Identification of Oxysterol 7 -Hydroxylase (Cyp7b1) as a Novel Retinoid-Related Orphan Receptor (ROR ) (NR1F1) Target Gene and a Functional Cross-Talk between ROR and Liver X Receptor (NR1H3)

The retinoid-related orphan receptors (RORs) and liver X receptors (LXRs) were postulated to have distinct functions. RORs play a role in tissue development and circadian rhythm, whereas LXRs are sterol sensors that affect lipid homeostasis. In this study, we revealed a novel function of ROR (NR1F1) in regulating the oxysterol 7 -hydroxylase (Cyp7b1), an enzyme critical for the homeostasis of cholesterol, bile acids, and oxysterols. The expression of Cyp7b1 gene was suppressed in the ROR null (ROR ) mice, suggesting ROR as a positive regulator of Cyp7b1. Promoter analysis established Cyp7b1 as a transcriptional target of ROR , and transfection of ROR induced the expression of endogenous Cyp7b1 in the liver. Interestingly, Cyp7b1 regulation seemed to be ROR -specific, because ROR had little effect. Reporter gene analysis showed that the activation of Cyp7b1 gene promoter by ROR was suppressed by LXR (NR1H3), whereas ROR inhibited both the constitutive and ligand-dependent activities of LXR . The mutual suppression between ROR and LXR was supported by the in vivo observation that loss of ROR increased the expression of selected LXR target genes, leading to hepatic triglyceride accumulation. Likewise, mice deficient of LXR and isoforms showed activation of selected ROR target genes. Our results have revealed a novel role for ROR and a functional interplay between ROR and LXR in regulating endoand xenobiotic genes, which may have broad implications in metabolic homeostasis. Retinoid-related orphan receptors (RORs, or NR1F1–3), including the , , and isoforms, were isolated based on their homology to the retinoid receptors (Jetten et al., 2001; Jetten and Joo, 2006). Each of the ROR isoforms has distinct tissue distribution patterns (Carlberg et al., 1994). ROR is widely distributed, with its expression detectable in the cerebellar Purkinje cells, liver, thymus, skeletal muscle, skin, lung, and kidney (Hamilton et al., 1996; Steinmayr et al., 1998). In contrast, ROR has a more tissue-specific distribution, expressing in the brain, retina and pineal gland (André et al., 1998a; Jetten et al., 2001). ROR is highly enriched in the thymus, but its expression is also detectable in the kidney, liver, and muscle (Medvedev et al., 1996; Jetten et al., 2001). The functional ligands of RORs remain elusive. It has been suggested that cholesterol and its sulfonated derivatives might function as ROR ligands (Kallen et al., 2002). However, to our knowledge, none of those have been convincThis work was supported in part by National Institutes of Health grants CA107011 and ES014626 (to W.X.) and by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health (to A.M.J.). H.G. is supported by postdoctoral fellowship PDF0503458 from the Susan G. Komen Breast Cancer Foundation. Normal human hepatocytes were obtained through the Liver Tissue Procurement and Distribution System, which was funded by National Institutes of Health grant N01-DK70004/HHSN267200700004C. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.040741. ABBREVIATIONS: ROR, retinoid-related orphan receptor; RORE, retinoid-related orphan receptor response element; LXR, liver X receptor; kb, kilobase(s); tk, thymidine kinase; Pcp2, Purkinje cell protein 2; Luc, luciferase; WT, wild type; bp, base pair(s); PCR, polymerase chain reaction; PEI, polyethylenimine; MEM, minimal essential medium; -Gal, -galactosidase; DMSO, dimethyl sulfoxide; RT, reverse transcription; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; LXRE, liver X receptor response element; SCR1, steroid receptor coactivator 1; VP, viral protein 16; DKO, double knockout; UAS, upstream activation sequence. 0026-895X/08/7303-891–899 MOLECULAR PHARMACOLOGY Vol. 73, No. 3 U.S. Government work not protected by U.S. copyright 40741/3305119 Mol Pharmacol 73:891–899, 2008 Printed in U.S.A. 891 at A PE T Jornals on A ril 2, 2017 m oharm .aspeurnals.org D ow nladed from ingly demonstrated to be physiological ROR agonists. RORs regulate gene expression by binding as monomers to the ROR response elements (ROREs) found in target gene promoters. A typical RORE is composed of a consensus AGGTCA halfsite preceded by an A/T-rich region (Giguère et al., 1994). ROR has also been shown to bind DNA as homodimers (Harding et al., 1997). Subsequent functional analyses, mainly through the creation and characterization of ROR-deficient mice, have revealed diverse physiological function of RORs. ROR / mice had cerebellar ataxia, a behavioral phenotype also observed in the Staggerer (sg/sg) mutant mice, which contained a natural deletion in the ligand binding domain of the ROR gene as a result of a frame shift (Hamilton et al., 1996; Steinmayr et al., 1998). The sg/sg mice exhibited vascular dysfunction, muscular irregularities, osteoporosis, and immuno abnormalities (Jarvis et al., 2002). The sg/sg mice developed severe atherosclerosis and hypolipoproteinemia when maintained on an atherogenic diet (Mamontova et al., 1998). ROR is thought to be involved in the processing of sensory information, because ROR / mice showed significant phenotypes in circadian behaviors and retinal degeneration (André et al., 1998b). ROR / mice lacked all lymph nodes and Peyer’s patches, and they had reduced numbers of thymocytes (Kurebayashi et al., 2000), suggesting that ROR plays an essential role in lymphoid organogenesis and thymopoiesis. Although both ROR and are expressed in the liver, their hepatic function is largely unknown. Both liver X receptor (LXR) and are nuclear receptors that can be activated by the endogenous oxysterols, such as 22(R)-hydroxycholesterol; and by synthetic agonists, such as T0901317 (TO1317) (Schultz et al., 2000) and GW3965 (Collins et al., 2002). LXRs exhibit diverse functions, ranging from cholesterol efflux to lipogenesis and anti-inflammation (Repa and Mangelsdorf al., 2002; Zelcer and Tontonoz, 2006). LXRs have also been explored as therapeutic targets for atherosclerosis (Tontonoz and Mangelsdorf, 2003), diabetes, and Alzheimer’s disease (Zelcer et al., 2007) in animal models. We have recently identified several novel LXR target genes. These include the bile acid-detoxifying sulfotransferase Sult2a9/2a1 (Uppal et al., 2007), estrogen sulfotransferase (Est/Sult1e1) (Gong et al., 2007), and fatty acid transporter Cd36 (J. Zhou and W. Xie, unpublished data). We showed that activation of Sult2a9/2a1 by LXR was associated with increased bile acid detoxification and alleviation of cholestasis (Uppal et al., 2007). In the same study, the expression of Cyp7b1 was found to be suppressed in LXR-activated mice, but the mechanism for this suppression is unknown (Uppal et al., 2007). Activation of Est/ Sult1e1 by LXR led to functional estrogen deprivation and inhibition of estrogen-dependent breast cancer growth (Gong et al., 2007). More recently, we showed that Cd36 is a LXR target gene and an intact expression of Cd36 plays an important role in the steatotic effect of LXR agonists (J. Zhou and W. Xie,