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(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
Brown adipose tissue (BAT) ( 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
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
pRSV-RAR
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
Figure 5:
RA stimulation of(-4551)UCP-CAT
expression in transiently transfected brown adipocytes and HepG2 cells.
Effect of RAR
Analysis of CAT activity was carried out as
described(43, 44) . Acetylation of
[
Purified, bacterially expressed RAR For the gel retardation assays,
oligonucleotides were end-labeled using
[
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
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
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 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 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 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.
Volume 270,
Number 10,
Issue of March 10, 1995 pp. 5666-5673
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
RETINOIC ACID IS A TRANSCRIPTIONAL ACTIVATOR OF THE MITOCHONDRIAL
UNCOUPLING PROTEIN GENE (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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) .
Materials
DNA-modifying enzymes and
poly(dI
dC) were purchased from Boehringer Mannheim or Promega.
[-
P]dCTP was from Amersham Corp., and D-threo-[1,2-
C]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), 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
), 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, 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/EBP(34) . The cDNA probes were labeled with
[-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.
genes(36) , respectively. The RAREmut oligonucleotide
corresponds to the mutated sequence AcGTCATGACgT, unable to bind RAR (37) .
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.
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-cTR) or the human form
(pRSV-hTR)(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-RAR and/or pRSV-RXR. In some experiments 1 µg of
pRSV-cTR or pRSV-hT alone or together
with 1 µg pRSV-RXR were transfected. 2 µg of
RSV--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. and/or
pRSV-RXR (see Fig. 5B), or 1 µg of
pRSV-cTR 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.
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.
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.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.
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) .-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(dIdC), 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.
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.
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
and 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.
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/EBP 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/EBP 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).
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 RAR or RAR plus RXR enhanced the RA responsiveness
of the (-4551)UCP-CAT 5-fold, whereas co-transfection of 1 µg
of RXR alone did not cause any substantial change in RA
responsiveness (Fig. 5A). No consistent effect of
RXR was observed even when higher amounts of pRSV-RXR 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
-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 RAR or RXR 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 RAR required for
maximal RA responsiveness was lower than the amount of RXR
required. When cotransfections included RAR plus RXR, 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. 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).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.
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 RAR
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 RAR and RAR-related Proteins in BAT Tissue
Nuclei or nuclear extracts from BAT. As shown in Fig. 7B (left), recombinant purified RAR
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.
(48, 49) , is unaltered by RA. ()Hence, the action of RA on ucp gene transcription
appears to be independent of the adrenergic pathways. 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. 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.
), 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).))
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/EBP probes, respectively.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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