Page 1 of 49Articles in PresS. Physiol Genomics (July 31, 2007). doi:10.1152/physiolgenomics.00098.2007
Gene expression profiling reveals a regulatory role for ROR and ROR in Phase I and Phase II Metabolism
Hong Soon Kang1*, Martin Angers1*, Ju Youn Beak1, Xiying Wu2, Jeffrey M. Gimble2, Taira Wada3, Wen Xie3, Jennifer B. Collins4, Sherry F. Grissom4, and Anton M. Jetten1
1
Cell Biology Section, 4Microarray Group
Division of Intramural Research The National Institute of Environmental Health Sciences National Institutes of Health Research Triangle Park, NC 27709 2
Stem Cell Laboratory
Pennington Biomedical Research Center Louisiana State University System 6400 Perkins Rd, Baton Rouge, LA 70808 3
Center for Pharmacogenetics and Department of Pharmaceutical Sciences
University of Pittsburgh Pittsburgh, PA 15213
*The first two authors contributed equally to this study
1 Copyright © 2007 by the American Physiological Society.
Page 2 of 49
Address correspondence to: Dr. Anton M. Jetten, Cell Biology Section, Division of Intramural Research, The National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, Tel: 919-541-2768; Fax: 919-541-4133; E-mail:
[email protected]
Abbreviations: ROR, retinoid-related orphan receptor; Sult, sulfotransferase; Cyp, cytochrome P450; RORE, ROR-response element; sg, staggerer; PAPS, 3’-phosphoadenosine 5’phosphosulfate; CT, circadian time
Running title: Regulation of metabolism by RORs
Key words: Liver, nuclear receptor, metabolism, sulfotransferase, staggerer mice, gene expression analysis, circadian rhythm
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ABSTRACT Retinoid-related orphan receptors alpha (ROR ) and gamma (ROR ) are both expressed in liver; however, their physiological functions in this tissue have not yet been clearly defined. The ROR 1 and ROR 1 isoforms, but not ROR 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 -deficient staggerer mice (ROR and ROR
sg/sg
ROR
-/-
sg/sg
), ROR
-/-
,
double knockout (DKO) mice by microarray analysis. DKO mice were
generated to study functional redundancy between ROR and ROR . These analyses demonstrated that ROR and ROR affect the expression of a number of genes. ROR and ROR are particularly important in the regulation of genes encoding several Phase I and Phase II metabolic enzymes, including several 3 -hydroxysteroid dehydrogenases (Hsd3b), cytochrome P450 (Cyp) enzymes, and sulfotransferases. In addition, our results indicate that ROR
and ROR each affect the
expression of a specific set of genes but also exhibit functional redundancy. Our study shows that ROR and ROR 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.
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THE RETINOID-RELATED ORPHAN RECEPTOR (ROR) subfamily of nuclear receptors consists of three members: alpha (ROR ), beta (ROR ), and gamma (ROR ), also referred to as NR1F1-3 or RORA-C, respectively (16, 17). Through alternative splicing and promoter usage each ROR gene generates several variants that are expressed in a tissue-specific manner and regulate specific target genes and physiological functions. These variants have been implicated in the regulation of distinct biological processes. ROR receptors preferably bind as monomers to specific ROR response elements (ROREs) (11, 29) in the regulatory region of target genes. Repression and activation of gene transcription by RORs is mediated through the recruitment of co-repressors and co-activator complexes, respectively, as has been demonstrated for other nuclear receptors (16, 17). Recent crystal structure analyses of RORs have provided evidence indicating that the transcriptional activity of RORs is ligand-dependent (20, 39). Cholesterol-sulfate and other (sulfated) lipid metabolites have been reported to function as agonists for ROR , while several retinoids were demonstrated to bind ROR and ROR and to act as partial antagonists (20, 39). Characterizations of mice deficient in the expression of RORs have implicated ROR , ROR , and ROR in the control of distinct physiological processes. These studies showed that ROR plays a critical role in the maturation and survival of Purkinje cells (4, 9, 12, 17). ROR has also been implicated in bone formation, lipid homeostasis, and in the regulation of several immune functions (7, 15, 30, 32, 37, 43). ROR , , and
all appear to have a role in the regulation of circadian
rhythm (2, 17, 34). Moreover, a recent study implicated ROR in the regulation of the S opsin gene in retinal cone photoreceptors (36). ROR t (also referred to as ROR 2) has been reported to play a critical role in the development of secondary lymphoid tissues and in thymopoiesis (10, 17, 25, 41, 42).
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While the expression of ROR is rather restricted and shows little overlap with those of the other two ROR receptors, ROR and ROR are co-expressed in many tissues, including brown fat, liver, and kidney (17). The latter raised the question whether there is any functional redundancy between these two receptors as has been demonstrated for several other nuclear receptors. To obtain greater insight into this question and the roles of RORs in liver, we generated ROR
and ROR
double knockout (DKO) mice and compared the gene expression profiles of livers from wild type (WT), ROR
sg/sg
, ROR
-/-
, and DKO mice by microarray analysis. These analyses showed that lack
of ROR and ROR expression affects the expression of several genes encoding Phase I and Phase II metabolic enzymes. In addition, our data demonstrated that ROR and ROR each can influence the expression of a specific set of genes but also exhibit functional redundancy and that they can affect gene expression in a positive as well as negative manner. Our study shows that ROR and ROR receptors affect the expression of several genes involved in the metabolism of steroids, bile acids, and xenobiotics suggesting that they may play an important role in the control of metabolic homeostasis and the detoxification and elimination of endogenous and exogenous compounds.
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MATERIALS AND METHODS Experimental Animals. Heterozygous C57BL/6 staggerer (ROR from Jackson Laboratories (Bar Harbor, ME). The staggerer (ROR mouse strain, contain a 6.5 kb deletion in the ROR ROR (13). C57BL/6 ROR
-/-
+/sg
sg/sg
+/sg
with ROR
-/-
) mice, a natural mutant
gene resulting in a functional knockout of
mice were described previously (25). ROR
were generated by crossing ROR
) mice were purchased
mice. ROR
sg/sg
ROR
-/-
(DKO) mice
genotyping was carried by
polymerase chain reaction (PCR) of tail DNA according to the instructions of Jackson Laboratories while genotyping of the ROR
/
mice was performed as previously described (25). Littermate WT
mice were used as controls. The animals were bred at NIEHS and were supplied ad libitum with NIH-A31 formula and water. Blood was collected by heart puncture and sera stored at -80 oC. The C57BL/6 and AKR/J mice used in the study were maintained on a constant 12 h light: 12 h dark cycle with the light cycle beginning at 6 AM. All animal protocols followed the guidelines outlined by the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the NIEHS and the Pennington Biomedical Research Center. RNA isolation. Tissues were collected from mice at the circadian time (CT) indicated. Tissues were rapidly dissected and parts either processed in RNA Later solution (Ambion, Austin, TX) or flash frozen in liquid nitrogen. Tissues were stored at -80 oC. Tissues were then homogenized in 4 ml RLT solution in presence of
-mercaptoethanol with a polytron PT 3000 (Brinkmann
Instruments, Westbury, NY). The homogenate was loaded into a QIAshredder column (Qiagen, Valencia, CA) and centrifugated at 12,000 × g for 3 min. The supernatant was collected and one volume 70% ethanol (50% for liver) added. The mixture was then loaded onto an RNeasy midi column and RNA isolated following the manufacturer’s instructions (Qiagen). The quality and
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Page 7 of 49
integrity of the RNA was assessed by bioanalyzer (Agilent, Santa Clara, CA) and agarose gel electrophoresis. Northern blot analysis. Northern blot analyses were performed as described (21). Briefly, 15 µg of total RNA were separated on a 1.2% agarose gel containing 0.5% formaldehyde in 1X MOPS buffer, then transferred onto a nylon membrane (Sigma, St. Louis, MO). After UV-cross-linking, the membrane was hybridized to [32P]-labeled probes for Sult1e1, Sult2a1, Igfbp1, Elovl3, Keg-1, Hsd3b5, and Cyp2b10. The membrane was then washed and exposed on Hyperfilm (Amersham Bioscience, NJ) at -70 oC. Microarray analysis. Gene expression analyses were conducted by the NIEHS Microarray Group (NMG) on Agilent mouse 20,000-oligo chips. Total RNA was isolated from livers of 8-12 weeks-old WT, DKO, ROR
sg/sg
, or ROR
-/-
mice around CT19. Each analysis was performed in
duplicate, employing a fluor reversal. In the case of WT and DKO mice, two independent experimental replicates were analyzed with a different subset of mice. Briefly, equal amounts of total RNA from individual male mice (5 WT vs 8 DKO in the first analysis, 4 WT vs 4 DKO in the second analysis, 4 WT vs 4 ROR -/-, and 4 WT vs 4 ROR
sg/sg
) were pooled for each genotype
group and then amplified using Agilent Low RNA Input Fluorescent Linear Amplification Kit. Starting with 0.5 µg of amplified total RNA, Cy3- or Cy5-labeled cRNA was produced according to manufacturer’s protocol. For each two-color comparison, 750 ng of each Cy3- and Cy5-labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit. Hybridizations were performed for 16 h in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Data were retrieved with the Agilent Feature Extraction software (v7.5), using defaults for all parameters. The Agilent Feature Extraction Software performed error modeling,
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Page 8 of 49
adjusting for additive and multiplicative noise. The resulting data were processed using the Rosetta Resolver® system (version 5.1) (Rosetta Biosoftware, Kirkland, WA). The Resolver system combines ratio profiles to create ratio experiments using an error-weighted average as described (45). P values are generated and propagated throughout the system and represent the probability that a given gene is significantly expressed. Genes with a p value less than 0.001 were considered statistically, differentially expressed. The signature genes were sorted into different categories with the help of the GeneSpring software. One microarray analysis was carried out with pooled RNA from livers of 1 female and 2 male WT mice vs 3 female DKO mice. Except for several genderspecific genes, this analysis confirmed the pattern of differentially expressed genes obtained with RNA from livers of male WT and DKO mice (data not shown). The microarray data discussed in this
study have
been
deposited
in
the
NCBI's
Gene
Expression
Omnibus
(GEO,
http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE7564. Quantitative Real-time PCR (QRT-PCR). SYBRG and TaqMan QRT-PCR analyses were performed to validate several of the genes identified by microarray analyses and to measure gene expression as a function of CT. Total RNA from 3 individual mice within each genotype, gender or CT group were analyzed as indicated. The RNA was reversed-transcribed using the High capacity cDNA archive kit according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). QRT-PCR reactions with 50 ng of cDNA were carried out in triplicate in a 7300 Real Time PCR system (Applied Biosystems) as follows: 2 min at 50 oC, 10 min at 95 oC, then 40 cycles each at 95 oC for 15 sec, and 60 oC for 60 sec. Pre-designed Assays-on-Demand
primers/probe sets
were purchased from Applied Biosystems: Mm00484132_m1 (Cyp4a14), Mm00484157_m1 (Cyp7b1), Mm00431814_m1 (ApoA4), Mm00468164_m1 (Elovl3), Mm00657677_mH (Hsd3b5).
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Page 9 of 49
Mm00833447_m1 (Igfbp1), Mm00649796_m1 (Slco1a1), Mm00499178_m1 (Sult1e1). Others primers and probes (Table 1) were designed using the ABI PrimerExpress 2.0 software and synthesized by Sigma/Genosys. All results were normalized relatively to the 18S transcript except in the circadian rhythm experiments where cyclophilin B was utilized instead. Sulfotransferase and steroid assays. Flash frozen liver tissues were homogenized with a polytron at 4 oC in 4 volumes of homogenizing buffer (250 mM sucrose, 5 mM HEPES, and 2 mM -mercaptoethanol, pH 7.4). The homogenates were centrifuged at 16,000 x g for 10 min at 4 oC. Subsequently the supernatants were collected and analyzed for sulfotransferase activity following a modified protocol adapted from (22). Briefly, reaction buffer (100 mM Tris, 10 mM MgCl2, 20 mM of substrate, and 3’-phosphoadenosine 5’-phosphosulfate (PAPS), pH 7.4) was added to protein extracts (100 µg) and incubated for 30 min at 37 oC. [2,4,6,7-3H]-labeled estradiol (PerkinElmer Life Sciences, Boston, MA) served as a substrate for Sult1e1 while [1,2,6,7-3H]-labeled dehydroepiandrosterone (DHEA; PerkinElmer Life Sciences) was used for Sult2a1. The enzymatic reactions were stopped by addition of 600 µl of Tris-HCl, pH 8.7, and 1.7 ml of dichloromethane. The samples were mixed thoroughly then centrifuged at 600 x g for 5 min. The supernatant was collected and the radioactivity determined in a scintillation counter. Estradiol and DHEA levels were determined by radioimmune assay (RIA). An estradiol RIA kit was purchased from Diagnostic Products Corp. (Los Angeles, CA) and a DHEA RIA kit was obtained from Diagnostic Systems Laboratory Inc. (Webster, TX). Assays were performed in an Apex Automatic gamma counter (ICN Micromedic Systems, Huntsville, AL). Transfection. Mouse primary hepatocytes were plated in 6-well dishes and maintained in Hepatocyte Maintenance Medium (HMM; Cambrex BioScience, Walkersville, MA) and then transfected with 4 µg of VP-ROR or ROR
expression plasmid DNA using Lipofectamine 2000
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(Invitrogen, Carlsbad, CA). VP-ROR
was created by fusing the VP16 activation domain of the
herpes simplex virus to the amino-terminal of human ROR . Compared to its wild type counterpart, VP-ROR
showed a similar affinity to prototypical RORE but exhibited substantially higher
activity on RORE reporter gene (data not shown). Hydrodynamic liver transfection in 4-week old CD-1 female mice was carried out as described previously (48). Mice were sacrificed and liver tissues harvested 6 h after the injection of VP-ROR or ROR plasmid DNA (5 µg). Total RNA was extracted and subjected to QRT-PCR analysis. Blood analysis. The levels of glucose, cholesterol, triglycerides, and HDL were determined using the Cobas Mira Classic Chemistry System (Roche Diagnostics Systems Inc., Montclair, NJ) and the chemical reagents for all assays were purchased from Equal Diagnostics (Exton, PA).
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RESULTS Generation and characterization of DKO mice. Previous studies have shown that ROR and ROR are expressed in several tissues (5, 17). Some tissues, including liver, express both receptors whereas other tissues, such as brain, express only ROR (Fig. 1A). In addition to the generation of different isoforms, each ROR gene produces multiple transcripts of different sizes that are generated by the use of alternative polyadenylation signals (17). Mouse liver expresses the ROR 1 and ROR 4 isoforms and only the ROR 1 isoform (5, 16). Figure 1B shows that ROR 1 and ROR 1 exhibit an oscillatory pattern of expression consistent with a circadian rhythm (46). Both ROR 1 and ROR 1 mRNA are optimally expressed between CT16 and CT0 and expressed at low levels between CT4 and CT12. The expression of ROR 4 mRNA did not display a significant oscillatory expression pattern. To obtain greater insight into the physiological functions of ROR and ROR receptors in liver and to determine whether ROR ROR
sg/sg
ROR
-/-
and ROR
have redundant functions, we generated
double knockout (DKO) mice (Fig. 2A). The DKO mice were viable and
exhibited all the main phenotypic characteristics previously found to be associated with ROR and ROR knockout mice including the staggerer phenotype, a characteristic of ROR absence of lymph nodes and a greatly reduced thymus, characteristics of ROR
sg/sg
-/-
mice, and the
mice (10, 12, 17,
25, 41). DKO mice weighed about 30% less than WT littermate controls similar to the 25% reduction in body weight observed for ROR
sg/sg
mice. No significant differences were observed in
relative liver weights between each group (Fig. 2B) and histopathological examination did not reveal any obvious morphological changes in the liver of either ROR
, ROR
sg/sg
-/-
or DKO mice
(data not shown). A significant reduction in blood triglyceride (35%) and cholesterol (25%) levels
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Page 12 of 49
was observed in DKO mice that was comparable to those observed in ROR triglyceride and cholesterol levels were not significantly changed in ROR
-/-
sg/sg
mice while
mice (Fig. 3). The level
of blood glucose was reduced by 25% in DKO mice compared to WT and although a 12% reduction in blood glucose was observed in ROR different from that of WT mice. ROR
sg/sg
-/-
mice, this reduction was not statistically
and WT mice did not exhibit a significant difference in
blood glucose levels (Fig. 3). ROR
and ROR have specific and redundant functions and act as positive and negative
regulators of gene expression. To obtain insight into the physiological functions of ROR receptors in liver, we compared the gene expression profiles from livers of WT, ROR
sg/sg
, ROR
-/-
, and DKO
mice by microarray analysis. Because ROR 1 and ROR 1 expression displayed a circadian rhythmicity, the tissues were collected at CT19, a time at which receptor expression is at its zenith. Differentially expressed genes were considered for further analysis if they were induced or repressed at least 1.5-fold or more in one of the 3 knockout groups and if their p-value was less than 0.001. The gene profiling analysis indicated that in mouse liver, many genes were regulated by ROR and/or ROR . The expression of 69 genes was changed in both ROR genes in both ROR
-/-
and DKO mice, 20 genes in ROR
sg/sg
, ROR
sg/sg
and DKO mice, 12
-/-
, and DKO mice while 797
genes were changed only in DKO mice. Table 2 shows a partial list of genes regulated by RORs in liver. The complete listing of all the changes in gene expression identified in the different comparisons is available at http://dir.niehs.nih.gov/microarray/jetten/home.htm. The genes most dramatically repressed or induced in liver from DKO mice, include genes encoding several sulfotransferases,
cytochrome
P450
enzymes,
microsomal
enzymes,
hydroxysteroid
dehydrogenases, glutathione transferases, and solute carriers. In addition, these data suggested that certain genes are regulated preferentially by either ROR or ROR while other genes are under the
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control of both receptors and indicates that ROR and ROR have specific as well as redundant functions in liver. The gene profiling analysis also showed that about 30% of the genes were induced while about 70% were down-regulated in livers from DKO-deficient mice. The latter indicates that RORs can function as positive as well as negative regulators of gene expression. Expression profiling analyses reveal novel biological functions of ROR receptor. RORs regulate the expression of genes encoding proteins with widely different functions including the cell adhesion protein cadherin 1, the mitochondrial enzyme creatine kinase (Ckmt2), the insulin growth factor binding protein 1 (Igfbp1), the lipophilic ligand carrier protein lipocalin 13 (Lcn13), the selenium binding protein 2 (Selenbp2), the asparagine synthetase (Asns) and the chemokine Ccl5, also named RANTES. Interestingly, among the genes up- or down-regulated in liver from DKO mice compared to WT littermates were many genes encoding Phase I and Phase II proteins involved in the metabolism of lipids, steroids, and xenobiotics. The Phase I genes regulated by RORs included several cytochrome P450 genes. The expression of Cyp2b9, Cyp2b10, Cyp39a1, Cyp4a14, and Cyp2b13 was induced whereas that of Cyp7b1, Cyp2f2, and Cyp8b1 was repressed in liver of ROR
sg/sg
, ROR
-/-
, and DKO mice. The expression of Cyp2b9, Cyp39a1, and Cyp7b1
appeared to be dependent on ROR
rather than ROR . Cyp7b1, Cyp39a1, and Cyp8b1 have
important functions in the biosynthesis of bile acids and their regulation by RORs suggests a role for these receptors in the control of bile acid biosynthesis. The expression of two other P450 genes, Cyp7a1 and Cyp27a1, with major roles in the metabolism of cholesterol into bile acids, was not significantly altered between livers of WT and ROR-deficient mice (data not shown). Cyp2b9 and Cyp2b10 play a role in the oxidation of various xenobiotics and drugs while Cyp4a10 and Cyp4a14 have been implicated in lipid hydroxylation (40). These observations suggest a role of RORs in the regulation of the metabolism of lipids, steroids, and xenobiotics.
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RORs also affect the expression of several genes encoding Phase II metabolic enzymes. These include several sulfotransferases (Sults), which catalyze the transfer of sulfonyl groups using 3’phosphoadenosine 5’-phosphosulfate (PAPS) as sulfate donor. The expression of Sult1e1 mRNA was greatly induced in liver of male DKO and ROR
sg/sg
mice (Table 2). Loss of ROR expression
had little effect on Sult1e1 expression. These observations suggest that ROR negatively regulates the expression of Sult1e1. Hydroxysteroid sulfotransferase Sult2a1 expression, which has been reported to be gender-specific and only expressed in liver from female mice (33), was greatly induced in liver from DKO mice suggesting that is regulated by both ROR and ROR . Sult2a1 catalyzes the sulfonation of procarcinogens, xenobiotics, hydroxysteroids, and bile acids, particularly lithocholic acid (LCA), while Sult1e1 catalyzes the sulfonation of estrogen and estrone (23). These observations suggest a role for RORs in the regulation of the metabolism of steroids, bile acids, and xenobiotics. Glutathione transferases form another group of Phase II metabolic enzymes that catalyze the conjugation of glutathione with a wide variety of xenobiotics generally resulting in their detoxification and elimination (28). ROR-deficiency inhibited the expression of several glutathione transferases while the expression of others was enhanced (Table 2). These results further support a role of RORs in the modulation of xenobiotic metabolism. Table 2 also shows that RORs regulate the expression of several members of the 3 hydroxysteroid dehydrogenase (Hsd3b) family. In mice the Hsd3b family consists of 6 members Hsd3b1-6. Hsd3b4 and Hsd3b5 are NADPH-dependent 3-ketosteroid reductases while Hsd3b2, Hsd3b3, and Hsd3b6 are NAD+-dependent dehydrogenases/isomerases (35). The expression of Hsd3b2-6 were all down-regulated in DKO mice (Table 2). Hsd3b5, which is expressed largely in liver of male mice, was the most affected by the loss of ROR expression. The expression of Hsd3b5
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and Hsd3b4 was down-regulated in both ROR - and ROR -deficient mice and almost totally repressed in DKO mice. These results suggest that Hsd3b4 and Hsd3b5 are positively regulated by both ROR
and ROR . Hsd3b family members catalyze either the biosynthesis of active steroid
hormones or the inactivation of steroid hormones (35). Therefore, their regulation by RORs suggests a role for these receptors in the control of steroid hormone metabolism. Northern blot and QRT-PCR analysis. The induction or repression of the expression of Sult1e1, Sult2a1, Igfbp1, Elovl3, Keg-1, Hsd3b5, and Cyp2b9/10 mRNAs in livers from ROR-deficient mice was verified by Northern blot analysis (Fig. 4A). In addition, the expression of several genes was quantified by QRT-PCR (Fig. 4B). To obtain an independent evaluation QRT-PCR analysis was performed on RNA samples that were different from the ones used in the microarray and Northern blot analysis. A good correlation was observed between QRT-PCR analysis and the data obtained by Northern blot and microarray analysis. The data confirmed that expression of Sult1e1 was induced in livers from both DKO and ROR
sg/sg
mice but not greatly altered in ROR
-/-
mice. This
suggests that Sult1e1 is affected preferentially by ROR . In contrast, the expression of Elovl3 was significantly decreased in DKO and ROR
-/-
mice and somewhat decreased in ROR
sg/sg
mice
suggesting that Elovl3 is modulated preferentially by ROR . Expression of Cyp7b1 was preferentially regulated by ROR . Hsd3b5 mRNA expression was repressed in both ROR ROR
-/-
sg/sg
and
mice but its expression was considerably more repressed in livers from DKO mice
suggesting that both ROR and ROR affect Hsd3b5 expression and that loss of both RORs results in the maximal repression of this gene. Inversely, the expression of Sult2a1, Igfbp1, and Cyp4a14 are induced to a greater extent in DKO mice than in single knockout mice suggesting that both receptors are involved in their negative regulation. The results with Hsd3b5, Cyp4a14, Sult2a1, and Igfbp1 expression are in agreement with the conclusion that ROR
and ROR exhibit redundant
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functions. The expression of several genes, including Sult2a1 and Elovl3, have been shown to be gender dependent. RORs affect the expression of those genes only in females or males (33). Increased Sult1e1 and Sult2a1 expression and activity in DKO mice. The induction of Sult1e1 and Sult2a1 expression in ROR-deficient mice was confirmed by QRT-PCR analysis (Fig. 5A). Sult2a1 activity was only detectable in livers from female mice as reported previously (33). The results further demonstrated that Sult1e1 was preferentially affected by ROR Sult2a1 was influenced by both ROR
expression while
and ROR . It also showed that RORs are negative
modulators of Sult1e1 and Sult2a1 expression. The latter was supported by observations showing that transfection of wild type ROR
or activated VP-ROR
suppressed the expression of
endogenous Sult1e1 and Sult2a1 expression in mouse hepatocytes compared to cells transfected with empty vector (Fig. 5B). To examine whether the increase in Sult1e1 and Sult2a1 expression was reflected in a corresponding increase in protein activity, we compared sulfotransferase activities in liver from WT, DKO,
ROR
-/-
,
and
ROR
sg/sg
mice
using,
respectively,
[3H]-labeled
estradiol
and
dehydroepiandrosterone as substrates. As shown in Fig. 5C, Sult1e1 enzyme activity was greatly increased in liver extracts from DKO and ROR
sg/sg
mice compared to those from WT and ROR
-/-
mice. Sult1e1 activity was increased in liver of both male and female mice. In contrast, Sult2a1 activity was enhanced in liver extracts from DKO and ROR ROR
sg/sg
-/-
mice and to a lesser extent in
mice. These results show that the induction in Sult1e1 and Sult2a1 enzyme activities
reflect the increased expression of their corresponding mRNAs as observed by QRT-PCR and Northern blot analysis. To determine whether the changes in the expression of steroid metabolizing enzymes in liver of ROR-deficient mice had any effect on blood steroid levels, we analyzed estrogen and DHEA in
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Page 17 of 49
blood from WT and ROR-deficient mice. This analysis indicated no significant differences in blood levels of estrogen and DHEA between male and female WT, ROR
sg/sg
, ROR
-/-
, and DKO mice
(data not shown). Circadian pattern of expression of ROR-regulated genes. Since ROR 1 and ROR 1 exhibit an oscillatory pattern of expression (Fig. 1B), one would expect that at least some genes regulated by these receptors show a similar or inverse oscillatory pattern of expression during circadian rhythm. We, therefore, examined the level of expression of Elovl3, Hsd3b5, and Sult1e1 in livers from WT male mice as a function of CT. As shown in Figure 6, the expression of Elovl3, which is positively regulated by RORs, exhibited a rhythmicity that was in phase with that of RORs, while the expression of Sult1e1, which is suppressed by RORs, was out of phase with ROR expression. Hsd3b5 did not exhibit a clear rhythmic pattern of expression.
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DISCUSSION The main objective of this study was to obtain insights into the physiological roles of ROR and ROR in liver. Liver expresses the isoforms ROR 1, ROR 4, and ROR 1 (5, 14) of which ROR 1 and ROR 1 exhibit an oscillatory pattern of expression consistent with a circadian rhythm (Fig. 1). ROR 1 and ROR 1 are maximally expressed between CT16 and CT0. To study their function and investigate the possibility of functional redundancy between these two receptors, we generated ROR
sg/sg
ROR
-/-
double knockout (DKO) mice. The DKO mice exhibited many of the
same phenotypic changes reported for the single knockout mice. The DKO mice exhibited reduced triglyceride and cholesterol levels as previously reported for ROR levels were not significantly changed in ROR
-/-
sg/sg
mice (32). Triglyceride
mice; however, these mice exhibited reduced blood
glucose levels. The latter was also observed in DKO mice. These observations suggest that RORs have distinct regulatory roles in triglyceride and glucose homeostasis. To obtain further insight into the physiological functions of ROR examined the gene expression profiles of livers from WT, ROR
sg/sg
and ROR in liver, we
, ROR
-/-
, and DKO mice by
microarray analysis (Table 2). Comparison of the gene expression profiles indicated that RORs affect the expression of a number of genes. This led to several important conclusions. Firstly, the gene expression profiles showed that some genes were down-regulated in livers from DKO mice compared to WT mice while other genes were induced in DKO mice. These data suggest that ROR
and ROR can influence gene expression in a positive as well as negative
manner. This concept is in agreement with conclusions from recent crystal structure studies indicating that ROR receptors are ligand-dependent transcription factors (20, 38). In addition, RORs have been reported to interact with co-repressors, such as NCoR and RIP140, as well as coactivators, including CBP and SRC1 (12, 17). Although it is likely that RORs control the expression 18
Page 19 of 49
of some of these genes by an indirect mechanism, the positive and negative regulation of the transcription of at least some genes may involve an interaction of RORs with these co-repressors and co-activators. As has been demonstrated for the regulation of gene expression by ROR
in
Purkinje cells, the promoter context of the RORE is important in determining what transcriptional mediators are recruited by RORs (12). This may also determine whether ROR induces or represses the expression of a gene. In addition, our gene profile analyses demonstrated that the expression of certain genes is affected preferentially by ROR or ROR while the expression of other genes is influenced by both receptors. For example, Sult1e1 and Cyp7b1 mRNA expression are affected preferentially in liver of ROR
sg/sg
mice while Elovl3 is suppressed preferentially in ROR
only slightly down-regulated in livers of ROR
sg/sg
and ROR
-/-
-/-
mice. In contrast, Hsd3b5 is
mice while it is dramatically
repressed in DKO mice. Similarly, repression of Cyp8b1 expression was only observed in livers of DKO mice. These observations suggest that there is a degree of functional redundancy between ROR and ROR . The latter may not be surprising since previous studies have shown that although ROR
and ROR receptors have distinct affinities for specific ROREs, they also can bind and
compete for the same RORE (11, 29). Thus the regulation of expression of specific genes by ROR and ROR depends on their affinity to the respective RORE present in the target gene. In addition, the promoter context of the RORE likely plays an important role as well in determining which ROR receptor binds which RORE (4, 9, 12, 13, 17). Most importantly, comparison of the gene expression profiles allowed us to identify categories of genes affected by the loss of ROR expression. This analysis suggested novel roles for RORs in the modulation of the expression of a number of Phase I and II enzymes in the liver (Fig. 7). The liver is the primary site of drug metabolism which has been classified as phase I and phase II
19
Page 20 of 49
reactions. Phase I reactions are primarily mediated by P450 microsomal enzymes catalyzing oxidation and hydroxylation and several other enzymes flavin monooxygenases, peroxidases, dehydrogenases, oxidases, etc. (47). Sulfation, glucuronidation, and glutathione conjugation are the major classes of phase II metabolism. Often compounds undergo phase I oxidation before undergoing phase II conjugation. Phase I and II enzymes play an important role in the detoxification and elimination of endogenous, e.g. steroids, bile acids as well as exogenous compounds, e.g. xenobiotics, drugs and environmental chemicals. However, Cyps also play a role in the generation of active metabolites. Several of the Phase I genes affected by RORs play a role in bile acid biosynthesis (Fig. 7). Cholesterol homeostasis is a balance between the dietary intake of cholesterol, the endogenous biosynthesis of cholesterol, and the disposal of cholesterol in the form of bile acids. The classic or neutral pathway of bile acid biosynthesis involves the conversion of cholesterol to 7 hydroxycholesterol by the rate-limiting enzyme Cyp7a1 and its further conversion by several other enzymes, including Cyp27a1 and Cyp8b1, into cholic acid (CA) and chenodeoxycholic acid (CDCA). The alternative (acidic) bile acid synthesis pathway involves Cyp27a1 and Cyp7b1 that mediate the 7 -hydroxylation of the oxysterols 25- and 27-hydroxycholesterol and several steroids. Our gene profiling analysis showed that RORs affect Cyp7b1 and Cyp8b1 expression suggesting a role for RORs in the control of both the neutral and the acidic pathway of bile acid biosynthesis. RORs did not significantly affect the expression of Cyp7a1 and Cyp27a1 (data not shown), which have major functions in bile acid biosynthesis. In addition to P450 enzymes involved in bile acid biosynthesis, RORs also influence the expression of a number of other Phase I enzymes, including members of the Cyp4a family, which catalyze
-hydroxylation of fatty acids and related
compounds facilitating their degradation (40), and members of the Cyp2b family, which are
20
Page 21 of 49
involved in the oxidation of xenobiotics (44). These results further support the conclusion that RORs affect the expression of Cyp7b1 and Cyp2b10 and play a role in the modulation of Phase I metabolic pathways. RORs also regulate the expression of a number of Phase II metabolic enzymes, including several glutathione transferases and sulfotransferases (47). Glutathione transferases catalyze the conjugation of glutathione with a wide variety of xenobiotics generally resulting in their detoxification and elimination (28). Sult1e1 is main enzyme for sulfonation of estradiol, estrone, and genestein. Sulfonation generally increases the aqueous solubility of substrates and causes a loss of biological activity because sulfonated steroids are unable to bind their (nuclear) receptors. Sult1e1 appears to be regulated preferentially by ROR (Fig. 7). Sult2a1 catalyzes the sulfonation of procarcinogens, xenobiotics, hydroxysteroids, and bile acids (23). Sult2a1 is important for the sulfonation of DHEA and LCA. Sult2a1 expression is affected by both ROR Exogenous expression of ROR
and ROR .
in mouse hepatocytes suppressed the expression of Sult1e1 and
Sult2a1 in agreement with the concept that RORs are negative modulators of the Sult1e1 expression. Another group of ROR-regulated genes involved in steroid metabolism are members of the Hsd3b and Hsd17b family (35). The expression of Hsd3b4 and Hsd3b5, which are involved in the inactivation of steroid hormones, is greatly diminished in DKO mice and appears to be regulated by both ROR and ROR . These observations strongly suggest that RORs modulate several aspects of steroid and bile acid metabolism. In this respect it is interesting to note that cholesterol sulfate has been reported to bind the ROR
receptor (19). Possibly other sulfated steroid or cholesterol
metabolites may function as ligands for RORs. Such an interaction could lead to repression of genes (e.g. sulfotransferases) regulated by RORs thereby creating a feedback control mechanism. To test this idea further, we analyzed the binding of estrogen- and estrone sulfate, products of Sult1e1, to
21
Page 22 of 49
ROR ; however, no binding of these compounds was detectable (Jetten, A.M., unpublished observations). ROR 1 and ROR 1, but not ROR 4, exhibit an oscillatory pattern of expression in liver consistent with a circadian rhythm. Therefore, one might expect that the expression of genes positively regulated by ROR 1 and ROR 1 is in phase with ROR expression and that the inverse is true for genes suppressed by RORs. This is supported by observations showing that the expression of Elovl3, which is positively regulated by ROR 1, is in phase with the circadian pattern of ROR 1 and that the expression of Sult1e1, which is negatively affected by ROR , is out of phase with the circadian pattern of expression of ROR 1 in agreement with the concept that it is under the control of ROR 1. The expression of Hsd3b5 did not exhibit a clear circadian pattern and its regulation might involve a different mechanism possibly involving ROR 4. These results are in agreement with previous observations showing a role for RORs in the regulation of gene expression during circadian rhythm (17). It is interesting to note that the phase of the circadian profile of RORs in liver is the inverse of that reported for the Rev-erb nuclear receptors which function as negative regulators of transcription (49). Consequently, Rev-erb receptors might be involved in the negative regulation of ROR 1 and ROR 1 expression. In addition, Rev-erb receptors have been reported to compete with RORs for binding to ROREs (3). Thus, during circadian rhythm ROR and Rev-erb receptors might function as positive and negative regulators of overlapping sets of genes. Likely not all genes identified in Table 2 are regulated by RORs by a direct mechanism; some may be controlled by an indirect mechanism possibly involving regulation by other transcription factors that themselves are controlled by RORs. Previous studies have identified several genes, including ApoA5, that are regulated directly by ROR (26, 27). In this study we show that Cyp7b1 expression is suppressed in DKO and ROR
sg/sg
mice. The Cyp7b1 gene contains an RORE at nt -
22
Page 23 of 49
952 in the upstream promoter region that was able to bind ROR activation by ROR
and mediate transcriptional
(Wada, T., et al., manuscript in preparation). In addition, ChIP analysis
showed that this ROR
binds ROR
in vivo suggesting that Cyp7b1 is a direct target gene of
ROR . Interestingly, many of the Phase I and II genes found to be affected by RORs have been previously reported to be targets for transcriptional regulation by other nuclear receptors, including the liver X receptor (LXR), constitutive androstane receptor (CAR or NR1I3), pregnane X receptor (PXR), hepatocyte nuclear factor 4
(HNF4 ), peroxisome proliferator-activated receptors
(PPARs), and farnesoid X receptor (FXR) (1, 6, 18, 24, 31, 33). For example, Sult2a1 expression is under the transcriptional control of CAR, PXR, and FXR, while CAR and PXR function, respectively, as a negative and positive regulator of Cyp2b10 expression, and HNF4
is a
transcriptional activator of several Cyps, including Cyp8b1. The effects of RORs on gene expression could involve regulation of the expression of one or more of these receptors, synthesis of receptor ligands or competition for the same DNA response element. Examination of CAR and LXR
expression showed no significance difference in their expression between WT and ROR-
deficient mice (Kang, HS, unpublished observations). Future studies have to determine whether there is any crosstalk between these receptors and RORs as has been recently demonstrated for CAR and HNF4 (8). In summary, our study demonstrates that ROR 1 and ROR 1 exhibit an oscillatory pattern of expression consistent with a circadian profile. Analysis of the gene expression profiles indicated that ROR and ROR affect downstream gene expression in a positive and negative manner. Some genes were modulated preferentially by ROR or by ROR while a number of genes were affected by both ROR
and ROR
suggesting functional redundancy. Most importantly, our study
demonstrates that RORs are able to modulate the expression a number of Phase I and Phase II
23
Page 24 of 49
metabolic enzymes, suggesting that expression of RORs can affect bile biosynthesis and the metabolism of steroids and xenobiotics.
ACKNOWLEDGMENTS: The authors would like to thank Dr. C. Teng for her valuable comments on the manuscript. We like to thank Laura Miller for her assistance with the mice. This research was supported by the Intramural Research Program of the NIEHS, NIH and by a CNRU Center Grant # 1P30 DK072476 entitled “Nutritional Programming: Environmental and Molecular Interactions” sponsored by NIDDK (J.M.G.).
24
Page 25 of 49
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Figure legends Fig. 1. Expression of the ROR and ROR . A. ROR and ROR are both highly expressed in liver. The expression levels of ROR
and ROR mRNA in several mouse tissues were examined by
Northern blot analysis. Expression of 18S rRNA served as loading control. (*) indicates transcripts of different size generated by the use of alternative polyadenylation signals. B. Circadian oscillation of the expression of ROR 1, ROR 4, and ROR 1 mRNA in liver. The mRNA expression was examined by QRT-PCR analysis and normalized relative to cyclophilin B. Data from a single 24 h period was double plotted. Male AKR/J mice used in the study were maintained on a constant 12 h light: 12 h dark cycle as indicated by the bar. CT indicates circadian time.
Fig. 2. Characterization of ROR ROR
sg/sg
, and ROR
-/-
sg/sg
ROR
-/-
double knockout (DKO) mice. A. Genotyping of DKO,
mice. Tail DNA was isolated and genotype examined by RT-PCR analysis.
PCR products were separated by the gel-electrophoresis. The size of the PCR products of the wild type (WT) and recombinant (Rec) allele for ROR are 318 bp and 450 bp, respectively, while those of the WT and recombinant allele for ROR are 380 bp and 430 bp, respectively. B. Average total body weight. C. Average relative liver and kidney weights of three-months old male WT, ROR and ROR
-/-
,
-/-
, and DKO mice (for each group, n=6).
Fig. 3. Comparison of the levels of triglycerides, cholesterol, HDL, and glucose in WT, ROR ROR
sg/sg
, and DKO mice. N=24 (WT), 17 (DKO), 6 (ROR
sg/sg
), and 13 (ROR
sg/sg
,
-/-
).
32
Page 33 of 49
Fig. 4. Lack of ROR
and ROR affects the expression of several genes. A. Livers from two
individual 8-12 weeks-old male or female mice within each genotype group were collected at CT19. RNA was isolated and subsequently examined by Northern blot analysis using radio-labeled probes for Sul1e1, Sult2a1, Cyp2b9/10, Hsd3b5, Igfbp1, Elovl3, Keg-1. B. Comparison of gene expression in livers from WT, DKO, ROR
sg/sg
, and ROR
-/-
mice by QRT-PCR analysis. Livers of three
individual 8-12 weeks-old male or female mice within each genotype group were collected at CT19. RNA was isolated and the expression of a selected group of differentially expressed genes identified by gene profiling analysis analyzed by QRT-PCR. (*) indicates p< 0.05 (compared to WT).
Fig. 5. Altered expression of Sult1e1 and Sult2a1 in DKO, ROR
sg/sg
, and ROR
-/-
mice. A. Livers
were collected at CT19 from four individual, 8-12 week-old, male or female mice within each genotype group and then analyzed by QRT-PCR. B. Suppression of Sult1e1 and Sul2a1 expression by exogeneous ROR
in normal hepatocytes. Mouse primary hepatocytes were transiently
transfected with empty vector or expression vectors for ROR
or VP-ROR
(black bars). Total
RNA was prepared 40 h after transfection. In a second experiment livers of female CD-1 mice were transfected with empty vector or expression vectors for ROR or VP-ROR by a hydrodynamic gene deliver method (gray bars). Six h after transfection mice were sacrificed and total liver RNA isolated. RNA was subjected to QRT-PCR analyses to detect the expression of endogenous Sult1e1 and Sult2a1 mRNA. The percent inhibition by ROR was calculated and plotted. C. Comparison of Sult1e1 and Sult2a1 activity in WT, ROR
sg/sg
, and ROR
-/-
, and DKO mice. Livers were collected
at CT19 from four individual, 8-12 weeks-old, male or female mice within each genotype group.
33
Page 34 of 49
Liver extracts were prepared and assayed for Sult1e1 and Sult2a1 activity as described in Materials and Methods. (*) indicates p< 0.05 (compared to WT).
Fig. 6. Circadian oscillation of the expression of Sult1e1, Hsd3b5, and Elovl3 mRNA in livers of WT male AKR/J mice. The mRNA expression was examined by QRT-PCR analysis and normalized relative to cyclophilin B. Data from a single 24 h period was double plotted. The mice used in the study were maintained on a constant 12 h light: 12 h dark cycle as indicated by the bar.
Fig. 7. Schematic view of the metabolic pathways affected in ROR
sg/sg
, ROR
-/-
, and DKO mice.
ROR and ROR influence several Phase I and Phase II metabolic pathways, including bile acid biosynthesis, steroid, xenobiotic, and fatty acid metabolism. GST, glutathione-S-transferase; GSH, glutathione.
34
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Table 1. List of primers and probes used for QRT-PCR.
Gene
Forward primer GAGGTATCTCAGTCACGAAG
Reverse primer
Probe (5’-6-FAM 3’-TAMRA)
ROR 1
AACAGTTCTTCTGACGAGGACAGG
NA
ROR 4
TGTGATCGCAGCGATGAAAG
AACAGTTCTTCTGACGAGGACAGG
NA
ROR 1
ACTACGGGGTTATCACCTGTG
GCCAAACTTGACAGCATCTC
NA
Sult2a1
CATCTCTTCTCCAAGTCTTTCTTCAGT
CAGACACAAGAATATCTCTCGGATTT
CCAAGGCCAAGGCGATCTATCTCATG
Cyp4a10
CCACCACAACCCGAAGG
GCTCCACAACAGAATCAGT
NA
Cyp8b1
AAGGCTGGCTTCCTGAGCTT
AACAGCTCATCGGCCTCATC
CGGCTACACCAAGGACAAGCAGCA AG
Cyp7a1
TGATCCTCTGGGCATCTCAAG
TTCCCTCCAGAGCTGAGCTCT
NA
18S
CGGCTACCACATCCAAGGAA
GCTGGAATTACCGCGGCT
TTCTGGCACCAGACTTGCCCTC
NA: not applicable
35
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Table 2. A partial list of genes repressed or induced in livers of male DKO, ROR mice. Functional Category Metabolism cytochrome P450
sulfotransferase
steroid
Gene Description
sg/sg
, and ROR
-/-
Gene Symbol
Genbank Accession#
Fold Change
Cyp2b9
NM_010000
Cytochrome P450 2b9
8.9
7.2
1.6
Cyp2b13
NM_007813
Cytochrome P450 2b13
6.9
4.1
1.3
Cyp4a14
NM_007822
Cytochrome P450 4a14
4.2
3.9
2.8
Cyp2b10
NM_009998
Cytochrome P450 2b10
3.1
1.2
1.2
Cyp39a1
NM_018887
Cytochrome P450 39a1
2.7
2.2
-1.3
Cyp4a10
NM_010011
Cytochrome P450 4a10
2.5
3.3
1.9
Cyp3a25
NM_019792
Cytochrome P450 3a25
2.4
1.4
1.3
Cyp3a41
NM_017396
Cytochrome P450 3a41
2.2
1.5
-1.1
Cyp2c38
NM_010002
Cytochrome P450 2c38
2.1
1.7
1.3
Cyp2a4
NM_009997
Cytochrome P450 2a4
2.1
1.8
-1.1
Cyp3a16
NM_007820
Cytochrome P450 3a16
2.1
1.5
-1.2
Cyp4a1
NM_201640
Cytochrome P450 4a1
2.1
3.1
2.0
Cyp3a11
NM_007818
Cytochrome P450 3a11
2.0
1.4
1.0
Por
NM_008898
P450 (cytochrome) oxidoreductase
2.0
2.3
1.1
Cyp2c37
NM_010001
Cytochrome P450 2c37
1.6
1.6
1.2
Cyp2e1
NM_021282
Cytochrome P450 2e1
1.6
1.8
1.4
Cyp4f15
NM_134127
Cytochrome P450 4f15
-1.7
-1.3
-1.2
Cyp2c70
NM_145499
Cytochrome P450 2c70
-1.9
-1.4
-1.4
Cyp2f2
NM_007817
Cytochrome P450 2f2
-2.0
-1.6
-1.2
Cyp8b1
NM_010012
Cytochrome P450 8b1
-4.6
-1.8
-1.6
Cyp7b1
NM_007825
Cytochrome P450 7b1
-7.4
-4.2
-1.5
Sult2a1
NM_009286
Sulfotransferase 2a1
43.5*
1.4
2.3
Sult1e1
NM_023135
Sulfotransferase 1e1
16.9
3.8
1.1
Sult1d1
NM_016771
Sulfotransferase 1d1
3.0
1.9
1.3
Sult1a1
NM_133670
Sulfotransferase 1a1
2.0
1.1
-1.1
Sult1c1
NM_026935
Sulfotransferase 1c1
1.7
1.3
1.0
Sult5a1
NM_020564
Sulfotransferase 5a1
-1.6
-1.4
-1.2
Sult3a1
NM_020565
Sulfotransferase 3a1
-1.6
-1.2
1.1
Nsdhl
NM_010941
NAD(P) dependent steroid dehydrogenase-like
7.8
-1.3
1.2
Hsd17b7
NM_010476
3.0
-1.3
-1.1
Hsd3b2
NM_153193
-1.7
-1.2
-1.4
Hsd3b6
NM_013821
Hydroxysteroid (17- ) dehydrogenase 7 Hydroxy- -5-steroid dehydrogenase 3- and steroid -isomerase 2 Hydroxy- -5-steroid dehydrogenase 3- and steroid -isomerase 6
-2.1
-1.2
-1.7
Hsd17b3
NM_008291
-2.2
-1.4
1.2
Hsd3b3
NM_001012306
-2.2
-1.2
-1.6
Hsd3b4
NM_008294
-5.4
-1.9
-1.5
DKO ROR
Hydroxysteroid (17- ) dehydrogenase 3 Hydroxy- -5-steroid dehydrogenase 3- and steroid -isomerase 3 Hydroxy- -5-steroid dehydrogenase 3- and steroid -isomerase 4
sg/sg
ROR
-/-
36
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lipid
fatty acid
glutathione
carbohydrate
other
Hsd3b5
NM_008295
Hydroxy- -5-steroid dehydrogenase 3- and steroid -isomerase 5
Cd36
NM_007643
Fdft1 Scd2
-15.8
-2.8
-1.9
Cd36 antigen
4.2
2.7
2.0
NM_010191
Farnesyl diphosphate farnesyl transferase 1
3.7
-1.4
1.1
NM_009128
Stearoyl-Coenzyme A desaturase 2
2.6
1.0
-1.1
Hmgcr
NM_008255
3-hydroxy-3-methylglutaryl-CoA reductase
2.5
-1.1
-1.2
Pck1
NM_011044
Phosphoenolpyruvate carboxykinase 1
2.3
1.2
-1.1
Acot2
NM_134246
Acyl-CoA thioesterase 2
2.2
1.7
1.1
Vldlr
NM_013703
Very low density lipoprotein receptor
2.1
1.1
1.1
Dhcr24
NM_053272
24-dehydrocholesterol reductase
2.0
1.2
1.1
Akr1d1
NM_145364
Aldo-keto reductase 1d1
1.6
1.2
-1.1
Acsl6
AK046027
Acyl-CoA synthetase long-chain 6
-1.9
-1.4
-1.2
Elovl6
NM_130450
Elongation of long chain fatty acids 6
3.9
1.3
1.0
Elovl7
NM_029001
Elongation of long chain fatty acids 7
-1.7
-1.1
1.2
Elovl3
NM_007703
Elongation of very long chain fatty acids-like 3
-7.7
-2.2
-6.3
Gstt3
NM_133994
Glutathione S-transferase theta 3
2.7
1.8
1.5
Mgst3
NM_025569
Microsomal glutathione S-transferase 3
1.9
1.2
1.2
Gstm4
NM_026764
Glutathione S-transferase mu 4
1.6
1.3
1.2
Gsta1
NM_008181
Glutathione S-transferase alpha 1
1.6
1.1
1.0
Gstm7
NM_026672
Glutathione S-transferase mu 7
-1.7
-1.1
1.1
Gstp1
NM_013541
Glutathione S-transferase pi 1
-1.9
-1.3
-1.2
Gyk
NM_008194
1.5
1.1
1.0
B3galt1
NM_020283
-1.6
-1.3
1.1
Ppp1r3c
NM_016854
Glycerol kinase UDP-Gal: GlcNAc 1,3-galactosyltransferase 1 Protein phosphatase 1, regulatory (inhibitor) subunit 3c
-2.2
-1.7
-1.8
Asns
NM_012055
Asparagine synthetase
4.9
1.1
-1.1
Ckmt2
NM_198415
3.7
1.1
-1.1
Hpgd
NM_008278
Creatine kinase, mitochondrial 2 Hydroxyprostaglandin dehydrogenase 15 (NAD)
2.7
1.4
1.1
Rdh9
NM_153133
Retinol dehydrogenase 9
1.8
1.5
1.1
Sulf2
NM_028072
Sulfatase 2
1.6
1.4
-1.1
Bhmt
NM_016668
Betaine-homocysteine methyltransferase
-1.7
-1.5
-1.2
Ddc
NM_016672
Dopa decarboxylase
-1.7
-2.4
-2.8
Nat1
NM_008673
N-acetyltransferase 1
-1.8
-1.4
1.1
Arsg
NM_028710
Arylsulfatase
-1.8
-1.5
-1.4
Acacb
NM_133904
Acetyl-Coenzyme A carboxylase
-2.0
1.0
-1.3
Cml5
NM_023493
Camello-like 5
-2.6
-2.3
-1.7
Sdro
NM_027301
Orphan short chain dehydrogenase/reductase
-3.0
-1.6
-1.7
Cml4
NM_023455
Camello-like 4
-3.5
-2.0
1.0
Slc1a2
NM_011393
Solute carrier 1a2
2.9
1.7
1.0
Slco1a4
NM_030687
Solute carrier organic anion transport 1a4
2.8
1.4
1.0
Solute carrier
37
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Slc16a7
NM_011391
Solute carrier 16a7
2.4
2.2
1.3
Slc39a5
NM_028051
Solute carrier 39a5
2.3
1.3
1.4
Slc16a10
NM_028247
Solute carrier 16a10
2.1
1.7
1.0
Slc15a4
NM_133895
Solute carrier 15a4
1.8
1.3
1.1
Slc2a13
NM_001033633
Solute carrier 2a13
-1.7
-1.4
1.1
Slc25a24
NM_172685
Solute carrier 25a24
-1.8
-1.2
1.0
Slc13a2
NM_022411
Solute carrier 13a2
-1.8
-1.5
-1.4
Slc29a4
NM_146257
Solute carrier 29a4
-1.9
-1.4
1.1
Slc41a2
NM_177388
Solute carrier 41a2
-2.0
-2.0
-1.4
Slc5a4a
NM_133184
Solute carrier 5a4a
-2.0
1.1
1.2
Slc2a3
NM_011401
Solute carrier 2a3
-2.0
1.0
-1.1
Slco1a1
NM_013797
Solute carrier organic anion transporter 1a1
-3.0
-1.5
-1.1
Slc30a10
NM_001033286
Solute carrier 30a10
-4.7
-1.5
-1.7
Nr1i3
NM_009803
1.5
1.5
1.2
Arntl
NM_007489
Nuclear receptor 1i3 (CAR) Aryl hydrocarbon receptor nuclear translocator-like
-2.0
-1.6
-1.2
Rorc
NM_011281
RAR-related orphan receptor c
-2.3
1.2
-2.8
Rora
NM_013646
RAR-related orphan receptor a
-3.0
-2.6
-1.3
Rhbg
NM_021375
Rh family, B glycoprotein
3.0
1.6
1.4
Abcg5
NM_031884
ATP-binding cassette g5
2.4
1.9
1.2
Abcd2
NM_011994
ATP-binding cassette d2
2.4
1.3
1.4
Apoa4
NM_007468
Apolipoprotein A-IV
2.1
1.4
1.1
Atp1b1
NM_009721
ATPase, Na+/K+ transporting,
1.8
1.2
-1.2
Lcn2
NM_008491
Lipocalin 2
1.7
1.3
2.2
Lcn12
NM_029958
Lipocalin 12
-2.0
-1.3
1.1
Kcnk5
NM_021542
Potassium channel k5
-2.4
-1.4
1.1
Aox3
NM_023617
Aldehyde oxidase 3
-2.6
-2.1
-1.4
Aqp8
NM_007474
Aquaporin 8
-2.9
-2.2
1.1
Mup5
NM_008649
Major urinary protein 5
-3.0
-1.9
1.5
Mup3
NM_001039544
Major urinary protein 3
-3.9
-1.7
1.3
Lcn13
NM_153558
Lipocalin 13
-5.1
-1.5
-1.9
Igfbp1
NM_008341
Insulin-like growth factor binding protein 1
5.7
-1.1
1.1
Transcription
Transport
1
Cell signaling Cish
NM_009895
Cytokine inducible SH2-containing protein
-1.9
-1.4
-3.5
Lphn1
NM_181039
Latrophilin 1
-2.1
1.0
1.2
Mc1r
NM_008559
Melanocortin 1 receptor
-2.8
-1.5
1.2
Ube2o
NM_173755
Ubiquitin-conjugating enzyme e2o
-2.1
-1.3
1.1
Usp9y
NM_148943
Ubiquitin specific protease 9, Y-linked
-2.1
-1.4
1.9
Usp53
NM_133857
Ubiquitin specific protease 53
-2.6
1.0
-1.3
Ubiquitin cycle
38
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Miscellaneous Ccl5
NM_013653
Chemokine (C-C motif) ligand 5
5.9
1.0
1.4
Lrtm1
NM_176920
Leu-rich repeats and transmembrane domains 1
4.3
1.4
1.7
Mt2
NM_008630
Metallothionein 2
3.7
5.9
5.8
Ccrn4l
NM_009834
Carbon catabolite repression 4-like
3.5
1.7
-1.3
Mt1
NM_013602
Metallothionein 1
3.5
2.4
2.1
Nnmt
NM_010924
Nicotinamide N-methyltransferase Growth arrest and DNA-damage-inducible 45
2.4
-1.1
-1.5
Gadd45b
NM_008655
2.3
2.0
1.0
Lpin2
NM_022882
Lipin 2
1.8
1.4
1.2
Keg1
NM_029550
-2.0
-1.6
-1.4
Ugcgl2
AK052077
Kidney expressed gene 1 UDP-glucose ceramide glucosyltransferase-like 2
-2.0
-1.2
1.0
Napsa
NM_008437
Napsin A aspartic peptidase
-2.1
-1.4
1.1
Ccnb1
NM_172301
Cyclin B1
-2.2
-1.3
1.2
Naalad2
NM_028279
N-acetylated alpha-linked acidic dipeptidase 2
-2.3
-1.3
1.2
S100a8
NM_013650
S100 calcium binding protein a8
-2.6
-1.3
-1.4
S100a9
NM_009114
S100 calcium binding protein a9
-2.7
-1.3
-1.5
Selenbp2
NM_019414
Selenium binding protein 2
-5.1
-3.1
-3.7
Fold change indicates ratio of DKO, ROR sg/sg, or ROR -/- over WT. (-) indicates genes down-regulated in ROR-deficient mice. (*) indicates fold change in Sult2a1 expression in livers from a mix of male/female DKO mice as described in Materials and Methods.
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