©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Regulatory Pathway of Brown Fat Thermogenesis
RETINOIC ACID IS A TRANSCRIPTIONAL ACTIVATOR OF THE MITOCHONDRIAL UNCOUPLING PROTEIN GENE (*)

(Received for publication, November 16, 1994; and in revised form, December 27, 1994)

Rosa Alvarez Javier de Andrés Pilar Yubero Octavi Viñas Teresa Mampel Roser Iglesias Marta Giralt Francesc Villarroya (§)

From the Unitat de Bioquímica i Biologia Molecular B, Departament de Bioquímica i Fisiologia, Universitat de Barcelona, 08028 Barcelona, Spain

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mitochondrial uncoupling protein (UCP) is responsible for the thermogenic function of brown fat, and it is a molecular marker of the brown adipocyte cell type. Retinoic acid (RA) increased UCP mRNA levels severalfold in brown adipocytes differentiated in culture. This induction was independent of adrenergic pathways or protein synthesis. RA stimulated ucp gene expression regardless of the stage of brown adipocyte differentiation. In transient transfection experiments RA induced the expression of chloramphenicol acetyltransferase vectors driven by 4.5 kilobases of the 5`-noncoding region of the rat ucp gene, and co-transfection of expression vectors for RA receptors enhanced the action of RA. Retinoic acid receptor alpha was more effective than retinoid X receptor in promoting RA action, whereas a mixture of the two was the most effective. The RA-responsive region in the ucp gene was located at -2469/-2318 and contains three motifs (between -2357 and -2330) of the consensus half-sites characteristic of retinoic acid response elements. This 27-base pair sequence specifically binds purified retinoic acid receptor alpha as well as related proteins from brown fat nuclei. In conclusion, a novel potential regulatory pathway of brown fat development and thermogenic function has been recognized by identifying RA as a transcriptional activator of the ucp gene.


INTRODUCTION

Brown adipose tissue (BAT) (^1)is the anatomical site for non-shivering thermogenesis. Heat production in the brown adipocyte is caused by the mitochondrial uncoupling protein (UCP), which permeabilizes the mitochondrial inner membrane to protons, thus uncoupling the respiratory chain and oxidative phosphorylation systems(1) . The ucp gene is under strict transcriptional regulation in relation to cell specificity, BAT development, and heat needs(2, 3, 4) . The ucp gene is only expressed in the brown adipocyte, and it constitutes a unique molecular marker that distinguishes this cell type from any other mammalian cell including the white adipocyte(5) . The main pathway of regulation of ucp gene expression described so far relies on the sympathetic nervous system acting upon the BAT in a physiological adaptive response to changes in the environmental temperature and diet (6, 7) . ucp gene transcription is stimulated by the sympathetic nervous system because of the action of norepinephrine on beta-1 and beta-3 adrenergic receptors in the surface of the brown fat cell(8, 9) . cAMP, and probably also T(3), are the main intracellular mediators of the norepinephrine action upon ucp gene transcription(6, 10) . Much less is known about the molecular signals involved in the regulation of ucp gene transcription in relation to cell specificity and differentiation, since they seem to be largely independent of the sympathetic action. During the development of most mammalian species, ucp gene transcription is switched on in late fetal life, and substantial levels of ucp gene expression are attained before birth, when the sympathetic nervous system is not fully developed(4) . Similarly, permanent exposure of brown preadipocytes in culture to adrenergic stimulators does not affect the differentiation-dependent expression of the ucp gene(8) .

The ucp genes of rat, mice, and humans have been isolated and cloned(11, 12, 13) . It has been established that a few thousand base pairs in the 5`-flanking regions of the rat and mice genes contain the main cis-acting regulatory elements of ucp gene transcription, including the elements responsible for cellspecificity and cAMP responsiveness(14, 15) . However, the molecular identity of the main transcription factors involved in regulating ucp gene expression is not known, and only members of the C/EBP family of transcription factors have been reported to transactivate the ucp gene promoter(16) .

The vitamin A-derivative retinoic acid (RA) plays an important role in development and differentiation of mammalian cells, and it is the only putative morphogen molecule identified so far in vertebrates(17, 18) . RA acts through nuclear receptors, which are members of the steroid/thyroid receptor superfamily and which behave as ligand-dependent transcription factors(19, 20) . RA receptors (RARs) bind to elements that activate transcription in response to all-trans-RA and 9-cis-RA, and retinoid X receptors (RXR) bind and activate transcription in response to 9-cis-RA(18, 20) . In different cell types of epithelial origin as well as in muscle cells, RA promotes cell differentiation (17, 21) . In contrast, the acquisition of the white adipocyte phenotype is blocked when preadipocytes are exposed to RA(22, 23, 24) . Similarly to white adipose tissue, BAT expresses high levels of cytosolic retinoid binding proteins (25) and accumulates substantial amounts of vitamin A derivatives(26) . In this study, we report that RA is a strong activator of ucp gene expression, acting through an RA-responsive region in the ucp gene. The action of RA is independent of the adrenergic pathways of regulation of ucp gene transcription. We suggest a critical role for RA in the development and regulation of the thermogenic function of BAT.


EXPERIMENTAL PROCEDURES

Materials

DNA-modifying enzymes and poly(dIbulletdC) were purchased from Boehringer Mannheim or Promega. [alpha-P]dCTP was from Amersham Corp., and D-threo-[1,2-^14C]chloramphenicol was from ICN. Tissue culture media and fetal calf serum (FCS) were obtained from Whittaker. All-trans-retinoic acid (RA),(-)-arterenol bitartrate (norepinephrine (NE)), triiodothyronine (T(3)), and insulin were purchased from Sigma.

Cell Culture

Isolation and culture of brown preadipocytes was performed as described(8, 27) . Three-week-old Swiss mice were killed, and interscapular, cervical, and axillary depots of BAT were removed. Precursor cells were isolated, plated on 60-mm Petri dishes (7500 cells/cm^2), and grown in 5 ml of Dulbecco's modified Eagle's medium (DMEM), Ham's F12 Medium (1:1) supplemented with 10% FCS, 20 nM insulin, 2 nM T(3), and 100 µM ascorbate (``differentiating'' medium). When indicated, cells were grown in a hormone-``depleted'' medium containing 10% charcoal-treated FCS(28) . Cells were exposed to all-trans-retinoic acid at the concentrations and times indicated in each experiment. Unless otherwise stated, 0.5 µM norepinephrine or 1 mM 8-bromo-cAMP (Sigma) were added for 4 h. As indicated in the corresponding experiment, cycloheximide (Sigma) was used at a dose of 5 µg/ml at day 7 of culture as reported for blocking protein synthesis in brown adipocytes in culture(29) . Prazosin and propranolol (Sigma) were added at 10 µM final concentration. HepG2 human hepatoma cells were grown in DMEM supplemented with 10% FCS.

RNA Isolation and Northern Blot Analysis

Total RNA was extracted by the single-step method using guanidine hydrochloride(30) . For Northern blot analysis, 20 µg of total RNA was denatured, electrophoresed on 1.5% formaldehyde-agarose gels, and transferred to nylon membranes (Hybond N, Amersham Corp.). 0.2 µg/ml of ethidium bromide was added to RNA samples in order to check equal loading of gels and transfer efficiency(31) . Hybridization and washing were carried out as reported(4) . Blots were hybridized to DNA probes corresponding to the full-length cDNA for rat UCP(2) , 0.5 kb of the cDNA for subunit II of cytochrome oxidase (COII)(32) , 0.6 kb of the cDNA for guinea pig lipoprotein lipase (33) or the full-length cDNA for rat C/EBPalpha(34) . The cDNA probes were labeled with [alpha-P]dCTP using the random oligonucleotide-primer method. Autoradiographies were quantified by densitometric scanning (Pharmacia Biotech Inc.) or by radioactivity counting (AMBIS, Inc.).

Plasmids

The plasmid(-4551)UCP-CAT contains the region -4551 to +110 of the rat ucp gene driving the promoterless chloramphenicol acetyltransferase (CAT) gene(14) . The plasmids (-3628)UCP-CAT,(-896)UCP-CAT, and(-157)UCP-CAT were constructed using the internal unique restriction sites SphI, HindIII, and BstXI, respectively, in (-4551)UCP-CAT. The internal deletions between nucleotides -2469/-53, -3608/-2318, and -2469/-2283 were carried out by digesting with BclI/NaeI, XbaI, and BclI/ApaI, respectively. The plasmid Bgl-Sph + Apa-BstX contains the fragment -3628/-2283 linked to(-157)UCP-CAT(14) . The -2469/-2318 region was placed upstream from the(-172)UCP-CAT using the compatible restriction sites XbaI and SpeI.

Oligonucleotides were chemically synthesized by Oligos, Inc. The UCP oligonucleotide corresponds to positions -2357 to -2330 of the ucp gene flanked by XbaI ends, and its sequence is depicted in Fig. 7. The DR-2 and DR-5 are 24-base pair double-stranded oligonucleotides corresponding to the RA response element (RARE) in the mouse cellular retinol-binding protein type I (35) and RARbeta genes(36) , respectively. The RAREmut oligonucleotide corresponds to the mutated sequence AcGTCATGACgT, unable to bind RAR (37) .


Figure 7: Electrophoretic mobility shift assay of the -2357/-2330 region of the rat ucp gene. A, synthetic oligonucleotide containing the indicated region of the rat ucp gene used as labeled probe in the gel shift assays. The upper arrows show the putative alignments of three motifs closely related to the AGGTCA idealized half-site for RAREs(18, 46) . B, the double-stranded oligonucleotide was end-labeled and incubated with either 15 fmol of RARalpha expressed and purified from E. coli (left) or 5 µg of protein from rat BAT (right). The competitor oligonucleotide DR-2, DR-5, or RAREmut (see ``Experimental Procedures'') was added to the binding reactions at a 50-fold molar excess. The arrows in B indicate the retarded bands specifically lost because of competition with DR-2.



pRSV-RARalpha and pRSV-RXRalpha are mammalian expression vectors that contain the alpha subtype of the human RAR or the alpha subtype of the human RXR, respectively, driven by the Rous sarcoma virus (RSV) promoter(38, 39) . Thyroid hormone expression vectors contain the chicken alpha form (pRSV-cT(3)Ralpha) or the human beta form (pRSV-hT(3)Rbeta)(40, 41) .

Transfection Assays

Murine primary brown adipocytes differentiated in culture were transiently transfected by the calcium phosphate precipitation method on day 7 of culture, when 80-90% of cells were already differentiated(16) . Each transfection contained between 5 and 15 µg of UCP-CAT vectors and included or did not include 1 µg of the expression vector pRSV-RARalpha and/or pRSV-RXRalpha. In some experiments 1 µg of pRSV-cT(3)Ralpha or pRSV-hT(3)beta alone or together with 1 µg pRSV-RXRalpha were transfected. 2 µg of RSV-beta-galactosidase was included in all the experiments to assess the efficiency of separate transfections. After transfection, the medium was replaced by DMEM:F12 containing 10% charcoal-treated FCS. The cells were incubated for 24 h with or without the addition of 1 µM RA. For each condition, at least three plates were pooled. The experiments were performed at least twice using independent DNA preparations of each construct.

HepG2 cells were transfected by calcium phosphate precipitation essentially as described(42) . Each transfection contained, if not otherwise indicated, between 5 and 15 µg of UCP-CAT vector, different amounts of pRSV-RARalpha and/or pRSV-RXRalpha (see Fig. 5B), or 1 µg of pRSV-cT(3)Ralpha or pRSV-hT(3)beta expression vectors alone or together with 1 µg of RXRalpha, and 2 µg of RSV-beta-galactosidase. The cells were incubated for 36-38 h in DMEM 10% charcoal-treated FCS medium with or without 1 µM RA. All transfections and CAT assays were performed in duplicate.


Figure 5: RA stimulation of(-4551)UCP-CAT expression in transiently transfected brown adipocytes and HepG2 cells. Effect of RARalpha and/or RXRalpha co-transfection. A, brown adipocytes differentiated in culture (day 7) were transiently transfected with 15 µg/plate of the(-4551)UCP-CAT plasmid. When indicated, 1 µg of either the expression vector for pRSV-hRARalpha (RAR) or pRSV-RXRalpha (RXR) or an equimolar mixture of pRSV-RARalpha plus pRSV-RXRalpha (RAR + RXR) was co-transfected. After transfection, cells were exposed or not exposed to 1 µM RA. B, HepG2 cells were transfected with 15 µg of(-4551)UCP-CAT together with increasing amounts of the expression vector pRSV-RARalpha (circle), pRSV-RXRalpha (), or pRSV-RARalpha plus pRSV-RXRalpha (bullet). Transfections were performed and data were analyzed as described under ``Experimental Procedures.'' For each cell type, results are expressed as -fold induction by 1 µM RA relative to untreated cells for each experimental situation. Bars in A and points in B are means of two independent experiments, each one performed in triplicate.



Analysis of CAT activity was carried out as described(43, 44) . Acetylation of [^14C]chloramphenicol was determined by thin layer chromatography and quantified by radioactivity counting (AMBIS, Inc.). The CAT activity was normalized for variation in transfection efficiency using the beta-galactosidase activity as a standard.

DNA Binding Experiments

Nuclear proteins were isolated from rat BAT as described(16) , and protein concentration was determined by the micromethod of Bio-Rad using bovine serum albumin as standard.

Purified, bacterially expressed RARalpha was a kind gift from H. H. Samuels. RARalpha purity was checked by silver staining of an SDS-polyacrylamide gel and quantitated by ligand binding assays(37) .

For the gel retardation assays, oligonucleotides were end-labeled using [alpha-P]dCTP and Klenow enzyme. The DNA probe (20-30,000 cpm) was incubated for 30 min at 25 °C with either 15 fmol of purified RARalpha or 5 µg of BAT tissue nuclear protein extract. Reactions were carried out in a final volume of 30 µl containing 25 mM Tris (pH 7.8), 0.5 mM EDTA, 88 mM KCl, 10 mM 2-mercaptoethanol, 0.5 µg of poly(dIbulletdC), 10% glycerol, and 0.05% Triton X-100. Samples were analyzed by electrophoresis at 4 °C for 60 min in nondenaturing 5% polyacrylamide gels in 1 times TAE (10 mM Tris, 7.5 mM acetic acid, 40 µM EDTA, pH 7.8). Gels were analyzed by autoradiography. In the competition experiments, a 50-fold molar excess of unlabeled oligonucleotide was included in each binding reaction.


RESULTS

RA Stimulates ucp Gene Expression in Primary Brown Adipocytes

The action of RA on UCP mRNA expression in cultured brown adipocytes was studied and compared with the effects of norepinephrine and cAMP, known to be able to stimulate UCP mRNA expression. As depicted in Fig. 1, 0.5 µM norepinephrine and 1 mM 8-bromo-cAMP treatment of differentiated brown adipocytes (day 7 of culture) led to a 20- and a 5-fold rise in UCP mRNA levels, respectively, consistent with previous reports(8) . Exposure to 1 µM RA caused an increase in UCP mRNA levels that was at least as marked as that caused by norepinephrine. A stimulating action of RA on UCP mRNA abundance was also observed in preadipocytes (day 4 of culture), which showed very low levels of basal UCP mRNA expression, and in late stages of brown adipocyte differentiation (day 10 of culture). The parallel determination of COII mRNA abundance (see Fig. 1, bottom) showed that changes in COII mRNA occur only as a consequence of the stage of brown adipocyte differentiation (higher at day 7 than at day 4 of culture). In contrast, COII mRNA expression did not respond to RA, thus indicating that the effect of RA is specific for UCP mRNA.


Figure 1: Effects of RA on ucp gene expression in brown adipocytes differentiating in culture. BAT precursor cells were isolated and grown in culture for 4, 7, or 10 days. On the indicated days, 1 µM RA was added 24 h before the cells were harvested for RNA extraction. Cells were also treated for 4 h with 0.5 µM norepinephrine (NE) or 1 mM 8-bromo-cAMP (cAMP). Untreated cells were used as controls (C). Three plates were pooled for each treatment, and 20 µg of total RNA were analyzed by the Northern blot hybridization procedure described under ``Experimental Procedures.'' The filters were hybridized first with the UCP cDNA probe, and thereafter a new hybridization was performed with the COII cDNA probe. Bars are means from at least two independent experiments. Examples of the Northern blot analyses are depicted in the bottom of the figure. Arrows indicate the position of the two UCP mRNA species in mice (1.6 and 1.9 kb) and the COII mRNA (0.8 kb).



Fig. 2depicts dose-response and time course curves for the action of RA on UCP mRNA expression. As depicted in Fig. 2A, maximum levels of RA stimulation of UCP mRNA levels were achieved when primary brown adipocytes were exposed to 1 µM RA, although 1 nM RA was enough to elicit a substantial rise in UCP mRNA abundance. The effects of RA were maximal after 24 h of exposure to RA (Fig. 2B). Comparison with COII mRNA expression showed the specificity of RA action for UCP mRNA expression.


Figure 2: Dose-response and time course curves for the effect of RA on ucp gene expression. Brown adipocytes differentiated in culture (day 7) were exposed to the indicated concentrations of RA for 24 h (A) or exposed to 1 µM RA (B) for the indicated times. Points are means from at least two independent experiments with triplicate plates. Representative Northern blots hybridized with the UCP and COII cDNA probes, as described in Fig. 1, are depicted in the bottom of the figure.



The Stimulation of ucp Gene Expression by RA Is Independent of Adrenergic Pathways or Protein Synthesis

Treatment of brown adipocytes with 10 µM propranolol plus 10 µM prazosin, a mixture of beta and alpha adrenergic inhibitors(8) , suppressed the norepinephrine-induced increase in UCP mRNA expression (see Fig. 3A) but did not affect the stimulation elicited by RA. The action of RA was also studied in the presence of the inhibitor of protein synthesis cycloheximide. As shown in Fig. 3B, cycloheximide treatment led to lower basal expression of UCP mRNA, but it did not affect the ability of RA to increase UCP mRNA. However, the absolute extent of the UCP mRNA rise achieved was lower than in non-cycloheximide-treated cells.


Figure 3: Effects of adrenergic inhibitors or cycloheximide on the action of RA on ucp gene expression. Brown adipocytes differentiated in culture (day 7) were used. A, cells were exposed to 0.1 µM norepinephrine (NE) or 1 µM RA for 12 h in the presence or absence of a mixture of 10 µM propranolol plus 10 µM prazosin (INH). In the Northern blot example, 20 µg of total RNA were loaded per lane except in the NE -INH lane (6 µg). B, cells were exposed to 1 µM RA for 12 h in the presence of 5 µg/ml cycloheximide (CHX). Treatments are indicated as +, whereas untreated cells are shown as -. For experimental and representation details, see the Fig. 1legend.



Long Term Exposure of Differentiating Brown Fat Precursor Cells to RA Increases UCP mRNA Levels Regardless of the Acquisition of the Adipocyte Phenotype

In order to gain insight into the relationships between RA action on ucp gene expression and differentiation of brown adipocytes, the effects of long term exposure to RA on UCP mRNA expression were studied by exposing brown preadipocytes (day 4 of culture) to RA until day 7 of culture. The study was performed by using the regular differentiating medium, which allows most cells to acquire the brown adipocyte morphology at day 7, or using a culture medium depleted of growth factors and hormones, including RA (hormone-depleted medium). In the presence of the differentiating medium, cultured cells differentiated into a brown adipocyte phenotype characterized by a dramatic change in cell morphology, caused by rounding up of the cells and lipid accumulation (Fig. 4A). Brown adipocyte differentiation between day 4 and 7 of culture (Fig. 4B) also resulted in the appearance of substantial levels of basal UCP mRNA expression and an increase in COII mRNA abundance, an indicator of overall mitochondriogenesis. There was also a rise in the expression of lipoprotein lipase mRNA and C/EBPalpha mRNA, molecular markers for adipocyte differentiation(45) . RA treatment did not change either the appearance of the adipocyte morphology or the rise in the non-BAT-specific markers of adipocyte differentiation and mitochondriogenesis. However, levels of UCP mRNA rose 7-fold (Fig. 4B). When cells where exposed to the hormone-depleted medium, the acquisition of the adipocyte morphology was completely blocked (see Fig. 4A, bottom). Basal levels of UCP mRNA did not rise with respect to those at day 4 of culture, and the mRNA levels for COII, lipoprotein lipase, and C/EBPalpha even decreased (Fig. 4B). The 3-day RA treatment did not overcome the lack of adipocyte differentiation, as estimated by cell morphology and the mRNA markers of mitochondriogenesis and adipogenesis, but it always increased UCP mRNA abundance (7-fold).


Figure 4: Effects of long term RA treatment on brown adipocyte differentiation and ucp gene expression. Brown adipocyte precursor cells were grown in culture for 4 days and treated thereafter with either the regular differentiating medium or the hormone-depleted medium, as described under ``Experimental Procedures.'' For each medium, half of the plates were supplemented with 1 µM RA. A, microphotographs of the cells on day 4 of culture or on day 7 after being cultured in the different media. B, five plates were pooled on day 4 and three on day 7 for each treatment, and 20 µg of total RNA was analyzed by Northern blot as described under ``Experimental Procedures.'' Bars are means of at least three independent experiments.



The 4.5-kb 5`-Noncoding Region of the Rat ucp Gene Contains RA Response Elements

The action of RA on CAT expression driven by the upstream 4.5 kb of the rat ucp gene was studied in transiently transfected primary brown adipocytes. As shown in Fig. 5A, RA increased the(-4551)UCP-CAT activity at least 2-fold. Co-transfection of 1 µg of the expression vector for RARalpha or RARalpha plus RXRalpha enhanced the RA responsiveness of the (-4551)UCP-CAT 5-fold, whereas co-transfection of 1 µg of RXRalpha alone did not cause any substantial change in RA responsiveness (Fig. 5A). No consistent effect of RXRalpha was observed even when higher amounts of pRSV-RXRalpha were co-transfected. When HepG2 hepatoma cells were transfected with the(-4551)UCP-CAT construct, basal expression of CAT was 10% of the activity found in transfected brown adipocytes using the beta-galactosidase activity for comparison. However, RA treatment resulted in a rise of more than 2-fold in (-4551)UCP-CAT expression. As shown in Fig. 5B, cotransfection of either RARalpha or RXRalpha enhanced RA responsiveness in a dose-dependent manner, and similar maximal effects of around 6-fold RA stimulation of CAT activity were observed at high concentrations of each transfected vector. However, the amount of RARalpha required for maximal RA responsiveness was lower than the amount of RXRalpha required. When cotransfections included RARalpha plus RXRalpha, a dramatic decrease was observed in the amount of transfected receptors needed for a maximal stimulation of(-4551)UCP-CAT activity in response to RA.

When either primary brown adipocytes or HepG2 cells were exposed to 100 nM T(3) no effect was observed on(-4551)UCP-CAT expression. Co-transfections of the expression vectors for thyroid hormone receptor alpha or beta, either alone or together with RXRalpha, were unable to confer T(3) responsiveness to the(-4551)UCP-CAT. However, in parallel transfection experiments, T(3) caused a significant stimulation of the expression of(-490)PEPCK-CAT, a CAT vector driven by the phosphoenolpyruvate carboxykinase gene promoter used as a T(3)-responsive positive control (42) (results not shown).

Elements between -2469 and -2318 in the Rat ucp Gene Are Required for RA Responsiveness

In order to determine the site in the 5`-region of the rat ucp gene responsible for RA action, the effects of RA on different deletion mutants of the(-4551)UCP-CAT were studied in transfected brown adipocytes and in HepG2 cells. As shown in Fig. 6, transfection of 5`-deletion mutants of the (-4551)UCP-CAT indicated that the RA-responsive site was located in the distal upstream region of the ucp gene, between -3628 and -896. Internal deletion mutants lacking either -2469/-53 or -3608/-2318 were unresponsive to RA, whereas a construct in which -3628/-2283 was placed immediately upstream of -157 retained RA responsiveness. These results indicated that the region between -2469 and -2318 was necessary for the stimulation by RA of the ucp gene expression. This was confirmed by the analysis of a construct in which only the -2469/-2283 region of the(-4551)UCP-CAT had been deleted, resulting in a lack of RA induction of CAT activity, and by placing the -2469/-2318 region in front of the(-172)UCP-CAT, thus conferring RA responsiveness to the minimal ucp promoter.


Figure 6: Effects of RA on the expression of transiently transfected 5`-deletion mutants of the (-4551)UCP-CAT. Brown adipocytes differentiated in culture (day 7) and HepG2 cells were transiently transfected with 15 µg/plate of (-4551)UCP-CAT or equivalent amounts of the deletion mutants illustrated on the left. 1 µg of the expression vector pRSV-hRARalpha was co-transfected. After transfection, cells were exposed or not exposed to 1 µM RA. Transfections were performed and data were analyzed as described under ``Experimental Procedures.'' Results are expressed as the -fold induction caused by RA on each transfected construct either in primary brown adipocytes (open bars) or HepG2 cells (dark bars). For the primary brown adipocytes data, bars are means of at least two independent experiments, each one done in triplicate. The HepG2 results shown are the mean ± S.E. for at least three independent transfection experiments, each performed in duplicate.



The -2357/-2330 Region of the Rat ucp Gene Binds RARalpha and RAR-related Proteins in BAT Tissue Nuclei

Analysis of the sequence between -2469 and -2318 required for RA responsiveness of the ucp gene indicated the presence of the AGGTCA sequence and two adjacent related motifs characteristic of previously reported consensus RAREs (18, 46) (see Fig. 7A; arrows indicate half-site-related motifs). The best alignment of these motifs is as three direct repeats with two or three base pair spacings. Electrophoretic gel mobility shift assays are shown in Fig. 7B. The DNA fragment depicted in Fig. 7A was used as labeled probe and incubated with either RARalpha or nuclear extracts from BAT. As shown in Fig. 7B (left), recombinant purified RARalpha interacted with the -2357/-2330 region of the ucp gene, as shown by the appearance of a main retarded band. Competition assays indicated that binding was specific since it was suppressed by an excess of oligonucleotides corresponding to previously characterized RAREs, whether they consisted in a direct repeat with a 2-base pair spacing (DR-2) or a 5-base pair spacing (DR-5). An excess of unlabeled oligonucleotide corresponding to a two-point mutated form of a RARE that is unable to bind RAR (RAREmut) (37) , had no effect. The incubation of this probe with BAT nuclear extracts yielded two main retarded bands in the gel shift assays (Fig. 7B, right). Competition with an excess of unlabeled oligonucleotide corresponding to the RAR-binding element DR-2 caused a loss of both retarded bands. A similar effect was observed using DR-5 (not shown). Inclusion of an excess of RAREmut did not affect the appearance of the two retarded bands.


DISCUSSION

We have identified RA as a powerful stimulator of ucp gene expression, capable of raising UCP mRNA to levels as high as those elicited by norepinephrine, the main inducer of ucp gene expression known to date. The action of RA on UCP mRNA levels is essentially independent of protein synthesis, and it does not depend on any putative physiological or artifactual stimulation of the adrenergic receptors. In addition, RA does not mimic the overall effects of norepinephrine upon gene transcription in BAT. For instance, the expression of other adrenergic-stimulated genes in BAT such as lipoprotein lipase (47) or C/EBPbeta(48, 49) , is unaltered by RA. (^2)Hence, the action of RA on ucp gene transcription appears to be independent of the adrenergic pathways.

The effects of RA on ucp gene expression occur irrespective of the stage of brown adipocyte differentiation. RA action is highly specific in stimulating the expression of the ucp gene, the only unequivocal molecular marker that differentiates the brown adipocyte from the white adipocyte phenotype. This is in contrast with the established action of RA as an inhibitor of white adipose cell differentiation (22) and as a repressor of the expression of marker genes for the white adipocyte differentiated phenotype(23, 24) . The specific action of RA provides evidence that the expression of the ucp gene is regulated independently of the overall program of adipose cell differentiation common to brown and white adipocytes.

Most of the biological actions of RA on gene expression occur via the regulation of gene transcription. The time course and dose-response curves for the RA effect on UCP mRNA expression are in the range of those observed for other genes where RA action is caused by an RA receptor-mediated stimulation of gene transcription(50, 51) . The stimulatory effect of RA on the transfected chimeric plasmid in which CAT expression was driven by the 4.5-kb 5`-flanking region of the rat ucp gene together with the potentiation of this effect by co-transfection with expression vectors for RA receptors indicates the presence of RAREs in the ucp gene. The finding that co-transfection of RARalpha enhanced the RA responsiveness of the ucp promoter whereas RXR was less effective is a characteristic response previously observed for genes whose responsiveness to RA occurs through RAR(52, 53) . In fact, in most of the RA-responsive genes studied so far, RXR has an auxiliary role of providing the heterodimerization partner for RAR(20) . The much higher sensitivity of the RA responsiveness of the(-4551)UCP-CAT to the co-transfection of RAR plus RXR strongly supports a main involvement of RAR-RXR heterodimers in RA action on the ucp gene. The ability of the transfected RXR receptor to affect RA responsiveness in HepG2 in contrast with the lack of effect in brown adipocytes might be related to differences in the expression of endogenous RXRalpha. Thus, HepG2 cells express substantial amounts of RARalpha but are especially devoid of RXRalpha(52, 54) . Conversely, BAT expresses constitutive levels of RARalpha and very high levels of RXRalpha, (^3)which explains the low sensitivity to exogenous RXRalpha of the brown fat cells.

Our results demonstrate that the -2469/-2318 region of the ucp gene is required for RA responsiveness. This region contains three potential RARE consensus half-sites in the -2357/-2330 sequence (see Fig. 7A), capable of binding RARalpha and RARE-binding proteins present in BAT nuclei. The putative alignments of these motifs include a 2-base pair spacing between two imperfect direct repeats and a 3-base pair spacing of the only fully homologous AGGTCA sequence with the adjacent motif. Characterization of the relative importance of the different elements of this complex region for RA responsiveness in the ucp gene is beyond the scope of this paper. A 2-base pair alignment of direct repeats is characteristic of several RAREs that depend on RAR/RXR heterodimers(20, 46) , whereas 3-base spacings have been more frequently found for vitamin D-responsive elements(46) . However, increasing evidence indicates that there is no single rule for the alignment of direct repeats in RAREs from mammalian RA-responsive genes(20, 55) . The lack of response of the(-4551)UCP-CAT to T(3) indicates that the RARE present in the ucp gene is specific for RA-mediated regulation and that it does not confer a promiscuous response mediated by related members of the steroid/thyroid receptor superfamily, such as the thyroid receptor. Despite the known positive action of thyroid hormones on ucp gene expression(9, 10) , the present results indicate also a lack of thyroid hormone-responsive elements in the ucp gene promoter.

The induction by RA of the(-4551)UCP-CAT is evidenced when transfected in brown adipocytes as well as in the HepG2 heterologous cell system. Therefore, elements involved in the cell-specific transcription of the ucp gene do not appear to be required for RA responsiveness. The rat ucp gene contains two main regulatory regions, a distal upstream one showing enhancer properties and a proximal one containing C/EBP and cAMP-responsive elements(14, 16, 56) . Present data indicate that the RARE is located in the distal upstream region of the rat ucp gene. The fact that the responsiveness of the rat ucp gene to norepinephrine is basically dependent on cAMPresponsive elements placed between -157 and -57, in the closely proximal region of the gene(56) , further supports the independence of the adrenergic and the RA-dependent regulations of ucp gene transcription.

In summary, RA action constitutes a novel, non-adrenergic, pathway of regulation of ucp gene expression with a potential relevance for the development and regulation of the thermogenic activity of BAT. Molecular mechanisms eliciting ucp gene expression in prenatal development, essential in most mammalian species to overcome the thermal stress after birth, are not known, but they are independent of adrenergic stimulation(4) . RA, a powerful regulator of vertebrate development and cell differentiation, would be a likely candidate to regulate ucp gene transcription during ontogeny. On the other hand, recent studies on transgenic mice with genetically ablated brown fat support a critical role for BAT in the regulation of energy balance and development of obesity(57) . The positive action of RA on BAT ucp gene expression, in contrast to the negative effects of RA on white adipose cell differentiation(22) , opens new perspectives for the development of molecules for the treatment of obesity and body weight disturbances.


FOOTNOTES

*
This work was supported by Grant PB92-0865 from DGICyT, Ministerio de Educación y Ciencia, Spain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Unitat de Bioquímica i Biologia Molecular, Departament de Bioquímica i Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avda Diagonal, 645. 08028-Barcelona, Spain. Tel.: 34-3-402-1525; Fax: 34-3-402-1559.

(^1)
The abbreviations used are: BAT, brown adipose tissue; UCP, uncoupling protein; RA, retinoic acid; CAT, chloramphenicol acetyltransferase; RAR, retinoic acid receptor; RXR, retinoid X receptor; T(3), triiodothyronine; C/EBP, CCAAT enhancer binding protein; FCS, fetal calf serum; COII, subunit II of cytochrome c oxidase; DR, direct repeat; RARE, retinoic acid response element; RSV, Rous sarcoma virus; DMEM, Dulbecco's modified Eagle's medium; kb, kilobase(s).

(^2)
J. Andrés, R. Alvarez, F. Villarroya, and M. Giralt, unpublished observations.

(^3)
R. Iglesias, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. D. Ricquier for kindly providing PSP4551 and UCP36 probe; Drs. H. H. Samuels and M. G. Rosenfeld for the retinoic acid receptor and thyroid hormone receptor expression vectors; Dr. R. W. Hanson for the(-490)PEPCK-CAT; and Drs. N. Glaichenhaus, S. Enerback, and S. L. McKnight for the COII, lipoprotein lipase, and C/EBPalpha probes, respectively.


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