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Endocrinology Vol. 141, No. 10 3799-3806
Copyright © 2000 by The Endocrine Society

ARTICLES

Circadian and Glucocorticoid Regulation of Rev-erb{alpha} Expression in Liver1

Inés Pineda Torra, Vladimir Tsibulsky, Franck Delaunay, Régis Saladin, Vincent Laudet, Jean-Charles Fruchart, Vladimir Kosykh and Bart Staels

U.325 INSERM, Département d’Athérosclérose (I.P.T., V.T., R.S., J.-C.F., B.S.), Institut Pasteur, 59019 Lille, and the Faculté de Pharmacie, Université de Lille II, 59006 Lille, France; CNRS UMR 5665 (F.D., V.L.), Ecole Normale Supérieure de Lyon, 69364 Lyon, France; and Institute of Experimental Cardiology (V.K.), Russian Cardiology Complex, Moscow, Russia

Address all correspondence and requests for reprints to: Bart Staels, U.325 INSERM, Institut Pasteur de Lille, 1 Rue Calmette BP245, 59019 Lille, France. E-mail: Bart Staels{at}pasteur-lille.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rev-erb{alpha} [NR1D1], a member of the nuclear receptor superfamily, is an orphan receptor that constitutively represses gene transcription. Rev-erb{alpha} has been shown to play a role in myocyte differentiation and to be induced during adipogenesis. Furthermore, Rev-erb{alpha} is a regulator of lipoprotein metabolism. It was recently shown that Rev-erb{alpha} messenger RNA (mRNA) levels oscillate diurnally in rat liver. Here, we report that the circadian rhythm of Rev-erb{alpha} in liver is maintained in primary cultures of rat hepatocytes. Because glucocorticoids have been shown to regulate other transcription factors with circadian expression, it was furthermore examined whether hepatic Rev-erb{alpha} expression is also regulated by glucocorticoids. Treatment of rats with dexamethasone resulted in a decrease of Rev-erb{alpha} mRNA levels by 70% after 6 h. Furthermore, dexamethasone decreased Rev-erb{alpha} expression in rat primary hepatocytes in a dose-dependent fashion. This effect was mediated by the glucocorticoid receptor because simultaneous addition of the glucocorticoid antagonist RU486 prevented the decrease in Rev-erb{alpha} mRNA levels by dexamethasone. Protein synthesis inhibition with cycloheximide markedly induced Rev-erb{alpha} mRNA levels; however, this induction was reduced by dexamethasone supplementation in both rat and human primary hepatocytes. Treatment with actinomycin D blocked the repression of Rev-erb{alpha} expression by dexamethasone in rat hepatocytes, suggesting that glucocorticoids regulate Rev-erb{alpha} expression at the transcriptional level. Transient transfection experiments further indicated that Rev-erb{alpha} promoter activity is repressed by dexamethasone in the presence of cotransfected glucocorticoid receptor. Taken together, these data demonstrate that Rev-erb{alpha} expression is under the control of both the circadian clock and glucocorticoids in the liver.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NUCLEAR RECEPTORS form a large family of ligand activated transcription factors (1). Yet, a number of members of this superfamily have no identified ligand and are referred to as orphan nuclear receptors. Rev-erb{alpha} (NR1D1) (2) together with Rev-erbß (NR1D2) (2) and the Drosophila homolog E75 [NR1D3] (2) belong to this orphan receptor subgroup (3). Rev-erb{alpha} binds either as a monomer to AGGTCA response elements or as a homodimer to a direct repeat of this core motif spaced by two nucleotides (Rev-DR2) preceded by a 5'-A/T-rich sequence (4, 5, 6, 7). Rev-erb{alpha} lacks the activation function (AF-2) present at the carboxy-terminal of the ligand binding domain of nuclear receptors and acts as a transcriptional repressor through direct interaction with the N-CoR family of corepressor proteins (8, 9, 10). Only two Rev-erb{alpha} natural target genes have been identified to date: the human Rev-erb{alpha} gene, which contains a Rev-DR2 site in its regulatory region thereby negatively regulating its own expression (7), and more recently the rat apolipoprotein (apo) A-I gene (11) that contains a monomeric site. In addition, Rev-erbß has been shown to down-regulate N-myc expression through a monomeric site (12).

Rev-erb{alpha} is abundantly expressed in skeletal muscle, brown fat, kidney, heart, brain, and liver (13). Evidence supporting a role for Rev-erb{alpha} in metabolic control and energy homeostasis comes from studies demonstrating that Rev-erb{alpha} messenger RNA (mRNA) expression is markedly induced during adipogenesis (14) and down-regulated during muscle differentiation (15). Furthermore, overexpression of Rev-erb{alpha} in myoblasts abolished differentiation completely (15). In addition to the observation that the rat apo A-I gene is a target for Rev-erb{alpha} (11), studies on a mouse model deficient for Rev-erb{alpha} identified this receptor as a transcriptional modulator of lipoprotein metabolism (16). Moreover, Rev-erb{alpha} is derived from opposite-strand transcription of the thyroid hormone (T3) receptor (TR{alpha}) genomic locus, which itself encodes TR{alpha}1 and the splice variant TR{alpha}2, a dominant negative regulator of T3 signaling (13, 17, 18). Thus a role for Rev-erb{alpha} as a modulator of T3 signaling has been suggested.

With the exception of the aforementioned studies, little is known concerning the regulation of Rev-erb{alpha} gene transcription. We recently reported that hypolipidemic drugs of the fibrate class induce human and rat Rev-erb{alpha} expression in liver through direct binding of the peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) (NR1C1) (2), another member of the nuclear receptor superfamily that mediates fibrate action on lipid and lipoprotein metabolism (19), to the Rev-DR2 site located in the promoter of the human Rev-erb{alpha} gene (20). PPAR{alpha} and Rev-erb{alpha} compete for binding to a subset of DR2 sites, thus featuring a cross-talk between these receptor signaling pathways (20). Previously, PPAR{alpha} mRNA expression was shown to follow a circadian rhythm in the liver (21) and to be transcriptionally regulated by glucocorticoids (22, 23). Other transcription factors display circadian expression patterns in the liver, such as the albumin D-site-binding protein (DBP) (24). Interestingly, DBP expression is under the negative control of glucocorticoids (24) and oscillating mRNA levels of certain hepatic enzymes have been recently shown to be under the control of DBP (25). Recently, it was also shown that Rev-erb{alpha} mRNA levels oscillate diurnally in rat liver (26). In the present study, we report that the circadian rhythm of Rev-erb{alpha} expression is maintained in primary cultures of rat hepatocytes. Furthermore, we investigated whether Rev-erb{alpha} expression is also subject to glucocorticoid hormone regulation in addition to diurnal variation. Our results demonstrate that glucocorticoids down-regulate Rev-erb{alpha} expression at the transcriptional level in a dose-dependent fashion and that this repression is mediated by the glucocorticoid receptor (GR).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Williams’ medium E, fatty acid free-BSA and dexamethasone were from Sigma (St. Louis, MO). DMEM and FCS were from Life Technologies, Inc. (Merelbeke, Belgium). Actinomycin D and cycloheximide were from Roche Molecular Biochemicals (Mannheim, Germany). Mifepristone (RU 486) was a kind gift from Roussel-UCLAF.

Animals and treatment
Male Sprague Dawley rats (150–200 g) were group-housed and had free access to water and food. For the circadian variation experiment, the animals were kept on a 12-h light, 12-h dark cycle (light from 0700–1900 h) for several weeks before they were killed at precise time points: 0400, 0800, 1200, 1600, 2000, and 2400. For each time point, the animals (n = 3) were decapitated under anesthesia. Livers were dissected immediately and frozen in liquid nitrogen. For the dexamethasone experiment, male Sprague Dawley rats received a single sc injection of dexamethasone (3.7 µg/g body mass, Solu-Decadron, Merck Sharp-Dome-Schribet, Paris, France) at 0800 h. Control animals were injected with vehicle. Control and treated animals (n = 3) were killed at 1400 h.

Isolation of rat and human hepatocytes
Rat hepatocytes were isolated by collagenase perfusion of livers from male Sprague Dawley rats as described (27). Hepatocytes (cell viability higher than 85% by trypan blue exclusion test) were cultured as monolayers (3 x 106 cells per 60 mm dish) in Williams’ medium E supplemented with FCS (10% vol/vol), glutamine (2 mM), and antibiotics at 37 C in humidified atmosphere of 5% CO2, 95% air. For the in vitro circadian variation experiment, rat liver was perfused at 1530 h and cells were seeded at 1730 h. After incubation for 4 h, medium was changed to fresh medium, and cells were harvested every 6 h at the indicated times. For the dexamethasone, cycloheximide, and RU 486 experiments, compounds were added to serum-free culture medium supplemented with fatty acid free-BSA (0.2% wt/vol) and cells were harvested 6 h later. For the actinomycin D experiment, cells were cultured for 2 h before the simultaneous addition of dexamethasone and actinomycin D to the medium. Human liver specimens were collected for transplantation at the Moscow Center and hepatocytes isolated as previously described (28). Donors were physically healthy people who died after traumatic brain injury. Permission to use the remaining untransplanted donor livers for scientific research purposes was obtained from the Ministry of Health of the Russian Federation. The procedure of hepatocyte isolation is described in detail elsewhere (29). For the dexamethasone experiment, hepatocytes isolated from a 30-yr-old man were incubated for 24 h before medium was changed and cells were treated with dexamethasone for 24 h in serum-free medium. For the cycloheximide experiment, hepatocytes isolated from an 18-yr-old woman were seeded, and 6 h later medium was replaced by serum-free medium containing compounds and cells were further incubated for 20 h. In this experiment, 6 h of preculture were enough for hepatocyte attachment to the cell culture dishes and for the formation of a confluent monolayer of cells. Cells were lysed in a guanidium-thiocyanate buffer and extracts were kept at -80 C before total cellular RNA was extracted as described (30).

RNA analysis
Northern blot analysis was performed as described (31) using human Rev-erb{alpha} (32), rat Rev-erb{alpha} (13), acidic ribosomal phosphoprotein 36B4 (33) and rat 28S (34) complementary DNA probes. Filters were hybridized to 1 x 106 cpm/ml of each probe as described (35) except Rev-erb{alpha} hybridizations that were performed using the Expresshyb Hybridization Solution (CLONTECH Laboratories, Inc., Palo Alto, CA) according to the manufacturer’s instructions. When indicated, ribosomal RNA was stained on the filters with methylene blue before hybridization to assess equal RNA loading and transfer (36).

Transient transfection experiments
The human hepatoma HepG2 cells (80 x 104 cells per well on a 24-well dish) were transfected using a cationic lipid, RPR 120535B, (kind gift of Aventis, Vitry, France) with a mixture of plasmids containing 100 ng of either the luciferase reporter pGL2 basic plasmid (Promega Corp., Madison, WI) or the pGL2 vector driven by a 1.7-kb fragment of the human Rev-erb{alpha} promoter with a mutated 5' of the Rev-DR2 site (pGL2 hRev-erb{alpha} {Delta}) (7), 1–100 ng of the GR{alpha} expression vector pMThGR{alpha} (37) and 100 ng of the pGKßgeobpA (38) as an internal control for transfection efficiency. All samples were complemented with pBSKS plasmid (Stratagene, La Jolla, CA) to a total amount of DNA (500 ng). After 2 h, cells were incubated with dexamethasone (10-6 M) or vehicle in medium containing 2% Ultroser (Biosepra SA, Villeneuve la Garenne, France). Luciferase and ß-galactosidase activities were assayed 36 h later. Transfection experiments were performed in triplicate and repeated at least three times.

Statistical analysis
Significant differences between groups were examined by the Mann-Whitney test. A value of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rev-erb{alpha} expression follows a circadian rhythm in liver in vivo and in vitro
To determine whether Rev-erb{alpha} expression exhibits significant circadian oscillations, rats kept on a 12-h light, 12-h dark cycle were killed at selected time points of the day. Rev-erb{alpha} mRNA levels oscillated during the day (Fig. 1Go, A and B), peaking at 1600 h and already decreasing 4 h later to reach a nadir at 2400 h. Similar results were obtained when the rats were fasted overnight before they were killed (data not shown), indicating that this RNA fluctuation occurs independently of food intake. These observations are in agreement with the data reported by Basalobre A. et al. (26). To evaluate whether Rev-erb{alpha} levels oscillate in primary cultures, Rev-erb{alpha} expression was examined in primary rat hepatocytes during a 52-h period after isolation. Rev-erb{alpha} mRNA oscillated in time, reaching a peak 10, 34, and 52 h after the cells were seeded (Fig. 1CGo). Therefore, primary rat hepatocytes retain the signals required for Rev-erb{alpha} to cycle. The period length of Rev-erb{alpha} mRNA first cycle in vitro was approximately of 24 h, which was similar to the circadian period observed in vivo (Fig. 1DGo). However, in the second cycle (34–52 h) the period seemed to shorten.



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  		Figure 1. Rev-erb{alpha} mRNA expression follows a circadian rhythm in rat liver and in rat primary hepatocytes. Total RNA (10 µg) was subjected to Northern blot analysis and hybridized to the indicated probes. A, Animals were killed at the indicated times as described in Materials and Methods. The light regimen of the animal house is depicted on top, black bars indicating hours of darkness. B, Graphic representation of oscillating Rev-erb{alpha} mRNA levels normalized to control 36B4 mRNA. The highest normalized values were arbitrarily set as 100. Results are the mean ± SD of three animals. C, Rat primary hepatocytes were cultured as described in Materials and Methods and harvested at the indicated times after the cells were seeded. D, 28S mRNA expression was measured and Rev-erb{alpha} mRNA levels were normalized to those of 28S and plotted relative to the time of culture. The highest normalized values were arbitrarily set as 100. Each point represents mean ± SD, n = 3. Statistically significant difference between Rev-erb{alpha} mRNA levels at 22 h (where levels are lowest) and the remainder time points is indicated by an asterisk. *, P < 0.05.

 
Dexamethasone regulates Rev-erb{alpha} gene expression in liver and in primary hepatocytes
It has been previously reported that some hepatic transcription factors displaying circadian expression patterns, such as PPAR{alpha} or DBP, are also regulated by glucocorticoids (21, 22, 23, 24). In view of the findings presented above, we hypothesized that Rev-erb{alpha} expression could be regulated by glucocorticoids. To test this, rats received a single injection of dexamethasone in the morning (0800 h) and 6 h later (1400 h) liver samples were dissected and Rev-erb{alpha} expression was analyzed. Basal Rev-erb{alpha} mRNA levels at the time the rats were killed were already high as expected from its circadian expression profile (Fig. 2AGo). Dexamethasone markedly down-regulated Rev-erb{alpha} expression by 70% (Fig. 2Go, A and B). To examine whether glucocorticoids act directly on the hepatocyte, Rev-erb{alpha} expression was examined in rat primary hepatocytes treated with increasing concentrations of dexamethasone. After incubation of the hepatocytes with various doses of dexamethasone for 6 h, a dose-dependent decrease of Rev-erb{alpha} mRNA levels was observed (Fig. 2Go, C and D). Low concentrations of dexamethasone already diminished Rev-erb{alpha} expression by 30%, the repression being maximal at the highest dexamethasone concentration used (up to 80%). Thus, glucocorticoids influence hepatic Rev-erb{alpha} expression by acting directly on the hepatocyte.



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  		Figure 2. Dexamethasone down-regulates Rev-erb{alpha} expression in rat liver and in rat primary hepatocytes. RNA (10 µg) was analyzed by Northern blot as described in Materials and Methods. A, Rats were injected sc either with vehicle (CON) or with 3.7 µg/g body mass dexamethasone (DEX). Animals were killed 6 h later. Autoradiograph showing Rev-erb{alpha} mRNA was exposed for 24 h. B, Quantification of 36B4-normalized Rev-erb{alpha} mRNA levels in livers of vehicle (CON) or dexamethasone injected (DEX) animals. Results are expressed as a percentage of the vehicle injected rats (means ± SD of three animals). Statistically significant difference between CON and DEX groups is indicated by an asterisk. *, P < 0.05. C, Isolated rat hepatocytes incubated with vehicle or the indicated concentrations of dexamethasone (DEX) for 6 h. Autoradiograph showing Rev-erb{alpha} mRNA was exposed for 6 h. D, Quantification of 36B4-normalized Rev-erb{alpha} mRNA levels in rat hepatocytes treated with either vehicle or various concentrations of dexamethasone, as indicated. Results are expressed as a percentage of vehicle treated cells as means ± SD, n = 3. Statistically significant difference between vehicle (black bar) and dexamethasone (white bars) treated groups is indicated by an asterisk. *, P < 0.05.

 
Dexamethasone down-regulates Rev-erb{alpha} gene expression via the GR
To investigate whether the effects of dexamethasone on Rev-erb{alpha} gene expression occur via the glucocorticoid receptor (GR), rat primary hepatocytes were incubated with dexamethasone in the presence or absence of the GR antagonist RU486. As previously observed, in the presence of increasing concentrations of dexamethasone alone, Rev-erb{alpha} mRNA levels were repressed in a dose-dependent fashion (Fig. 3AGo). RU486 alone did not have a significant effect on Rev-erb{alpha} expression. By contrast, addition of RU486 to dexamethasone-treated hepatocytes almost completely abolished the dexamethasone-induced repression of Rev-erb{alpha} mRNA (Fig. 3Go, A and B). These data indicate that, in rat primary hepatocytes, the GR is involved in the down-regulation of Rev-erb{alpha} gene expression by dexamethasone.



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  		Figure 3. RU486 antagonizes the dexamethasone-induced repression of Rev-erb{alpha} mRNA levels. Total RNA (10 µg) was subjected to Northern blot analysis and hybridized to the indicated probes. A, Rat primary hepatocytes treated with vehicle or various concentrations of dexamethasone as indicated in the presence or absence of 10-5 M RU486. Autoradiograph showing Rev-erb{alpha} mRNA was exposed for 6 h. B, Quantification of 36B4-normalized Rev-erb{alpha} mRNA levels in rat hepatocytes treated as indicated above. Results are expressed as a percentage of vehicle-treated cells as means ± SD, n = 3. Statistically significant difference between dexamethasone and dexamethasone plus RU 486 groups at each dexamethasone concentration is indicated by an asterisk. *, P < 0.05.

 
Dexamethasone down-regulates cycloheximide-induced Rev-erb{alpha} expression
It has been previously reported that Rev-erb{alpha} mRNA levels are induced 100-fold by the protein synthesis inhibitor cycloheximide in 235–1 rat pituitary cells (18). Because a structurally unrelated protein synthesis inhibitor had similar effects on Rev-erb{alpha} expression, the authors concluded that this induction was due to inhibition of the synthesis of one or various labile proteins that constitutively repress Rev-erb{alpha} mRNA levels (18). To determine whether cycloheximide induces Rev-erb{alpha} mRNA in rat hepatocytes, as well as whether dexamethasone could influence this effect, hepatocytes were cultured for 6 h with or without dexamethasone in the presence or absence of cycloheximide. Cycloheximide alone induced Rev-erb{alpha} mRNA levels more than 25-fold over control levels in rat primary hepatocytes (Fig. 4Go, A and B). Furthermore, dexamethasone was still able to decrease cycloheximide-induced Rev-erb{alpha} mRNA levels by 45% (Fig. 4Go, A and B), showing that ongoing protein synthesis is not required for Rev-erb{alpha} mRNA repression thus suggesting that preexist GR mediates this effect.



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  		Figure 4. Dexamethasone down-regulates cycloheximide-induced Rev-erb{alpha} expression. Total RNA (10 µg) was subjected to Northern blot analysis and hybridized to the indicated probes. A, Rat primary hepatocytes treated with vehicle (CON) or 10-6 M dexamethasone (DEX) in the presence or absence of 10 µg/ml cycloheximide (CHX) for 6 h. Autoradiograph showing Rev-erb{alpha} mRNA was exposed for 3 h. B, Graphic representation of Rev-erb{alpha} mRNA levels in rat hepatocytes treated as indicated above normalized to 36B4. Results are expressed as a percentage of vehicle-treated cells as means ± SD, n = 3. Statistically significant difference between CON and DEX, CON and CHX groups, and between CHX and CHX+DEXA groups is indicated by an asterisk. *, P < 0.05.

 
Dexamethasone down-regulates the expression of Rev-erb{alpha} in human primary hepatocytes
Next, we studied whether human Rev-erb{alpha} mRNA expression is also regulated by glucocorticoids. Hence, primary cultures of human hepatocytes were incubated in the presence of dexamethasone for 24 h. Consistent with our previous findings in rat hepatocytes, albeit to a lower extent, a decrease in human Rev-erb{alpha} mRNA levels of about 60% was observed (Fig. 5Go, A and B). The ability of dexamethasone to inhibit the cycloheximide-induced Rev-erb{alpha} expression was also examined in primary human hepatocytes. Cycloheximide induced Rev-erb{alpha} 3-fold (Fig. 5Go, C and D), and upon dexamethasone treatment Rev-erb{alpha} mRNA levels almost decreased to the levels detected in cells treated with vehicle only. Similar results were obtained when experiments were performed in the presence of hydrocortisone (data not shown). Altogether, these observations indicate that both human and rat Rev-erb{alpha} gene expression are regulated by glucocorticoids.



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  		Figure 5. Dexamethasone down-regulates Rev-erb{alpha} gene expression in primary human hepatocytes. Total RNA (10 µg) was subjected to Northern blot analysis using the indicated probes. A, Human hepatocytes were incubated with vehicle or 10-6 M dexamethasone (DEX) for 24 h. Autoradiograph showing Rev-erb{alpha} mRNA was exposed for 72 h. Methylene blue staining of ribosomal RNA was performed to assess equal loading and transfer. B, Quantification of Rev-erb{alpha} mRNA levels in human hepatocytes treated with either vehicle (CON) or dexamethasone (DEX). Results are expressed as a percentage of vehicle treated cells as means ± SD, n = 3. Statistical comparison between CON and DEX groups is shown. *, P < 0.05. C, Human hepatocytes treated with vehicle (CON) or 10-6 M dexamethasone (DEX) in the presence or absence of 10 µg/ml cycloheximide (CHX) for 20 h. Autoradiograph showing Rev-erb{alpha} mRNA was exposed for 72 h. Methylene blue staining of ribosomal RNA was performed to assess equal loading and transfer. D, Graphic representation of Rev-erb{alpha} mRNA levels. Results are expressed as a percentage of vehicle-treated cells as means ± SD, n = 3. Statistical comparisons between CON and CHX and between CHX and CHX plus DEXA groups are shown. *, P < 0.05.

 
Dexamethasone decreases Rev-erb{alpha} expression at the transcriptional level
To investigate whether the effect of glucocorticoids on Rev-erb{alpha} mRNA expression occurs at the transcriptional level, hepatocytes were incubated with dexamethasone in the presence or absence of the RNA polymerase inhibitor actinomycin D. Addition of actinomycin D, together with dexamethasone, inhibited the repression of Rev-erb{alpha} mRNA by dexamethasone (Fig. 6Go), suggesting that ongoing transcription is required for the dexamethasone effect.



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  		Figure 6. The transcriptional inhibitor actinomycin D prevents the dexamethasone-induced repression of Rev-erb{alpha} mRNA levels. RNA (10 µg) was analyzed by Northern blot as described in Materials and Methods. Rat primary hepatocytes preincubated for 2 h and treated thereafter with vehicle (CON) or 10-6 M dexamethasone (DEX) with or without 5 µg/ml actinomycin D (Act D) for 6 h. Due to the overall mRNA down-regulation observed in the actinomycin D-treated cells, Act. D and actinomycin D plus dexamethasone (Act. D + DEX) signals were obtained after exposing the autoradiography for 6 h, whereas vehicle and dexamethasone signals were obtained after 2 h of exposure.

 
Dexamethasone represses human Rev-erb{alpha} promoter activity.
Finally, we examined whether glucocorticoids regulate the activity of the human Rev-erb{alpha} promoter. Rev-erb{alpha} was previously shown to bind to the Rev-DR2 site located in its promoter, thus repressing its own transcription (7). Because this repression may mask that of glucocorticoids, a promoter fragment bearing a mutation in the Rev-DR2 site (pGL2-pRev-erb{alpha} {Delta}) was used to study the repressive effect of dexamethasone on Rev-erb{alpha} transcriptional activity in HepG2 cells. As expected, in the absence of cotransfected GR, dexamethasone did not influence the human Rev-erb{alpha} activity because HepG2 cells do not contain considerable amounts of endogenous GR (Fig. 7Go). In the presence of dexamethasone, cotransfection of increasing concentrations of GR resulted in a dose-dependent repression of up to 50% of Rev-erb{alpha} promoter activity. By contrast, dexamethasone did not influence the activity of the promoterless basic pGL2 vector plasmid, indicating that the repression observed was specific for the Rev-erb{alpha} promoter. These data further demonstrate that the effect dexamethasone exerts on Rev-erb{alpha} expression is due to a repression of Rev-erb{alpha} gene transcription.



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  		Figure 7. Dexamethasone represses human Rev-erb{alpha} promoter activity. HepG2 cells transfected with either the pGL2 basic or the pGL2 hRev-erb{alpha} {Delta} reporter construct in the presence of increasing concentrations (1, 3, 10, 30, and 100 ng) of pMT-hGR{alpha} expression vector. Cells were treated with 10-6 M dexamethasone (DEX) or vehicle (CON) for 36 h. Values (mean ± SD) represent luciferase activity relative to ß-galactosidase activity. The experiment was repeated in triplicate at least three times. Statistical comparisons between dexamethasone treated without GR cotransfection and dexamethasone plus GR cotransfection groups are shown. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Despite the increasing evidence supporting a role for Rev-erb{alpha} in metabolic control and energy homeostasis, little is known concerning the factors regulating the expression of this orphan receptor. In the present study, we aimed to investigate the regulation of Rev-erb{alpha} expression. Our findings are consistent with previously reported observations that hepatic Rev-erb{alpha} expression follows a circadian rhythm (26). Interestingly, the circadian rhythmicity of Rev-erb{alpha} mRNA levels was maintained in vitro in primary hepatocytes kept in culture for more than 2 days, indicating no external signal (e.g. light or food intake) is responsible for the oscillation of Rev-erb{alpha} mRNA levels. Thus, the circadian expression of Rev-erb{alpha} must be controlled by an independent endogenous oscillator present in the hepatocyte. In addition, these primary cells do not proliferate under the culture conditions used, making it unlikely that changes in cell density account for the observed oscillations of Rev-erb{alpha} mRNA expression levels.

In mammals, an internal circadian clock, localized in the suprachiasmatic nuclei (SCN) of the hypothalamus, controls rhythmic physiology and behavior such as hormone secretion, body temperature, sleep-wake cycles, and locomotor activity. The circadian clock is controlled by clock genes that interact to generate a molecular oscillator. This oscillator is synchronized to the external time cues by input signals and regulate peripheral circadian rhythms via output signals (39, 40). The observation that clock genes, are also expressed in peripheral mammalian tissues as well as in explanted tissue cultures (41, 42, 43), strongly suggests that mammals also posses several circadian clocks outside the SCN. The capacity of rat fibroblasts or NIH-3T3 cells to retain circadian properties in culture either after receiving a serum shock or following activation of the MAPK cascade was recently reported (26, 44). Our data showing rhythmic expression of Rev-erb{alpha} in isolated hepatocytes extend these findings to primary hepatocytes and indicate that the circadian clock of isolated cells remain synchronized in vitro in the absence of any treatment probably as a result of in vivo synchronization before isolation. Thus, in the serum-shocked rat fibroblasts experiment reported by Basalobre et al. (26), the serum is likely to synchronize already cycling cells rather than initiating and driving circadian gene expression. Our data show that the period of Rev-erb{alpha} mRNA oscillations in vitro shortens with time, suggesting that the signal for the proper synchronization of the hepatocyte clock might be missing in the culture medium. Synchronizing signals for mammal peripheral circadian clocks have not been identified yet, but it has been suggested that to control the different circadian outputs, the SCN may not only emit separate signals for each one of them but may also send a synchronizing signal to the peripheral clocks that would then be able to autonomously regulate the circadian outputs (45). In zebrafish, peripheral organs such as heart and kidney have been recently shown to contain circadian oscillators that are directly entrained by light (46).

A number of transcription factors follow a diurnal variation in tissues other than the SCN. DBP and the thyrotroph embryonic factor (TEF) have been shown to oscillate diurnally in liver and kidney (24, 47). Furthermore, these transcription factors dictate the hepatic circadian transcription of genes encoding enzymes governing liver metabolic pathways, such as the gene encoding cholesterol 7{alpha}-hydroxylase (CYP7) (47, 48, 49) and the coumarin 7-{alpha} hydroxylase (Cyp2a5) and steroid 15{alpha}-hydroxylase (Cyp2a4) genes (25). Thus, one would anticipate that expression of Rev-erb{alpha} target genes might oscillate diurnally. To date, only a few target genes for Rev-erb{alpha} have been reported. Due to the stability of apo AI mRNA, with a half-life of several hours, it is unlikely that apo AI gene expression is affected by Rev-erb{alpha} diurnal variation. Nevertheless, it is tempting to speculate that other currently unknown Rev-erb{alpha} target genes may be influenced by Rev-erb{alpha} circadian variation.

We initially studied Rev-erb{alpha} expression as a function of circadian time because Rev-erb{alpha} gene expression is regulated by PPAR{alpha} and PPAR{alpha} itself follows a circadian rhythm in liver. However, the amplitude of PPAR{alpha} variation is smaller than that of Rev-erb{alpha} (21) and the phase of Rev-erb{alpha} mRNA oscillation precedes the one of PPAR{alpha} mRNA oscillation by about 2 h (21). Thus Rev-erb{alpha} circadian expression is unlikely to be driven by PPAR{alpha}. Because glucocorticoids regulate the expression of oscillating liver transcription factors such as DBP (24), we evaluated whether the expression of Rev-erb{alpha} is regulated by glucocorticoids. Rev-erb{alpha} mRNA levels were shown to be down-regulated by dexamethasone in the liver and in both rat and human primary hepatocyte cultures. Because Rev-erb{alpha} mRNA levels cycle in vitro both in the presence or absence of glucocorticoids in the culture medium (data not shown), it seems unlikely that glucocorticoids drive the circadian oscillation of Rev-erb{alpha} mRNA but instead could for instance participate in the synchronization of its expression. Alternatively, the regulation of Rev-erb{alpha} by glucocorticoids may reflect a hormonal control independent of the circadian expression of the gene. The effect exerted by dexamethasone on Rev-erb{alpha} expression levels was mediated by GR because incubation of hepatocytes with the glucocorticoid antagonist RU486 abolished the repression of Rev-erb{alpha} gene expression by dexamethasone. Interestingly, dexamethasone decreased cycloheximide-induced Rev-erb{alpha} expression, suggesting that the dexamethasone effect did not require ongoing protein synthesis and that it might be mediated by preexist GR. Notably, the cycloheximide induction of Rev-erb{alpha} mRNA levels in rat hepatocytes was comparable to the one previously reported in 235–1 pituitary and GH3 cells (18). In those cell lines, cycloheximide-induced Rev-erb{alpha} mRNA was shown to be due to both increased transcriptional rate and increased mRNA stability (18). Addition of actinomycin D to cycloheximide-treated rat primary hepatocytes also abrogated the cycloheximide-induced Rev-erb{alpha} expression (data not shown), indicating that in this hepatocyte model the up-regulation of Rev-erb{alpha} expression by cycloheximide is exerted at the transcriptional level. When hepatocytes were incubated in medium containing cycloheximide plus actinomycin D in the presence of dexamethasone Rev-erb{alpha} mRNA levels were similar to the levels observed in cells not treated with dexamethasone (data not shown) that further confirms that mRNA synthesis is essential for the glucocorticoid effect on Rev-erb{alpha} expression. The regulation of Rev-erb{alpha} by glucocorticoids also occurs in humans as dexamethasone significantly represses, albeit to a lower extent, Rev-erb{alpha} mRNA levels in human primary hepatocytes.

In addition to actinomycin D experiments, transient transfection assays using a reporter gene driven by the proximal Rev-erb{alpha} gene promoter demonstrated that glucocorticoids regulate Rev-erb{alpha} expression at the transcriptional level. Several mechanisms by which GR can down-regulate transcription have been documented, and different types of negative glucocorticoid response elements (nGREs) within the promoter of target genes have been identified. First, simple negative GREs that interact directly with the GR without the assistance of other sequence-specific regulators (50, 51). Second, composite GREs (cGREs), which are capable of interacting with the receptor protein as well as other additional factors resulting in either activation or repression (52, 53, 54). Third, tethering GREs, in which GR interferes with transcriptional activators already bound to the DNA. In this case, direct interaction between the GR and the DNA is not required for the GR-mediated repression (55, 56, 57, 58). Fourth, binding of GR to competitive GREs may lead to interference with the basal transcriptional machinery (59). All these possible mechanisms illustrate the complexity of GR signaling. The promoter architecture of the human Rev-erb{alpha} gene has been poorly characterized to date. Only the Rev-DR2 site binding both Rev-erb{alpha} and PPAR{alpha} has been studied in detail (7, 20). Computer-aided analysis of the Rev-erb{alpha} promoter did not identify putative sequences resembling known negative GREs. This suggests that there might be a negative GRE in the Rev-erb{alpha} promoter that is different to those already identified. Further studies will be required to characterize the exact molecular mechanism of Rev-erb{alpha} repression by glucocorticoids.

The physiological consequence of Rev-erb{alpha} regulation by glucocorticoids remains to be elucidated. To date, Rev-erb{alpha} has been involved in processes such as adipogenesis and muscle differentiation. It will be of interest to study the effect of Rev-erb{alpha} repression by glucocorticoids in these processes. Interestingly, apoAI gene expression is negatively regulated by Rev-erb{alpha} and it is transcriptionally induced by glucocorticoids (60). The induction of apo AI gene transcription by dexamethasone requires the presence of GR and a labile cell-specific protein (60). Thus, by lowering the expression of Rev-erb{alpha}, glucocorticoids may indirectly enhance apo AI mRNA levels.

Taken together these data indicate that in addition to the circadian regulation, Rev-erb{alpha} is also negatively regulated at the transcriptional level by glucocorticoids in liver. The variation of Rev-erb{alpha} expression is likely to have important consequences in the downstream physiological and cellular processes governed by Rev-erb{alpha}.


   Acknowledgments
 
We would like to thank the technical assistance of Philippe Poulain.


   Footnotes
 
1 Support of grants from INSERM (PECO-NEI 212806C) (to V.T.) and the European Community (no. ERBFMBICT983214) (to I.P.T.) is kindly acknowledged. Back

Received March 7, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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