Retinoic acid-related orphan receptor γ directly regulates neuronal PAS domain protein 2 transcription in vivo.

Retinoic acid-related orphan receptors (RORs) and the basic helix-loop-helix-PAS transcription factor Npas2 have been implicated in the control of circadian rhythm. In this study, we demonstrate that RORγ directly regulates Npas2 expression in vivo. Although the rhythmicity of Npas2 mRNA expression was maintained in RORγ(-/-) mice, the peak level of expression was significantly reduced in several tissues, while loss of RORα had little effect. Inversely, overexpression of RORγ in hepatoma Hepa1-6 cells greatly induced the expression of Npas2. RORγ-activated Npas2 transcription directly by binding two ROREs in its proximal promoter. ChIP analysis demonstrated that RORγ was recruited to this promoter in the liver of wild-type mice, but not RORγ-deficient mice. Activation of Npas2 correlated positively with chromatin accessibility and level of H3K9 acetylation. The activation of Npas2 by RORγ was repressed by co-expression with Rev-Erbα or addition of the ROR inverse agonist T0901317. Npas2 expression was also significantly enhanced during brown adipose differentiation and that this induction was greatly suppressed in adipose cells lacking RORγ. Our results indicate that RORγ and Rev-Erbα are part of a feed-back loop that regulates the circadian expression of Npas2 suggesting a regulatory role for these receptors in Npas2-dependent physiological processes.

Mfsd2a encodes a novel major facilitator superfamily domain-containing protein highly induced in brown adipose tissue during fasting and adaptive thermogenesis.

This study describes the identification of Mfsd2a (major facilitator superfamily domain-containing protein 2a), a novel mammalian major facilitator superfamily domain-containing protein, and an additional closely related protein, Mfsd2b. Most intron/exon junctions are conserved between the two genes, suggesting that they are derived from a common ancestor. Mfsd2a and Mfsd2b share a 12 transmembrane alpha-helical domain structure that bears greatest similarity to that of the bacterial Na(+)/melibiose symporters. Confocal microscopy demonstrated that Mfsd2a localizes to the endoplasmic reticulum. Mfsd2a is expressed in many tissues and is highly induced in liver and BAT (brown adipose tissue) during fasting. Mfsd2a displays an oscillatory expression profile in BAT and liver, consistent with a circadian rhythm. Although the basal level of Mfsd2a expression is relatively low in mouse BAT, it is greatly induced during cold-induced thermogenesis and after treatment with betaAR (beta-adrenergic receptor) agonists. This induction is totally abolished in beta-less (betaAR-deficient) mice. These findings indicate that Mfsd2a is greatly up-regulated in BAT during thermogenesis and that its induction is controlled by the betaAR signalling pathway. The observed induction of Mfsd2a expression in cultured BAT cells by dibutyryl-cAMP is in agreement with this conclusion. The present study suggests that Mfsd2a plays a role in adaptive thermogenesis.

Identification of oxysterol 7alpha-hydroxylase (Cyp7b1) as a novel retinoid-related orphan receptor alpha (RORalpha) (NR1F1) target gene and a functional cross-talk between RORalpha 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 RORalpha (NR1F1) in regulating the oxysterol 7alpha-hydroxylase (Cyp7b1), an enzyme critical for the homeostasis of cholesterol, bile acids, and oxysterols. The expression of Cyp7b1 gene was suppressed in the RORalpha null (RORalpha(sg/sg)) mice, suggesting RORalpha as a positive regulator of Cyp7b1. Promoter analysis established Cyp7b1 as a transcriptional target of RORalpha, and transfection of RORalpha induced the expression of endogenous Cyp7b1 in the liver. Interestingly, Cyp7b1 regulation seemed to be RORalpha-specific, because RORgamma had little effect. Reporter gene analysis showed that the activation of Cyp7b1 gene promoter by RORalpha was suppressed by LXRalpha (NR1H3), whereas RORalpha inhibited both the constitutive and ligand-dependent activities of LXRalpha. The mutual suppression between RORalpha and LXR was supported by the in vivo observation that loss of RORalpha increased the expression of selected LXR target genes, leading to hepatic triglyceride accumulation. Likewise, mice deficient of LXR alpha and beta isoforms showed activation of selected RORalpha target genes. Our results have revealed a novel role for RORalpha and a functional interplay between RORalpha and LXR in regulating endo- and xenobiotic genes, which may have broad implications in metabolic homeostasis.

Gene expression profiling reveals a regulatory role for ROR alpha and ROR gamma in phase I and phase II metabolism.

Retinoid-related orphan receptors alpha (ROR alpha) and gamma (ROR gamma) are both expressed in liver; however, their physiological functions in this tissue have not yet been clearly defined. The ROR alpha1 and ROR gamma 1 isoforms, but not ROR alpha 4, show an oscillatory pattern of expression during circadian rhythm. To obtain insight into the physiological functions of ROR receptors in liver, we analyzed the gene expression profiles of livers from WT, ROR alpha-deficient staggerer (sg) mice (ROR alpha(sg/sg)), ROR gamma(-/-), and ROR alpha(sg/sg)ROR gamma(-/-) double knockout (DKO) mice by microarray analysis. DKO mice were generated to study functional redundancy between ROR alpha and ROR gamma. These analyses demonstrated that ROR alpha and ROR gamma affect the expression of a number of genes. ROR alpha and ROR gamma are particularly important in the regulation of genes encoding several phase I and phase II metabolic enzymes, including several 3beta-hydroxysteroid dehydrogenases, cytochrome P450 enzymes, and sulfotransferases. In addition, our results indicate that ROR alpha and ROR gamma each affect the expression of a specific set of genes but also exhibit functional redundancy. Our study shows that ROR alpha and ROR gamma receptors influence the regulation of several metabolic pathways, including those involved in the metabolism of steroids, bile acids, and xenobiotics, suggesting that RORs are important in the control of metabolic homeostasis.

Modulatory role for retinoid-related orphan receptor alpha in allergen-induced lung inflammation.

RATIONALE: Nuclear receptors play a critical role in the regulation of inflammation, thus representing attractive targets for the treatment of asthma. OBJECTIVE: In this study, we assess the potential regulatory function of retinoid-related orphan receptor alpha (RORalpha) in the adaptive immune response using ovalbumin (OVA)-induced airway inflammation as a model. METHODS: Allergen-induced inflammation was compared between wild-type (WT) and staggerer (RORalpha(sg/sg)) mice, a natural mutant strain that is deficient in RORalpha expression. MEASUREMENTS AND MAIN RESULTS: Despite robust increases in OVA-specific IgE, RORalpha(sg/sg) mice developed significantly less pulmonary inflammation, mucous cell hyperplasia, and eosinophilia compared with similarly treated WT animals. Induction of Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, was also significantly less in RORalpha(sg/sg) mice. Microarray analysis using lung RNA showed increased expression of many genes, previously implicated in inflammation, in OVA-treated WT mice. These include mucin Muc5b, the chloride channel calcium-activated 3 (Clca3), macrophage inflammatory protein (MIP) 1alpha and 1beta, eotaxin-2, serum amyloid A3 (Saa3), and insulin-like growth factor 1 (Igf1). These genes were induced to a greater extent in OVA-treated WT mice relative to RORalpha(sg/sg) mice. CONCLUSIONS: Our study demonstrates that mice deficient in RORalpha exhibit an attenuated allergic inflammatory response, indicating that RORalpha plays a critical role in the development of Th2-driven allergic lung inflammation in mice, and suggests that this nuclear receptor should be further evaluated as a potential asthma target.

In vivo DNase I-mediated footprinting analysis along the human bradykinin B1 receptor (BDKRB1) gene promoter: evidence for cell-specific regulation.

By applying in vivo dimethyl sulphate and UV light type C-footprinting analysis, we previously showed that specific DNA sequences in the -1349/+42 core promoter region of the inducible human BDKRB1 (bradykinin B1 receptor) gene correlated with its transcriptional activity. In the present study we used the highly sensitive DNase I in vivo footprinting approach to delineate more precisely the functional domains of the BDKRB1 gene promoter in human SMCs (smooth muscle cells). Human lymphocytes that do not express a functional BDKRB1 were also studied as a reference using dimethyl sulphate, UV light type C and DNase I treatments. An obvious difference was found in the DNase I-footprinting patterns between cellular systems that express a functional BDKRB1 (SMCs) in comparison with human lymphocytes, where randomly distributed nucleosome-like footprinting patterns were found in the bulk of the core promoter region studied. Gel-shift assays and expression studies pointed to the implication of the YY1 and a TBP/TFIIB (TATA-box-binding protein/transcription factor IIB) transcription factor in the regulation of BDKRB1 gene expression in SMCs and possible YY1 involvement in the mechanisms of nuclear factor kappaB-mediated regulation of the receptor expression. No significant changes in the promoter foot-printing pattern were found after treatment with interleukin-1beta or serum (known BDKRB1 gene inducers), indicating that definite regulatory motifs could exist outside the BDKRB1 gene core promoter region studied.