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Molecular Endocrinology 13 (3): 400-409
Copyright © 1999 by The Endocrine Society

Fibrates Increase Human REV-ERB{alpha} Expression in Liver via a Novel Peroxisome Proliferator-Activated Receptor Response Element

Philippe Gervois1, Sandrine Chopin-Delannoy1, Abdessamad Fadel, Guillaume Dubois, Vladimir Kosykh, Jean-Charles Fruchart, Jamila Najïb, Vincent Laudet and Bart Staels

U.325 INSERM Département d’Athérosclérose (P.G., A.F., G.D., J.-C.F., J.N., B.S.) Institut Pasteur de Lille and The Faculté de Pharmacie Université de Lille II 59019 Lille, France
Endocrino’s Group (S.C.-D.) CNRS UMR 319 Institut de Biologie de Lille 59019 Lille, France
Cardiology Research Complex (V.K.) 721552 Moscow, Russia
E.N.S. (V.L.) 69364 Lyon cedex 07, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fibrates are widely used hypolipidemic drugs that act by modulating the expression of genes involved in lipid and lipoprotein metabolism. Whereas the activation of gene transcription by fibrates occurs via the nuclear receptor peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) interacting with response elements consisting of a direct repeat of the AGGTCA motif spaced by one nucleotide (DR1), the mechanisms of negative gene regulation by fibrates and PPAR{alpha} are largely unknown. In the present study, we demonstrate that fibrates induce the expression of the nuclear receptor Rev-erb{alpha}, a negative regulator of gene transcription. Fibrates increase Rev-erb{alpha} mRNA levels both in primary human hepatocytes and in HepG2 hepatoblastoma cells. In HepG2 cells, fibrates furthermore induce Rev-erb{alpha} protein synthesis rates. Transfection studies with reporter constructs driven by the human Rev-erb{alpha} promoter revealed that fibrates induce Rev-erb{alpha} expression at the transcriptional level via PPAR{alpha}. Site-directed mutagenesis experiments identified a PPAR response element that coincides with the previously identified Rev-erb{alpha} negative autoregulatory Rev-DR2 element. Electromobility shift assay experiments indicated that PPAR{alpha} binds as heterodimer with 9-cis-retinoic acid receptor to a subset of DR2 elements 5' flanked by an A/T-rich sequence such as in the Rev-DR2. PPAR{alpha} and Rev-erb{alpha} bind with similar affinities to the Rev-DR2 site. In conclusion, these data demonstrate human Rev-erb{alpha} as a PPAR{alpha} target gene and identify a subset of DR2 sites as novel PPAR{alpha} response elements. Finally, the PPAR{alpha} and Rev-erb{alpha} signaling pathways cross-talk through competition for binding to those response elements.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fibrates are hypolipidemic drugs that lower plasma cholesterol and triglycerides (1). Fibrates exert their effects primarily via the liver by regulating the expression of several genes implicated in lipid metabolism. On the one hand, fibrates stimulate the expression of the human apo A-I (2), rat lipoprotein lipase (3), rat acyl-CoA synthetase (4), rat acyl-CoA oxidase (5), rat multifunctional enzyme (6), and human muscle-type carnitine palmitoyltransferase I (7) genes in the liver. On the other hand, fibrates repress the expression of the rat apo A-I (8), rat apo A-IV (9), human, rat, and mouse apo C-III (10, 11, 12, 13), rat hepatic lipase (14), and rat lecithin-cholesterol acyl transferase (15) genes in the liver. Fibrates have been shown to activate specific receptors, termed peroxisome proliferator-activated receptors (PPARs), belonging to the nuclear receptor gene superfamily (16, 17, 18). So far, three different PPAR forms, {alpha}, ß({delta}), and {gamma}, have been identified, of which the PPAR{alpha} form mediates the effects of fibrates on liver gene expression (13, 19). After activation, PPARs heterodimerize with the 9-cis-retinoic acid receptor (RXR) and subsequently bind to DNA on specific response elements termed peroxisome proliferator response elements (PPRE), located in regulatory regions of target genes, thereby modulating their transcriptional activity. All PPREs identified so far consist of the juxtaposition of two derivatives of the canonical hexamer sequence PuGGTCA spaced by one nucleotide and commonly called direct repeat 1 (DR1).

Whereas PPAR{alpha} mediates fibrate action on lipoprotein metabolism through PPREs identified in the regulatory sequences of positively regulated genes, the mechanisms of negative gene regulation by fibrates are unclear. Studies using PPAR{alpha} knockout mice demonstrated that PPAR{alpha} is a mediator of the negative regulation by fibrates, at least with respect to the mouse apo A-I and apo C-III genes (13). Fibrates may repress transcription by interfering negatively with the expression and activity of positive transcription factors, such as hepatocyte nuclear factor-4 (HNF-4) (11, 20). However, not all fibrate-regulated genes are under transcriptional control by HNF-4. For instance, although fibrates repress rat apo A-I gene transcription, HNF-4 is not considered to be a major regulator of apo A-I gene transcription (21, 22). Alternatively, fibrates may actively repress transcription by activating a negative transcription factor. Interestingly, we recently identified in the rat apo A-I gene promoter a response element for the nuclear receptor Rev-erb{alpha}, an orphan receptor of the nuclear receptor family that acts as a negative transcription factor (23). Furthermore, we have shown that Rev-erb{alpha} gene expression is induced by fibrates in rat liver, indicating that Rev-erb{alpha} may be a mediator of negative gene transcription by fibrates.

The goal of the present study was to determine whether fibrates also regulate human Rev-erb{alpha} expression and to investigate the molecular mechanisms involved. Our results demonstrate that fibrates increase Rev-erb{alpha} expression in human hepatocytes and in HepG2 cells. Furthermore, we show that the induction of Rev-erb{alpha} gene expression occurs at the transcriptional level in hepatocytes and is mediated by PPAR{alpha}. Finally, we demonstrate that PPAR{alpha} binds to a DR2 site coinciding with the Rev-DR2 site in the human Rev-erb{alpha} promoter (24), which constitutes a novel PPAR{alpha} response element mediating a cross-talk between the PPAR{alpha} and Rev-erb{alpha} pathways.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fibrates Increase Rev-erb{alpha} mRNA Expression and Protein Synthesis in Human Liver Cells
The regulation of Rev-erb{alpha} by fibrates was analyzed in human primary hepatocytes. Treatment of cells with fenofibric acid or Wy 14,643 induced a pronounced increase of Rev-erb{alpha} mRNA levels, whereas control 36B4 mRNA levels did not change (Fig. 1AGo). In addition, in HepG2 cells treatment with Wy 14,643 increased Rev-erb{alpha} mRNA levels in a dose-dependent fashion (Fig. 1BGo). To analyze whether the induction of Rev-erb{alpha} mRNA by fibrates is associated with increased synthesis of Rev-erb{alpha} protein, HepG2 cells were cultured for 24 h in the presence of Wy 14,643 or vehicle, labeled with 35S-methionine, and Rev-erb{alpha} was subsequently immunoprecipitated. Compared with control, treatment with Wy 14,643 resulted in a significant increase in Rev-erb{alpha} protein synthesis (Fig. 1CGo). By contrast, as a control, apolipoprotein E secretion was not influenced by fibrate treatment (data not shown). These experiments demonstrate that fibrates increase Rev-erb{alpha} mRNA levels as well as protein synthesis in human hepatocytes.



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  		Figure 1. Fibrates Increase Rev-erb{alpha} mRNA Expression and Protein Synthesis in Primary Human Hepatocytes and HepG2 Cells

Total RNA (10 µg) was subjected to Northern blot analysis using hRev-erb{alpha} (top panel) or 36B4 (bottom panel) cDNA probes as described in Materials and Methods. A, Human hepatocytes were isolated and treated for 24 h with 100 µM fenofibric acid, 50 µM Wy 14,643, or vehicle (DMSO). B, HepG2 cells were treated for 24 h with increasing concentrations of Wy 14,643 (0, 10, 50, 100, 150, 200, 250, 500 µM). C, HepG2 cells were treated for 24 h with 500 µM Wy 14,643 or DMSO. Left, RNA analysis from the same plates used for immunoprecipitation experiments. Right, Cell lysates were subjected to immunoprecipitation using serum depleted of anti-Rev-erb{alpha} antibody (A83 ads) or polyclonal anti-Rev-erb{alpha} antibody (A83 spe) as described in Materials and Methods.

 
Fibrate Induction of Rev-erb{alpha} Gene Expression Occurs at the Transcriptional Level via PPAR{alpha} Interacting with the Rev-DR2 Site of the Human Rev-erb{alpha} Promoter
To investigate whether the effect of fibrates on Rev-erb{alpha} expression occurred at the transcriptional level, the 1.7 kb containing Rev-erb{alpha} promoter was transiently transfected in HepG2 cells in the presence of a human PPAR{alpha} expression vector (pSG5hPPAR{alpha}) or empty vector (pSG5) (Fig. 2AGo). Rev-erb{alpha} promoter-driven luciferase activity increased significantly after cotransfection with PPAR{alpha}, an effect that was increased in the presence of fenofibric acid (Fig. 2AGo), indicating that Rev-erb{alpha} gene transcription is increased by PPAR{alpha}. Two putative nuclear receptor-binding sites containing AGGTCA-like motifs were previously identified in the human Rev-erb{alpha} promoter and called distal (Rd) and proximal sites (Rp) (24). To delineate whether one of these putative binding sites mediated PPAR{alpha} transactivation, unilateral deletion and site- directed mutagenesis experiments were performed. Hence, a 0.7-kb 5'-deletion of the Rev-erb{alpha} promoter containing only the Rp site was transfected in the presence or absence of human PPAR{alpha} (Fig. 2AGo). This deleted Rev-erb{alpha} promoter construct was induced by PPAR{alpha}. Since both the 1.7-kb and 0.7-kb Rev-erb{alpha} promoter constructs responded to the same extent to PPAR{alpha}, we hypothesized the existence of a PPRE located near the Rp site of the human Rev-erb{alpha} promoter (24). Thus, to determine the role of this site in the transcriptional regulation of Rev-erb{alpha} by PPAR{alpha}, we explored the influence of PPAR{alpha} on various mutations around this region (Fig. 2AGo). Mutations affecting either the 5'-AGGTCA motif (pGL2hRev-erb{alpha} {Delta}) or the A/T-rich region (pGL2hRev-erb{alpha} CCC) of the Rp site resulted not only in a loss of Rev-erb{alpha} promoter inductibility by PPAR{alpha}, but also in an increase in baseline reporter activity (Fig. 2AGo). These results indicate that the PPAR{alpha} response element colocalizes with the proximal Rev-erb{alpha} binding site, referred to as Rev-DR2 (24).



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  		Figure 2. Fibrate Induction of Rev-erb{alpha} Gene Expression Occurs at the Transcriptional Level via PPAR{alpha} Interacting with the Rev-DR2 Site of the Human Rev-erb{alpha} Promoter

A, Effects of PPAR{alpha} on the expression of human Rev-erb{alpha} promoter containing a wild-type or mutated Rev-DR2. B, Effects of PPAR{alpha} on wild-type or mutated human Rev-erb{alpha} Rev-DR2 cloned in two copies upstream of the heterologous SV40 promoter. Luc, luciferase reporter gene. HepG2 cells were transfected with the indicated reporter constructs, in the presence of cotransfected pSG5hPPAR{alpha} or pSG5 vector. Cells were treated with fenofibric acid (FF) (10 µM) or vehicle (DMSO), and luciferase activity was measured as described in Materials and Methods. The Rev-DR2 half-site direct repeat sequences are indicated by arrows. Rd and Rp putative nuclear receptor-binding sites are shown as solid and shaded boxes, respectively. The mutated nucleotides in the Rp site are underlined.

 
To ascertain that the Rev-DR2 site could function as a PPAR-responsive element, we performed transient transfection experiments using wild-type and mutated versions of the Rev-DR2 site cloned in front of the heterologous SV40 promoter (Rev-DR2 SV40, M5'Rev-DR2 SV40, and M3'Rev-DR2 SV40) (Fig. 2BGo). Upon cotransfection with pSG5hPPAR{alpha} in HepG2 cells, it was evident that the Rev-DR2 could transmit PPAR{alpha} responsiveness to the heterologous SV40 promoter, an effect that was enhanced in the presence of fenofibric acid. By contrast, PPAR{alpha} did not activate the SV40 promoter. Furthermore, PPAR{alpha} did not induce the activity of the SV40 promoter driven by the Rev-DR2 site mutated in its 5'-half-site (Fig. 2BGo), confirming the importance of this motif in the structure of the PPAR{alpha}-responsive element. Interestingly, mutation of the 3'-half-site of the DR2 also abolished transactivation by PPAR{alpha} (Fig. 2BGo), indicating that the 3'-AGGTCA half-site is also implicated in mediating induction of human Rev-erb{alpha} gene transcription by PPAR{alpha}. Taken together, these data strongly argue that the human Rev-erb{alpha} promoter contains a bona fide PPAR-responsive element that coincides with the Rev-DR2 site, which is constituted of two AGGTCA motifs separated by two nucleotides (DR2) and 5'-flanked by an A/T-rich region (Rev-DR2).

PPAR Binds as a Heterodimer with RXR to a DR2 Site Containing an A/T-Rich 5'-Flanking Region, but Not to a Standard DR2 Site
To investigate direct interaction of PPAR{alpha} with the Rev-DR2 site, we performed electromobility shift assays (EMSAs) using in vitro synthesized PPAR{alpha} and RXR{alpha} protein. RXR{alpha} or PPAR{alpha} alone did not bind to the Rev-DR2 site oligonucleotides (Fig. 3BGo, lanes 10–12 and 14 and lanes 15–17 and 19). Furthermore, PPAR{alpha} did not bind to an oligonucleotide containing a monomer binding site for Rev-erb{alpha} (G8A) (Fig. 3BGo, lane 18) (24), indicating that PPAR{alpha} cannot bind as a monomer. By contrast, binding was observed when PPAR{alpha} was incubated in the presence of RXR with the Rev-erb{alpha} promoter Rev-DR2 as well as the consensus Rev-DR2 sites (direct repetition of the AGGTCA motif separated by two nucleotides), which both contain an A/T-rich region at their 5'-extremities (Fig. 3BGo, lanes 5 and 9). This binding was specific since it was competed out by excess of unlabeled oligonucleotide (Fig. 3Go, panel C, lanes 4–8, and panel D, lanes 4–8).



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  		Figure 3. PPAR{alpha} Binds as a Heterodimer with RXR{alpha} to a DR2 Site Containing an A/T-Rich 5'-Flanking Region, but Not to a Standard DR2 Site

A, Sequences of the different response elements used as probes or as competitors. The 5'-flanking A/T-rich region and half-site sequences are indicated. The mutated sequences are underlined. B, Gel retardation assays were performed on the indicated end-labeled oligonucleotides in the presence of in vitro translated hPPAR{alpha} and mRXR{alpha} or unprogrammed lysate (Unprog. L) (B–E). Competition experiments for binding of PPAR{alpha}/RXR{alpha} to the human Rev-erb{alpha} promoter Rev-DR2 (prom Rev-DR2) (C) or consensus Rev-DR2 (cons Rev-DR2) (D) and (E) oligonucleotides were performed with 10- and 100-fold molar excess of indicated cold oligonucleotide. PPAR/RXR heterodimer or Rev-erb{alpha} complexes are indicated by arrows.

 
Next, we characterized the structural requirements for PPAR/RXR binding by performing competition EMSAs on the promoter (Fig. 3CGo) as well as on the consensus (Fig. 3DGo) oligonucleotides. PPAR/RXR binding to wild-type promoter Rev-DR2 (Fig. 3CGo, lane 4) or to consensus Rev-DR2 (Fig. 3DGo, lane 4) oligonucleotides could not be competed either by the promoter Rev-DR2 site carrying a mutation in the 5'-half-site (M5') (24) or in the 3'-half-site (M3') (Fig. 3Go, C and D, lanes 9, 11, and 12). Interestingly, at high excess (100-fold) of competitor, the 3'-half-site-mutated oligonucleotide started competing for PPAR/RXR binding, indicating that the 3'-part of the Rev-DR2 site is of lesser importance (Fig. 3Go, C and D, lane 10). Similar results were obtained with an oligonucleotide containing the promoter Rev-DR2 site completely lacking the second half-site (1/2A) (Fig. 3Go, C and D, lanes 13 and 14), whereas an oligonucleotide lacking the promoter Rev-DR2 A/T-rich sequence and 5'-half-site (1/2B) did not compete at all (Fig. 3Go, C and D, lanes 15 and 16). These results indicate that 5'- and 3'-half-sites are both implicated in PPAR/RXR binding, with the 5'-half-site, to which PPAR{alpha} presumably binds (25), being most important.

To investigate the role of the 5'-A/T-rich flanking sequence in PPAR/RXR binding to a DR2 site, competition experiments were performed with oligonucleotides in which the 5'-flanking sequence was substituted by C nucleotides (9–11C and 7–9C; Fig. 3AGo). Neither 9–11C nor 7–9C oligonucleotides competed for PPAR/RXR binding to the consensus Rev-DR2 sequence (Fig. 3EGo), indicating absolute requirement of the 5'-A/T-rich flanking sequence for PPAR/RXR binding to a DR2 site.

To ensure that Rev-DR2 sites are high-affinity response elements for PPAR{alpha}, we compared the relative affinities of PPAR/RXR binding to either DR1 or Rev-DR2 sites by competition EMSA (Fig. 4Go). Using oligonucleotides labeled to similar specific activities and under identical experimental conditions, a higher intensity shift with PPAR/RXR was obtained on the Rev-DR2 oligonucleotide compared with the naturally occurring DR1 PPRE site of the human apo A-II promoter, which has been shown to drive its regulation by fibrates (26) (Fig. 4Go, lanes 1 and 10). When increasing amounts of unlabeled oligonucleotide were added, cold Rev-DR2 oligonucleotide competed more efficiently than cold DR1 oligonucleotide for binding of PPAR/RXR to the Rev-DR2 site (Fig. 4Go, lanes 2–5 and 6–9). Reciprocally, binding of PPAR/RXR to labeled DR1 oligonucleotide was more rapidly competed by cold Rev-DR2 than by cold DR1 oligonucleotide (Fig. 4Go, lanes 11–14 and 15–18).



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  		Figure 4. PPAR{alpha} Binds with Similar Affinities to Natural DR2 and DR1 PPAR Response Element

Gel retardation assays were performed on end-labeled RevDR2 (prom Rev-DR2) and apo A-II PPRE DR1 oligonucleotides (29 ) in the presence of in vitro transcribed/translated PPAR{alpha} and RXR{alpha} protein. Competition experiments were performed by adding 1-, 5-, 10-, and 50-fold molar excess of indicated oligonucleotide.

 
These binding experiments demonstrate that PPAR{alpha} binds as a heterodimer with RXR, but not as monomer, to DR2 sites containing a Rev-erb{alpha}-type 5'-flanking region and that Rev-DR2 constitutes a novel PPAR{alpha}-binding site of higher affinity than the natural apo A-II DR1 PPRE site.

Rev-DR2 Mediates a Cross-Talk between PPAR{alpha} and Rev-erb{alpha}
To test directly whether PPAR{alpha} and Rev-erb{alpha} could functionally compete on a Rev-DR2 element, transient cotransfection experiments were performed. As expected, Rev-erb{alpha} was able to repress Rev-DR2-driven SV40 promoter activity (24) (Fig. 5AGo). Cotransfection of PPAR{alpha} in increasing proportions against a constant amount of Rev-erb{alpha} led to a progressive abolishment of Rev-erb{alpha}-mediated repression resulting in a transcriptional activation of the reporter gene at a 3:2 ratio of PPAR{alpha} to Rev-erb{alpha}, respectively, as evidenced by Western blot analysis of transfected cell extracts (Fig. 5AGo and inset). Furthermore, in the absence of cotranfected Rev-erb{alpha}, reporter transcription activity was even further enhanced by PPAR{alpha} (Fig. 5AGo). Thus, PPAR{alpha} and Rev-erb{alpha} are able to functionally cross-compete for the same Rev-DR2 element.



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  		Figure 5. Rev-DR2 Mediates a Cross-Talk between PPAR{alpha} and Rev-erb{alpha}

A, PPAR{alpha} relieves the repressive activity of Rev-erb{alpha} on Rev-DR2-driven transcription. HepG2 cells were transfected with the Rev-DR2 SV40pGL2 reporter construct (Fig. 2BGo), in the presence of pSG5hPPAR{alpha} and/or pSG5hRev-erb{alpha} expression vector. Increasing amounts of pSG5hPPAR{alpha} (0.5-fold, 1-fold, 1.5-fold) were added to a constant amount of pSG5hRev-erb{alpha}. Cells were treated with fenofibric acid (FF) (10 µM) or vehicle (DMSO), and luciferase activities were measured as described in Materials and Methods and expressed relative to control set as 1. Proteins extracted from HepG2 cells transfected with Rev-erb{alpha} and 1.5-fold excess of PPAR{alpha} were analyzed by immunoblotting (inset). B, Competition experiments for binding of PPAR{alpha}/RXR{alpha} or Reverb{alpha} to the human Rev-erb{alpha} promoter Rev-DR2 (prom Rev-DR2) oligonucleotide. Competition was performed with 1-, 5-, 10-, and 50-fold molar excess of indicated cold oligonucleotide. PPAR/RXR heterodimer and Rev-erb{alpha} monomer or homodimer complexes are indicated by arrows.

 
Finally, to estimate the relative affinities of PPAR/RXR and Rev-erb{alpha} binding to a Rev-DR2 site, EMSAs were performed using Rev-DR2 as probe (Fig. 5BGo). As expected, PPAR/RXR formed a heterodimeric complex whereas Rev-erb{alpha} bound both as monomer and as heterodimer (Fig. 5BGo, lanes 1 and 6). When competition was performed using cold Rev-DR2 oligonucleotide, PPAR/RXR binding decreased in a manner similar to Rev-erb{alpha} monomer (Fig. 5BGo, compare lanes 2–5 and 7–10). However, Rev-erb{alpha} homodimer binding appeared slightly more sensitive to competition with unlabeled Rev-DR2 (Fig. 5BGo, lanes 7–10). Altogether, these results indicate that PPAR/RXR binds to Rev-DR2 sites with similar affinity as Rev-erb{alpha} monomer, whereas Rev-erb{alpha} homodimers appear to bind with higher affinity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present report we studied the regulation of Rev-erb{alpha} by fibrates in human liver cells and the molecular mechanisms involved. Our results on human primary hepatocytes and HepG2 cells demonstrate that fibrates induce Rev-erb{alpha} mRNA expression, an effect that is associated with induction of Rev-erb{alpha} protein synthesis in HepG2 cells. In addition, transfection studies revealed that the regulation of Rev-erb{alpha} expression by fibrates occurs at the transcriptional level via PPAR{alpha}. Using deleted and mutated Rev-erb{alpha} promoter constructs, we localized the fibrate-responsive region in the human Reverb{alpha} promoter to the previously identified negative Rev-erb{alpha} autoregulation site. Moreover, mutations in the Rev-DR2 site of the Rev-erb{alpha} promoter abolished basal Rev-erb{alpha}-mediated repression as well as PPAR{alpha}-mediated activation. EMSA experiments proved that fibrate signaling occurs through direct interaction of PPAR/RXR heterodimers to the Rev-DR2 site of the human Rev-erb{alpha} promoter. Since all PPREs described so far consist of the juxtaposition of the degenerated hexamer AGGTCA sequence separated by one nucleotide (DR1) (4, 6, 11, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38), these results represent the first demonstration of a DR2 site as a PPAR-responsive element. Interestingly, a specific structure of the DR2 is required for high-affinity PPAR/RXR binding. In addition to the 5'- and 3'-AGGTCA half-sites, the 5'-flanking region is required for binding of PPAR/RXR to a DR2 site. Thus, several fundamental characteristics of protein-DNA interaction, such as the contact of the receptor with the 5'-A/T-rich flanking sequences of the response element, are conserved among a number of the superfamily members. Taken together, our data suggest that nuclear receptors are more flexible for recognition of responsive elements than previously anticipated.

Rev-erb{alpha} belongs to a subfamily of orphan receptors that are repressors of target gene transcription (for review see Ref. 39). Rev-erb{alpha} appears to be ubiquitously expressed (40, 41), but its functions are ill defined. Several observations suggest a role for Rev-erb{alpha} in metabolic control and energy homeostasis. First, Rev-erb{alpha} mRNA levels increase during differentiation of preadipocytes into adipocytes (42). Second, Rev-erb{alpha} has been suggested to act as a modulator of thyroid hormone signaling (40, 41, 43, 44). Indeed, Rev-erb{alpha} has been shown to bind a subset of thyroid hormone-response elements (45). Interestingly, a significant level of cross-talk exists also between peroxisome proliferator and thyroid hormone-signaling pathways (46, 47, 48, 49, 50, 51, 52). Our present data identify Rev-erb{alpha} as a fibrate target gene and reveal the existence of cross-talk between the PPAR{alpha} and Rev-erb{alpha}-signaling pathways. This cross-talk is governed via two mechanisms (see Fig. 6Go for overview). First, PPAR{alpha} induces Rev-erb{alpha} expression by interfering with the negative autoregulatory loop of Rev-erb{alpha} expression via the Rev-DR2 site. Therefore, genes regulated by Rev-erb{alpha}, such as N-myc (53) and rat apo A-I (23), will be negatively regulated by PPAR{alpha} via an indirect mechanism. Second, PPAR{alpha} and Rev-erb{alpha} may compete for binding to similar DR2 sites. Rev-erb{alpha} itself is an example of a gene containing a response element recognized by both PPAR{alpha} and Rev-erb{alpha}. Hence, target genes containing Rev-DR2 sequences to which PPAR{alpha}, as heterodimer with RXR, and Rev-erb{alpha} compete for binding will be derepressed by fibrates. By contrast, genes carrying monomeric Rev-RE, to which Rev-erb{alpha} binds exclusively as monomer, will be further repressed after fibrate treatment. Therefore, whether a gene will be predominantly regulated by PPAR{alpha} or Rev-erb{alpha} will depend on the relative levels of ligands for each receptor, the relative concentrations of each receptor, and on the structure of the target gene DR2 sequence that determines the relative binding affinities of PPAR{alpha} and Rev-erb{alpha}. The fact that PPAR{alpha} and Rev-erb{alpha} bind to similar DR2 subset sites is most likely due to the similarity of their T/A boxes (25), which are involved in the recognition of the 5'-half-site extension (24, 45, 54, 55, 56). Therefore, PPAR{alpha} could repress genes by inducing Rev-erb{alpha} while simultaneously activating its own target genes via either DR1 or DR2 sites.



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  		Figure 6. Scheme Describing the Mechanism Implicated in the Regulation of Rev-erb{alpha} Expression by Fibrates

It is conceivable that an equilibrium exists between inducible activation of the Rev-erb{alpha} promoter by PPAR/RXR heterodimer and its repression by Rev-erb{alpha}. This allows a fine tuning of Rev-erb{alpha} mRNA and protein levels and hence of Rev-erb{alpha} target gene expression.

 
In a previous study, we demonstrated that differences between human and rat apo A-I gene regulation in response to fibrates are due to a combination of two distinct mechanisms implicating the nuclear receptors PPAR{alpha} and Rev-erb{alpha} (23). Our data indicated that the species-distinct regulation of apo A-I gene expression by fibrates is due to sequence differences in cis-acting elements. In man, apo A-I transcription is induced via PPAR{alpha} binding to a positive PPRE located in the A site footprint (2). This site is not conserved in rats, resulting in a lack of binding of PPAR to the rat apo A-I A promoter site. By contrast, rat apo A-I gene transcription is repressed by Rev-erb{alpha}, the expression of which is induced by fibrates and which binds to a Rev-RE site adjacent to the TATA-box in the rat, but not in the human apo A-I gene promoter. The identification of rat apo A-I as a target gene for Rev-erb{alpha} suggests an implication of Rev-erb{alpha} in lipoprotein metabolism (23). Although the rat apo A-I Rev-RE is not conserved in man, Rev-erb{alpha} expression is controlled by fibrates both in rats and in man, which may point to a role for this nuclear receptor as a modulator in lipid and lipoprotein metabolism and possibly in atherosclerosis susceptibility in both species. It will be of interest, therefore, to identify target genes involved in lipid metabolism that are also under control of Rev-erb{alpha} in man.

In conclusion, our data indicate that the human Rev-erb{alpha} gene is regulated at the transcriptional level by fibrates in liver. Furthermore, this regulation is mediated by PPAR{alpha}, which binds to a novel response element consisting of a 5'-A/T-rich preceded DR2 sequence. Finally, we provide evidence that PPAR{alpha} and Rev-erb{alpha} bind to the same regulatory site, indicating the existence of a cross-talk between PPAR{alpha} and Rev-erb{alpha}-signaling pathways.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
Human hepatocytes, isolated by collagenase perfusion, and HepG2 cells were cultured exactly as described previously (12).

RNA Analysis
RNA extraction and Northern blot analysis were performed as described (3) using human Rev-erb{alpha} (43) and human acidic ribosomal phosphoprotein 36B4 (57) cDNA probes.

Construction of Recombinant Plasmids and Transfection
Cloning of the human Rev-erb{alpha} promoter fragments into pGL2 promoterless or SV40pGL2 reporter vectors (Promega, Madison, WI) and site-directed mutagenesis of Rev-erb{alpha} response elements were as described (24). Human hepatoma HepG2 cells were obtained from European Collection of Animal Cell Culture (Porton Down, Salisbury, UK). Cells were grown in DMEM, supplemented with 2 mM glutamine and 10% (vol/vol) FCS, in a 5% CO2 humidified atmosphere at 37 C. Stimuli were dissolved in dimethylsulfoxide (DMSO). Control cells received vehicle only. All transfections were performed with a mixture of plasmids containing reporter (2 µg) and expression vectors (0.3 to 1 µg). The luciferase activity in cell extracts was determined using a luciferase assay system (Promega) following the supplier’s instruction. Transfection experiments were performed in triplicate and repeated at least three times.

In Vitro Translation and EMSAs
pSG5hPPAR{alpha}, pSG5mRXR{alpha}, and pSG5hRev-erb{alpha} were in vitro transcribed with T7 polymerase and translated using the rabbit reticulocyte lysate sytem (Promega). EMSAs with Rev-erb{alpha}, PPAR{alpha}, and/or RXR{alpha} were performed exactly as described previously (2, 58). For competition experiments, increasing amounts of indicated cold probe were added just before the labeled oligonucleotide. The complexes were resolved on 5% polyacrylamide gels in 0.25x TBE buffer (90 mM Tris-borate, 2.5 mM EDTA, pH 8.3) at 4 C. Gels were dried and exposed overnight at -70 C to x-ray film (XOMAT-AR, Eastman Kodak, Rochester, NY).

Coimmunoprecipitation from Cell Extracts
HepG2 cells incubated in DMEM + 0.2% BSA were treated with fenofibric acid (0.5 mM) or vehicle (DMSO) for 24 h. Cells were subsequently washed in PBS and incubated in methionine-free DMEM supplemented with 35S-labeled methionine (0.1 mCi/ml medium) for 5 h. Cells were lysed in 1 ml RIPA buffer [20 mMTris, pH 7.5, 150 mM sodium chloride, 2 mM EDTA, 1% (wt/vol) sodium deoxycholate, 1% (vol/vol) Triton X-100, 0.25% (wt/vol) SDS]. Lysates were centrifuged at 100,000 x g for 30 min, and the supernatant was subsequently incubated with polyclonal anti-Rev-erb{alpha} antibody (S. Chopin-Delannoy and V. Laudet, manuscript in preparation) overnight at 4 C in RIPA buffer. Immune complexes were collected using protein A-Sepharose (Pharmacia, Piscataway, NJ) and washed six times in RIPA buffer. Protein complexes were separated on 10% SDS-polyacrylamide gels under reducing conditions. Gels were dried and exposed at -70 C to BIOMAX-MS film (Kodak).


   ACKNOWLEDGMENTS
 
We thank Olivier Chassande, Jean-Marc Vanacker, and Franck Delaunay for critical reading.


   FOOTNOTES
 
Address requests for reprints to: Dr. Bart Staels, U.325 INSERM, Département d’Athérosclérose, Institut Pasteur, 1 Rue Calmette, 59019 Lille, France. E-mail Bart.Staels{at}pasteur-lille.fr

This research was sponsored by grants from INSERM, Fondation pour la Recherche Médicale, and the Région Nord-Pas de Calais.

1 Both authors have equally contributed to this work. Back

Received for publication July 13, 1998. Revision received November 5, 1998. Accepted for publication November 23, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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