훾 have been implicated in the control manner leading to increased apoptosis. The connection of energy homeostasis and the regulation of several lipid and between ROR훼 and p53 was supported by a report demon- glucose metabolic genes [3, 22, 24, 161–169]. Regulation strating that treatment of hepatocellular carcinoma cells with of energy homeostasis is a complex process that involves an ROR훼 agonist enhanced p53 stability [156]. This increase multiple interrelated glucose and lipid metabolic pathways was shown to involve elevated SOX4 transcription, a gene in many organs and is controlled by the circadian clock, critically involved in MDM2-dependent regulation of p53 gut microbiota, and by the endocrine, immune and nervous stability. Another role for ROR훼 in cancer was revealed systems [170–173]. This has made it difficult to determine

AgiAl Publishing House | http://www.agialpress.com/ 8 Nuclear Receptor Research whether the metabolic changes observed in ROR-deficiency phosphoenolpyruvate carboxykinase (Pepck) and glucose- are cause or effect. ROR훼-deficient (staggerer) mice were 6 phosphatase (G6pc), which play a role in gluconeoge- shown to be protected against high fat diet (HFD)-induced nesis, fibroblast growth factor 21 (Fgf21), which is an metabolic syndrome as indicated by reduced weight gain, important regulator of glucose and lipid homeostasis, and adiposity and hepatic steatosis, and improved insulin sensi- genes involved in triglyceride synthesis and storage, such tivity [161, 174, 175]. Adipocytes in ROR훼-deficient mice as glycerol-3-phosphate acyltransferase (Gpam), perilipin fed a high fat diet accumulated considerably less lipid and 2 (Plin2), monoacylglycerol O-acyltransferase 1 (Mogat1), the infiltration of inflammatory macrophages and expression and cell death-inducing DFFA-like effector a (Cidea)[154, of several inflammatory genes, including interleukin 6 (Il6), 166, 174, 184]. In addition, the hepatic expression of Toll-like receptor 8 (Trl8), and chemokine (C-C motif) several genes involved in sterol and bile acid metabolism, ligand 8 (Ccl8), were greatly diminished [174]. Interleukin- including cytochrome P450 8b1 (Cyp8b1), Cyp7b1, and 1 receptor antagonist (Il1rn) was among the genes most sulfotransferase Sul1b1 were significantly diminished in dramatically repressed in white adipose tissue (WAT) of ROR훼-deficient mice [153, 167, 174, 185]. However, the ROR훼-deficient mice. This gene has been implicated in the hepatic expression of sulfotransferase Sult1e1 was found to regulation of obesity and insulin resistance, suggesting that be dramatically induced in both male and female ROR훼-, the reduced susceptibility to metabolic syndrome in ROR훼- but not in ROR훾-deficient mice, whereas Sult2a1, known to deficient mice might at least in part be attributed to Il1rn sulfonate bile acids, hydroxysteroid dehydroepiandrosterone, repression [174, 176]. WAT-associated inflammation plays a and related androgens, was increased in both ROR훼- and critical role in the development of metabolic syndrome [172, ROR훾-deficient mice, but only in female mice [167]. In con- 177]. The reduced inflammation observed in ROR훼-deficient trast, in cultured human hepatocytes and hepatoma HepG2 mice might be in part responsible for the improved insulin cells, exogenous expression of ROR훼 induced SULT2A1, sensitivity in these mice. A role for ROR훼 in the regulation while ROR훼 knockdown with siRNAs decreased its expres- of insulin sensitivity is supported by a study showing an sion [153]. Moreover, overexpression of ROR훼 inhibited association between a single nucleotide polymorphism in LXR and SREBP expression as well as lipid accumulation ROR훼 (rs7164773) and an increased risk for type 2 diabetes in these cells [186]. Adenovirus-mediated overexpression of in the Mexico Mestizo population [178]. A recent study ROR훼 in liver also reduced triglyceride levels in mice fed showed that the expression of several thermogenic genes, a high fat diet. The cause of the discrepancy between the such as uncoupling protein 1 (Ucp1) and deiodinase 2 (Dio2), observations in ROR훼-deficient mice and those in HepG2 markers of brown adipose tissue (BAT), was enhanced and liver overexpressing ROR훼 has yet to be understood. in adipose tissue from ROR훼-deficient mice. This was ChIP and promoter analysis indicated that many metabolic associated with increased expression of the histone-lysine genes, including G6pc, Adfp, Cyp7b1, citrate synthase N-methyltransferase 1 (Ehmt1), a gene that controls BAT (Cs), Cyp2c8, Fgf21, secreted protein, acidic, cysteine-rich specification and maintenance [175, 179]. The greater cold- (Sparc), Sult1b1, and Sult2a1, were directly regulated by tolerance of ROR훼-deficient mice appears to be related to ROR훼 in HepG2 cells [153, 154, 166, 174, 185, 187, 188]. the increased expression of these genes, leading to increased ROR훼 cistrome data [165] revealed that in liver, ROR훼 was oxygen consumption and heat generation from lipid oxidation recruited to ROREs in several genes important in glucose that likely contributes to the improved energy homeostasis homeostasis and lipid metabolism, including G6pc, Fasn, and insulin-sensitivity observed in these mice. Both ROR훼 Pepck1, Apoa1, and Elovl3, indicating that ROR훼 positively and ROR훾 have been shown to be induced during adipocyte regulates the transcription of these metabolic genes by differentiation in 3T3-L1 cells [180]; however, exogenous binding ROREs in their regulatory region. expression of ROR훼 inhibits adipocyte differentiation in ROR훼-deficient mice also display metabolic changes in 3T3-L1 cells, as indicated by the reduced induction of fatty skeletal muscle that are accompanied by alterations in the acid binding protein 4 (Fabp4), perilipin 1 (Plin1) and fatty expression of several genes [169]. Glucose uptake in skeletal acid synthase (Fasn)[181]. muscle of ROR훼-deficient mice was enhanced and found to In addition to WAT, loss of ROR훼 induces changes in be associated with increased phosphatidylinositol 3-kinase gene expression in macrophages and liver. Disruption of signaling and Glut4 expression [161, 169]. Expression of ROR훼 in macrophages leads to diminished expression of a dominant-negative ROR훼 in skeletal muscle C2C12 cells cholesterol 25-hydroxylase (Ch25h), which converts choles- and in skeletal muscle in mice was reported to down- terol to 25-hydroxycholesterol, and reduced phagocytosis regulate the expression of carnitine palmitoyltransferase-1 [182, 183]. Interestingly, addition of 25-hydroxycholesterol (Cpt1), caveolin 3 (Cav3), and Abca1 encoding proteins was able to reverse the inhibition of phagocytosis in ROR훼- involved in 훽-oxidation and cholesterol homeostasis, and of deficient macrophages suggesting a link between oxysterol Srebp1c and its downstream targets, Fas and Scd1/2l, which metabolism and the regulation of phagocytosis. In the liver, are involved in lipogenesis [163, 189]. Promoter analysis the expression of a large number of genes related to lipid indicated that Cav3 and Cpt1 were directly regulated by and glucose metabolism were found to be down-regulated ROR훼. Expression of a dominant-negative ROR훼 in skeletal in ROR훼-deficient mice fed a HFD [174]. These included muscle induced mild hyperglycemia and glucose intolerance

AgiAl Publishing House | http://www.agialpress.com/ Nuclear Receptor Research 9 and attenuated insulin-mediated phosphorylation of Akt2. HFD. Lipid and glucose metabolic genes are under a The latter contrasts with the increase in Akt2 expression and complex control and involve regulation by other transcrip- phosphorylation observed in ROR훼-deficient sg/sg mice. tion factors, including several nuclear receptors, such as ROR훾 also plays a role in the regulation of glucose Rev-Erb, PPAR, LXR, and CAR. Since some of these metabolism and insulin sensitivity [164, 165, 168, 190, receptors interact with similar binding sites, the transcrip- 191]. ROR훾-deficient mice were significantly more insulin tional control of several of lipid and glucose metabolic sensitive and glucose tolerant than WT mice. The eug- genes likely involves interplay between different nuclear lycemic clamp test revealed that hepatic glucose production receptor signaling pathways. The best known example of was considerably reduced in ROR훾-deficient mice, whereas this is the competition of Rev-Erbs with RORs for the ectopic expression of ROR훾 in ROR훾-deficient liver tissue same binding sites. Comparison of the ROR훼 and ROR훾 or primary hepatocytes increased glucose production [165]. cistromes from liver indicated that although many genes Moreover, the conversion of exogenously administered pyru- were selectively regulated by either ROR훼 or ROR훾, vate to glucose was significantly lower in ROR훾−/−. The several genes, including G6pc, Apoa2, Elovl5, and Cry1, reduced hepatic gluconeogenesis in ROR훾-deficient mice were regulated by both ROR훼 and ROR훾, indicating some may be at least partly responsible for the improved insulin redundancy between the two RORs in regulating these genes sensitivity and glucose tolerance observed in these mice [165, [165]. 190]. Loss of ROR훾 significantly decreased peak expression of several glucose (e.g., G6pase, Pklr, Glut2, PPAR훿) and lipid (e.g., Insig2a, Elovl3, Cyp8b1, Sult1e1) metabolic genes 7. RORs and Circadian Rhythm [165, 167, 168, 192]. Conversely, exogenous expression of ROR훾 in ROR훾−/− liver tissue by adenovirus significantly It has been well established that the regulation of the increased the expression of G6pase, Pepck, Gck, Gckr, circadian rhythm is interconnected with the diurnal control of Ppar훿, Pcx, and Klf15. [165]. Together, these observations behavior, metabolic activities, immune responses, and many indicated that ROR훾 is an important modulator of hepatic other physiological functions. For example, the circadian gluconeogenesis and glycolysis. ChIP-Seq analysis not only clock has been shown to regulate the diurnal expression of uncovered the consensus sequence of the in vivo RORE, but many lipid and glucose metabolic genes as well as immune also revealed that ROR훾 is recruited to the regulatory region response genes [170, 193–195]. It therefore not surprising of a number of metabolic genes involved in glycolysis and that disruption of the circadian rhythm has been linked gluconeogenesis, including G6pase, Pepck, Pklr, Ppar훿, Gck, to increased risk for metabolic diseases, including obesity, Gckr, Glut2, Gys2, Dlat, Pcx, and Klf15 [165]. These data diabetes, and liver steatosis, as well as several inflammatory indicated that ROR훾 positively regulates the transcription and neuropsychiatric disorders [170, 171, 196–201]. In mam- of these metabolic genes by binding ROREs in their regu- mals, the suprachiasmatic nucleus (SCN) serves as the central latory region. Promoter analysis further supported that the circadian pacemaker that integrates light-dark cycle input and expression of several of these genes was directly regulated by synchronizes the autonomous oscillators in peripheral tissues ROR훾. The observations further suggested that the decreased [170, 171, 196, 197]. The molecular clock machinery consists expression of these genes is at least in part responsible for the of several transcription/translation feedback loops in which reduced gluconeogenesis and lower glycogen accumulation the heterodimeric complex consisting of brain and muscle and consequently for the improved insulin sensitivity and ARNT-like (Bmal1) and circadian locomotor output cycles glucose tolerance observed in ROR훾 null mice. A role for kaput (Clock) or its paralog neuronal PAS domain protein 2 ROR훾 in the regulation of insulin resistance is supported (Npas2) form the positive regulatory loop of the oscillator, by studies showing that the level of ROR훾 expression whereas two cryptochrome (Cry) and three period proteins positively correlates with adiposity and insulin resistance in (Per) are part of the negative control mechanism. The nuclear human obese patients [190, 191]. These observations suggest receptors Rev-Erb훼 and 훽 (NR1D1/2) further regulate the that ROR훾 antagonists might be beneficial in controlling core loop by repressing the transcription of several clock glucose homeostasis and in the management of metabolic genes, including Bmal1, Clock and Npas2 (Figure 3). diseases. RORs are associated with the circadian clock at several In addition to gluconeogenesis, ROR훾 regulates hepatic different levels (Figure 3). First, RORs exhibit a rhythmic lipid metabolism. Loss of ROR훾 reduced the expression of pattern of expression in several tissues. In particular, ROR훾 a number of lipid metabolic genes, including the insulin- exhibits a robust oscillatory pattern of expression in liver, induced gene 2a (Insig2a), elongation of very long chain brown adipose tissue (BAT), pancreatic 훽 cells, kidney, fatty acids-like (Elovl3), Sult2a1, Cyp7b1, and Cyp8b1 [153, and small intestines (jejunum), with peak expression around 167, 168, 185]. ChIP and promoter analysis showed that Zeitgeber Time (ZT) 16-18, whereas ROR훼 exhibits no to several of these genes are directly regulated by ROR훾. moderate oscillation in the SCN and several other tissues The changes in the expression of these genes were asso- [94, 192, 202–205]. ROR훽2 displays a rhythmic expression ciated reduced levels of triglycerides, cholesterol, and bile pattern in mouse SCN, pineal gland and retina, with a max- acids in liver and blood in ROR훾-deficient mice fed a imum at ZT18 [122, 203, 206, 207]. Several studies showed

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Glucose and lipid Metabolic syndrome metabolic genes Insulin resistance Bmal1-Clock/Npas2 ROR Inflammatory genes Autoimmune disease Circadian clock gene Neuropsychiatric disorders (Cry, Npas2, G6pase, Gck, Insig2a, Elovl5, Il17, Il23R) Per1-3 Cry1/2 Rev-Erb

Figure 3: ROR훼 and ROR훾 function as intermediaries between the circadian clock and its regulation of glucose/lipid metabolic and inflammatory gene expression. RORs are linked to the circadian clock at different levels: a) ROR expression is regulated by the circadian clock machinery, including Bmal1, Clock, Rev-Erbs and Cry1; b) RORs are involved in the modulation of clock gene expression, including Npas2, Clock and Rev-Erb, and participate in the regulation of the rhythmic expression of glucose and lipid metabolic genes as well as inflammatory genes; c) Deficiency in ROR훼 or ROR훽 causes changes in the circadian behavior, which might be linked to neuropsychiatric disorders, while deficiency in ROR훾 leads to increased insulin sensitivity and glucose tolerance and a lower risk of developing diabetes. that RORa and RORc are regulated by Bmal1/Clock and Rev- are directly regulated by RORs. The transcriptional mediator, Erb. This is supported by data indicating that Bmal1, Clock RIP140, has been shown to be recruited by ROR훼 to the and Rev-Erb훼/훽 were recruited to the E-box and RORE, Bmal1 promoter, suggesting that it is involved in mediating respectively, in the proximal RORc promoter in mouse liver the transactivation of Bmal1 by ROR훼 [222]. [192, 208, 209]. Moreover, Bmal1 and Clock were able to One might predict that the rhythmic expression of RORs induce activation of the RORc promoter in reporter assays leads to a rhythmic expression of ROR target genes. Indeed, [192, 210]. The RORc gene contains two E-box binding sites several studies demonstrated that, in addition to clock genes, for Bmal/Clock [192, 204, 208, 211]. Mutation of either E1 ROR훾 also participates in the diurnal regulation of several or E2 significantly reduced the activation, while the double metabolic genes. Loss of ROR훾 significantly decreased mutation totally abolished this induction by Clock/Bmal1. peak expression of several glucose (e.g., G6pase, Pklr, The activation of the RORc promoter by Clock/Bmal1 was Glut2, PPAR훿) and lipid (e.g., Insig2a, Elovl3, Cyp8b1, repressed by Cry1 and correlated with changes in chromatin Sult1e1) metabolic genes [165, 167, 168, 192]. ChIP analysis accessibility at the RORc promoter. Rev-Erbs, rather than showed a ZT-dependent association of ROR훾 with ROREs Bmal1, regulate the rhythmic expression of RORc [210]. This in several of these genes. The transcriptional mediator, is supported by data showing that in Bmal1 KO mice, the Prospero-related homeobox 1 (Prox1), which functions as hepatic expression of ROR훾 is greatly enhanced particularly a co-repressor of RORs as well as several other nuclear at ZT4-8, thereby largely abolishing the robust rhythmic receptors, was shown to participate in the diurnal regulation expression pattern of ROR훾 [210]. The increase in ROR훾 of hepatic lipid/glucose metabolism by RORs [223, 224]. mRNA expression appeared largely due to the loss of Rev- ROR훾-deficient mice exhibited a significantly greater insulin Erb expression in Bmal1 KO liver, which subsequently sensitivity and glucose tolerance than WT mice particularly abolished the repression of RORc by Rev-Erb at ZT4-8. at ZT4-6. Moreover, the conversion of exogenously adminis- A second association between RORs and the circadian tered pyruvate to glucose was significantly lower in RORc−/− clock is their participation in the diurnal regulation of a mice particularly at ZT4-6. Together these findings suggested number of clock genes, including Bmal1, Npas2, Clock, that ROR훾 participates in the diurnal regulation of hepatic Rev-Erb훼, and Cry1 [1, 3, 24, 171, 192, 202, 211, 221]. lipid metabolism, gluconeogenesis and insulin sensitivity. Exogenous expression of ROR훾, as well as of ROR훼, These studies further suggest that ROR훾 functions as an in Hepa1-6 cells enhanced the endogenous expression of intermediary between the circadian clock machinery and its Cry1, Bmal1, E4bp4, Clock, Npas2, and Rev-Erb훼, whereas regulation of glucose and lipid metabolism. treatment with an ROR훾 antagonist inhibited their induction Recent observations uncovered a connection between [24, 192, 214]. ROREs have been identified in these clock RORs and the circadian control of immune functions. ROR훾t genes [24, 192, 211, 214, 218]. Reporter gene and mutation was found to play a role in the diurnal regulation of Th17 analysis indicated that RORs are involved in the transcrip- differentiation by the circadian clock [225]. In Th17 cells, tional regulation of these genes [24, 192, 214–217]. Rev- ROR훾t is expressed at significantly higher levels at daytime Erbs, which can compete with RORs for RORE binding, than at nighttime. This diurnal pattern of expression was inhibited this activation. ChIP-Seq and ChIP-QPCR analyses found to be related to an increase in the daytime expression further supported the association of RORs with these ROREs of Rev-Erb by Bmal1/Clock, which results in repression of in vivo, consistent with the conclusion that these clock genes NFIL3 transcription. Since NFIL3 functions as a repressor of

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ROR훾t transcription, its repression during daytime alleviates a series of related ROR agonists and inverse agonists, such its inhibition of ROR훾t transcription leading to enhanced as SR2211 and SR1001 (Figure 4), which do not bind ROR훾t expression. Another study demonstrated that in the LXR [25, 237, 243]. Some of these compounds interacted ileum, ROR훼 regulates the diurnal expression of several with both ROR훼 and ROR훾, while others were ROR훼- or genes associated with TLR signaling [94]. Analysis of ROR훾-selective [25, 234, 237]. Ursolic acid, a pentacyclic gene expression profiles of mucosal biopsies from healthy triterpene acid found in many plants, and several vitamin D individuals and patients with inflammatory bowel diseases metabolites, including 20-hydroxyvitamin D, were shown to (IBD) showed that the expression of several circadian genes, exhibit ROR훾 antagonist activity [35, 244, 245]. Evidence including ARNTL2. NPAS2, PER1, and RORA, was up- was provided suggesting that these vitamin D metabolites regulated in IBD patients, consistent with a role for these were able to bind the ROR훾 LBD. A high throughput screen proteins in this pathophysiology [226]. Together, these for ROR훾 ligands led to the identification of the cardiac studies indicate that ROR훼 and ROR훾 function as a link glycoside, digoxin (Figure 4), and several of its analogs as between the circadian clock and its regulation of various ROR훾 antagonists [246]. Subsequently, other investigators inflammatory pathways and provide a possible mechanism by set out to discover additional ROR훾 ligands [32]. This led which disruption of the circadian rhythm is associated with to the identification of several series of high affinity ROR훾 an increased risk of inflammatory diseases. inverse agonists, including various sulfonamides, such as Clinical studies have indicated an important association GSK3038548A and GNE-3500 (Figure 4)[43, 240–242, 247, between abnormalities in circadian rhythms and patients 248]. For a comprehensive review of small molecule ligands with mood and neuropsychiatric disorders. Alterations in that interact with and modulate ROR receptors, we refer to circadian behavior observed in mice deficient in either ROR훼 several recent reviews [2, 25, 32, 249]. or ROR훽 receptor [122, 124, 216] and associations between Recently, the connection between sterols and their mod- SNPs in RORA and RORB with an increased risk for sev- ulation of ROR activity was further strengthened by studies eral neuropsychiatric disorders, including autism spectrum showing a link between the cholesterol biosynthetic pathway (ASD) and bipolar disorder, schizophrenia, depression, and (Figure 5A) and the regulation of ROR훾 activity [34, 35]. posttraumatic stress syndrome [106–117, 129, 130], would These studies demonstrated that several intermediates of the be consistent with a link between disturbance in the circadian cholesterol biosynthetic pathway were able to function as rhythm and these pathologies. endogenous agonists of ROR훾t. Zymosterol and desmosterol were among the most effective sterols activating ROR훾, exhibiting EC50s of 0.11 and 0.08 휇M, respectively, while 8. ROR (ant)agonists cholesterol exhibited a much lower affinity for ROR훾. These sterols enhanced ROR훾 transcriptional activity as well as There has long been debate about whether RORs function the recruitment of coactivators. In addition, these sterols as constitutively active receptors or whether their activity were able to enhance Th17 differentiation and increase the is regulated by (endogenous) ligands that function as an expression of IL-17A [34, 35]. Characterization of lipid- agonist or active antagonist (referred to as inverse agonist) bound ROR훾 complexes immunoprecipitated from mam- or neutral antagonist [227]. Kallen, Stehlin-Gaon, and co- malian cells supported the concept that cholesterol biosyn- workers provided the first evidence for the hypothesis that thetic intermediates function as endogenous ROR훾 ligands RORs function as ligand-dependent transcription factors [33, [35]. The connection between ROR훾 and sterol metabolism 228, 229]. Crystal structure analysis revealed that cholesterol was further supported by studies showing that changes and cholesterol sulfate (Figure 4) bind the ligand-binding in the expression of enzymes involved in the cholesterol pocket of ROR훼 and act as ROR훼 agonists [33, 228]. Simi- biosynthetic pathway were able to modulate ROR훾 activity. larly, several retinoids were found to interact with the ligand- For example, ROR훾 transcriptional activity was lost in binding pocket of ROR훽 and to function as inverse agonists Fdft1-deficient cells lacking squalene synthase, an enzyme of ROR훽 as well as ROR훾 [229]. Subsequent studies iden- acting upstream in the cholesterol biosynthetic pathway [35]. tified a series of oxysterols as ligands for ROR훼 and ROR훾 Treatment with azole-type fungicides, such as ketoconazole [25, 32, 230–233]. For example, 7훼-hydroxycholesterol and and clotrimazole, which inhibit the sterol 14훼-demethylase 24(R)-hydroxycholesterol (Figure 4) were shown to function cytochrome P450, Cyp51a1, an enzyme upstream in the as inverse agonists, while 25-hydroxycholesterol, 20(훼)- cholesterol biosynthetic pathway (Figure 5A), caused a hydroxycholesterol, 22(R)-hydroxycholesterol, and 7훼 and dramatic reduction in zymosterol and desmosterol levels and 7훽 27-hydroxycholesterol act as agonists in mammalian cells. a decrease in ROR훾-mediated transactivation, Th17 differ- A search for additional ROR ligands led to the discovery entiation, and IL-17 expression [34, 250]. Moreover, ROR훾- of a number of other small molecule modulators of ROR훾 mediated transactivation is greatly diminished in mammalian [25, 32, 234–242]. The synthetic LXR agonist T0901317 cells made deficient in Cyp51a1 by shRNA knockdown was found to interact with both ROR훼 and ROR훾 and to act or germline deletion. Interestingly, several physiological as an inverse agonist [234]. Through chemical modification processes that were impaired in ROR훾-deficient mice were of T0901317, Burris and Griffin and coworkers identified also affected in Cyp51a1−/− mice [35]; branchial lymph

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Figure 4: Chemical structure of several ROR훼/훾 inverse agonists and (ant)agonists. T0901317, SR1001, and 7훼-hydroxycholesterol function as inverse agonists of both ROR훼 and ROR훾; cholesterol, cholesterol sulfate, and 25-hydroxycholesterol act as ROR훼 and/or ROR훾 agonists; all other compounds have been reported to function as an inverse agonist or antagonist of ROR훾. node anlagen were absent in 75% of Cyp51a1−/− mice and differentiation. For example, an increase in Th17 cells and the number of IL17RA+ and CD4+ Lti cells was reduced. IL-17A under hypercholesterolemic conditions might at least In a separate study, mice deficient in the mitochondrial in part be due to an increase in endogenous sterol levels and sterol 27-hydroxylase (Cyp27A1), a key enzyme in bile their subsequent activation of ROR훾t, while low cholesterol acid synthesis and the production of 27-hydroxy cholesterol, diet might do the inverse. This hypothesis is supported by exhibit a reduction in CD4+ and 훾훿+ T cells and a reduced a study reporting that patients with chronic hepatitis C, capacity for Th17 differentiation [233]. These similarities which is associated with increased levels of Th17 cells, when in phenotypic changes are consistent with a link between placed on a normocaloric, low cholesterol diet showed a the cholesterol biosynthetic pathway and ROR훾 activation. significant reduction in Th17 cells and IL-17 levels [252]. The role of cholesterol synthesis and ROR훾 activity in Th17 Furthermore, treatment with statins, inhibitors of cholesterol cells was further supported by observations showing that synthesis, lead to a reduction in Th17 differentiation and IL- Th17 differentiation is associated with increased cholesterol 17 production [253]. uptake and biosynthesis and an accumulation of desmosterol Several of the sulfated conjugates, such as desmosterol that subsequently enhances ROR훾t activation and Th17 sulfate, have also been shown to activate ROR훾 at levels two- differentiation. In addition, activation of the TCR pathway, fold higher than the unsulfated sterols. In this context, it is which results in activation of SREBP in favor of sterol- interesting to note that Th17 cell differentiation is accompa- sulfate and cholesterol synthesis, might synergize with ROR훾 nied with an increase in the expression of the sulfotransferase, in promoting Th17 differentiation and IL-17 synthesis [34, Sult2B1, and reduced expression of the sulfotransferase, STS 251]. Together, these studies suggest that changes in the [34]. This would be consistent with increased synthesis of cholesterol biosynthetic pathway and the level of cholesterol desmosterol sulfate and ROR훾 activation and stimulation of intermediates by diet or cholesterol-lowering drugs might Th17 differentiation. Oxysterols exhibit a much lower affinity control ROR훾 activation and as a consequence influence for ROR훾 and appear to play a lesser role in modulating physiological processes regulated by ROR훾, including Th17 ROR훾 activity in Th17 cells; however, this may depend on

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A.

B. Cholesterol biosynthesis e.g. desmosterol

Co-activator complex Basic Transcriptional CBP Machinery NCOA1 Peripheral ROR circadian clock Target Gene TATA Prox1 RORE TRANSCRIPTION

Antagonists e.g., e.g. T091317, Consensus RORE Cry1, Npas2, GSK3038548A, Gck, Insig2a, 20S(OH)D3 Elovl5, G6pc, Cyp8b1, Ppar

Figure 5: Metabolites of the cholesterol biosynthetic pathway function as endogenous ROR훾 agonists. A. Shown is a schematic view of the cholesterol synthetic pathway. Zymosterol and desmosterol are among the ROR훾 agonists with the highest affinity. Deficiency in Fdft1 or Cyp51A1, enzymes acting upstream in the cholesterol biosynthetic pathway, inhibit the synthesis of downstream ROR훾 agonists subsequently leading to reduced ROR훾t activation and Th17 differentiation. FDT1, Farnesyl-Diphosphate Farnesyltransferase 1; SQLE, Squalene Epoxidase; LSS, Lanosterol Synthase; TM7SF2, Transmembrane 7 Superfamily Member 2 (C-14 Sterol Reductase); FAXDC2/SC4MOL, Fatty Acid Hydroxylase Domain Containing 2/Methylsterol Monooxygenase 1; NSDHL, NAD(P) Dependent Steroid Dehydrogenase-Like; HSD17B7, Hydroxysteroid (17-Beta) Dehydrogenase 7; EBP, Emopamil Binding Protein (Sterol Isomerase); SC5D, Sterol-C5-Desaturase; DHCR7, 7-Dehydrocholesterol Reductase; DHCR24, 24-Dehydrocholesterol Reductase. B. Schematic view of ROR훾-mediated transcriptional activation of target genes by endogenous sterol agonists and its inhibition by antagonists. The circadian clock regulates ROR훾 expression and as a consequence the expression of ROR훾 target genes. Prox1 modulates ROR훾 transcriptional activity. The in vivo consensus RORE derived from ChIP-Seq analysis using liver tissue and an anti-ROR훾 antibody, is shown.

AgiAl Publishing House | http://www.agialpress.com/ 14 Nuclear Receptor Research the cell type and the type of oxysterol. Interestingly, Cyp7b1 affect this pathway, including cholesterol-rich or -low diets, and 3훽-hydroxysteroid dehydrogenases (3훽HSDs), which are environmental agents, such as the azole-type fungicides, and involved in the hydroxylation or dehydrogenation of sterols, drugs that control cholesterol levels, such as lovastatin. Most have been reported to be regulated by ROR훼 and ROR훾 importantly, by inhibiting ROR훾 transcriptional activity and [167, 185] and therefore might affect the formation of certain thereby reducing Th17 generation and IL-17A/F production, (oxy)sterols and as a consequence the activation of ROR훾 ROR훾 inverse agonists may provide a novel strategy in [233]. the treatment of various pathologies in which ROR훾 is Many of the ROR (ant)agonists have been shown to bind implicated, including inflammatory, metabolic, endocrine, the ligand-binding pocket within the LBD of RORs [2, 32, 33, and autoimmune diseases [1, 2, 13, 25, 257, 258]. Similarly, 228–232]. As has been reported for other nuclear receptors, ROR훼 antagonists might affect pathologies by inhibiting the agonist binding induces a conformational change in the ROR generation of ILC2 cells and other physiological functions LBD and realignment of helix 12 that allows release of and be useful in the management of inflammatory, metabolic, co-repressor complexes and promotes recruitment of co- and neuropsychiatric disorders [1, 2, 13, 25, 108, 113– activator complexes, which then mediate the transcriptional 117, 174]. This concept is supported by reports showing activation by RORs [1]. Particularly, the PLYKELF sequence that by inhibiting Th17 differentiation and IL-17 production, within the C-terminal activation function (AF) plays a critical ROR훾 inverse agonists suppress Th17 responses in mice role in ROR transactivation activity and mutations in or and ameliorate the development of experimental autoim- deletion of this motif result in a dominant-negative ROR mune encephalomyelitis and imiquimod-induced cutaneous [254, 255]. Conversely, binding of ROR antagonists and inflammation [43, 244, 246, 259]. The beneficial effects of inverse agonists, such as 25-hydroxycholesterol, inhibits ROR훾 antagonists may not only be mediated through the the interaction with co-activators and promote interac- inhibition of IL-17A and IL-17F synthesis in Th17 cells, tion with co-repressors (Figure 5B). A number of co- but also by repressing the synthesis of these and other + repressors and coactivators have been identified that mediate cytokines in ROR훾t innate lymphoid cells (ILC3), and + ROR-dependent transcriptional activation, including NCOR, ROR훾t 훾훿 T cells, which also play a critical role in several SRC1/2, and RIP140 [36, 222, 256]. autoimmune and inflammatory diseases [34, 77, 260, 261]. ROR inverse agonists can inhibit ROR-induced tran- Attenuating ROR훼/훾 activity by antagonist treatment might scriptional activation through different mechanisms. Certain also be beneficial for the management of metabolic dis- inverse agonists, such as TMP920, have been reported to eases, including metabolic syndrome and insulin resistance inhibit ROR훾 binding to ROREs, whereas the ability of [161, 165, 174, 178, 190, 191]. Recently, the ROR훼/훾 ROR훾 to bind DNA target sites was mostly preserved with inverse agonist SR1001 was shown to suppress insulitis other inverse agonists, such as TMP778 and GSK805 [43]. and prevent hyperglycemia in a mouse model of type 1 Interestingly, ROR훾 cistrome analysis revealed that the latter diabetes [262]. Together, these studies reinforce the potential compounds stabilized ROR훾t binding to a number of new of ROR antagonists in the management of autoimmune genomic sites [43]. The distinct effects by various ligands disease, neuropsychiatric and metabolic disorders, and other are likely related to the induction of different conformational pathologies. changes in ROR훾 that influence its affinity for different ROREs as well as its interaction with other transcriptional Acknowledgements mediators. In addition to Th17 related genes, such as Il17 [35, 43, 244, 246], ROR훾 inverse agonists have been reported This research was supported by the Intramural Research to inhibit the expression of a number of ROR훾 target genes, Program of the NIEHS, NIH (Z01-ES-100486). including the clock genes, Bmal1, Cry1, and Npas2, and several glucose and lipid metabolic genes, such as G6pase, Insig2a, Elovl3, Gck, and PPAR훿 [24, 165, 168, 192]. ROR훼 References inverse agonists and agonists were shown to, respectively, suppress or induce the expression of the ROR훼 target genes, [1] A. M. Jetten, Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular G6pase, Fgf21, CS, and Npas2 [166, 187, 231]. metabolism, Nuclear Receptor Signaling, 7, Article ID e003, (2009). [2] L. A. Solt and T. P. Burris, Action of RORs and their ligands 9. 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