From the Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
Received for publication, July 18, 2002, and in revised form, October 29, 2002
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ABSTRACT |
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Brown adipose tissue expresses the
thermogenic uncoupling protein-1 (UCP-1), which is positively regulated
by peroxisome proliferator-activated receptor (PPAR) agonists and
retinoids through the activation of the heterodimers PPAR/retinoid X
receptor (RXR) and retinoic acid receptor (RAR)/RXR and binding
to specific elements in the ucp-1 enhancer. In this study
we show that in fetal rat brown adipocyte primary cultures the PPAR Brown adipose tissue
(BAT)1 is the main site for
non-shivering thermogenesis in small mammals. This function relies on
the presence of the tissue-specific uncoupling protein-1 (UCP-1), which
is located in the mitochondrial inner membrane and stimulates heat
production by uncoupling oxidative phosphorylation from the respiratory
chain (1). UCP-1 expression is under complex regulation. An enhancer
element in the 5'-flanking region of the ucp-1 gene has been
described with putative binding sites for the thyroid hormone receptor
(THR), retinoic acid (RA) receptors (RAR and RXR), and the peroxisome
proliferator-activated receptor (PPAR) (2, 3). Although We have previously reported (11, 12) that the PPAR Materials--
Wy14643 was from Biomol Research Laboratories
(Plymouth, UK). Rosiglitazone was kindly provided by Dr. S. A. Smith (GlaxoSmithKline, Harlow, UK). PD169316 and PD98059 were
purchased from Calbiochem-Novabiochem. Myelin basic protein
(MBP), BSA, Bt2cAMP, 9-cis-RA and
all-trans-RA were from Sigma. Fetal calf serum (FCS),
phosphate buffered saline (PBS), culture media, and Trizol were from
Invitrogen. Nylon membranes were GeneScreenTM (PerkinElmer
Life Sciences). Autoradiographic films were Kodak X-O-MAT/AR
(Eastman Kodak). [ Cell Culture--
Fetal brown adipocytes were obtained from
interscapular brown adipose tissue of 20-day-old Wistar rat fetuses and
isolated by collagenase dispersion as described (23). Isolated cells were plated at 1.5 × 106 cells/60-mm tissue culture
dishes in 2.5 ml of minimal essential medium (MEM) with Earle's salts
supplemented with 10% FCS, the presence of serum being essential for
the attachment of cells to the plastic surface of the dishes. After
4-6 h of culture at 37 °C, cells were rinsed twice with PBS, and a
70% confluent monolayer was observed under inverse light microscopy.
Cells were maintained for 20 h in a serum-free MEM supplemented
with 0.2% (w/v) BSA before compound addition. Then, cells were further
incubated for different times (from 1 to 24 h) in serum-free
BSA-MEM supplemented or not with Rosi or Wy14643 or retinoids in either
the presence or absence of several chemical inhibitors (PD169316 or
PD98059 or H89).
RNA Extraction and Analysis--
For Northern-blot analysis of
RNA, at the end of the culture time the cells were washed twice with
ice-cold PBS and lysed directly with Trizol following the protocol
supplied by the manufacturer for total RNA isolation (11). Total
cellular RNA (15 µg) was submitted to Northern blot analysis,
i.e. electrophoresed on 0.9% agarose gels containing 0.66 M formaldehyde, transferred to GeneScreenTM
membranes using a VacuGene blotting apparatus (LKB-Pharmacia) and
cross-linked to the membranes by UV light. Hybridization was in 0.25 mM NaHP04, pH 7.2, 0.25 M NaCl, 100 µg/ml denatured salmon sperm DNA, 7% SDS, and 50% deionized
formamide containing denatured 32P-labeled cDNA
(106 cpm/ml) for 24 h at 42 °C as described
(24). Complementary DNA labeling was carried out with
[ Transfection Conditions--
The plasmid constructs used for
transfection were Western Blotting--
Cells were lysed in the lysis buffer (25 mM Hepes, 0.3 M NaCl, 20 mM
glycerolphosphate, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM dithiothreitol, 0.1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, pH
7.5), and the cellular proteins (30 µg) were submitted to SDS-PAGE,
transferred to Immobilon membranes, and blocked using 5% nonfat dried
milk in 10 mM Tris-HCl and 150 mM NaCl, pH 7.5, and incubated overnight with several antibodies as indicated in each
case in 0.05% Tween 20, 1% nonfat dried milk in 10 mM
Tris-HCl, and 150 mM NaCl, pH 7.5. Immunoreactive bands were visualized using the enhanced chemiluminiscence (ECL-Plus) Western
blotting protocol (Amersham Biosciences).
p38MAPK Activity Assay--
Cells were extracted with lysis
buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1%
Triton X-100, 2 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin, and
25 µg/ml aprotinin) and immunoprecipitated with an anti-p38MAPK
antibody as described (26). Immune complexes were washed five times
with ice-cold lysis buffer with 0.5 M NaCl and two times
with kinase buffer (35 mM Tris, pH 7.5, 10 mM
MgCl2, 0.5 mM EGTA, 1 µM
Na3VO4). The kinase reaction was performed in buffer containing 1 µCi of [ Gel Shift Assay--
Nuclear extracts were prepared as described
previously (25). The UCP-1-PPRE double-stranded oligonucleotide,
chemically synthesized by Sigma (Genosys), corresponds to positions
Data Analysis--
Results are means ± S.E.
(n = 6 or 8) for duplicate dishes from three or four
independent experiments, respectively. Statistical significance was
tested with a one-way analysis of variance followed by the protected
least significant different test. p values less than 0.01 were considered significant. In experiments using x-ray films
(Hyperfim), different exposure times were used to ensure that the bands
were not saturated.
Fetal rat brown adipocytes, when isolated and allowed to attach to
the plastic dishes for 4 h in 10% FCS-MEM, express adipogenic (FAS) and thermogenic (UCP-1) markers. After 20 h of culture in the absence of serum, UCP-1 mRNA levels decrease dramatically, facilitating the study of its up-regulation by several
hormones/signals. Under these conditions, treatment for 24 h with
noradrenaline or triiodothyronine or 9-cis-RA or
insulin-like growth factor-1 is able to induce UCP-1 gene expression
(27) (25). We have previously used the same culture conditions to
demonstrate that Rosi per se and in the absence of any other
exogenously added signal increases UCP-1 and UCP-3 mRNA levels in a
time- and dose-dependent manner and induces activation of
the full promoter UCP-1-CAT (11) (12). To explore the possible
involvement of the extracellular signal regulated kinases (ERKs),
p38MAPK, and/or PKA on UCP-1 induction by Rosi, we used chemical
inhibitors for those kinases as follows: PD98059 (PD), 20 µM, to inhibit ERKs; PD169316 (PD*), 800 nM,
as a p38MAPK inhibitor; and H89, 10 µM, to inhibit PKA activity. The efficiency of PD* and PD to inhibit p38MAPK activity and
p44/p42MAPK phosphorylation at the doses used was tested in Fig. 3,
B and C. Furthermore, H89 at 10 µM
completely blocked UCP-1 mRNA induction as well as FAS mRNA
repression by Bt2cAMP (Fig.
1B). Fetal rat brown
adipocytes were treated for 24 h with or without 10 µM Rosi either in the absence or presence of the inhibitors. At the end of the culture period, total RNA was extracted and analyzed for UCP-1 expression by Northern blot (Fig.
1A). As expected, Rosi increased UCP-1 mRNA levels by
4-fold, and this effect was unmodified in the presence of PD or
H89. However, the inhibition of p38MAPK activity with PD*
resulted in a nearly complete blockade of UCP-1 induction by Rosi
without a significant effect on the basal UCP-1 mRNA levels found
in untreated cells. To validate whether the increase in UCP-1
expression produced by Rosi in a p38MAPK-dependent manner
was independent of differentiation-inducing properties of Rosi or
p38MAPK (28), the expression of the adipogenic marker FAS was also
analyzed (Fig. 1A). Rosi treatment for 24 h did not
induce FAS mRNA levels and did not increase intracellular lipid
content (data not shown), indicating that the effects of Rosi are
related to specific thermogenic gene expression rather than to a
differentiation program. Furthermore, no changes on FAS mRNA
expression were observed regardless of the presence or absence of the
inhibitors (Fig. 1A).
agonist rosiglitazone (Rosi), as well as retinoic acids
9-cis-retinoic acid and all-trans-retinoic acid
also have "extragenic" effects and induce p44/p42 and p38 mitogen-activated protein kinase (p38MAPK) activation. The latter is
involved in UCP-1 gene expression, because inhibition of p38MAPK activity with PD169316 impairs the ability of Rosi and retinoids for
UCP-1 induction. The inhibitory effects of PD169316 are mimicked by the
antioxidant GSH, suggesting a role for reactive oxygenated species
(ROS) generation in the increase of UCP-1 expression in response either
to Rosi or 9-cis-retinoic acid. Thus, we propose that Rosi
and retinoids act as PPAR/RXR and RAR/RXR agonists and also activate
p38MAPK. These two coordinated actions could result in a high increase
of transcriptional activity on the ucp-1 enhancer and hence
on thermogenesis. PPAR
and
agonists but not retinoids also
increase UCP-3 expression in fetal brown adipocytes. However, the
regulation of UCP-3, which is not involved in thermogenesis, seems to
differ from UCP-1 given the fact that is not affected by p38MAPK inhibition.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic
stimulation is the main physiological pathway that induces
thermogenesis and UCP-1 expression in BAT, it is likely to be mediated
by the induction of the PPAR
coactivator (PGC-1), a master
coactivator that binds PPAR
, PPAR
, THR, RAR, and RXR (4, 5). BAT
also expresses the recently described UCP-2 and UCP-3, the latter being
restricted to two thermogenic tissues (BAT and skeletal muscle). UCP-3
has shown to uncouple respiration when expressed ectopically in yeast
and mammalian cells (6, 7), and a role for UCP-3 in the regulation of energy expenditure has been reported (8) as a response to nutritional states, with a key role for PPAR
and PPAR
ligands as positive regulators of UCP-3 gene expression (9, 10).
agonist,
rosiglitazone (Rosi), increased UCP-1 and UCP-3 mRNA levels in
fetal rat brown adipocytes prior to the acquisition of the catecholaminergic neuronal input. In contrast, the PPAR
ligand Wy14643 induced UCP-3 but not UCP-1 expression, although it was able to
transactivate the
4551UCP-1-chloramphenicol acetyltransferase (CAT)
reporter gene (11, 12). The effect of Rosi on UCP-1 mRNA levels is
mediated by increasing the transcription of the gene through the
binding of the PPAR/RXR heterodimer to the PPAR response element (PPRE)
described in the enhancer (2). PPREs have also been described in the
5'-flanking region of the human ucp-3 gene (13, 14). Among
the coactivators that bind to PPAR, PGC-1 is highly expressed in BAT
and plays a key role in adaptive thermogenesis. Because recent reports
have described modulation either of PPAR
, PPAR
, and PGC-1
activity by phosphorylation via p44/p42 mitogen-activated protein
kinase (MAPK) (15, 16), p38MAPK (17, 18), and/or protein kinase A (PKA)
(19), we decided to explore the involvement of those kinases in the
UCP-1 and UCP-3 induction by PPAR
and
agonists. This paper
describes p38MAPK and p44/p42MAPK activation triggered either by Rosi
or 9-cis-RA or all-trans-RA treatment in fetal
rat brown adipocytes, with p38MAPK implication in UCP-1 but not UCP-3 expression.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP,
[
-32P]ATP, [14C]chloramphenicol, and the
multiprimer DNA-labeling system kit were purchased from Amersham
Biosciences. All other reagents used were of the purest grade
available. The cDNAs used as probes were UCP-1, UCP-3, and fatty
acid synthase (FAS) (12, 20, 21). The transfection MBS mammalian
transfection kit was from Stratagene (La Jolla, CA). The plasmid
constructs used for transfection experiments were
4551UCP-1-CAT,
kindly provided by D. Ricquier (Meudon, France) (22) and
pCMV
-galactosidase supplied by Stratagene. The anti-phospho- and anti-(p44/p42MAPK and p38MAPK) antibodies were from Cell Signaling (Beverly, MA). PPAR
antibody "sc-1984" was from Santa Cruz Biotechnology.
-32P]dCTP to a specific activity of 109
cpm/µg of DNA by using multiprimer DNA-labeling system kit. For serial hybridization with different probes, the blots were stripped and
subsequently rehybridized as needed in each case. Membranes were
subjected to autoradiography, and relative densities of the hybridization signals were determined by densitometric scanning of the
autoradiograms in a laser densitometer (Amersham Biosciences).
4551UCP-1-CAT, where the CAT reporter gene is under
the control of a 4551 full-length 5'-flanking region of rat UCP-1, and
pCMV
-galactosidase, a viral promoter-driven expression of the
reporter gene
-galactosidase. Fetal primary brown adipocytes were
cultured for 24 h in the presence of 10%FCS-MEM and then
transiently transfected according to the calcium phosphate-mediated
protocol with 10 µg of DNA-CAT together with 2 µg of DNA
gal
(to monitor transfection efficiency) as described previously (25).
After 4 h of incubation, cells were shocked with 3 ml of 15%
PBS-glycerol for 2 min, washed, and then fed with serum-free BSA-MEM
medium (either in the absence or presence of Rosi ± PD169316) for
24 h. The cells were then harvested, and the lysates were prepared
for CAT and
-galactosidase activity assays. CAT activity was
determined by incubating 50 µl of cell extracts with 0.25 µCi of
[14C]chloramphenicol and 0.5 mM acetyl
coenzyme A in 0.25 M Tris (pH 7.8) at 37 °C for 12 h, and samples were then submitted to thin layer chromatography. The
amount of acetylated substrate was directly quantified with a
radioimaging device (Fujifilm BAS-1000). CAT enzyme activity was
expressed as a percentage of acetylated [14C]chloramphenicol normalized to the internal control,
-galactosidase (assayed according to the Stratagene
protocol). Routinely, we performed direct
-galactosidase
staining on parallel dishes, and observation under inverse light
microscopy demonstrated that blue cells were mainly brown adipocytes
with their characteristic multilocular fat droplets phenotype.
-32P]ATP, 60 µM ATP, and 1 µg of MBP as a substrate for 30 min at 30 °C, and it was terminated by the addition of 4× SDS-PAGE sample buffer followed by boiling for 5 min at 95 °C. Samples were resolved in 12% SDS-PAGE, and gels were dried out and subjected to autoradiography.
2485 to
2458 of the rat ucp-1 gene, and its sequence is
5'-GTGGGTCAGTCACCCTTGATCACACTGC-3'. The gel mobility shift assay was
performed essentially as described previously (26). The gel was dried
and subjected to autoradiography and also quantified directly with a
radioimaging device.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Rosi induction of UCP-1 gene expression is
dependent on p38MAPK activity but independent of p44/p42MAPK and PKA
activities. Fetal brown adipocytes, after 20 h of serum
deprivation in BSA-MEM medium, were cultured for 24 h in the
absence or presence of 10 µM Rosi ± 20 µM PD or 800 nM PD* or 10 µM
H89 (A) or for 6 h in the absence or presence of 0.5 mM Bt2cAMP
((Bu)2cAMP) ± 10 µM H89 (B). Total RNA was extracted
from the different conditions, and 15 µg of it was submitted to
Northern-blot analysis and hybridized with labeled UCP-1 and FAS
cDNAs. A final hybridization with the 18 S rRNA cDNA was
performed for normalization. Representative autoradiograms and
densitometric analysis of UCP-1 mRNA levels after standardization
using the 18 S rRNA signal are shown. Results (arbitrary densitometric
units) are means ± S.E. (n = 8). Statistical
significance was tested as described under "Experimental
Procedures." The differences between values in the presence of Rosi
versus control are represented by *, and the differences
between values in the presence of Rosi plus inhibitors
versus Rosi are represented by . For * and
,
p < 0.01.
We also performed transient transfections with 4551UCP-1-CAT (where
the CAT reporter gene is under the control of a 4551-bp full-length
5'-flanking region of the rat UCP-1 promoter) to test whether the
inhibition was produced on the transcription process. Fetal primary
brown adipocytes were transiently co-transfected with 10 µg of
4551UCP-1-CAT together with 2 µg of pCMV
-galactosidase for
internal control of transfection. Upon transfection, cells were
cultured for 24 h in a serum-free BSA-MEM medium in the absence or
presence of Rosi with or without PD169316. At the end of the culture
period, cells were collected and assayed for CAT activity. As depicted
in Fig. 2A, Rosi increased
4551UCP-1-CAT activity 3-fold, and this stimulation was totally
precluded when the p38MAPK inhibitor was present in the culture medium.
These results indicate that p38MAPK but not p44/p42MAPK or PKA activity
is necessary for Rosi-induced UCP-1 gene transcription. To check
whether the inhibition of p38MAPK was affecting the levels of the
PPAR
protein, a direct Western blot was performed (Fig.
2B). Similar PPAR
protein content was detected in brown
adipocytes either in the absence or presence of PD*. Furthermore, we
checked whether nuclear protein binding to the PPRE of the UCP1
enhancer was affected by the inhibition of p38MAPK, either in the
presence or absence of Rosi. Nuclear extracts from cells cultured for
24 h with or without Rosi and/or PD* were used in gel mobility
shift assays using UCP-1-PPRE double-stranded oligonucleotide as a
probe (Fig. 2C). Protein binding to DNA was observed in
control brown adipocytes, and this binding was essentially unmodified
either by the presence of Rosi or PD*. This band seems to be specific,
because it disappears in competition experiments with a 100-fold molar
excess of unlabeled PPRE probe.
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Many ligands for nuclear receptors act not only on gene expression
activating nuclear receptors, but also affect cytosolic signaling
pathways (29-31). To check the possibility that Rosi could be inducing
p38MAPK activation, we treated brown adipocytes with Rosi for different
period of time, and cell lysates were analyzed for dual phosphorylation
(Thr-180/Tyr-182) of p38MAPK by Western blot with a polyclonal
antibody. No phosphorylation of p38MAPK was detected in control cells
cultured for 20 h in a serum-free BSA-MEM medium. However, Rosi
treatment for 1 h induced p38MAPK phosphorylation, which was
maintained up to 6 h (Fig. 3A). The changes observed in
the amount of phospho-p38MAPK reflect changes in the activity, because
the protein levels of p38MAPK are similar in all the conditions.
Furthermore we performed p38MAPK activity assay with protein lysates
from control cells and cells treated with Rosi for 3 h and
obtained similar results. Rosi increased MBP phosphorylation 3-fold in
anti-p38MAPK immunoprecipitates, and this activation was prohibited in
cells cotreated with Rosi and PD*, validating the use of this chemical
compound as a p38MAPK inhibitor (Fig. 3B). Western blot
analysis with anti-phospho-p44/p42MAPK antibody revealed that the
presence of Rosi for 3 h also increases p44/p42MAPK activity,
which is further elevated after 6 h (Fig. 3C). This
p44/p42MAPK activation is inhibited by PD, validating the use of this
chemical compound in brown adipocytes. 0.1% Me2SO, the
vehicle used, did not produce any effect (data not shown). These data
clearly show that Rosi, besides being a PPAR agonist, stimulates
kinase activities that can modulate its effects on gene expression.
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Although p38MAPK activation, but not p44/p42MAPK, is involved in UCP-1
gene induction by Rosi, this kinase does not play any role in UCP-3
expression either induced by Rosi or by the PPAR agonist Wy14643 as
is assumed from the results obtained by Northern-blot analysis and
represented in Fig. 4, where it is shown
that UCP-3 expression is induced in cells treated with Rosi or Wy14643
regardless of the presence of the p38MAPK inhibitor PD169316.
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Because PPARs bind to DNA as heterodimers taking RXR as partner, and
RXR ligands also increase UCP-1 gene transcription, we wanted to check
the possibility that p38MAPK activation was also involved in UCP-1
induction by RXR ligands. Brown adipocytes were cultured for 24 h
with 9-cis-RA (agonist for RXR and RAR) or
all-trans-RA (RAR agonist) in the absence or presence of the
p38MAPK inhibitor (PD169316). Northern-blot analysis for UCP-1
expression revealed that 9-cis-RA treatment produced a huge
increase in UCP-1 mRNA levels (12-fold) that was inhibited 50% in
the presence of PD169316. The RAR agonist also induced UCP-1 expression
but to a lesser extent than 9-cis-RA, this effect also being
precluded by inhibition of p38MAPK. As previously published (12), none
of the retinoids had any effect on UCP-3 expression (Fig. 4). Because
p38MAPK activation also seemed to be involved in UCP-1 induction by
retinoic acids, we decided to examine whether 9-cis-RA and
all-trans-RA also activated p38MAPK; this was monitored by a
Western blot of phosphorylated p38MAPK. As shown in Fig.
5, 9-cis-RA induced p38MAPK
phosphorylation with a timing pattern similar to Rosi. p44/p42MAPK
activation was also observed upon 9-cis-RA stimulation.
Regarding all-trans-RA, it also activated p44/p42MAPK and
p38MAPK but less strongly than 9-cis-RA.
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A very recent paper, which showed that MAPK activation by the PPAR
agonists 15-deoxy-
12,14-prostaglandin J2
(dPGJ2) and ciglitazone is produced through a mechanism involving
reactive oxygenated species (ROS) (32), prompted us to examine whether
ROS generation could be involved in UCP-1 induction by Rosi and/or
retinoic acids. Brown adipocytes were or were not pretreated 30 min
with an antioxidant agent, reduced GSH, before the addition of
Rosi or 9-cis-RA and further cultured for 24 h. UCP-1
expression was analyzed by Northern-blot, and the results depicted in
Fig. 6 show that the presence of GSH inhibited the increase in UCP-1 mRNA levels elicited by either Rosi
or 9-cis-RA.
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DISCUSSION |
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Nuclear PPARs activate the transcription of multiple genes involved in lipid metabolism as well as the thermogenic protein UCP-1 and the recently described UCP-2 and UCP-3. PPARs are activated by ligand binding, which induces conformational changes leading to the recruitment of several coactivators (33). Furthermore, some agonists for PPARs, including the prostaglandin 15d-PGJ2 and the thiazolidinediones ciglitazone, pioglitazone, and troglitazone, have shown PPAR-independent effects, activating different cytosolic signaling pathways (ERK or c-Jun NH2-terminal kinase or p38MAPK) depending on the cellular system (30, 32, 34). It has also been reported that retinoic acids induce ERKs and p38MAPK activation in some cellular systems (29, 31, 35). To our knowledge, the results presented in this study describe for the first time p44/p42 and p38MAPK activation by 9-cis-RA and all-trans-RA as well as Rosi in fetal rat brown adipocytes. Kinetics for both activations are different. Meanwhile p38MAPK activation is rapid and sustained, whereas p44/p42MAPK phosphorylation delays and happens gradually. The biological effects of p44/p42MAPK activation are unknown. Phosphorylation by p44/p42MAPK can modulate PPAR activity, both in a positive and negative manner (15, 16). Nevertheless, from our results we conclude that this kinase is not involved in UCP-1 gene expression, but other effects remain unexplored. On the contrary, the timing of p38MAPK phosphorylation by Rosi fits tightly to the time frame for UCP-1 gene induction (11), and inhibition of p38MAPK activity prevents UCP-1 mRNA increase by this thiazolidinedione or retinoids. This effect seems to be specific for the thermogenic protein UCP-1 and not related to the differentiation-inducing properties of Rosi or p38MAPK, because the expression of the adipogenic gene FAS remains unmodified by either the presence of Rosi or PD* in fetal rat brown adipocytes.
p38MAPK activation via PKA in -adrenergic UCP-1 gene induction in
post-natal mouse brown adipocytes has been reported (36). We have
discarded the implication of PKA in UCP-1 induction by Rosi in fetal
rat brown adipocytes, because H89 did not produce any effect. However,
it is possible that different mechanisms operate in fetal as compared
with post-natal brown adipocytes differentiated in culture. ROS
generation has been involved in p38MAPK activation by ciglitazone and
15d-PGJ2 (32). Although we have not measured ROS formation by Rosi, the
fact that the antioxidant GSH prevents UCP-1 induction by this
thiazolidinedione in a similar fashion as that of the p38MAPK inhibitor
suggests a role for ROS in the action of Rosi in fetal brown
adipocytes. These are preliminary results that obviously deserve future study.
The inhibition of rosiglitazone-induced UCP-1 by PD* is not mediated by
changes either in PPAR levels or in nuclear protein binding to
UCP-1-PPRE. However, we cannot exclude the possibility that PD* could
be blocking the binding of Rosi to PPAR
, although it does not seem
likely because other effects of Rosi (such as UCP-3 induction) are not
modified by the presence of PD*. PPAR, RXR, and RAR bind PGC-1, a
master coactivator involved in thermogenesis and UCP-1 expression.
PGC-1 has been shown to be susceptible to phosphorylation by p38MAPK,
resulting in a positive regulation of its transcriptional activity (18,
37). Therefore, it is tempting to speculate that Rosi and retinoids
would act by two convergent mechanisms: 1) binding to specific nuclear
receptors; and 2) switching on signaling pathways (p38MAPK) that
positively modulate coactivators involved in activating transcription
of specific genes such as UCP-1. In contrast to our results, Oberkofler et al. (38) have reported that the inhibition of p38MAPK
signaling decreases the stimulatory effects of PPAR
agonists
treatment on UCP-1 transcription without reducing the response to
thiazolidinediones or retinoic acids. This study was performed in the
human brown adipocyte cell line PAZ6 co-transfected with expression
vectors for h-PGC-1 and UCP-1-reported plasmids, whereas our
experimental conditions are closer to a physiological situation because
the primary culture is used with no exogenously expressed protein. These differences or even cell type (human versus rat) could
explain the contrasting results. In any case, both studies show that
p38MAPK activity regulates PPAR/RXR action on UCP-1 gene transcription.
Regarding UCP-3, we show that both Rosi and Wy14643 (PPAR and
PPAR
agonists respectively) induce UCP-3 expression, but the mechanism seems to differ from UCP-1 because p38MAPK inhibition does
not affect the response. This could imply that there is no participation of PGC-1 in UCP-3 gene expression as has been reported in
white adipose or skeletal muscle cells ectopically expressing PGC-1 (4,
39) or even that the PPRE found in the ucp-3 promoter is not
functional and that other response elements are responsible for Rosi-
or Wy14643-induced UCP-3 gene expression as was reported for UCP-2
(40). In conclusion, Rosi or retinoid treatment activates p44/p42MAPK
and p38MAPK, the latter being involved in UCP-1 but not in UCP-3 gene
expression in fetal rat brown adipocytes.
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ACKNOWLEDGEMENT |
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We thank the Cooperation in the Field of Scientific and Technical Research (COST) B17 Action (Insulin Resistance, Obesity and Diabetes Mellitus in the Elderly) from the European Commission.
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FOOTNOTES |
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* This work was supported by Direccion General de Investigacion Cientifica, Ministerio de Ciencia y Tecnologia (Spain) Grants PM1998-0082 and BMC2002-01322.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this experimental work.
§ Recipient of a postdoctoral fellowship from the Comunidad Autonoma de Madrid.
¶ Recipient of a postgraduate fellowship from the Ministerio de Educacion y Cultura.
To whom correspondence should be addressed. Tel.:
34-913941858; Fax: 34-913941779; E-mail: mlorenzo@farm.ucm.es.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M207200200
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ABBREVIATIONS |
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The abbreviations used are:
BAT, brown adipose
tissue;
BSA, bovine serum albumin;
CAT, chloramphenical
acetyltransferase;
ERK, extracellular signal-regulated kinase;
FAS, fatty acid synthase;
FCS, fetal calf serum;
MAPK, mitogen-activated
protein kinase;
MBP, myelin basic protein;
MEM, minimal essential
medium;
PBS, phosphate-buffered saline;
PD, PD98059;
PD*, PD169316;
PGC-1, PPAR coactivator;
PKA, protein kinase A;
PPAR, peroxisome
proliferator-activated receptor;
PPRE, PPAR response element;
RA, retinoic acid;
RAR, RA receptor;
ROS, reactive oxygenated species;
Rosi, rosiglitazone;
RXR, retinoid X receptor;
UCP, uncoupling
protein.
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