Download 1 sirt1 is involved in glucocorticoid-mediated control of

JBC Papers in Press. Published on September 20, 2007 as Manuscript M707114200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M707114200
SIRT1 IS INVOLVED IN GLUCOCORTICOID-MEDIATED
CONTROL OF UNCOUPLING PROTEIN-3 GENE
TRANSCRIPTION
Ramon Amat, Gemma Solanes, Marta Giralt, Francesc Villarroya*
From Departament de Bioquimica i Biologia Molecular and Institut de Biomedicina
(IBUB), Universitat de Barcelona and CIBER Fisiopatologia de la Obesidad y Nutrición,
Instituto de Salud Carlos III, Avda Diagonal 645, 08028-Barcelona, Spain
Running title SIRT1 controls uncoupling protein-3 gene
*Address correspondence to: Francesc Villarroya, Departament de Bioquimica i Biologia
Molecular, Facultat de Biologia, Universitat de Barcelona. Avda Diagonal 645, 08028Barcelona, Spain. Tel. 34 934021525, FAX 34 934021559, E mail: [email protected]
transporter
(p300) or negatively (histone deacetylases)
expressed preferentially in skeletal muscle.
the action of glucocorticoids on UCP3 gene
UCP3 lowers mitochondrial membrane
transcription via histone acetylation or
potential and protects muscle cells against
deacetylation
an overload of fatty acids, as well as it
Among them, SIRT1 acts as a major
probably reduces excessive production of
repressor of UCP3 gene expression in
reactive
Accordingly,
response to glucocorticoids. The action of
UCP3 gene transcription is highly sensitive
SIRT1 requires its deacetylase activity and
to fatty acid-dependent stimulation, but
results in histone deacetylation in the
also to other unrelated stress signals. In
UCP3 promoter. Moreover, it involves a
the present study, glucocorticoids are
specific impairment of association of p300
identified as major inducers of UCP3 gene
with the glucocorticoid receptor. Agents
transcription in muscle. Glucocorticoids
activating SIRT1, such as resveratrol,
activate the transcription of the UCP3
repress UCP3 gene expression. The control
gene through a glucocorticoid receptor-
of SIRT1 activity via the cell’s metabolic
binding site in the promoter region.
redox status points to a novel regulatory
Glucocorticoids are capable of inducing
pathway of UCP3 gene transcription in
UCP3 gene transcription independently
response to metabolic and stress signaling
from the myogenic regulatory factor
in muscle cells.
mitochondrial
membrane
oxygen
species.
processes,
respectively.
MyoD, in contrast with the transcriptional
activation of the UCP3 gene through other
Uncoupling protein-3 (UCP3) is a
nuclear hormone receptors. An interplay
member of the family of the mitochondrial
1
Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
Downloaded from www.jbc.org by on September 23, 2007
of regulatory factors modulate positively
Uncoupling protein-3 (UCP3) is a
preferentially
protective mechanism when levels of free
expressed in skeletal muscle. The precise
fatty acids are high in order to favor fatty acid
physiological function of UCP3 is still a
oxidation
matter of debate. Biochemical studies using
mitochondria
isolated mitochondria indicated that the
production.
membrane
transporters
or
fatty
acid
without
export
excess
from
ROS
UCP3 gene induction may be under
with proton permeabilization and a reduction
the control not only of stress-related signals
in mitochondrial membrane potential, of
arising from fatty acid overload or thyroid
which the main physiological role would be
effects, but also of signals of wider origin.
to protect against excessive production of
Thus, UCP3 mRNA is up-regulated in muscle
reactive oxygen species (ROS). In fact, ROS
in response to experimentally-induced sepsis
themselves or by-products of ROS act as
(10), lipopolysaccharide (LPS) injection (11)
activators of the proton conductance activity
or cancer cachexia (12) and these effects
of UCP3 (1). On the other hand, several
apparently occur independently of fatty acid
studies
increased
or thyroid actions. Glucocorticoids may play
expression of UCP3 in muscle cells favors
a role in the induction of UCP3 gene
fatty acid oxidation (2;3). These observations
expression in muscle in response to stress.
are
Thus, injection of dexamethasone induces
have
established that
consistent
with
the
transcriptional
regulation of the UCP3 gene, which is
UCP3
extremely sensitive to fatty acids (4-6). The
glucocorticoids
human and mouse UCP3 gene promoters
mediate
contain
induction of UCP3 gene expression (10).
peroxisome
proliferator-activated
mRNA
most
expression
have
of
been
the
(13)
proposed
and
to
sepsis-mediated
elements
Glucocorticoids are known to play a major
that mediate responsiveness to fatty acids
role in the response of skeletal muscle to
(7;8).
provide
catabolic conditions such as sepsis, severe
responsiveness to thyroid hormones (9). The
injury or burn, and glucocorticoids are
myogenic factor MyoD acts as a permissive
considered important mediators of muscle-
transcription factor for PPAR-dependent and
wasting and mitochondrial dysfunction in
thyroid-dependent regulation of UCP3 gene
such conditions (14).
receptors
(PPAR)α/δ-responsive
These
same
elements
In the present study we identify
transcription in skeletal muscle (7). Although
the
high
sensitivity
of
UCP3
glucocorticoids
gene
as
direct
activators
of
transcription to fatty acids does not provide
transcription of the UCP3 gene and we report
any
specific
that glucocorticoid-dependent stimulation of
physiological role of UCP3, it suggests that
UCP3 gene transcription is involved in the
this would be related to fatty acid handling by
response to LPS. Acetylation processes
muscle cells. Current hypotheses are that
mediated by co-activators such as p300,
UCP3 gene expression would be induced as a
which bears histone acetylase activity, as well
direct
evidence
for
the
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uncoupling activity of UCP3 is associated
Cell
as deacetylation mediated by class I histone
Culture:
Mouse
myoblastic
deacetylases are involved in the control of
C2C12 and rat myoblastic L6 cells were
UCP3 gene transcription in response to
obtained from the American Type Culture
glucocorticoids. Here we establish that the
Collection (Manassas, VA). L6 cells were
silent information regulator 2 mammalian
grown in DMEM (Dulbecco's modified
ortholog SIRT1, is also involved in mediating
Eagle's medium) containing 10% fetal bovine
negative regulation of UCP3 gene expression.
serum (FBS) and C2C12 cells in DMEM
SIRT1 is a member of the class III histone
supplemented with 10% horse serum. C2C12
deacetylases
recently
cells were induced to differentiate using
recognized as being involved in multiple
DMEM supplemented with 2% horse serum.
biological
caloric
Treatments with 100 nM dexamethasone, 100
restriction,…) which are all associated with
µM resveratrol, 10 mM nicotinamide (NAM)
energy
(Sigma, St Louis, MO) and 50 µM sirtinol
that
has
processes
metabolism
been
(aging,
(15;16).
SIRT1
is
(Calbiochem,
implicated in myogenic differentiation (17).
San Diego, CA) were performed in C2C12
Thus, as SIRT1 activity is under the control
myotubes at day 4 of differentiation.
of the NAD/NADH ratio, it constitutes an
energy
sensing
Biosciences
Analysis of UCP3 mRNA levels:
capable
of
Northern blot analyses of 20 µg of total RNA
regulation
in
from skeletal muscle samples were performed
response to the metabolic energy status of
using standard methods and a fragment of the
cells.
mouse
mediating
molecule
EMD
transcriptional
UCP3
cDNA
as
probe
(5).
Hybridization signals were quantified using a
EXPERIMENTAL PROCEDURES
PhosphorImager
(Molecular
Dynamics,
Image Quant software). For cells, quantitative
Animals
and
treatments:
Male
UCP3
mRNA
expression analysis
was
Sprague Dawley rats, 8-10 weeks of age,
conducted using a two-step reaction and
(Taconic) were injected intraperitoneally with
reagents from Applied Biosystems (Foster
LPS (Sigma, St Louis, MO) (5 mg/ kg),
City, CA). First, 1µg of RNA was transcribed
mifepristone (RU-486, Biomol, Plymouth,
into
PA) (30 mg/kg), dexamethasone (Sigma, St
Transcription Reagents. Second, a TaqMan
Louis, MO) (10mg/kg), adrenocorticotropic
Real-Time PCR reaction was performed
hormone (ACTH, Peninsula Laboratories,
within the ABI PRISM 7700HT sequence
Belmont, CA) (1 mg/kg) or saline between 9-
detection system using the TaqMan Universal
10 a.m. Six hours after treatment, rats were
PCR Master Mix and the standardized
sacrificed and gastrocnemius skeletal muscle
primers
was obtained.
(Mm00494074). Appropriate controls lacking
cDNA
for
using
mouse
TaqMan
UCP3
Reverse
mRNA
RNA, primers, or reverse transcriptase were
3
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expressed in skeletal muscle and it has been
included in each set of experiments. Each
deacetylase activity through a change of
sample was run in duplicate, and the mean
histidine 363 to tyrosine were generated
value of the duplicates was used to calculate
using
the mRNA expression. The quantity of the
mutagenesis kit (Stratagene, La Jolla, CA).
transcript for UCP3 in each sample was
Complementary oligonucleotides containing
normalized to that of the endogenous
the
reference
GCCATGATGAAAGTCTCGAGAACTAG
transcript
(β2-microglobulin,
a
Quick-change
desired
site-directed
mutation,
(5’-
Mm00437762) using the comparative (2-
CATGTAGTAGTTGATCAACC-3’
for
∆CT)
2mUCP3-mutGRE-Luc
5’-
method,
according
to
the
and
GGATCCTTCAGTGTTATGGTTCCTTTG
manufacturer’s instructions.
CAACAGCATCTTGCC-3'
Plasmid constructs: The plasmid
for
SIRT1
H363Y) were used. The mutated plasmids
promoter from -1946 to + 65 bp cloned into
were checked by direct DNA sequencing.
pGL3 basic
(2mUCP3-Luc) has been
The plasmid GRE-luc (18) is a vector that
described previously (9). Deleted constructs
contains the luciferase reporter gene under
were obtained from 2mUCP3-Luc by PCR
the control of the consensus glucocorticoid
reaction using the following 5' primers: 5'-
response element (GRE).
GGGTACCGCTGGGGGAAGGGAAAGGG
Transient
GG-3' (-615 to -594)
Transfection
Assays.
to generate -
Transfection experiments were carried out in
5'-
L6 cells at 50% confluence using FuGene
615mUCP3-Luc;
GGGGTACCTGCTTCCTAGAGATGTGG-
Transfection Reagent (Roche Diagnostics,
3' (-440 to 419) to generate -440bp mUCP3-
Barcelona, Spain). Each transfection point
Luc; 5'CCTGCCTCTTGTCAAGTGATC 3'
was assayed in duplicate or triplicate in a six-
(-100 to -79) to generate -100bp mUCP3-
well plate and contained 1.5 µg of luciferase
Luc. The complementary 3' primer was 5'-
reporter vector, 0.3 µg of the mammalian
GGGAAGCTTCCATTCCAAGGTAGCAG
expression vectors myc-GR (19), pCMV-
CAGG-3', +35 to +65 downstream of the
MyoD (20), prSV-human TRβ1 (21), pCMV-
transcription initiation site, according to
p300
GenBank/EMBL
pcM/HDAC-1 and pcM/HDAC-H141A (23),
data
(Accession
and
pCMV-p300∆HAT
AB011070). The resulting DNA products
pcx-P/CAF
were cloned into pGL3-basic (Promega,
pcDNA3-SRC1 (25), Tat-interactive protein-
Madison, WI), which contains the cDNA for
60Kda Tip60 (26), pCMV-PGC1α (27),
firefly luciferase as a reporter gene. All
pCMV-PGC1β (provided by A. Vidal-Puig),
fragments were sequenced by the dideoxy
pCMV-SIRT1 (Upstate Biotechnology, Lake
method.
Placid,
The
point-mutation
construct
(2mUP3-mutGRE-Luc) and a version of the
SIRT1
expression
plasmid
devoid
and
NY)
or
pcx-P/CAF∆HAT
(22),
pCMV-SIRT3
(24),
(Open
Biosystens, Huntsville, AL). The pRL-CMV
of
4
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construct bearing the mouse UCP3 gene
expression vector for the sea pansy (Renilla
presence of the glucocorticoid receptor (GR)
reniformis) luciferase was used as an internal
expression vector, which expresses a GR-
transfection control (Promega, Madison, WI).
Myc fusion protein, and were treated with
Cells
dexamethasone (100 nM). Protein–DNA
were
incubated
for
48
h
after
cross-linking
treated for 24h before harvest with 100 nM
formaldehyde (final concentration 1%) for
dexamethasone or 50 nM T3. Firefly
30’. Then, 1.25 mM glycine was added. The
luciferase and Renilla luciferase activities
cells were washed twice with ice-cold PBS
were
Designs
and were centrifuged, and re-suspended in
Luminometer (TD 20/20) using the Dual
lysis buffer (50 nM Hepes-KOH pH 7.4, 140
Luciferase Reporter assay system (Promega,
mM NaCl, 1 mM EDTA, 10 % glycerol, 0.5
Madison, WI). Luciferase activity elicited by
mM EGTA, 0.25 % Triton X-100, 0.5 %
UCP3 promoter constructs was normalized
Igepal) supplemented with protein inhibitors.
for variation in transfection efficiency using
Another centrifugation was performed and
Renilla luciferase as an internal standard.
the pellets were re-suspended in wash buffer
measured
in
a
Turner
was
achieved
by
adding
Western blot analysis. Cell extracts
(10 mM Tris-HCl ph 8, 200 mM NaCl, 1 mM
from myogenic cells were prepared by
EDTA, 0,5 mM EGTA) and incubated for 10
homogenization in a buffer containing 100
min at room temperature. The samples were
mM Tris (pH 8.5), 250 mM NaCl, 1% Igepal
then centrifuged and the pellets were re-
CA-630 (Sigma St Louis, MI), 1 mM EDTA,
suspended in 2 ml RIPA buffer (10 nM Tris-
a cocktail of protease inhibitors (Complete-
HCl pH 8, 140 mM NaCl, 1 mM EDTA, 5 %
Mini, Roche Diagnostics Applied Science,
glicerol, 0,5 mM EGTA, 0,1 % sodium
Barcelona,
deoxycholate, 0,1 % SDS, 1 % Triton X-
Spain)
and
0.1
%
phenylmethylsulphonyl fluoride. Proteins (20
100).
µg/lane) were separated by 10% or 8% SDS-
sonicated, centrifuged for 15’ at 4ºC and
PAGE gel electrophoresis and transferred to
incubated overnight at 4ºC with 5 µg of Myc
Immobilon-P
(Millipore).
antibody or 2 µg of the p300 antibody. The
Immunological detection was performed with
immunocomplex was collected by binding to
specific antibodies against HDAC1 (7028
60 µl protein G-sepharose slurry. After
Abcam, Cambridge, UK), p300 (Santa Cruz,
incubation for 1 h at 4ºC, the sepharose beads
CA, SC-585), SIRT1 (07-131 Upstate, Lake
were collected by centrifugation and washed
Placid, NY), c-Myc (a gift from C.Caelles,
5 times at 4ºC with 1 ml of RIPA buffer
IRB, PCB, Barcelona, Spain) or β-actin
supplemented with protein inhibitors. The
(Sigma, A5441, St Louis, MI).
pellets were then eluted in 100 µl of TE, 0.5
membranes
The
re-suspended-pellets
were
immunoprecipitation
% SDS, and 200 µg /ml proteinase K for 4
(ChIP) assay. L6 cells were transfected with
hours at 55ºC. Finally, another incubation at
2mUCP3-luc or 2mUCP3GREmut-luc in the
65ºC for 6 hours was performed. After phenol
Chromatin
5
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transfection and, when indicated, they were
/ chloroform extraction, the DNA was used
with 1 ml of buffer containing 50 mM Tris-
for PCR analysis.
HC1, 0.15 M NaCl, 5 mM EDTA, and 1 %
ChIP analysis of acetyl-histone H3 and
Nonidet P-40 (pH 7.4) prior to resuspending
acetyl-histone H4 was performed using the
the precipitates in sample buffer. Western-
supplied reagents (Acetyl-Histone H3 and H4
blot of immunoprecipitated extracts was
Immunoprecipitation, ChIp, Assay kits) from
performed as already indicated, except for
Upstate Biotechnology (Lake Placid, NY).
p300 analysis in which case was used one-
The primers for amplifying a 690-bp
step IP-Western Kit (GenScript Corporation,
fragment from -615 to + 65 bp encompassing
NJ, USA), as indicated by supplier. To check
the GRE of the mouse UCP3 promoter were
for similar amounts of p300 protein in
5'-
myogenic cells (p300 input), western-blot
GGGGTACCGCTGGGGGAAGGGAAAGG
was performed using equal amounts of
GGG-3'
protein extracts before immunoprecipitation.
(forward)
and
5'-
SIRT1
gene
GCAGG-3’ (reverse). The PCR reaction was
transfer. An adenoviral vector driving the
performed for 25 cycles at 95ºC for 30 s,
expression of murine SIRT1 cDNA was
66ºC for 30 s, and 72ºC for 1 min 30 s. The
constructed (Ad5-CMV-SIRT1, CEBATEG,
PCR products were electrophoresed in a 1 %
Barcelona). C2C12 myotubes were infected
agarose gel, visualized by ethidium bromide
with the adenoviral vector or AdCMV-GFP
staining and quantified by densitometric
(control) at a multiplicity of infection of 20 or
analysis (Phoretics 1D Softward, Phoretic
50 for 3 h in DMEM medium. Further culture
International Ltd, UK).
of cells was conducted with regular culture
Immunoprecipitation Whole cell lysates were
medium and conditions for the C2C12
prepared by lysing L6 myoblasts in 50 mM
myotubes (see above). This treatment led to
Tris- HCl, 0.1 M NaCl, 10 mM EDTA, 1%
an efficiency of transduction of about 90% on
Nonidet P-40 (pH 7.4) containing a protease
the basis of GFP fluorescence. Western blot
inhibitor cocktail (Complete Mini, Roche,
analysis of SIRT1 expression was determined
Mannheim, Germany). GR-Myc in whole cell
as described above and equal loading of
lysate fraction was immunoprecipitated using
protein
a mouse monoclonal anti-Myc. 1 mg of
immunodetection of β-actin. C2C12 cells
protein
were
was
used
for
the
samples
studied
48
was
h
assessed
after
by
AdSIRT1
immunoprecipitation. After 2 h of mixing at
transduction, when the increase in the 110
4ºC with 1 µg of anti-myc antibody, 40 µl of
kDa immunoblot signal for SIRT1 protein
packed Protein A/G PLUS-Agarose (Santa
was around 3-4 fold with respect to controls.
Cruz Biotechnology) was added and the
Statistical analyses were performed using the
samples were mixed at 4ºC overnight.
Student’s t test.
Immunoprecipitates were washed four times
6
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Adenoviral-mediated
GGGAAGCTTCCATTCCATTAGGTAGCA
(Fig 1B) that was in the range of that elicited
RESULTS
by other bona-fide activators of the UCP3
Glucocorticoids induce UCP3 gene
gene, such as thyroid hormone T3 (9). The
expression in skeletal muscle. In order to
effect of dexamethasone was suppressed by
determine the capacity of glucocorticoids to
exposure to the GR antagonist RU-486.
induce UCP3 gene expression “in vivo”, rats
Similar results were obtained using the rat
were
of
myogenic cell line L6 (data not shown).
dexamethasone. The results indicated a
These findings confirm the positive action of
dramatic induction of UCP3 mRNA levels in
glucocorticoids on UCP3 gene expression.
treated
with
a
single
dose
Effects of dexamethasone on UCP3
was found when rats had been injected with
promoter activity. In order to establish
ACTH in order to induce a rise in
whether UCP3 gene induction in response to
endogenous glucocorticoids. Further analysis
glucocorticoids is due to direct transcriptional
of the involvement of glucocorticoids in the
activation, a construct containing 2kb of the
control of UCP3 gene expression was
UCP3 promoter region driving expression of
performed using LPS injections. A single
the luciferase reporter gene was transfected
injection of LPS caused a dramatic induction
into
of UCP3 mRNA levels, as previously
dexamethasone did not significantly modify
reported (11) whereas concomitant injection
basal
with the GR inhibitor RU-486 significantly
transfection with an expression vector driving
diminished the ability of LPS to induce UCP3
expression of GR conferred to the UCP3
gene expression (Fig 1A, right). These results
promoter a high sensitivity to activation by
indicated
the
dexamethasone (around 30-fold induction)
activation of GR, are involved in the control
(Fig 2). To date, hormonal regulation of
of UCP3 gene expression.
UCP3 gene transcription by known activators
that
glucocorticoids,
via
L6
myogenic
promoter
cells.
activity.
Addition
However,
of
co-
Effects of dexamethasone on UCP3
such as thyroid hormones or PPAR activators
mRNA expression in myotubes. UCP3 mRNA
required the co-transfection of a MyoD
levels were barely detectable in C2C12
expression vector (7). Effectively, in the
myoblasts, though they became higher when
absence of transfected MyoD the UCP3 gene
C2C12 cells differentiated into myotubes.
promoter remained insensitive to T3 despite
However, the UCP3 mRNA levels remained
the dramatic activation by glucocorticoids
much lower than in mouse skeletal muscle
(Fig
(around two-orders of magnitude lower), but
conferred to the UCP3 gene sensitivity to
were nonetheless detectable using real-time
activation by T3 and enhanced the activity of
PCR.
the UCP3 promoter in the presence of
Exposure
dexamethasone
led
of
to
a
myotubes
to
modest
but
2).
transfected
appears
significant induction of UCP3 mRNA levels
7
Co-transfection
GR
and
therefore
with
MyoD
dexamethasone.
that
It
glucocorticoid-
Downloaded from www.jbc.org by on September 23, 2007
skeletal muscle (Fig 1A, left). A similar result
dependent regulation of the UCP3 gene,
between –573 to –559 compatible with the
differently to other hormonal regulators, does
structure of a consensus GRE (Fig 3B). A
not necessarily require MyoD. Identical
version of the UCP3 promoter construct in
results were obtained in parallel experiments
which point mutations were introduced in the
using C2C12 myogenic cells (data not
putative GRE displayed a loss of most of the
shown).
responsiveness to dexamethasone. These
The
transfection
requirement
to
of
GR
show
responsiveness
was
also
co-
results
to
indicate
that
most
of
the
investigated,
responsiveness of the UCP3 gene promoter to
considering the existence of endogenous GR
glucocorticoids relies on the presence of the -
expression in myogenic cells (28). A
573/-559 sequence. However, the presence of
construct in which luciferase is driven by two
minor but significant responsiveness to
copies
dexamethasone, both in the deleted construct
dexamethasone
of
responsive
a
consensus
element
glucocorticoid
(GRE-Luc)
lacking this GRE and in the mutated version,
was
indicates
that
the
presence
exposed to dexamethasone in the absence or
additional
presence of co-transfected GR. The results
glucocorticoids in the proximal region of the
indicated that dexamethasone significantly
promoter cannot be excluded.
elements
of
minor
sensitive
to
induced the GRE reporter construct only
In order to establish whether the GRE
when GR had been co-transfected, (Fig 2).
at –573/-559 binds the GR, L6 cells were
This indicated that the requirement for co-
transfected with the 2mUCP3 promoter
transfected GR is not a particular feature of
construct
the UCP3 gene, but was shared by standard
expression vector for a Myc-tagged version
responsiveness to glucocorticoids in this
of GR. ChIP using the c-Myc antibody
transient transfection experimental setting.
revealed enrichment of the 630 bp PCR
and
co-transfected
with
the
The effect of glucocorticoids on
product of the UCP3 promoter sequence
UCP3 gene transcription is mediated by a
encompassing the GRE (Fig 3C). Parallel
glucocorticoid-responsive element in the
experiments
promoter region. In order to establish
promoter construct corresponded to the point-
whether the action of dexamethasone is due
mutated GRE version showed a lack of
to the presence of a GRE in the UCP3
enrichment. This demonstrates that GR binds
promoter, serial deletion constructs of the
to the –573/-559 region of the UCP3
mouse UCP3 gene were analyzed. The results
promoter.
in
which
the
transfected
indicated that deletion of the region from –
Acetylation processes mediate the
615 to –440 in the promoter led to a drop of
action of glucocorticoids on UCP3 gene
or around 75% in the
transcription.
responsiveness to
Previous
studies
on
the
dexamethasone (Fig 3A). Sequence analysis
hormonal, MyoD-dependent, regulation of
of this region revealed the presence of a site
UCP3 gene transcription have underlined the
8
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transfected into L6 cells and the cells were
involvement
of
These
co-regulator-driven
results
indicate
that
acetylation
acetylation processes. In order to determine
processes driven by p300, and potentially
whether glucocorticoid-dependent activation
down-regulated by deacetylation via type I
is also mediated via acetylation processes,
HDACs, are involved in the mechanisms of
expression vectors for co-regulators bearing
transcriptional control of the UCP3 gene by
histone
glucocorticoids.
acetylation
activity
were
co-
In order to examine this proposal
constructs and checked for their capacity to
directly, the UCP3 gene promoter was
alter the responsiveness to dexamethasone.
transfected in the presence or not of co-
Among all the co-activator expression vectors
transfected GR plus dexamethasone, and the
tested (SRC-1, Tip60, PCAF, PGC-1α, PGC-
extent of histone-3 and histone-4 acetylation
1β,
enhanced
in the proximal UCP3 promoter region was
glucocorticoid-dependent activation of the
determined by ChIP using anti-acetylated
UCP3 promoter (Fig 4A) despite the fact that
histone-3 or acetylated histone-4 antibodies.
they were all capable of co-activating known
The results indicated that the action of
target genes in the context of myogenic cells
glucocorticoids resulted in an increase in
(see Supplementary material). Moreover, co-
acetylation of histones binding to the
transfection with a mutated form of p300
proximal promoter region of the UCP3 gene
devoid of histone acetyltransferase activity
(Fig 4B). Co-transfection with the HDAC1
(p300∆HAT), significantly reduced the extent
expression vector reduced the extent of
of activation by dexamethasone, indicating
histone-3 and histone-4 acetylation in the
that glucocorticoids were unable to fully
UCP3 promoter and suppressed the effect
activate transcription of the UCP3 gene in the
driven by glucocorticoids.
with
p300)
the
only
UCP3
p300
SIRT1 is involved in the acetylation-
presence of an excess of mutated p300. No
effects were observed with parallel assays
mediated
using a ∆HAT version of PCAF (see
glucocorticoid-dependent activation of UCP3
Supplementary material). Moreover, when
gene transcription. The experiments above
cells were co-transfected with a vector
indicated that, despite the repressive effects
driving type I histone deacetylase (HDAC1),
of HDAC1 on the UCP3 promoter in its
the action of dexamethasone was severely
response to dexamethasone, even the highest
reduced
co-
doses of co-transfected HDAC1 were unable
transfection with a mutated form of HDAC1
to fully suppress the glucocorticoid effects. In
in which a histidine has been mutated to
order to test whether another type of histone
aspartic
deacetylase could be involved, cells were
(Fig
acid
4A).
Conversely,
(H141A),
thus
blunting
processes
influencing
in
transfected with a vector driving expression
enhancement of the action of glucocorticoids.
of SIRT1, the parental type of class III
deacetylase
activity
(23),
resulted
histone deacetylases or sirtuins. The results
9
Downloaded from www.jbc.org by on September 23, 2007
promoter
transfected
indicated
that
SIRT1
dramatically when SIRT1 was co-transfected
over-expression
(Fig 5B).
significantly reduced the activation of the
SIRT1 interferes with the interaction
This effect was specific to SIRT1 as parallel
of GR and p300 in the UCP3 gene promoter.
experiments indicated that SIRT3, another
Although the action of SIRT1 in reducing the
member of the sirtuin family, had no effect
extent of histone acetylation in the UCP3
on glucocorticoid-mediated regulation of the
gene promoter could be attributed to its
UCP3 gene transcription (see Supplementary
intrinsic histone deacetylase activity, it may
material).
was
also be the end-point of SIRT1 effects upon
dependent on the deacetylase activity of
specific components of the GR-associated
SIRT1, as it was not observed when an
machinery that induces transcription of the
expression vector for a mutated form of
UCP3 gene. The effects of SIRT1 on the
SIRT1 devoid of acetylase activity (29)
interaction between GR and p300 were
(mutation of histidine 363 to tyrosine,
analyzed by immunoprecipitation in L6
H363Y) and driving equivalent amounts of
myogenic cells. It was observed that SIRT1
expressed protein respect to the wild-type
transfection
SIRT1 was employed for co-transfection. The
interaction, and this effect was dependent on
enhancement
dexamethasone-induced
the deacetylase activity of SIRT1, as the
UCP3 promoter activity by p300 was also
SIRT1H363Y mutated construct had no
blunted by SIRT1, whereas the repression
effect (Fig 6A). It was also determined
due to p300∆HAT was further intensified.
whether SIRT1 specifically influenced the
Remarkably, co-transfection with HDAC1
binding of p300 in association with GR, to
plus SIRT1 expression vectors led to a full
the UCP3 gene. ChIP assays using p300
suppression of the capacity of dexamethasone
antibody indicated that GR+dexamethasone
to induce UCP3 promoter activity. This
dramatically increased p300 binding to the
additive effect suggests the participation of
UCP3 gene promoter. SIRT1, however,
both type of deacetylases in the control of
reduced such interaction (Fig 6B). The same
UCP3
result was observed in non-transfected cells,
Moreover,
of
transcription
the
in
effect
response
to
dramatically
reduced
such
by amplifying the endogenous UCP3 gene
glucocorticoids.
check
(not shown). This result establishes that the
whether the effects of SIRT1 were associated
repressive action of SIRT1 on glucocorticoid-
with modifications in the acetylation status of
mediated
histones binding to the UCP3 promoter
transcription involves specific interference
region. It was observed that the induction in
with the recruitment of p300 to the GR
binding of acetylated histone-3 and acetylated
interacting with the UCP3 promoter region.
ChIP
was
performed
to
The
histone-4 to the UCP3 promoter region when
induction
SIRT1
of
UCP3
activator
gene
resveratrol
inhibits glucocorticoid-mediated UCP3 gene
dexamethasone was present was reduced
10
Downloaded from www.jbc.org by on September 23, 2007
UCP3 promoter by dexamethasone (Fig 5A).
transcription. As the activity of sirtuin
glucocorticoids to increase UCP3 mRNA
deacetylases is under the control of activating
levels (Fig 8B). Treatment of C2C12
or repressing molecules, cells transfected
myotubes with resveratrol also blocked
with the UCP3 promoter construct were
glucocorticoid-dependent increases in UCP3
exposed to dexamethasone in the presence of
mRNA levels (Fig 8C). Similar results were
resveratrol, an activator of SIRT1. The results
recorded when L6 myotubes were used (data
indicated a dramatic impairment in the
not shown).
responsiveness to dexamethasone (Fig 7),
DISCUSSION
consistent with the repressive role of SIRT1.
However, potential effects of resveratrol not
The results of this study indicate that
considering the multiple targets of action of
glucocorticoid action is a relevant component
this reagent. In any case, this was a bona-fide
of the mechanisms of induction of UCP3
effect on the UCP3 promoter activity and was
gene transcription in response to LPS and
not due to any potential artifactual effect of
also indicate that treatment with exogenous
resveratrol on luciferase activity (30), as
glucocorticoid can induce “in vivo” the
other transfected plasmid constructs in which
expression of UCP3 mRNA. This effect is
luciferase expression is driven by basal
dependent on GR activation, as evidenced by
promoters (TK-Luc, for instance) were
the inhibitory action of the GR antagonist
unaffected by resveratrol treatment. The
RU-486.
effects of sirtinol and NAM, two compounds
glucocorticoids in vivo are consistent with
acting as
were also
previous reports (10;13). The positive effects
determined. Although they tended to increase
of glucocorticoids on UCP3 gene expression
the responsiveness to glucocorticoids, no
are also observed, although to a lower extent,
statistically
in differentiated myogenic cells in culture.
couldn’t
be
SIRT1 repressors,
significant
differences
were
These
The
observed.
observed
analysis
of
effects
of
transcriptional
regulation of the UCP3 gene indicated that
In order to establish whether the
observed repressive effects of SIRT1 on the
glucocorticoids
transfected UCP3 gene promoter occur also
transcription
in the endogenous UCP3 gene, SIRT1 was
glucocorticoid-responsive
over-expressed in C2C12 myotubes by the
which binds GR in the UCP3 gene promoter.
use of an adenoviral-driven SIRT1 expression
Remarkably, whereas all the hormonal-
vector. Enhancement of SIRT1 levels in
dependent regulation of the UCP3 gene
myotubes to around 3-4-fold those of basal
reported to date and involving members of
endogenous levels (Fig 8A) resulted in a
the nuclear hormone receptor super-family
dramatic reduction of basal UCP3 levels and
(thyroid/retinoid/PPAR) requires the action
completely
of MyoD (7;9), the effects of glucocorticoids
suppressed
the
capacity
of
11
activate
mostly
UCP3
through
element
gene
a
(GRE)
Downloaded from www.jbc.org by on September 23, 2007
excluded
involving SIRT1
via GR did not require MyoD. These findings
has been reported to be capable of mediating
identify a novel molecular mechanism of
positive
regulation
transcription
transcription of some genes (33). The present
explaining several observations of UCP3
findings seem to exclude such a possibility
gene induction under the action of physio-
for the UCP3 gene and indicate that, similarly
pathological
to fatty-acid dependent regulation, HDAC1 is
of
UCP3
gene
conditions
of
stress
upon
effects
skeletal muscle, such as the LPS treatment
a
negative
used in the present study or other conditions
transcription.
on
regulator
GR-dependent
of
UCP3
gene
in which glucocorticoid action upon skeletal
The present study shows for the first
muscle is known to be enhanced (14), such as
time that a SIRT1 deacetylase is involved in
sepsis (10), burn trauma (31) or cancer
UCP3 gene regulation. Moreover, it is shown
cachexia (12).
that the repressive action of SIRT1 involves,
together with potential direct effects on
of regulation of UCP3 gene transcription by
histone de-acetylation, effects mediated by its
fatty acids indicated that p300 plays a major
interaction with the GR-p300 complex.
role in linking PPAR-mediated fatty acid
SIRT1, via its deacetylase activity, is capable
actions at the UCP3 gene promoter with
of disrupting such interaction and therefore
acetylation of basal transcription factors and
inhibiting glucocorticoid effects. This is
enhancement of transcription (7). The data in
consistent with the capacity of SIRT1 to
the present study indicate that p300 histone
deacetylate p300 and, by this modification,
acetylase activity is involved in mediating the
lower the capacity for trans-activation of
action of GR-dependent activation of UCP3
target genes (34). However, further research
gene transcription. This is consistent with the
will be required to establish whether GR
relevance of p300 as part of the overall
itself can be acetylated and its transcriptional
machinery of catabolic response in skeletal
activity be influenced by such modification,
muscle to glucocorticoids (32). Moreover, the
similarly to that which occurs for the
present findings indicate that a high level of
androgen receptor (35).
to
The recognition of SIRT1 as a
glucocorticoids is blunted by combined over-
negative regulator of the UCP3 gene in
expression of HDAC1 and SIRT1, indicating
response to glucocorticoids has multiple
that
both type I and type III histone
implications in relation to the molecular
deacetylases are involved in the negative
mechanisms of control of UCP3 gene
control of UCP3 gene transcription. Both
transcription and the physiological control of
histone deacetylases appear to be capable of
muscle cell function. First, the results of the
diminishing the extent of acetylation of
present study constitute the first evidence that
histones binding to the proximal promoter
glucocorticoid-dependent gene transcription
region of the UCP3 gene. Recently, HDAC1
is under the negative control of the SIRT1
UCP3
transcription
in
response
12
Downloaded from www.jbc.org by on September 23, 2007
Previous studies on the mechanisms
protein. Further research will be required to
myogenic cells, which are devoid of MyoD
establish
participates
(37). Recently, SIRT1 has been reported to
systematically in GR-mediated regulation of
be capable of activating the expression of
gene transcription. However, the preliminary
several
data indicate only a minor effect of SIRT1 on
oxidative functions in muscle cells (38),
dexamethasone-induced
transcriptional
essentially through the de-acetylation of
activity of an artificial plasmid construct
PGC-1α, which acts as a co-activator of those
driven by a consensus GRE (unpublished
genes. The fact that PGC-1α does not behave
observations),
SIRT1-
as a co-activator of the UCP3 gene may
mediated repression may be related to the
explain why there is not such positive effect,
particular architecture of regulatory elements
but instead an opposite, repressive effect, of
in the UCP3 gene promoter region.
SIRT1 on the UCP3 gene.
whether
SIRT1
suggesting
that
Second, SIRT1 has been reported to
genes
The
related
SIRT
to
family
mitochondrial
of
histone
deacetylases provides molecular mechanisms
myogenic differentiation, including gene
for transcriptional control in response to
transcription
late
changes in energy metabolism, due to the
myogenic differentiation (17). The present
control of SIRT1 activity by the NAD/NADH
findings are consistent in general terms with
ratio (15;16). Mitochondrial activity and the
this observation, as UCP3 is a very late
extent of coupling is expected to influence
marker of myogenic differentiation (5;36).
cellular metabolism and the redox status of
However, the inhibitory action of SIRT1 on
the cell and, for instance, UCP2 has been
UCP3 gene expression should not be viewed
shown to reduce NADH levels (39). It has
as a simple consequence of negative effects
been recently reported that SIRT1 represses
on myogenic differentiation, as they can take
UCP2 gene transcription in pancreatic beta
place when SIRT1 levels or activity are
cells (40). UCP2 is a protein highly similar to
experimentally
already-
UCP3, expressed mainly in tissues in which
differentiated myotubes. The negative action
UCP3 is not present and, as far as currently
of SIRT1 on myogenic differentiation has
known, having similar functions in relation to
been proposed to involve de-acetylation of
mitochondrial uncoupling and control of ROS
MyoD, mediated indirectly via the interaction
production. The present findings showing a
and subsequent deacetylation of the co-
repressive action of SIRT1 on the expression
activator P/CAF (17). In the present case,
of the UCP3 gene are consistent with a
MyoD acetylation does not appear to be the
concept of a broad role of SIRT1 as a
mediator of SIRT1-dependent repression. The
repressor of UCP genes in different tissues.
effects of glucocorticoids on the UCP3 gene
Concerning the biological significance of
do not require MyoD and the inhibitory
this action, it might be hypothesized that a
action of SIRT1 is observed even in L6
transient rise in the NAD/NADH ratio due to
of
marker
increased
genes
in
of
13
Downloaded from www.jbc.org by on September 23, 2007
act negatively on the overall program of
sudden enhancement of muscle catabolism
only in response to glucocorticoid stimulation
could enhance SIRT1 activity. This would
but also as a more general mechanism.
therefore repress the expression of UCP3
Research is under way to examine this
which, in turn, would lower the NAD/NADH
possibility. Finally, the property of SIRT1
ratio thus providing a feed-back regulatory
that it can be modulated by chemicals like
system prone to the maintenance of stable
resveratrol (activator) or sirtinol (inhibitor),
levels of NAD/NADH. In fact, a potential
and the concomitant effects observed on
role of UCP2 and UCP3 in the control of
UCP3 gene expression in muscle cells, raises
NAD/NADH
been
the possibility of exploring the use of
suggested previously (41). Moreover, it has
chemicals modulating SIRT1 activity as tools
been proposed that SIRT1 activity is lowered
to modulate UCP3 gene expression. This
when there is reduction in the NAD/NADH
would
ratio due to a shift from carbohydrate-
associated with the expected biological
mediated metabolism to utilization of fatty
actions of UCP3, such as reduction of
acids (40), a metabolic situation associated
intramuscular fatty acid accumulation or
with enhanced UCP3 gene transcription (4,5).
lowering of ROS production in muscle under
Thus, the fact that negative effects of SIRT1
acute metabolic stress conditions.
homeostasis
had
promote
therapeutic
approaches
glucocorticoid-dependent
Acknowledgements: We thank Drs S. Kato,
stimulation raises the possibility that the
M. Martinez-Balbas, C. Caelles, M.C. Harter,
negative effects of SIRT1 on UCP3 gene
M.E. Harel-Bellan, A. Vidal-Puig and P.
transcription and the modulation of this effect
Puigserver for kindly supplying expression
by
vectors
absence
the
of
NAD/NADH
ratio
acts
as
a
homeostatic mechanism in muscle cells not
.
FOOTNOTES
This work was supported by Grant SAF2005-01722 (Ministerio de Educación y Ciencia, Spain).
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FIGURE LEGENDS
Figure 1. Effects of glucocorticoids on UCP3 mRNA expression in rat skeletal muscle and
C2C12 myotubes. A, Rats were injected intraperitoneally with 5 mg/ kg LPS, 30 mg/kg RU486, 10 mg/kg dexamethasone, 1 mg/kg ACTH or equivalent volumes of saline and were
studied 6 hours after treatment. B, . Differentiated C2C12 myogenic cells (day 4) were treated
with 100 nM dexamethasone (Dexa), 10 nM RU-486, or 50nM T3 for 24 hours. Bars are means
+ SEM of 5 independent experiments and statistically significant differences with respect to
controls are shown as *P<0.01.
Figure 2. Effects of dexamethasone or T3 in the presence or absence of MyoD on the UCP3
gene promoter activity. Transient transfection assays included 1.5 µg of the constructs
reporter genes. When indicated 0.3 µg of MyoD, thyroid hormone receptor β1 (TR) or GR
expression vectors were co-transfected. Cells were treated with 100 nM dexamethasone or
50nM T3 for 24h. Results are expressed as fold induction of luciferase activity with respect to
basal promoter values, and are the means + SEM of at least 3 independent experiments
performed in triplicate. Statistically significant differences due to dexamethasone or T3 are
shown as **P < 0.001 or *P<0.01. All samples co-transfected with MyoD-expression vector
showed significantly higher differences than non-cotransfected ones (P<0.001).
Figure 3. A glucocorticoid response element between -573 and -559 bp is responsible for
dexamethasone activation of the UCP3 gene promoter. A, deletion and point-mutation
analysis of the mouse UCP3 promoter in response to 100 nM dexamethasone in the presence of
co-transfected GR expression vector. The results are expressed as the fold-induction of
luciferase activity with respect to basal promoter values, and are the means + SEM of at least 3
independent experiments performed in triplicate. For each construct, statistically significant
differences due to dexamethasone are shown as *P<0.05, and those with respect to 2mUCP3Luc activity at a given condition as #. B, sequence of the -573 to -559 region of the UCP3 gene
promoter and comparison with a consensus GRE. In lower case are shown the point-mutations
introduced to generate the 2mUCP3-mutGRE-Luc construct. C, ChIP analysis of GR binding to
the mouse UCP3 promoter. ChIP was performed with 2mUCP3-Luc and 2mUCP3-mutGRELuc constructs. The experiment was performed in the presence of co-transfected Myc-tagged
GR expression vector and 100 nM dexamethasone. A representative example of ChIP analysis
is shown in the left. The arrow indicates the 630 bp PCR product from UCP3 gene
17
Downloaded from www.jbc.org by on September 23, 2007
containing 1946 bp of the mouse UCP3 (2mUCP3-Luc) promoter region or GRE-Luc as
encompassing the -573/-559 region. Control + means the positive control (plasmid
amplification). Bars are the means + SEM of the fold-induction in the intensity of the amplified
PCR product relative to 2mUCP3 in three independent experiments and statistically significant
difference with respect to 2mUCP3 is shown as *P<0.01 or **P<0.001.
.
.
Figure 4. Effects of p300 and HDAC1 on dexamethasone-dependent activation of the
UCP3 gene promoter. A, Co-transfection experiments were performed as described in
Experimental procedures, and when indicated, 0.3 µg of expression vectors per well were
included in the experiment. The results are expressed as fold-induction of luciferase activity
with respect to basal promoter values, and are means + SEM of at least 4 independent
experiments performed in triplicate. Statistically significant differences due to dexamethasone
cotransfected or not with a given expression vector by # P<0.05. In the right, examples of
Western blot analysis showing equal abundance of HDAC1 and H141, and of p300 and
p300∆HAT after transfection. B, ChIP analysis of the histone H3 and histone H4 acetylation
status at the UCP3 promoter after activation with dexamethasone. Effects of HDAC1. A
representative example of ChIP analysis is shown in the left. The PCR amplification product
corresponds to a 630 bp fragment of the UCP3 gene promoter (see Experimental procedures).
Input corresponds to the PCR of cleared chromatin before immunoprecipitation. Bars are the
means + SEM of the fold-induction in the intensity of the amplified PCR product relative to
2mUCP3 in at least three independent experiments and statistically significant difference due to
GR+ dex for condition is shown as *P<0.05 and that due to HDAC-1 by # **P<0.05.
Figure 5. Effects of SIRT1 on dexamethasone-mediated activation of the UCP3 gene
promoter, and functional interaction with p300 and HDAC1. A, Effect of SIRT1, SIRT1
H363Y, SIRT3, p300, p300∆HAT and HDAC1 on the dexamethasone dependent
responsiveness of 2mUCP3-Luc. Cotransfection experiments were performed as described in
Experimental procedures and, when indicated, 0.3 µg SIRT1, p300, p300∆HAT, HDAC1 or
H141A expression vectors per well were included. The results are expressed as fold induction of
luciferase activity with the co-transfected GR plus dexamethasone (GR + dex) compared to
basal promoter values (-), and are the means + SEM of at least 4 independent experiments
performed in triplicate. Statistically significant differences due to dexamethasone are shown as
18
Downloaded from www.jbc.org by on September 23, 2007
are shown as *P<0.05 and those comparing the effect of dexamethasone between cells
*P<0.05. and those due to the co-transfection of a given expression vector are shown as
#P<0.05. In the bottom, examples of Western blot analysis showing equal abundance of SIRT1
and SIRT1 H363Y after transfection are shown. B, ChIP analysis of the effects of SIRT1 on
histone H3 and H4 acetylation status at the UCP3 promoter after activation with
dexamethasone. A representative example of ChIP analysis is shown in the left, as in Fig 5.
Bars are the means + SEM of the fold-induction in the intensity of the amplified PCR product
relative to 2mUCP3 in at least three independent experiments and statistically significant
difference due to GR+ dex for condition is shown as *P<0.05 and that due to SIRT1 by #
**P<0.05.
Figure 6. Effects of SIRT1 on the interaction of p300 with the glucocorticoid receptor. A,
Immunoprecipitation analysis of the interaction of GR with p300 and the effects of SIRT1. L6
SIRT1 H363Y as indicated. Cell extracts were immunoprecipitatated using an antibody directed
against the myc-tag in the GR and Western-blotted (WB) with p300 antibody (top panel) or
anti-myc antibody (middle panel). Whole cell extracts were probed with the p300 antibody to
indicate equivalent p300 input before immunoprecipitation. B, ChIP analysis of the effects of
SIRT1 on p300 binding to the UCP3 promoter after activation with dexamethasone. A
representative example of ChIP analysis is shown in the left. The PCR amplification product
corresponds to a 630 bp fragment of the UCP3 gene promoter (see Experimental procedures).
Input corresponds to the PCR of cleared chromatin before immunoprecipitation. Bars are the
means + SEM of the fold-induction in the intensity of the amplified PCR product relative to
2mUCP3 in the absence of GR + dex in at least three independent experiments. Statistically
significant difference due to GR+ dex is shown as *P<0.05 and that due to SIRT1 by #
**P<0.05.
Figure 7. Effect of resveratrol, sirtinol or nicotinamide (NAM) on the dexamethasonedependent activity of the UCP3 gene promoter. Results are expressed as fold-induction of
luciferase activity in the presence of GR and dexamethasone with respect to basal promoter
values, and are the means + SEM of at least 3 independent experiments performed in triplicate.
Statistically significant differences due to 100 nM dexamethasone treatment are shown as
*P<0.05. Statistically significant differences due to 100 µM resveratrol, 50 µM sirtinol or 10
mM NAM treatment are shown by #.
19
Downloaded from www.jbc.org by on September 23, 2007
cells were transiently transfected with expression vectors for GR-myc, p300 and SIRT1 or
Figure 8. Effect of SIRT1 and resveratrol on UCP3 mRNA expression in C2C12 myotubes.
A, Example of an immunoblot for SIRT1 after transduction of differentiated C2C12 myotubes
with 50 (+) or 100 (++) Ad-SIRT1 M.O.I. β-actin immunoblot is shown as loading control. B,
Effects of dexamethasone on UCP3 mRNA levels in C2C12 myotubes over-expressing SIRT1
(50 M.O.I). C, Effects of treatment with 100 µM resveratrol for 24h on UCP3 mRNA levels in
C2C12 myotubes. Bars are means + SEM and statistically significant differences due to
dexamethasone are shown as *P<0.05, and those due to SIRT1 (B) or to resveratrol (C) as
#P<0.05
Downloaded from www.jbc.org by on September 23, 2007
20
A
Fig. 1
8
*
20
*
18
UCP3 mRNA
(fold increase versus saline)
UCP3 mRNA
(fold increase versus saline)
7
6
*
5
4
3
2
1
16
14
12
*
#
10
8
6
4
2
0
Control
Dex
B
ACTH
Control
2,5
*
2
UCP3 mRNA
(fold induction)
*
1,5
#
1
0,5
0
Dex
RU-486
T3
-
+
-
+
-
+
+
-
+
LPS
LPS+RU-486
Downloaded from www.jbc.org by on September 23, 2007
0
Fig 2
5
80
*
*
4.5
3.5
50
3
*
40
2.5
*
2
**
30
1.5
20
1
10
0
0.5
--
Dex
--
Dex
GR
--
T3
--
Dex
TR
-MyoD
--
Dex
GR
--
T3
TR
--
Dex
--
Dex
GR
+MyoD
2mUCP3-Luc
GRE-Luc
0
GRE-Luc activity
(Fold induction over basal)
4
60
Downloaded from www.jbc.org by on September 23, 2007
2mUCP3-Luc activity
(Fold induction over basal)
70
Fig 3
A
2mUCP3-Luc (-1946bp)
GRE
Luc 5
GR
-615mUCP3-Luc
Luc 4
-440mUCP3-Luc
Luc 3
-100mUCP3-Luc
Luc 2
**
**
*#
-
Luc 1
GR + dex
*#
0
5
10
15
20
25
30
Luciferase activity
(Fold induction)
B
GRE consensus
UCP3 GRE
UCP3 mutGRE
AGAACA NNN TGTTCT
TGATGA AAG TGTTCT
TGATGA AAG TcTcga
-573
-559
6
GR-Myc Ab
-
+
-
+
2m
UC
2m
-m UC
ut P
GR 3
Co E
nt
ro
No l +
DN
A
630 bp
P3
*
5
Relative intensity
C
4
3
2
1
0
GR-myc-Ab
-
+
2mUCP3
-
+
2mUCP3mutGRE
Downloaded from www.jbc.org by on September 23, 2007
2mUCP3-mutGRE-Luc
GRE
GR
*#
Fig 4
A
#
50
*
45
#
GR
*
GR + dex
40
HDAC1 H141
*
35
HDAC1 Ab
65kDa
30
#
25
*
20
p300
p300
∆HAT
p300 Ab
#
15
300kDa
*
10
5
0
--
p300
p300∆
∆HAT
HDAC1
H141A
B
7
2 mUCP3- Luc
6
Input
GR +
:
dex
- + - +
HDAC1
Anti-acetylHistone H4
Input
GR +
:
dex
- +
- +
HDAC1
5
Relative intensity
Anti-acetylHistone H3
*
*
4
3
2
GR +
:
dex
#
#
1
* *
* *
-
+
-
+
HDAC1
Anti-acetylHistone H3
-
+
-
+
HDAC1
Anti-acetylHistone H4
Downloaded from www.jbc.org by on September 23, 2007
2mUCP3-Luc activity(Fold induction)
-
Fig 5
A
40
30
2mUCP3-Luc activity
(fold induction)
-
*
35
*
GR+dex
*
25
#
*
20
#
15
*
#
10
*
#
5
-
SIRT1 SIRT1 H363Y SIRT3
SIRT1 +
SIRT1 +
p300∆HAT HDAC1
SIRT1
H363Y
SIRT1
+
SIRT1 +
p300
Downloaded from www.jbc.org by on September 23, 2007
0
++
+
++
SIRT1 Ab
110kDa
B
2 mUCP3- Luc
6
Input
GR +
:
dex
-
+
-
+
SIRT1
Anti-acetylHistone H4
Input
GR +
:
dex
-
+
SIRT1
+
*
*
5
Relative intensity
Anti-acetylHistone H3
4
#
3
*
2
#
*
1
GR +
:
dex
-
+
-
+
SIRT1
Anti-acetylHistone H3
-
+
-
+
SIRT1
Anti-acetylHistone H4
Fig 6
A
SIRT
1
H363
Y
SIRT
1
Myc-GR- + p300
WB: Anti p300
WB: Anti Myc-GR
Downloaded from www.jbc.org by on September 23, 2007
p300 input
B
2 mUCP3- Luc
Anti-p300
Input
GR + dex:
SIRT1:
-
+
-
+
+
Relative intensity
5
*
4
3
2
#
1
GR +
:
dex
-
+
+
SIRT1
Anti-p300
*
15
#
*
0
Downloaded from www.jbc.org by on September 23, 2007
NAM
Sirtinol
Resveratrol
-
*
-GR + dex
*
30
2mUCP3-Luc activity
(fold induction)
Fig 7
Fig 8
A
Ad
GFP
Ad-SIRT1
+ ++
110 kDa (SIRT1)
41 kDa (β-actin)
B
UCP3 mRNA
(fold induction)
GFP
SIRT1
1.2
0.8
0.4
#
#
Dexamethasone
C
*
UCP3 mRNA
(fold induction)
2.0
1.0
#
#
Dexamethasone
Control
Resveratrol
Downloaded from www.jbc.org by on September 23, 2007
*
1.6