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RORα switches transcriptional mode of ERRγ that results in transcriptional repression of CYP2E1 under ethanol-exposure

Increased cytochrome P450 2E1 (CYP2E1) expression is the main cause of oxidative stress, which exacerbates alcoholic liver diseases (ALDs). Estrogen-related receptor gamma (ERRγ) induces CYP2E1 expression and contributes to enhancing alcohol-induced liver injury. Retinoic acid-related orphan receptor alpha (RORα) has antioxidative functions; however, potential cross-talk between ERRγ and RORα in the regulation of CYP2E1 has not been studied. We report that RORα suppressed ERRγ-mediated CYP2E1 expression. A physical interaction of RORα with ERRγ at the ERRγ−response element in the CYP2E1 promoter was critical in this suppression. At this site, coregulator recruitment of ERRγ was switched from coactivator p300 to the nuclear receptor corepressor 1 in the presence of RORα. Cross-talk between ERRγ and RORα was demonstrated in vivo, in that administration of JC1–40, a RORα activator, significantly decreased both CYP2E1 expression and the signs of liver injury in ethanol-fed mice, and this was accompanied by coregulator switching. Thus, this non-classical RORα pathway switched the transcriptional mode of ERRγ, leading to repression of alcohol-induced CYP2E1 expression, and this finding may provide a new therapeutic strategy against ALDs.


INTRODUCTION
Alcoholic liver disease (ALD) is the common liver disease caused by alcohol abuse, which increases global burden of liver disease-related mortality (1). Chronic alcohol abuse triggers liver injury, which manifests as a broad spectrum of hepatic disorders including steatosis, alcoholic steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma (2). Alcohol is converted to acetaldehyde by two oxidative pathways, namely, alcohol dehydrogenase and the microsomal ethanol oxidizing system (3,4). With chronic alcohol consumption, cytochrome P450 2E1 (CYP2E1) is a key microsomal enzyme that induces the generation of reactive oxygen species (ROS) which exacerbate alcohol-induced oxidative stress (4). Feeding a liquid diet containing ethanol to CYP2E1-expressing transgenic mice or to mice infused with an adenovirus encoding CYP2E1 produced higher serum transaminase levels and damaged histological features, further suggesting that CYP2E1 plays a critical role in ALDs (5,6). Therefore, understanding the molecular mechanism of gene expression and modulation of the enzymatic activity of CYP2E1 could provide a potential therapeutic strategy for controlling alcoholic liver injury.
Retinoic acid-related orphan receptor alpha (ROR␣; NR1F1) regulates diverse target genes associated with energy homeostasis including the regulation of diverse lipid and cholesterol metabolic pathways (7). Cholesterol derivatives, including cholesterol sulfate (CS), act as endogenous ligands that fit in the ligand-binding pocket of ROR␣, while synthetic ROR␣ agonists such as SR1078 and JC1-40 accomplish the cellular functions of ROR␣ (8)(9)(10). Coregulators of transcription factors such as p300 and nuclear receptor corepressor (NCoR) interact with and regulate the transcriptional activity of ROR␣ positively or negatively (11,12). Previously, we reported that ROR␣ and its ligands protect against the progression of non-alcoholic steatohepatitis (NASH) probably through activating AMP-activated protein kinase (AMPK) and by repressing liver X receptor alpha (LXR␣)-mediated lipogenesis (8). Further, ROR␣ reduced the formation of ROS and 4-hydroxynonenal (4-HNE), a lipid peroxidation marker, in the hepatocytes and Kupffer cells of mice with NASH induced by a methioninecholine deficient diet (13). Although these studies suggested a potential protective role of ROR␣ in ALDs, its function in the regulation of CYP2E1 has not been studied.
Chronic alcohol abuse increases endocannabinoid levels and results in alcohol-induced hepatic lipid accumulation and oxidative stress via cannabinoid 1 (CB1) receptor activation (14,15). Recently, CYP2E1 was identified as the target of estrogen-related receptor gamma (ERR␥ ; NR3B3) that was induced by CB1 receptor signaling under exposure to alcohol (16). ERR␥ belongs to the NR3B subfamily, which is known to play a role in regulating bioenergetics pathways such as hepatic gluconeogenesis and iron metabolism (17,18). ERR␥ recognizes the DNA sequence, 5 -TNAAGGTCA-3 , termed the ERR␥ -response element (ERRE) as monomer, homodimer or heterodimer (19). Unlike other classic ligand-dependent nuclear receptors, ERR␥ is active constitutively without ligands, and the transcriptional status of ERR␥ is mainly regulated by docking of coregulator such as CREB-binding protein/p300, peroxisome proliferator-activated receptor ␥ coactivator-1␣, and nuclear receptor-interacting protein 140 (20)(21)(22). Downstream target genes of ERR␥ are mainly involved in the metabolism of glucose, lipids and iron, including those encoding phosphoenolpyruvate carboxykinase, glucose-6phosphatase (G6Pase), lipin-1 and hepcidin (17,18,23). Recently, a specific inverse agonist of ERR␥ , GSK5182, was demonstrated to ameliorate chronic alcohol-induced liver injury through inhibition of CYP2E1-mediated ROS production in mice, indicating a new function of ERR␥ in the control of alcohol-mediated oxidative stress in the liver (16).
Here, we report that ROR␣ suppressed the CB1 receptorand the ERR␥ -mediated induction of CYP2E1 expression at the transcriptional level. Interestingly, ROR␣ interacted directly with ERR␥ to induce the differential recruitment of coregulators at the ERRE site in the upstream promoter of CYP2E1. This process switched the transcriptional mode of ERR␥ from an activator to a repressor of CYP2E1 transcription. Finally, administration of the thiourea derivate JC1-40, an activator of ROR␣, dramatically protected mice from alcohol-induced oxidative stress and liver injury. Together these results suggest that ROR␣ and its ligands might provide a potential therapeutic strategy against ALDs.

Cell culture and reagents
Hepatocytes were isolated from 8-9 weeks old, male C57BL6/N mouse (Charles River Laboratories, Wilmington, MA) by perfusion of liver using collagenase type IV (Sigma-Aldrich, St Louis, MO), as described previously (13). Hepatocytes were cultured in M199 Medium (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (FBS), 23 mM HEPES and 10 nM dexamethasone. Chang liver cell line (CCL-13 TM ), a human hepatoma cell line, was obtained from American Type Culture Collection (ATCC, Rockville, MD), and cultured in Dulbecco's Modified Eagle's Medium (Hyclone) supplemented with 10% FBS. The Chang liver cells possess a similar gene expression profile for hepatocytes markers such as albumin and CYP3A4 with the normal human hepatocytes (24). The cells were grown in an incubator with 5% CO 2 and 95% air at 37 • C.

Reporter gene analysis
Chang liver cells were transfected with a plasmid mixture containing reporter plasmids, eukaryotic expression vectors, and/or si-RNAs together with pCMV-␤-galactosidase using the Polyfect (Qiagen) or Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol. At the end of experiments, cells were lysed and harvested using luciferase lysis buffer (Promega, Madison, WI). Luciferase activity in whole cell lysates was measured using LB9508 luminometer (Berthold, Bad Wildbad, Germany) and normalized to ␤-galactosidase activity.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was prepared using Easy-Blue reagents (IN-tRON Biotechnology, Seoul, Korea) according to the man- ufacturer's protocol and cDNA was synthesized as previously described (8). qRT-PCR was performed using an ABI StepOnePlus TM Real-time PCR system (Applied Biosystem, Foster City, CA) using specific primers (13,16,26,27). Relative mRNA level of target gene was analyzed by the equation 2 −Ct (Ct = Ct of target gene minus Ct of ␤-actin) and presented with a level of control group as 1. Detailed method of qRT-PCR was described previously (13,28).

In situ proximity ligation assays (PLAs)
To detect protein-protein interactions with high selectivity and sensitivity, PLAs were performed using the Duolink II Detection Reagent Red (Sigma) according to manufacturer's protocol. Briefly, cells grown in 8-well chamber slides were transfected with FLAG-ERR␥ and MYC-ROR␣. Cells were fixed and incubated with primary antibodies against FLAG and MYC. The slides were incubated with PLA probes and the subsequent ligation and rolling circle amplification were performed. The PLA signals were visualized by a confocal microscope (Carl Zeiss, New York, NY).

Animals, treatments and histological analysis
Eight-week-old male C57BL/6 mice were gradually habituated to a liquid Lieber-DeCarli liquid diet (Dyets, Bethlehem, PA) with 5% (v/v) ethanol over a period of 2 weeks, then maintained on the 5% ethanol diet (36% ethanolderived calories) (n = 12) for 5 weeks as described previously (29). Diet consumption was recorded daily and isocaloric amounts of a non-ethanol-containing diet (n = 8) were dispensed to pair-fed animals. After 3 weeks of feeding with a 5% ethanol diet, mice from both the ethanol-fed and pair-fed groups were administered JC1-40 at a dose of 10 mg/kg/day in 0.2 ml 0.5% carboxymethyl cellulose by oral gavage for 2 weeks. All groups of mice maintain a similar body weight within 5% difference after treatment. At the end of treatment, liver tissue was rapidly excised and portions of the liver were stored for further analysis of protein and mRNA, or fixed in 10% formalin for histopathologi- For histological examination, a 3 m section of paraffinembedded tissue was stained routinely with hematoxylin and eosin (H&E). Immunohistochemistry was performed using anti-CYP2E1 (Millipore, Darmstadt, Germany) and anti-4-hydroxynonenal (4-HNE) (JalCA, Shizuoka, Japan) antibodies. Activities of glutamate pyruvate transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) were measured using Fuji DRI-CHEM 3500s serum biochemistry analyzer (Fujifilm, Kanagawa, Japan), and amounts of hepatic triglyceride (TG) were measured using the EnzyChrom TM Triglyceride Assay Kit (Bio Assay Systems, Hayward, CA).

Statistics
All values were expressed as means ± SD. Statistical analysis was performed using non-parametric Mann-Whitney U test for simple comparisons or Kruskal-Wallis ANOVA for multiple comparisons. P < 0.05 was considered as statistically different. Specified analysis was described in the figure legends.

ROR␣ represses the ERR␥-mediated induction of CYP2E1 expression
CYP2E1 is a key enzyme causing alcohol-induced oxidative stress and liver injury. To illustrate the pathophysiological role of ROR␣ in ALDs, we examined whether ROR␣ affected expression level of CYP2E1 under ethanol exposure. Since alcohol-induced liver injury is associated with CB1 receptor activation, we employed a synthetic CB1 receptor agonist, ACEA, to mimic alcohol-induced CYP2E1 gene expression (14)(15)(16). Treatment of ROR␣ agonists such as CS, SR1078 and JC1-40, largely decreased the expression level of CYP2E1 protein as well as transcripts that were induced by ACEA treatment in mouse primary hepatocytes (8,9). Expression level of a known transcriptional activator of CYP2E1, ERR␥ , increased after ACEA treatment, but it was not affected by the ROR␣ ligand treatment. Similarly, the CYP2E1 induction caused by adenovirus-mediated ERR␥ gene transfer was decreased by treatment with these ROR␣ ligands ( Figure 1A) (16). The similar pattern of CYP2E1 repression was observed at both transcript-and protein-levels when ROR␣ was exogenously introduced by infusion of Ad-ROR␣, suggesting that the ERR␥ -induced CYP2E1 gene transcription might be a target of ROR␣ during chronic ethanol consumption (Figure 1B). Our observation that knockdown of either ROR␣  or ERR␥ gene transcription using si-RNA decreased the ROR␣ agonist-induced CYP2E1 repression indicates the involvement of ROR␣ in the ERR␥ -mediated induction of CYP2E1 transcription ( Figure 1C). Knockdown of ROR␣ completely abolished the CS-, JC1-40-or SR1078-induced transcriptional activity of the RORE reporter, indicating that the effect of these compounds was mediated through ROR␣ (Supplementary Figure S2).

Expression of CYP2E1 is controlled by ROR␣-induced coregulator switch on the ERRE located in the upstream promoter
Previously, we reported an ERRE in the mouse CYP2E1 gene promoter that upregulates the gene expression in response to alcohol exposure (16) (Figure 2A). Therefore, we tested whether the ROR␣-induced suppression of CYP2E1 gene would be mediated through this ERRE. The activity of a reporter encoding the CYP2E1 gene promoter (-910 to +218 bp) was increased after transient expression of ERR␥ , but was significantly reduced after overexpression of ROR␣ or following JC1-40 treatment in Chang liver cells. The activity of a promoter encoding a mutated ERRE was not changed by either ERR␥ or ROR␣, further suggesting the involvement of this ERRE in the ROR␣-mediated repression of the CYP2E1 promoter (Figure 2B). A reporter encoding the consensus ERRE sequence found in the small heterodimer partner promoter was also largely suppressed in the presence of ROR␣ (Figure 2C) (30). Indeed, ChIP analysis performed on the CYP2E1 promoter region revealed that the levels of histone modifications such as AcH3K9, MeH3K9, and MeH3K27, markers of transcriptional activation or repression, were altered when ROR␣ was expressed by transient overexpression, suggesting that ROR␣ changed transcriptional mode of ERR␥ -ERRE from activation to repression (Figure 2D). Since transcriptional coregulators provide epigenetic control mechanisms for transcriptional switch, we tested whether p300 and NCoR1 were involved in the repressive function of ERR␥ on the CYP2E1 promoter. Consistently, coactivator p300 bound to the ERRE after treatment with ACEA or following Ad-ERR␥ infusion; however, this binding disappeared when cells were treated with ROR␣ agonists or Ad-ROR␣. In contrast, the DNA binding of NCoR1 at this promoter region was increased by activation of ROR␣ ( Figure 2E). Together, these results indicate that ROR␣ represses CYP2E1 gene expression through reg- Luciferase activity that normalized to the corresponding ␤-galactosidase activity was converted to fold activity with no treatment as 1. The data represent mean ± SD. *P < 0.05 versus EV; # P < 0.05 versus ERR␥ ; = P < 0.05 versus ERR␥ with ROR␣ (n = 3). (E) Whole cell lysates obtained from the Chang liver cells transfected with FLAG-ERR␥ , ERR␥ -AF2, MYC-ROR␣ and/or ROR␣-AF2 were IP and probed using the indicated antibodies by WB. The level of proteins in the cell lysates was analyzed by WB as input. Representatives of at least three independent experiments with similar results are shown. ulation of the ERRE in upstream promoter by recruitment of NCoR1 instead of p300.

Physical interaction of ROR␣ and ERR␥ is required for the coregulator switch
To characterize further the cross-talk between ERR␥ and ROR␣, we examined whether these receptors interacted physically. As shown in Figure 3A, a specific interaction of ROR␣ with ERR␥ was demonstrated by reciprocal immunoprecipitation using specific antibodies. In situ proximity ligation assays (PLAs) further confirmed the interaction of ROR␣ and ERR␥ in the nucleus ( Figure 3B). The interaction of ROR␣ and ERR␥ was observed in the presence of ACEA, which further increased by CS, but decreased by SR3335, an inverse agonist of ROR␣ ( Figure 3C) (31). A deletion mapping study showed that the interaction between ROR␣ and ERR␥ was mediated through the LBDs of both receptors ( Figure 3D and E). The ERR␥ -LBD fused to GAL4 DNA binding domain activated the reporter encoding upstream activating sequences, i.e. Gal4-tk-Luc. However, this reporter activity was largely decreased by coexpression of the full-length as well as the LBD of ROR␣, but not by the LBD-deleted mutant, indicating that the ROR␣-induced repression of ERR␥ was mediated by a physical interaction of these receptors through their LBDs ( Figure 3F).
Furthermore, we observed that ERR␥ interacted with coactivator p300 when cells were treated with ACEA, whereas cotreatment with CS reduced the ERR␥ -p300 interaction. In contrast, CS treatment increased the binding of ERR␥ to the corepressor NCoR1 ( Figure 4A). The ERR␥ -NCoR1 binding disappeared in the absence of ROR␣, indicating that this binding was mediated by ROR␣ ( Figure 4B). Consistently, the ROR␣-mediated repression of ERRE activity was recovered by overexpression of p300 or by knockdown of NCoR1 ( Figure 4C). Indeed, NCoR1 repressed the ERRE activity only when ROR␣ was coexpressed, demonstrating that the repressive function of ERR␥ -NCoR1 required ROR␣ ( Figure 4D). Since nuclear receptors recruit cofactors through AF2 region, we examined the cofactor switch with the AF2 deleted mutants of ERR␥ and ROR␣. ERR␥ interacted with ROR␣ regardless of the presence of AF2 and vice versa. ERR␥ interacted with NCoR1 in the presence of ROR␣, but not of ROR␣-AF2, suggesting that ERR␥ recruited NCoR1 which bound to ROR␣, since ERR␥ did not interact with NCoR1 by itself ( Figure 4E, left). Consistently, ERR␥ -AF2 interacted with NCoR1 only when wild-type ROR␣ was present ( Figure 4E, right). Together, these results indicate that ROR␣ represses the transcriptional function of ERR␥ by recruitment of the corepressor NCoR1.

Administration of JC1-40 decreases hepatic CYP2E1 expression and protects against ethanol-induced liver injury in mice
Finally, we tested whether activation of ROR␣ would decrease the expression of CYP2E1 and attenuate the degree of liver injury in mice induced by chronic ethanol exposure. Both the ethanol-fed and pair-fed control groups were administered JC1-40, an activator of ROR␣, at 10 mg/kg per day for 2 weeks. Chronic ethanol feeding resulted in alcoholic liver injury; the numbers and sizes of hepatic lipid droplets were enhanced and the level of 4-HNE was increased when assessed by H&E staining and immunohistochemistry, respectively ( Figure 5A and B). The ratio of liver weight to body weight, the serum levels of GPT and GOT, and the amount of hepatic TG were increased in the ethanol-fed group ( Figure 5C-E and Supplementary Figure  S3). However, importantly, the administration of JC1-40 dramatically lowered all these liver injury parameters (Figure 5A-E and Supplementary Figure S3).
Consistent with the results obtained from in vitro experiments, the hepatic level of protein as well as mRNA for CYP2E1 were increased in the ethanol-fed group, but decreased in the JC1-40-treated group ( Figure 6A-C). Also we observed an interaction between ERR␥ and ROR␣, especially in the JC1-40-treated liver tissues of ethanol-fed mice ( Figure 6C). Expression level of ROR␣ protein was significantly decreased the ethanol-fed group, whereas it was largely increased in the JC1-40 treated groups (Figure 6C). These results indicate the protective role of ROR␣, which might be associated with suppression of CYP2E1 expression. Finally, in vivo DNA binding of p300 in the CYP2E1 promoter was significantly increased in livers of the ethanol-fed group, but largely decreased in the JC1-40 administered group. In contrast, the DNA binding of NCoR1 was significantly increased in the JC1-40 treated group ( Figure 6D). Together, these results demonstrated the presence of ROR␣ induced coregulator switch at the ERRE site in the CYP2E1 gene promoter in vivo, which was correlated with ROR␣-induced protection from oxidative stressinduced liver injury after chronic alcohol exposure (Figure 6E).

DISCUSSION
Nuclear receptors bind to a specific DNA element in target gene promoters as monomers, homodimers or heterodimers to perform transcriptional regulation (32). To date, ERR␥ has been shown to form homodimers or heterodimers with ERR␣ (33). Here, we showed for the first time that ERR␥ binds to ROR␣ on the ERRE half-site, resulting in transcriptional repression of specific target genes such as that encoding CYP2E1 ( Figure 6E). Because ROR␣ does not alter the promoter activity of CYP2E1 in the absence of ERR␥ , this ROR␣ action mechanism is non-classical and clearly different from the classical pathway involving a specific DNA binding-mediated regulation of target gene transcription ( Figure 2B). Especially, NCoR1 was recruited to the ERRE site when ROR␣ repressed ACEA-induced ERR␥ activity ( Figure 2E and 6D). A similar type of nonclassical pathway was reported in the regulation of the LXR␣-dependent LXR-response element (LXRE) activation by estrogen receptor alpha (ER␣) (34). ER␣ inhibited transcription of LXR␣ target genes such as those encoding sterol regulatory element-binding protein 1 and stearoyl CoA desaturase 1 through direct interaction with LXR␣, resulting in repression of the LXR␣-dependent hepatic lipid accumulation in mice (34). During this process, ER␣ replaced p300 to NCoR1 at the LXR␣ bound LXRE in the same way as ROR␣ switches the coregulator in the ERRE. Similarly, ROR␣ suppressed the transcriptional activity of CCAAT/enhancer-binding protein ␤ via physical interaction, causing dissociation of p300 from the relevant transcription factor (35). Previously, we showed that ROR␣ decreased the transcriptional activation of LXR␣ resulting in disruption of the autoregulatory activation loop of LXR␣ (8). It would be interesting to determine the target receptors or proteins for the non-classical ROR␣ pathway described here, which might include LXR␣.
The potential counteracting function of ROR␣ against ERR␥ has been noted in the regulation of the Lipin-1 gene of which the product plays an important role in metabolic processes. ERR␥ induced transcriptional activation of Lipin-1 expression in AML12 mouse liver cells (23). Meanwhile, hepatic expression of Lipin-1 was enhanced in the ROR␣ sg/sg mice, which displayed loss of ROR␣ function. This observation suggests that loss of the non-classical function of ROR␣ increased the ERR␥ -mediated hepatic Lipin-1 expression in these mice (36). However, in the case of G6Pase, a key enzyme of gluconeogenesis, both ERR␥ and ROR␣ act as activators through ERRE and RORresponse element in the promoter region of this gene, respectively (17,37). When we examined regulation of these downstream target genes in our experimental model, Ad-ROR␣ infusion repressed the ERR␥ -mediated Lipin-1 induction, whereas it increased the G6Pase level, which is similar to the previous reports (Supplementary Figure S4).
Nucleic Acids Research, 2016, Vol. 44, No. 3 1103 These observations suggest that the non-classical ROR␣ pathway might operate for specific target sites in specific target promoters. Further studies on the target genes of the non-classical ROR␣ pathway and on what determines the action mode of ROR␣ in the target sites are warranted.
Interestingly, expression level of ROR␣ protein was significantly downregulated in livers of the ethanol-fed groups in this study ( Figure 6B). Hepatic expression level of ROR␣ was lower in patients with ALDs to compare with that of normal, indicating that our finding obtained from mice is potentially relevant to the ROR␣ regulation in human ALDs (38) (Supplementary Figure S5). However, the mechanism of how expression of hepatic ROR␣ is downregulated under ethanol exposure is not known. Previously, ROR␣ was shown to sense cellular stress and expression level of ROR␣ was changed by various types of cellular stress including ultraviolet irradiation and H 2 O 2 treatment (39). Therefore, ethanol-induced cellular stress such as hepatic oxidative stress, endoplasmic reticulum stress response, lipotoxicity and inflammation, may lead to alterations in ROR␣ expression level. Especially, c-JUN, a downstream of stress responsive c-Jun N-terminal kinases (JNK) signaling pathway, prevented ROR␣ binding to RORE and suppressed transcriptional activity of ROR␣ in hepatic steatosis (40). Since ROR␣ expression is induced by its own transcriptional function, hepatic stresses including that activates the JNK signaling may cause disruption of the autoregualtion of ROR␣ expression (8). Further understandings on the molecular mechanism of ROR␣ downregulation under ethanol exposure may help to illustrate pathogenesis of ALDs.
Ethanol feeding increases amount of endocannabinoids such as 2-arachidonylglycerol, which is a CB1R agonist, in the stellate cells (15). The hepatic endocannabinoid system is the major contributor of pathogenesis of ALD since activating CB1 receptor results in induction of CYP2E1 mRNA in the hepatocytes (16). Interestingly, ethanol treatment induced expression of CYP2E1, but not of CB1R and ERR␥ in the hepatocytes (Supplementary Figure S6A). Similarly, ethanol did not increase DNA binding of ERR␥ or p300 on the CYP2E1 promoter, which is contrast to ACEA (Supplementary Figure S6B). Together these results indicate that the cross-talk between ERR␥ and ROR␣ functions mainly in the CB1R-ERR␥ axis of the hepatocytes that triggered by paracrine effect of endocannabinoids secreted from the stellate cells.
In clinics, patients with severe ALDs are treated with regimens such as anti-inflammatory corticosteroids and pentoxyfilline (2,41). Unfortunately, however, these pharmacological treatments show insufficient effectiveness and sensitivity (2,41). Here, we showed that treatment with a ROR␣ ligand decreased the ERR␥ -induced CYP2E1 expression and contributed to the repression of alcohol-induced oxidative stress and liver injury in ethanol-fed mice. In addition, the human CYP2E1 promoter contains an ERRE, and ROR␣ was found to repress ERR␥ -mediated CYP2E1 expression in human Chang liver cells (16) (Supplementary Figure S7). In addition, further, several other lines of evidence have demonstrated that ROR␣ possesses other beneficial functions that could improve the symptoms of ALDs. For example, ROR␣ activates AMPK which plays an important role in regulating sterol regulatory elementbinding protein 1-mediated lipid synthesis under conditions of alcohol exposure (8,42). ROR␣ inhibits the transcriptional activity of LXR␣ causing auto/paracrine activation of monocyte chemoattractant protein 1, an essential chemokine causing alcohol-induced liver injury (8,29,43). Administration of ROR␣ ligands protects against hepatic oxidative stress through the induction of antioxidant enzymes including superoxide dismutase 2 and glutathione peroxidase 1 (13). In conclusion, ROR␣ might possess multitargeting ability against important pathogenic events during the progression of ALDs, and agonists of ROR␣ could contribute to the development of useful new therapeutic strategies to improve the outcomes for affected patients.

SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.