(Received for publication, November 16, 1994; and in revised form, December 27, 1994)
From the
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 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
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.
Brown adipose tissue (BAT) ()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
-1 and
-3 adrenergic receptors in the
surface of the brown fat cell(8, 9) . cAMP, and
probably also T
, 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.
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 RAR
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
RAR 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-RAR and pRSV-RXR
are mammalian
expression vectors that contain the
subtype of the human RAR or
the
subtype of the human RXR, respectively, driven by the Rous
sarcoma virus (RSV) promoter(38, 39) . Thyroid hormone
expression vectors contain the chicken
form
(pRSV-cT
R
) or the human
form
(pRSV-hT
R
)(40, 41) .
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-RAR and/or
pRSV-RXR
(see Fig. 5B), or 1 µg of
pRSV-cT
R
or pRSV-hT
expression
vectors alone or together with 1 µg of RXR
, and 2 µg of
RSV-
-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 RAR and/or RXR
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-hRAR
(RAR) or pRSV-RXR
(RXR) or an
equimolar mixture of pRSV-RAR
plus pRSV-RXR
(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-RAR
(
),
pRSV-RXR
(
), or pRSV-RAR
plus pRSV-RXR
(
).
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
[C]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
-galactosidase activity as a standard.
Purified, bacterially expressed RAR
was a kind gift from H. H. Samuels. RAR
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
[-
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 RAR
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(dI
dC), 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
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.
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.
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.
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.
When either primary brown adipocytes or HepG2 cells
were exposed to 100 nM T no effect was observed
on(-4551)UCP-CAT expression. Co-transfections of the expression
vectors for thyroid hormone receptor
or
, either alone or
together with RXR
, were unable to confer T
responsiveness to the(-4551)UCP-CAT. However, in parallel
transfection experiments, T
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
-responsive positive control (42) (results not
shown).
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-hRAR 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.
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/EBP(48, 49) , is unaltered by RA. (
)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 RAR 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 RXR
. Thus, HepG2 cells express
substantial amounts of RAR
but are especially devoid of
RXR
(52, 54) . Conversely, BAT expresses
constitutive levels of RAR
and very high levels of RXR
, (
)which explains the low sensitivity to exogenous RXR
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 RAR 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
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.