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The retinoic acid receptor-related orphan receptors ␣ and ␥ (ROR␣ (NR1F1) and ROR␥ (NR1F3)) are orphan nuclear receptors and perform critical roles in regulation of development, metabolism, and immune function. Cholesterol and cholesterol sulfate have been suggested to be ROR␣ ligands, but the physiological significance is unclear. To date, no endogenous ROR␥ ligands have been described. Here, we demonstrate that 7-oxygenated sterols function as high affinity ligands for both ROR␣ and ROR␥ by directly binding to their ligand-binding domains In the late 1980s, as the canonical domain structure and conserved sequence of members of the nuclear hormone receptor (NHR) 3 superfamily became apparent, several laboratories began to isolate additional members of this superfamily that had no identified ligands. Many of these so-called orphan receptors still have no identified ligands. The first member of the ROR subfamily of receptors (ROR␣) was identified in the early 1990s based on sequence similarities to the retinoic acid receptor and the retinoid X receptor, hence the name "retinoic acid receptor-related orphan receptor" (1, 2). The highly similar receptors, ROR␤ and ROR␥, were identified soon after (3, 4).

The nuclear hormone receptor, REV-ERB, plays an essential role in adipogenesis. Rev-erbalpha expression is induced in 3T3-L1 cells during adipogenesis, and overexpression of this receptor leads to expression of adipogenic genes. We recently demonstrated that the porphyrin heme functions as a ligand for REV-ERB, and binding of heme is required for the receptor's activity. We therefore hypothesized that REV-ERB ligands may play a role in regulation of adipogenesis. We detected an increase intracellular heme levels during 3T3-L1 adipogenesis that correlated with induction of aminolevulinic acid synthase 1 (Alas1) expression, the rate-limiting enzyme in heme biosynthesis. If the increase in Alas1 expression was blocked, adipogenesis was severely attenuated, indicating that induction of expression of Alas1 and the increase in heme synthesis is critical for differentiation. Inhibition of heme synthesis during adipogenesis leads to decreased recruitment of nuclear receptor corepressor to the promoter of a REV-ERB target gene, suggesting alteration of REV-ERB activity. Treatment of 3T3-L1 cells with a synthetic REV-ERB ligand, SR6452, resulted in induction of adipocyte differentiation to a similar extent as treatment with the peroxisomal proliferator-activated receptor-gamma agonist, rosiglitazone. Combination of SR6452 and rosiglitazone had an additive effect on stimulation of adipocyte differentiation. These results suggest that heme, functioning as a REV-ERB ligand, is an important signaling molecule for induction of adipogenesis. Moreover, synthetic small molecule ligands for REV-ERB are effective modulators of adipogenesis and may be useful for treatment of metabolic diseases.

The retinoic acid receptor-related orphan receptor α and γ (RORα [NR1F1] and RORγ [NR1F3]) are members of the nuclear hormone receptor superfamily. These 2 receptors regulate many physiological processes including development, metabolism and immunity. We recently found that certain oxysterols, namely the 7-substituted oxysterols, bound to the ligand binding domains (LBDs) of RORα and RORγ with high affinity, altered the LBD conformation and reduced coactivator binding resulting in suppression of the constitutive transcriptional activity of these two receptors. Here, we show that another oxysterol, 24S-hydroxycholesterol (24S-OHC), is also a high affinity ligand for RORα and RORγ (K i ∼ 25 nM). 24S-OHC is also known as cerebrosterol due to its high level in the brain where it plays an essential role as an intermediate in cholesterol elimination from the CNS. 24S-OHC functions as a RORα/γ inverse agonist suppressing the constitutive transcriptional activity of these receptors in cotransfection assays. Additionally, 24S-OHC suppressed the expression of several RORα target genes including BMAL1 and REV-ERBα in a RORdependent manner. We also demonstrate that 24S-OHC decreases the ability of RORα to recruit the coactivator SRC-2 when bound to the BMAL1 promoter. We also noted that 24(S), 25-epoxycholesterol selectively suppressed the activity of RORγ. These data indicate that RORα and RORγ may serve as sensors of oxsterols. Thus, RORα and RORγ display an overlapping ligand preference with another class of oxysterol nuclear receptors, the liver X receptors (LXRα [NR1H3] and LXRβ [NR1H2]).

The mammalian clock is regulated at the cellular level by a transcriptional/translational feedback loop. BMAL1/CLOCK (or NPAS2) heterodimers activate the expression of the PERIOD (PER) and CRYPTOCHROME (CRY) genes acting as transcription factors directed to the PER and CRY promoters via E-box elements. PER and CRY proteins form heterodimers and suppress the activity of the BMAL1/CLOCK (or NPAS2) completing the feedback loop. The circadian expression of BMAL1 is influenced by retinoic acid receptor-related orphan receptor ␣ (ROR␣) and REV-ERB␣, two nuclear receptors that target a ROR-response element in the promoter of the BMAL1 gene. Given that BMAL1 functions as an obligate heterodimer with either CLOCK or NPAS2, it is unclear how the expression of the partner is coordinated with BMAL1 expression. Here, we demonstrate that NPAS2 is also a ROR␣ and REV-ERB␣ target gene. Using a ChIP/microarray screen, we identified both ROR␣ and REV-ERB␣ occupancy of the NPAS2 promoter. We identified two functional ROREs within the NPAS2 promoter and also demonstrate that both ROR␣ and REV-ERB␣ regulate the expression of NPAS2 mRNA. These data suggest a mechanism by which ROR␣ and REV-ERB␣ coordinately regulate the expression of the positive arm of the circadian rhythm feedback loop.Circadian rhythms are the natural ϳ24-h cycles that are conserved across a wide variety of organisms, including Arabidopsis, Drosophila, and mammals. In mammals, these rhythms are entrained by light signals and are controlled by the "master clock" in the suprachiasmatic nucleus (SCN) 2 in the brain. In the periphery, semi-autonomous clocks can be entrained to signals from the SCN and signals from other cues, including nutrient status (1). The mammalian circadian clock is controlled by a transcriptional/translational feedback loop involving several key proteins. The expression of these key proteins includes brain and muscle ARNT (aryl hydrocarbon receptor nuclear translocator)-like 1 (BMAL1), circadian locomotor output kaput (CLOCK), PERIOD (PER), and CRYPTOCROME (CRY). BMAL1 and CLOCK are members of the basic helix-loop-helix-PAS family of transcription factors and form a heterodimer that targets E-box DNA elements in the promoters of the PER and CRY genes and activates their transcription. The CRY and PER proteins heterodimerize, and as the levels of this protein complex accumulate it effectively inhibits the activity of BMAL1/CLOCK limiting the expression of the PER and CRY genes. This feedback loop results in the oscillations in expression of BMAL1/CLOCK and CRY/PER that follow a circadian pattern (2, 3).Two classes of nuclear receptors play a critical role in modulation of the mammalian circadian clock. The retinoic acid receptor-related orphan receptors (RORs) and REV-ERBs modulate expression of the BMAL1 gene through two conserved ROR-response elements (ROREs) located within the BMAL1 promoter. Expression of BMAL1 is modulated by competition between RORs and REV-ERBs for binding the BMAL1 promoter and either activation (ROR) or repression (RE...

Circadian rhythms are regulated at the cellular level by transcriptional feedback loops leading to oscillations in expression of key proteins including CLOCK, BMAL1, PERIOD (PER), and CRYPTOCHROME (CRY). The CLOCK and BMAL1 proteins are members of the bHLH class of transcription factors and form a heterodimer that regulates the expression of the PER and CRY genes. The nuclear receptor REV-ERBα plays a key role in regulation of oscillations in BMAL1 expression by directly binding to the BMAL1 promoter and suppressing its expression at certain times of day when REV-ERBα expression levels are elevated. We recently demonstrated that REV-ERBα also regulates the expression of NPAS2, a heterodimer partner of BMAL1. Here, we show that REV-ERBα also regulates the expression another heterodimer partner of BMAL1, CLOCK. We identified a REV-ERBα binding site within the 1st intron of the CLOCK gene using a chromatin immunoprecipitation – microarray screen. Suppression of REV-ERBα expression resulted in elevated CLOCK mRNA expression consistent with REV-ERBα's role as a transcriptional repressor. A REV-ERB response element (RevRE) was identified within this region of the CLOCK gene and was conserved between humans and mice. Additionally, the CLOCK RevRE conferred REV-ERB responsiveness to a heterologous reporter gene. Our data suggests that REV-ERBα plays a dual role in regulation of the activity of the BMAL1/CLOCK heterodimer by regulation of expression of both the BMAL1 and CLOCK genes.

The four R-spondins (RSPO1-4) and their three related receptors LGR4, 5 and 6 (LGR4-6) have emerged as a major ligand-receptor system with critical roles in development and stem cell survival through modulation of Wnt signaling. Recurrent, gain-of-expression gene fusions of RSPO2 (to EIF3E) and RSPO3 (to PTPRK) occur in a subset of human colorectal cancer. However, the exact roles and mechanisms of the RSPO-LGR system in oncogenesis remain largely unknown. We found that RSPO3 is aberrantly expressed at high levels in approximately half of the Keap1-mutated lung adenocarcinomas. This high RSPO3 expression is driven by a combination of demethylation of its own promoter region and deficiency in Keap1 instead of gene fusion as in colon cancer. Patients with RSPO3-high tumors (~9%, 36/412) displayed much poorer survival than the rest of the cohorts (median survival of 28 vs. 163 months, logrank test p < 0.0001). Knockdown of RSPO3, LGR4, or their signaling mediator IQGAP1 in lung cancer cell lines with Keap1 deficiency and high RSPO3-LGR4 expression led to reduction in cell proliferation and migration in vitro, and knockdown of LGR4 or IQGAP1 resulted in decrease in tumor growth and metastasis in vivo. These findings suggest that aberrant RSPO3-LGR4 signaling potentially acts as a driving mechanism in the aggressiveness of Keap1-deficient lung adenocarcinomas.

The retinoic acid receptor-related orphan receptor α (RORα) is a member of the nuclear receptor superfamily of transcription factors that plays an important role in regulation of the circadian rhythm and metabolism. Mice lacking a functional RORα display a range of metabolic abnormalities including decreased serum cholesterol and plasma triglycerides. Citrate synthase (CS) is a key enzyme of the citric acid cycle that provides energy for cellular function. Additionally, CS plays a critical role in providing citrate derived acetyl-CoA for lipogenesis and cholesterologenesis. Here, we identified a functional RORα response element (RORE) in the promoter of the CS gene. ChIP analysis demonstrates RORα occupancy of the CS promoter and a putative RORE binds to RORα effectively in an electrophoretic mobility shift assay and confers RORα responsiveness to a reporter gene in a cotransfection assay. We also observed a decrease in CS gene expression and CS enzymatic activity in the staggerer mouse, which has a mutation of in the Rora gene resulting in nonfunctional RORα protein. Furthermore, we found that SR1001 a RORα inverse agonist eliminated the circadian pattern of expression of CS mRNA in mice. These data suggest that CS is a direct RORα target gene and one mechanism by which RORα regulates lipid metabolism is via regulation of CS expression.