Dominant Negative Regulation by c-Jun of Transcription of the Uncoupling Protein-1 Gene through a Proximal cAMP-Regulatory Element: A Mechanism for Repressing Basal and Norepinephrine-Induced Expression of the Gene before Brown Adipocyte Differentiation
Pilar Yubero1,
MaJosé Barberá1,
Rosa Alvarez,
Octavi Viñas,
Teresa Mampel,
Roser Iglesias,
Francesc Villarroya and
Marta Giralt
Departament de Bioquímica i Biologia Molecular
Universitat de Barcelona 08028-Barcelona, Spain
 |
ABSTRACT
|
---|
The brown fat uncoupling protein-1
(ucp-1) gene is regulated by the sympathetic nervous
system, and its transcription is stimulated by norepinephrine, mainly
through cAMP-mediated pathways. Overexpression of the catalytic subunit
of protein kinase A stimulated a chloramphenicol acetyltransferase
expression vector driven by the 4.5-kb 5'-region of the rat
ucp-1 gene. Mutant deletion analysis indicated the presence
of the main cAMP-regulatory element (CRE) in the proximal region
between -141 and -54. This region contains an element at -139/-122
able to confer enhancer and protein kinase A (PKA)-dependent activity
to the basal thymidine kinase promoter. The potency of this
element was much higher in differentiated than in nondifferentiated
brown adipocytes. Gel shift analyses indicated that a complex array of
proteins from brown fat nuclei bind to the -139/-122 element, among
which CRE-binding protein (CREB) and Jun proteins were identified. In
transfected brown adipocytes, c-Jun was a negative regulator of basal
and PKA-induced transcription from the ucp-1 promoter
acting through this proximal CRE region. A double-point mutation in the
-139/-122 element abolished both PKA- and c-Jun-dependent regulation
through this site, and overexpression of CREB blocked c-Jun repression.
Thus, an opposite action of these two transcription factors on the
-139/-122 CRE is proposed. c-Jun content in brown adipocytes
differentiating in culture correlated negatively with both
ucp-1 gene expression and the acquisition of the brown
adipocyte morphology. These findings indicate that c-Jun provides a
molecular mechanism to repress the basal and cAMP-mediated expression
of the ucp-1 gene before the differentiation of the brown
adipocyte.
 |
INTRODUCTION
|
---|
Brown adipose tissue is a major site for nonshivering
thermogenesis in mammals. Its thermogenic function relies on a
mitochondrial proton conductance pathway due to the presence of the
uncoupling protein-1 (UCP-1), which releases as heat the energy from
substrate oxidation normally conserved in the form of ATP (1).
Transcription of the ucp-1 gene occurs only in the brown fat
cell, and it is under a complex regulation by hormonal signals and
during cell differentiation (2, 3, 4, 5, 6). The 5'-noncoding regions of the
ucp-1 genes from rat and mouse contain most of the elements
for transcriptional regulation assembled in two main regions. There is
an upstream enhancer, involved in multihormonal regulation by retinoic
acid, thyroid hormones, and agonists of peroxisome
proliferator-activated receptor-
(PPAR
) (6, 7, 8, 9), as well as a
proximal regulatory region including a silencer, CAAT/enhancer binding
protein (C/EBP)-regulated sites, and basal promoter elements (3, 5).
Although potential elements for differentiation-dependent
ucp-1 gene expression, such as C/EBP (5) or PPAR
(9),
have been reported, the regulatory elements for tissue-specific
ucp-1 gene expression have not been identified. Studies
performed so far using transgenic mice show brown fat-specific gene
expression when both the enhancer and proximal regulatory regions are
present in a ucp-1 construct transgene (7).
Control of brown fat thermogenesis in response to heat demands depends
upon the release of norepinephrine from sympathetic terminals
innervating the tissue. Norepinephrine activates transcription from the
ucp-1 gene, and cAMP has been proposed as the main
intracellular mediator of this action (2, 3). Furthermore, chronic
cAMP-dependent protein kinase A (PKA) overactivity occurring in the
brown fat of mice carrying a targeted disruption of the RIIß subunit
of PKA causes an enhanced expression of the ucp-1 gene (10).
Attempts to define the regulatory sites responsible for norepinephrine
stimulus of transcription in the mouse ucp-1 gene have
yielded a complex pattern in which multiple putative cAMP-regulatory
elements (CREs), widespread in the enhancer and proximal regions,
appear to be involved (4). Neither the specific role of these CREs nor
the trans-acting factors involved in the norepinephrine
stimulus of ucp-1 gene transcription have yet been
determined. On the other hand, there is a complex interaction between
the transcriptional regulation of the ucp-1 gene due to
brown adipocyte differentiation and in response to norepinephrine.
Brown adipocyte differentiation is associated with a rise in basal
ucp-1 gene expression and an increase in its responsiveness
to norepinephrine action (11). Although changes in the abundance of
ß-adrenergic receptor subtype occur in association with the
differentiation of the brown fat cell, intracellular cAMP generation in
response to norepinephrine is similar before and after brown adipocyte
is terminally differentiated (12, 13). Then, intracellular mechanisms
must act in association with brown adipocyte differentiation to provide
both high basal expression and full responsiveness of the
ucp-1 gene to cAMP.
In recent years, a substantial increase in our understanding of the
mechanisms of cAMP stimulation of mammalian gene transcription has been
achieved. Initially, CREs were identified in several gene promoters and
generally consist in small variations of a palindromic sequence
TGACGTCA (14, 15). Subsequently, proteins that bound to these CREs were
identified, and a whole family of transcription factors, the
CREB/activating transcription factor (ATF) family, is now known to
mediate most of the cAMP responsiveness of different genes. Several of
these proteins are activated by PKA-dependent phosphorylation (16, 17).
These CREB/ATF proteins are members of the larger basic-leucine zipper
(bZIP) family of transcription factors (18). Other proteins from the
bZIP family such as c-Jun and c-Fos or the C/EBP proteins, despite
being known to mediate distinct biological signals in relation to cell
proliferation or differentiation, also participate in the cAMP
responsiveness of transcription of several genes (19, 20, 21). In fact,
members of the bZIP family of transcription factors act as dimers, and
a complex array of heterodimers may be formed between CREB/ATF proteins
and other bZIP proteins (18).
c-Jun is a transcription factor that has a pivotal role in the
mediation of signal transduction pathways by a wide variety of stimuli.
In general, c-Jun promotes progression of the cell cycle (22) and
mediates the proliferative response to growth factors in multiple cell
types (23). c-Jun and c-Fos, the products of the c-jun and
c-fos protooncogenes, interact with specific DNA sequences
to modulate gene transcription. c-Jun/c-Jun homodimers and c-Jun/c-Fos
heterodimers often act through a sequence element, named AP-1 site,
that mediates transcriptional response to multiple signal transduction
pathways (for review see Ref. 24). In addition, c-Jun is able to
interact with other proteins, forming heterodimers with members of the
CREB/ATF family such as ATF-2, ATF-3, and ATF-4 (25, 26). c-Jun homo-
and heterodimers have been reported to interact with CREs, thereby
affecting positively or negatively the cAMP responsiveness of several
gene promoters (20, 27, 28).
The aim of this study was to establish the main regulatory elements and
transcription factors that determine the cAMP responsiveness of the rat
ucp-1 gene. The main CRE was recognized in the proximal
region of the rat ucp-1 promoter (at -139/-122), and its
activity was shown to be dependent upon the stage of brown adipocyte
differentiation. CREB and Jun proteins from brown fat nuclei were
identified as binding UCP-CRE. c-Jun was found to be a powerful
repressor of the basal and PKA-induced transcription of the
ucp-1 gene, acting through this CRE. Furthermore, c-Jun
expression was found to correlate negatively with ucp-1 gene
expression in differentiating brown fat cells. We propose a dominant
negative role for c-Jun in the molecular mechanisms that mediate
adrenergic responsiveness of ucp-1 gene expression linked to
brown fat differentiation.
 |
RESULTS
|
---|
The Rat ucp-1 Gene Is Activated by PKA due to a Major
Responsive Site in the Proximal 5'-Noncoding Region
Primary cultures of differentiated brown adipocytes were
transfected with (-4551)UCP-CAT and then incubated with norepinephrine
or cAMP effectors before harvesting and analysis of chloramphenicol
acetyl transferase (CAT) activity (see Fig. 1A
). Norepinephrine (0.1 µM)
caused an almost 4-fold increase in the expression of (-4551)UCP-CAT.
Forskolin (10 µM), a direct activator of the adenylate
cyclase catalytic subunit, also elicited a marked stimulation.
3-Isobutyl-1-methylxanthine (IBMX) (0.5 mM), a
phosphodiesterase inhibitor, had a similar effect. Furthermore, 1
mM 8-bromo-cAMP, a nonmetabolizable cAMP derivative, caused
a 3-fold rise of the transfected (-4551)UCP-CAT activity. To determine
whether PKA, whose cAMP-mediated activation is elicited by all these
agents, is a major effector of (-4551)UCP-CAT activity, brown
adipocytes were cotransfected with SR
-PKA, a vector driving the
expression of the catalytic subunit of PKA. This resulted in a
significant (P < 0.001) increase in (-4551)UCP-CAT
expression similar to that achieved by the previously tested agents.
These results indicate that the 4.5-kb 5'-noncoding region of the rat
ucp-1 gene contains PKA-responsive elements.

View larger version (16K):
[in this window]
[in a new window]
|
Figure 1. Effects of Norepinephrine, cAMP Effectors, and
Overexpression of the Catalytic Subunit of PKA on the Expression of
(-4551)UCP-CAT. Deletion Mutant Analysis of the PKA Effect
A, Primary brown adipocytes differentiated in culture were transfected
with 15 µg/plate of the (-4551)UCP-CAT plasmid and exposed or not to
0.1 µM norepinephrine (NE), 10 µM
forskolin, 0.5 mM IBMX, or 1 mM 8-Br-cAMP.
Other plates included in the transfection 3 µg of the SR -PKA, the
expression vector for the catalytic subunit of PKA. Results are
expressed as -fold induction relative to untreated cells. B, Brown
adipocytes were transiently transfected with 15 µg/plate of
(-4551)UCP-CAT or equivalent amounts of the deletion mutants
illustrated on the left. Transfections included (PKA) or
not (Basal) 3 µg/plate of the SR -PKA expression vector. Results
are expressed as the relative CAT activity with respect to the basal
value for (-4551)UCP-CAT, which is set to 1. Bars are
means of three independent experiments, each performed in triplicate.
SEMs did not exceed 10% of the means.
|
|
To determine the sites in the 5'-region of the rat ucp-1
gene responsible for this PKA-mediated stimulation, the effect of the
transfected plasmid SR
-PKA was determined on deletion mutants of the
(-4551)UCP-CAT transiently transfected into primary cultures of brown
adipocytes. The study of mutants with progressively longer 5'-deletions
of (-4551)UCP-CAT indicated that PKA responsiveness was lost only when
the proximal region between -141 and -54 was suppressed (Fig. 1B
).
The presence of sequences between -2494 and -172 in the absence of
the -141/-54 region was also able to support a 2-fold responsiveness
to PKA, markedly lower than that elicited when this proximal region was
present. These data indicate the presence of multiple PKA responsive
elements in the rat ucp-1 gene but also the greater response
due to the proximal -141/-54 region. On the other hand, all the
constructs in which the -141/-54 region was deleted showed lowered
basal expression with respect to others in which this region was
present, even though the activity of those constructs was always
significantly (P < 0.05) higher than the background
activities elicited by a transfected promoterless CAT vector (not
shown).
The -139/-122 Element of the Rat ucp-1 Gene Has
Enhancer Activity and Confers PKA Responsiveness to a Heterologous
Promoter
Our previous analysis on the DNA protein-binding domains in the
proximal region of the rat ucp-1 gene identified a major
DNaseI footprint site protected by rat brown fat nuclear proteins at
-139/-122, within the PKA-responsive region (29). A similar
observation has been reported using nuclear extracts from Syrian
hamster brown adipose tissue (30). To test the ability of the
-139/-122 sequence from the ucp-1 gene promoter to
function as PKA-response element, a series of heterologous CAT vectors
were generated by ligating one, two, or three copies of this sequence
(UCP-CRE oligonucleotide) to the herpes simplex virus (HSV)
thymidine kinase promoter. As shown in Fig. 2
, one copy of the UCP-CRE confers a 4-fold
induction by PKA to the unresponsive tk-CAT gene. Addition of more
copies of this UCP-CRE resulted in a higher responsiveness to PKA (a
12-fold and a 32-fold induction for the (UCP-CRE)2-tk-CAT and
(UCP-CRE)3-tk-CAT, respectively). Furthermore, basal activity of these
last plasmids indicates that UCP-CRE also shows enhancer properties in
differentiated brown adipocytes.

View larger version (14K):
[in this window]
[in a new window]
|
Figure 2. Basal and PKA-Induced Expression of Chimeric
Constructs Containing Multiple Copies of the -139/-122 Element
Upstream from the Basal Thymidine Kinase Promoter
Primary brown adipocytes differentiated in culture were transfected
with 5 µg/plate of the chimeric CAT constructs depicted on the
left with (PKA) or without (Basal) 3 µg/plate of
SR -PKA. The UCP-CRE oligonucleotide corresponds to the -139/-122
sequence of the rat ucp-1 gene (see Materials and
Methods). Results are expressed as the fold-induction of
activity with respect to the basal activity of the thymidine
kinase (tk)-CAT construct, which is set to 1.
Bars are means of two independent experiments, each
performed in triplicate.
|
|
The -139/-122 Element in the Rat ucp-1 Gene Binds
CREB and c-Jun Proteins from Brown Adipose Tissue Nuclei
Electrophoretic mobility shift assays were undertaken using the
UCP-CRE oligonucleotide as a labeled probe and protein extracts from
rat interscapular brown fat nuclei. A complex pattern of DNA-protein
binding complexes was formed as revealed by the presence of at least
four retarded bands (named A, B1, B2, and C) (Fig. 3A
). The appearance of B1 and B2 as two
separate bands was particularly evident in long time runs of the
electrophoreses (see Fig. 3
, B, C, and D). Excess of the unlabeled
UCP-CRE probe suppressed the appearance of the C and B bands but not
the A band, which was therefore considered as nonspecific. The C and B
bands also disappeared when incubations included as competitor an
excess of an oligonucleotide corresponding to a well characterized CRE
such as the CRE-I (-94/-77) in the rat phosphoenolpyruvate
carboxykinase (PEPCK) gene (see Fig. 3A
). A similar result was obtained
using the rat somatostatin gene CRE (S-CRE) oligonucleotide as
competitor (not shown). An excess of an unrelated oligonucleotide (the
GA-rich sequence from the stromelysin ETS-binding site, see
Materials and Methods), used as negative control, was
without effect. This indicated that CRE-binding proteins were the main
proteins binding specifically to the -139/-122 element in the rat
ucp-1 gene. To further assess this, the UCP-CRE-labeled
probe was incubated with partially purified recombinant CREB-1 (CREB),
a representative member of the CREB/ATF family of transcription factors
mediating cAMP stimulation of gene transcription (see Fig. 3B
). This
resulted in the appearance of a single retarded band in the gel-shift
assay. The specificity of binding was shown by the efficient
competition of an excess of either the rat PEPCK-CRE-I or rat S-CRE.
The mobility of the complex formed by CREB was identical to the C band
formed by the brown fat nuclear extract. The identity of CREB was
confirmed by incubating the brown fat nuclear extract and the UCP-CRE
probe with an antibody against CREB (not shown).

View larger version (52K):
[in this window]
[in a new window]
|
Figure 3. Electrophoretic Mobility Shift Assays of the
Nuclear Proteins that Interact with the -139/-122 Element in the Rat
ucp-1 Gene
A double-stranded oligonucleotide corresponding to the -139/-122
region of the rat ucp-1 gene (UCP-CRE) was used as
labeled probe. A, The probe was incubated with 5 µg of nuclear
protein extracts from rat brown adipose tissue (BAT), and competitors
were added at a 100-fold molar excess relative to probe concentration.
PEPCK-CRE-I is an oligonucleotide corresponding to the -94/-77 site
in the rat PEPCK gene, and ETS is a GA-rich oligonucleotide used as
negative control (see Materials and Methods).
Arrows indicate the mobilities of the major protein-DNA
complexes formed. B, Gel-mobility shift assay of the interaction of the
-139/-122 UCP-CRE probe with CREB. The labeled probe was incubated
with 1 µg of partially purified E. coli-expressed CREB
as described (45 ). Competitors were PEPCK-CRE-I as in panel A or S-CRE,
an oligonucleotide corresponding to the -60/-29 site in the rat
somatostatin gene. C, Gel-mobility shift assays of the interaction of
the -139/-122 UCP-CRE probe with c-Jun. The labeled probe was
incubated with 1 fpu (footprint protection unit) of purified
recombinant c-Jun. Competitors were as in panel B. D, Effects of
anti-Jun serum on the protein-DNA complexes formed between brown fat
nuclear proteins (BAT) and the -139/-122 UCP-CRE probe. Brown fat
nuclear protein extract (5 µg) was incubated with 0.2 µl or 1 µl
of anti-Jun (56 ) or preimmune (control) serum before incubation with
the labeled probe.
|
|
To investigate whether other bZIP transcription factors not belonging
to the CREB/ATF family but potentially related to cAMP-signaling
pathways could bind the -139/-122 UCP-CRE, the labeled probe was
incubated with purified C/EBPß (21, 31). Results indicated that the
UCP-CRE was unable to bind C/EBPß efficiently (not shown). The amount
of C/EBPß used in the assays in which binding did not occur was
enough to induce at least a 30% retardation of labeled probes such as
the -457/-440 and -335/-318 C/EBP binding sites in the
ucp-1 gene (5) or the -94/-77 PEPCK-CRE-I (21). Further
experiments were performed to assess whether c-Jun proteins could bind
to the -139/-122 UCP-CRE. Incubation of the -139/-122 probe with
recombinant purified c-Jun showed significant binding, indicated by the
appearance of a retarded band with similar mobility to the B1 band (see
Fig. 3C
). Binding was specific as shown by the competition of an excess
of PEPCK-CRE-I, which is known to bind c-Jun homodimers (20), but not
by competition with the negative control ETS. When brown fat nuclear
extracts were incubated with antibodies specific to Jun proteins, both
B bands disappeared specifically. A nonspecific increase in the
intensity of retardation at levels close to the mobility of the
nonspecific A band was observed in the presence of either the anti-Jun
serum or the preimmune (control) serum (see Fig. 3D
). This result
indicates that Jun proteins are components of the protein complexes
binding the UCP-CRE and originating the B bands.
c-Jun, but not c-Fos, Represses Basal and PKA-Induced
ucp-1 Gene Promoter Activity
To examine the functional significance of the binding of c-Jun to
the main proximal CRE in the ucp-1 gene, transient
cotransfection analysis of the CAT-driven ucp-1 promoter was
undertaken. However, as recently pointed out (32), plasmid constructs
derived from pUC contain an artifactual AP-1 binding site that hampers
their use for studying c-Jun or c-Fos action. As this was the case for
the (-4551)UCP-CAT and derived mutant deletion series, new constructs
were obtained by deleting this AP-1 site in the former (-2494)UCP-CAT
(see Materials and Methods for details). The construct in
which the -2494/+110 fragment of the ucp-1 gene drives CAT
expression and has the AP-1 site deleted showed a similar response to
PKA (>6-fold stimulation) to that of the former version (Fig. 4
). Cotransfection into primary brown
adipocytes of 3 µg of an expression vector in which the entire open
reading frame of c-Jun is transcribed from the cytomegalovirus (CMV)
promoter significantly (P < 0.01) diminished
AP1(-2494)UCP-CAT expression. Moreover, the ability of PKA to
stimulate
AP1(-2494)UCP-CAT was almost completely suppressed by
CMV-c-Jun cotransfection (Fig. 4
). Lower amounts of CMV-c-Jun resulted
in a weaker inhibitory effect and a 10-fold lower amount of
cotransfected expression vector (0.3 µg) reduced by only 20% the
basal and PKA-induced activity of
AP1(-2494)UCP-CAT (not shown).
Parallel experiments using the CMV-c-Fos vector showed no effect on the
basal expression of the ucp-1 promoter, and this vector also
failed to suppress the PKA-induced expression of the ucp-1
promoter. The effects of an equivalent mixture of transfected CMV-c-Jun
and CMV-c-Fos were essentially indistinguishable from the action of the
corresponding amount (one half) of the single CMV-c-Jun expression
vector.
The domains of c-Jun required for repression of the ucp-1
gene transcription were examined using mutant forms of rat c-Jun cDNA
with mutations in known functional domains. The mutant Jun
L3 encodes
a c-Jun protein defective in dimerization due to a leucine-to-valine
substitution at leucine 3 within the leucine zipper domain. The Jun
BR is a deletion mutant lacking amino acids 260266 within the
DNA-binding domain (see Materials and Methods). Both
mutations significantly (P < 0.05) attenuated the
ability of CMV-c-Jun to inhibit basal and PKA-induced expression of the
ucp-1 promoter expression (Fig. 4
).
The Proximal CRE Is Required for the Inhibitory Effect of c-Jun on
the Rat ucp-1 Gene Promoter
Deletion mutants from
AP1(-2494)UCP-CAT were obtained and
transiently transfected into brown adipocytes differentiated in culture
to assess whether the proximal CRE region that binds Jun proteins was
responsible for the repressing effect of c-Jun on the ucp-1
gene promoter. Deletion of most of the 5'-noncoding region of the
ucp-1 gene did not affect the ability of c-Jun to inhibit
basal and PKA-induced expression of the ucp-1 gene promoter
(Fig. 5
). The presence of 141 bp upstream
from the transcription start site was enough to retain the inhibition
by c-Jun of the ucp-1 gene promoter expression. Conversely,
an internal deletion in which only the -172/-54 region of
AP1(-2494)UCP-CAT had been eliminated was enough to suppress any
inhibitory action of c-Jun, despite a previous report on an AP-1
binding site at -2422 (7). Neither the basal nor the PKA-stimulated
expression present in this construct caused by sequences upstream from
-172 was affected by c-Jun expression. The construct in which the
whole region upstream from -54 had been deleted was insensitive to the
inhibitory action of c-Jun. These results indicate that, indeed, the
-141/-54 region, in which the Jun binding site is present, is
required for the c-Jun action inhibiting ucp-1 gene promoter
expression.
To determine whether the isolated -139/-122 UCP-CRE can confer
c-Jun-dependent repression, the (UCP-CRE)2-tk-CAT plasmid was
transfected into differentiated brown adipocytes (see Fig. 6C
). Both basal and PKA-stimulated expression
of this heterologous construct were significantly (P <
0.01) blocked by c-Jun expression. In contrast, two copies of a
double-point mutant version of the UCP-CRE (mutUCP-CRE, see Fig. 6A
)
were unable to confer either PKA- or c-Jun-dependent responsiveness to
the neutral thymidine kinase promoter (Fig. 6C
). These
results are consistent with the lack of capacity of mutUCP-CRE to bind
either CREB or Jun proteins present in protein extracts from brown fat
nuclei as shown in Fig. 6B
. The only protein-(mutUCP-CRE) binding
complex formed was the nonspecific A band. When used as unlabeled
competitor, the mutUCP-CRE oligonucleotide was also unable to compete
for the B and C bands formed with UCP-CRE as labeled probe (not
shown).

View larger version (30K):
[in this window]
[in a new window]
|
Figure 6. Expression of Chimeric Constructs Containing Either
the -139/-122 UCP-CRE or a Double Point Mutant Version Upstream from
the Thymidine Kinase Promoter in Differentiated Brown
Adipocytes. Effects of Cotransfection of c-Jun or CREB Expression
Vectors upon Basal and PKA Responsiveness
A, Sequence of the double-stranded oligonucleotides corresponding to
the -139/-122 region of the rat ucp-1 gene (UCP-CRE)
or to its double-point mutant derivative version (mutUCP-CRE) in which
the two changed bases are shown in lowercase. B,
Electrophoretic mobility shift assay. Wild-type and mutated forms of
UCP-CRE were used as labeled probes, and an equal amount of each one
was incubated with 5 µg of nuclear protein extracts from rat brown
adipose tissue. Arrows indicate the mobilities of the
major protein-DNA complexes formed. C, Brown adipocytes differentiated
in culture were transiently transfected with 5 µg/plate of the
(UCP-CRE)2-tk-CAT or the (mutUCP-CRE)2-tk-CAT plasmids, in which two
copies of either the -139/-122 UCP-CRE or its mutant derivative were
placed upstream from the thymidine kinase promoter in a
modified ( AP1)pBLCAT2 vector (see Materials and
Methods). Transfections included (+) or not (-) 3 µg of the
SR -PKA expression vector (PKA), and/or 3 µg of the CMV-c-Jun
expression vector (Jun), and/or 3 µg of the RSV-CREB expression
vector (CREB). Results are shown relative to the basal expression of
(UCP-CRE)2-tk-CAT, which is set to 1. Bars are means of
two to three independent transfection experiments, each performed in
triplicate. SEMs did not exceed 10% of the means.
|
|
To further analyze the functional interaction between the bZIP proteins
found to bind the UCP-CRE, the expression vectors for CREB and/or c-Jun
were cotransfected with the (UCP-CRE)2-tk-CAT plasmid. As depicted in
Fig. 6C
, although transfection of Rous sarcoma virus (RSV)-CREB alone
was without effect, when cotransfected with CMV-c-Jun, CREB was able to
block repression by c-Jun of (UCP-CRE)2-tk-CAT activity. The ability of
CREB to antagonize the inhibitory effect of c-Jun was even higher when
the influence of both on the PKA-induced expression of the plasmid was
analyzed. These results point to a functional competitive interaction
of CREB and c-Jun upon the UCP-CRE.
Basal Enhancer and PKA Responsiveness Conferred by the -139/-122
UCP-CRE Depends on Brown Adipocyte Differentiation
Brown adipose tissue precursor cells were cultured for 7 days in
conditions leading to differentiated brown adipocytes or in a
hormone-depleted culture medium known to impair adipocyte
differentiation (6). Only the former led to the appearance of the brown
adipocyte morphology, characterized by rounding up of the cells and
accumulation of lipid droplets (see Fig. 7A
).
High levels of UCP1 mRNA expression were also present as a phenotypic
feature of the differentiated cells with respect to the
nondifferentiated cells (Fig. 8B
).

View larger version (75K):
[in this window]
[in a new window]
|
Figure 7. Expression and PKA Responsiveness of the Chimeric
Construct (UCP-CRE)2-tk-CAT in Differentiated and Nondifferentiated
Cultured Brown Adipocytes
Primary brown adipocyte precursor cells were grown in culture for 4
days and treated thereafter with either regular differentiating medium
or nondifferentiating medium, as described in Materials and
Methods. A, Microphotographs of the cells on day 7 after being
cultured in the different media. Magnification 40x. B, Differentiated
or nondifferentiated brown adipocytes were transiently transfected with
5 µg/plate of the (UCP-CRE)2-tk-CAT including (+) or not (-) 3 µg
of the SR -PKA expression vector (PKA). Results are expressed as the
fold-induction of activity with respect to the basal activity of the
empty thymidine kinase-CAT vector ( AP1)pBLCAT2 in
each cell type, which is set to 1. Bars are means of two
independent experiments, each performed in triplicate.
|
|

View larger version (40K):
[in this window]
[in a new window]
|
Figure 8. Electrophoretic Mobility Shift Assay of the
-139/-122 Region of the ucp-1 Gene with Nuclear
Protein Extracts from Differentiated or Nondifferentiated Cultured
Brown Adipocytes. Northern and Western Blot Analyses of
c-Jun Expression in Cultured Brown Adipocytes
Cells were grown in culture as described in the legend of Fig. 7 . A,
The -139/-122 UCP-CRE probe was incubated with 5 µg of nuclear
protein extracts from either differentiated or nondifferentiated brown
adipocytes. Arrows indicate the mobilities of the major
protein-DNA complexes formed. B, Northern blot analysis of 15 µg of
total RNA from differentiated or nondifferentiated brown adipocytes.
Northern blots were probed with the rat UCP1 and the rat c-Jun cDNA
probes. The sizes of the detected transcripts are depicted on the
right. C, Nuclear protein extracts (5 µg) from either
differentiated or nondifferentiated brown adipocytes were analyzed by
Western blot using specific antisera for CREB (Santa Cruz Biochemicals)
or Jun (56 ) proteins. The sizes of the detected proteins are indicated
on the right.
|
|
To analyze whether the differentiation state of brown adipocytes can
affect either enhancer or PKA-responsive activity of the -139/-122
element of the ucp-1 gene promoter, we transfected
(UCP-CRE)2-tk-CAT into either differentiated or nondifferentiated
primary brown adipocytes. As depicted in Fig. 7B
, UCP-CRE did not show
enhancer activity in nondifferentiated cells (only 1.5-fold with
respect to the empty vector (
AP1)pBLCAT2) in contrast to its ability
to confer a 4-fold induction of activity to basal thymidine
kinase in differentiated cells. The PKA-responsiveness conferred
by the UCP-CRE showed dramatic differences when assessed in
nondifferentiated (a 3-fold induction by PKA) as compared with
differentiated cells (12-fold). Therefore, it is concluded that the
activity of the UCP-CRE is dependent upon the stage of brown adipocyte
differentiation.
Negative Correlation of c-Jun Abundance with Respect to the
Expression of the ucp-1 Gene and the Differentiation of the
Brown Adipocyte
To analyze whether endogenous c-Jun abundance could be involved in
determining the differentiation-dependent activity of UCP-CRE, we
performed gel-shift analysis using nuclear protein extracts from either
differentiated or nondifferentiated brown adipocytes. As shown in Fig. 8A
, the c-Jun-related B bands were more intense in the gel shift when
extracts from nondifferentiated cells were tested while the C band
predominated in extracts from differentiated brown adipocytes.
Likewise the expression of c-Jun was assessed in both differentiated
and nondifferentiated cells. As shown in Fig. 8B
, c-Jun mRNA showed a
pattern of expression similar to that found in other murine cells,
i.e. two mRNA species of 3.2 and 2.7 kb (33). c-Jun mRNA
levels in nondifferentiated brown fat cells were 4-fold those in
differentiated brown adipocytes. Accordingly, c-Jun protein content was
higher (5-fold) in nondifferentiated than in differentiated brown
adipocytes (Fig. 8C
). In contrast, CREB abundance in differentiated
brown adipocytes was 2-fold that in nondifferentiated cells. Thus, the
changes in the relative abundance of c-Jun and CREB during the
differentiation of brown fat cells in culture may account for the
differentiation-dependent basal and PKA-inducible ucp-1 gene
transcriptional activity found in these cells.
 |
DISCUSSION
|
---|
Norepinephrine has been classically recognized as the main inducer
of brown fat thermogenesis; it acts mainly by stimulating expression of
the ucp-1 gene at the transcriptional level (2, 3). The use
of three different agents to increase the level of cAMP as well as
cotransfection of a catalytic subunit of PKA expression vector in brown
adipocytes resulted in a similar induction of transcription from the
4.5-kb ucp-1 gene promoter to that seen with norepinephrine.
Thus, we show for the first time that the adrenergic regulation of the
ucp-1 gene transcription via norepinephrine is mediated by
cAMP stimulation of PKA. This is consistent with the enhanced
expression of ucp-1 observed in the brown fat of transgenic
mice with chronic PKA overactivity due to the targeted disruption of
the RIIß subunit of PKA (10). Furthermore, using a series of deletion
mutants of the ucp-1 promoter, we have identified a major
PKA-responsive region in the ucp-1 gene promoter located at
-141/-54. This region contains a CRE motif at -139/-122, able to
bind CREB, that is sufficient to confer PKA responsiveness to a neutral
promoter.
We have also found that the -141 to -54 proximal region of the rat
ucp-1 gene is crucial for the basal transcriptional activity
of the ucp-1 promoter. This is consistent with the presence
of deoxyribonuclease I (DNase I) hypersensitivity in this region (34).
Furthermore, the -139/-122 CRE has enhancer properties in
differentiated brown adipocytes (where the endogenous ucp-1
gene is highly expressed) but not in nondifferentiated cells (which
have a much lower expression of the endogenous ucp-1 gene).
Therefore, in addition to being the main cAMP-responsive element
identified in the rat ucp-1 gene, the -139/-122 element
probably accounts for the basal promoter activity of the gene
characteristic of the differentiated brown adipocyte. In the mouse
ucp-1 gene, the presence of several putative CRE sequences
along the gene has been proposed (4). The 5'-GCGCGTCA-3' core of the
-139/-122 UCP-CRE (antisense strand) is identical to a CRE sequence
in the proximal region of the mouse gene that was also claimed to be
important for basal expression (4). Present data fully establish the
relevance of this proximal CRE in both basal and cAMP stimulation of
transcription from the ucp-1 gene promoter. On the other
hand, although our present results demonstrate a major role for this
proximal CRE in the rat ucp-1 gene, the presence of other
CRE sequences upstream from the ucp-1 gene promoter should
be considered. In the mouse gene, a distal CRE located in the enhancer
region was proposed to confer most of the norepinephrine responsiveness
(4). The fact that the distal CRE in the mouse and the corresponding
sequence in the rat are less conserved than the proximal CRE may
explain the differences in the relative roles of these sites in these
species.
Present results demonstrate that c-Jun expression in brown adipocytes
represses basal transcription from the ucp-1 gene promoter.
Furthermore, c-Jun completely blocks the induction of transcription
from the ucp-1 promoter by the catalytic subunit of PKA. The
-141/-54 region responsible for repression by c-Jun colocalizes with
the main cAMP-responsive region in the rat ucp-1 gene
promoter. Furthermore, the isolated UCP-CRE confers c-Jun-dependent
repression to the neutral tk gene promoter. Both the
DNA-binding and the leucine zipper domains of c-Jun are required to
mediate its inhibitory effect on ucp-1 gene transcription.
These findings, along with c-Jun binding to the UCP-CRE, are consistent
with a model in which c-Jun blocks ucp-1 transcription by
interacting directly with the ucp-1 promoter via formation
of functional transcriptional complexes either alone or with other(s)
member(s) of the CREB/ATF family. Involvement of c-Jun/c-Fos
heterodimers in mediating this effect is not likely because of the lack
of effect of c-Fos cotransfection on ucp-1 gene
transcription. Any potential effect mediated by c-Jun/c-Fos
heterodimers would be enhanced by cotransfection of both expression
vectors, which does not appear to be the case for the repression of
c-Jun upon ucp-1 gene transcription. Although the
involvement of other Jun-related proteins, such as Jun-B or Jun-D,
cannot be ruled out, preliminary data indicate that Jun-B is
uneffective in repressing ucp-1 gene transcription (P.
Yubero, F. Villarroya, and M. Giralt, unpublished observations). Also,
our present findings rule out C/EBPs as components of the brown fat
nuclear protein complexes interacting with the -139/-122 CRE, even
though C/EBPß is overexpressed in rat brown fat under noradrenergic
stimulus (35), and C/EBPß binds and transactivates several CRE
reporter gene constructs (31, 36). On the other hand, neither the two
C/EBP responsive elements present in the proximal regulatory region of
the ucp-1 gene (5) nor the cis-acting sequences
in the enhancer region of the gene (6, 7, 8, 9) are required for the negative
regulation by c-Jun. However, whether this negative regulation by c-Jun
through the UCP-CRE affects the stimulation of the ucp-1
gene transcription by retinoic acid (6, 7), thyroid hormones (8), or
agonists of PPAR
(9) remains to be determined.
From our present results, a model in which an opposite action of CREB
and c-Jun in regulating basal and PKA responsiveness of transcription
from the ucp-1 gene through direct competition by binding to
the -139/-122 CRE is proposed. Support for this hypothesis comes from
several lines of evidence. Both PKA and c-Jun-dependent regulation are
reproduced by the isolated UCP-CRE but are lost when a double-point
mutation that abolishes binding is introduced in this element.
Furthermore, overexpression of CREB blocks c-Jun repression
consistently with a direct competition for binding to the same site in
the UCP-CRE. Effects of CREB alone on PKA responsiveness were hardly
observed in cotransfection assays, probably because of the high
constitutive expression of CREB in brown fat cells (Ref. 37 and Fig. 8C
), similarly to what has been described for CREB-responsive genes in
other cell types (38). In an analogous manner to the present findings
on the ucp-1 gene, other studies have shown that c-Jun is
able to repress transcription via CRE sites, as for instance in the
human insulin gene (27) and the
- and ß-subunit genes for human CG
(28). In contrast, the c-jun gene promoter is negatively
regulated by CREB and positively regulated by c-Jun (39). Thus, a
single cis-element provides positive or negative regulation
depending upon the relative abundance of active transcription factors
binding to this site.
On the basis of these results, we propose that the expression of the
ucp-1 gene and its responsiveness to cAMP are modulated by
the relative abundance of c-Jun in the brown adipocyte. We report here
that c-Jun content correlates inversely with the acquisition of the
terminally differentiated brown adipocyte phenotype, as assessed by
both cell morphology and ucp-1 gene expression. In
differentiating white adipocytes, a transient increase in the
expression of c-jun occurs in association with the mitotic
clonal expansion before terminal differentiation (40). Furthermore,
exposure of 3T3-L1 adipocytes to agents that inhibit their
differentiation process, such as tumor necrosis factor-
or retinoic
acid, results in a persistent rise in the expression of c-Jun mRNA (41, 42). The higher expression of c-Jun mRNA and protein in
nondifferentiated compared with differentiated brown adipocytes
suggests a parallel role for c-Jun in brown adipocytes, although the
cell cycle status during their differentiation is unknown. Further
research is also necessary to identify the specific mechanisms
down-regulating c-Jun abundance, and perhaps c-Jun activity, in
association with brown adipocyte differentiation.
The induction of the expression of the ucp-1 gene, which is
repressed in nondifferentiated cells, is the key event in the
acquisition of the differentiated phenotype of the brown fat cell both
during development (43) and in cell culture (11, 44). Furthermore,
terminal brown adipocyte differentiation is associated with an
enhancement in the responsiveness of ucp-1 gene expression
to the adrenergic stimulus both in vivo (43) and in primary
cultured brown adipocytes (11). This differentiation-dependent
modulation of the ucp-1 gene transcription is reproduced
when analyzing the activity of the UCP-CRE, thus indicating that this
element can support these regulatory effects. Furthermore, the action
of c-Jun as a dominant inhibitor of basal and cAMP-induced
transcription from the ucp-1 promoter provides the first
evidence of a molecular mechanism by which expression of the
ucp-1 gene can be differentially regulated by norepinephrine
in undifferentiated vs. terminally differentiated brown
adipocyte cells. Although the involvement of the upstream
enhancer-regulatory region cannot be ruled out, a schematic overview of
differences in the transcriptional regulation of the ucp-1
gene associated with brown adipocyte differentiation is proposed on the
basis of the present findings on the proximal regulatory region (see
Fig. 9
).

View larger version (25K):
[in this window]
[in a new window]
|
Figure 9. Model for the Differentiation-Dependent Regulation
of the ucp-1 Gene Transcription through Its Proximal
Regulatory Region
The proximal regulatory region of the rat ucp-1 gene
contains the CRE at -139/-122 and two C/EBP-responsive elements at
-457/-440 and -335/-318 (5 ). In nondifferentiated cells, low levels
of basal and cAMP-induced ucp-1 gene expression are
explained by c-Jun repression together with low transactivating
activity by C/EBP proteins due to its low expression in
nondifferentiated brown adipocytes when compared with the
differentiated cells (6 ). In contrast, differentiated brown adipocytes
have high levels of basal ucp-1 gene expression, and the
gene is fully responsive to the noradrenergic stimulus. At the
transcriptional regulatory level, this can be explained by C/EBP
transactivation and the release of c-Jun inhibition allowing
CREB-dependent regulation of the UCP-CRE.
|
|
 |
MATERIALS AND METHODS
|
---|
Materials
DNA-modifying enzymes and poly(deoxyinosinic-deoxycytidylic)acid
were purchased from Boehringer Mannheim (Indianapolis, IN) or Promega
(Madison, WI). [
-32P]dCTP was from Amersham (Arlington
Heights, IL) and
D-threo-[1,2-14C]chloramphenicol was from ICN
(Cleveland, OH). Tissue culture media and FCS were obtained from
Biowhittaker (Verviers, Belgium). T3, insulin,
norepinephrine (arterenol bitartrate), 8-bromo-cAMP, forskolin, and
IBMX were from Sigma (St. Louis, MO).
Oligonucleotides and Plasmids
Oligonucleotides were chemically synthesized by Boehringer
Mannheim. The UCP-CRE double-stranded oligonucleotide corresponds to
positions -139 to -122 of the rat ucp-1 gene, and its
sequence is 5'-GGGAGTGACGCGCGTCTG-3', flanked by XbaI ends.
The mutated version mutUCP-CRE corresponds to the sequence
5'-GGGAGTGTGGCGCGT-CTG-3' also flanked by
XbaI ends. The PEPCK-CRE-I and S-CRE are double-stranded
oligonucleotides corresponding to the -94/-77 and -60/-29 CREs of
the rat phosphoenolpyruvate carboxykinase (45) and rat somatostatin
(46) gene promoters, respectively. ETS is an oligonucleotide
corresponding to the -208/-192 GA-rich region in the stromelysin
promoter used as negative control in the DNA binding experiments
(47).
(-4551)UCP-CAT, a pSP73-derived plasmid containing the region -4551
to +110 of the rat ucp-1 gene driving the promoterless CAT
gene, was kindly provided by Dr. D. Ricquier (3). The plasmids
(-2494)UCP-CAT, (-896)UCP-CAT, (-141)UCP-CAT and (-54)UCP-CAT were
constructed using the internal restriction sites AatII,
HindIII, BstXI, and NaeI in
(-4551)UCP-CAT, respectively. The internal deletions between
nucleotides -172/-54 and -2469/-54 were carried out by digesting
with SpeI/NaeI and
BclI/NaeI, respectively.
Plasmids derived from (-4551)UCP-CAT but lacking the artifactual AP-1
site (32) present upstream from the polylinker of pSP73 (position 8449
in (-4551)UCP-CAT)(3) were constructed as follows:
AP1(-2494)UCP-CAT was obtained by eliminating the fragment between
8348 and 2058 in (-4551)UCP-CAT by digestion and further religation
using the AatII sites at those positions. The plasmid
(
-172/-54)(-2494)UCP-CAT containing the internal deletion between
nucleotides -172 and -54 was obtained using the unique
SpeI and NaeI sites in
AP1(-2494)UCP-CAT.
AP1(-141)UCP-CAT and
AP1(-54)UCP-CAT were obtained by digestion
and further religation of the original plasmids using the
AatII and BglII sites corresponding to the former
8348 and 8690 positions in (-4551)UCP-CAT.
The heterologous (UCP-CRE)-tk-CAT vectors in which copies of the
-139/-122 sequence of the ucp-1 gene are placed upstream
from the HSV thymidine kinase promoter were generated by
cloning one, two, or three copies (direct repeats) of the synthetic
double-stranded oligonucleotide UCP-CRE into the XbaI site
of a version of pBLCAT2 in which the artifactual AP-1 site (position 32
in pBLCAT2) (48) had been previously deleted by an
AatII/HindIII digestion and further religation.
The mutant version (mutUCP-CRE)-tk-CAT was generated by cloning two
copies of the mutUCP-CRE double-stranded oligonucleotide as a direct
repeat into the XbaI site of the (
AP1)pBLCAT2
plasmid.
SR
-PKA is an expression vector for the catalytic subunit of PKA
transcribed from the SR
promoter (49). Construction of pRSV-CREB,
the mammalian expression vector for full-length CREB-1, has been
described (21). Expression plasmids driving c-Fos and various forms of
c-Jun were kindly provided by Dr. T. Curran. pCMV-c-Jun and pCMV-c-Fos
are mammalian expression vectors that contain the rat cDNAs of c-Jun
and c-Fos, respectively, driven by the cytomegalovirus promoter (50, 51). pCMV-Jun
L3 is a CMV-driven expression vector containing a
leucine-to-valine amino acid substitution at leucine 3 within the
leucine zipper domain, which disrupts dimerization (50). pCMV-Jun
BR
contains amino acid 260266 deletion within the DNA-binding domain
that disrupts DNA binding (50).
Cell Culture and Transfection Assays
Isolation and culture of brown preadipocytes was performed as
described (6, 11). Three-week-old Swiss mice were killed and
interscapular, cervical, and axillary depots of brown fat were removed.
Precursor cells were isolated, plated on 60-mm petri dishes (7500
cells/cm2), and grown in 5 ml DMEM-Hams F12 medium (1:1)
supplemented with 10% FCS, 20 nM insulin, 2 nM
T3, and 100 µM ascorbate (regular
differentiating medium). When indicated, cells were grown in a
hormone-depleted (nondifferentiating) medium containing 10%
charcoal-treated FCS (6).
Murine primary brown adipocytes differentiated in culture were
transiently transfected by the calcium phosphate precipitation method
on day 7 of culture, when 8090% of cells had already differentiated
(5). Each transfection contained between 5 and 15 µg of UCP-CAT
vectors and included or not the indicated amounts of expression
vectors. When indicated 0.1 µM norepinephrine, 1
mM 8-Br-cAMP, 0.5 mM IBMX, or 10
µM forskolin were added after transfection.
RSV-ß-galactosidase (2 µg) was included in all the experiments to
assess the efficiency of separate transfections. The cells were
incubated for 24 h and, for each condition, at least three plates
were pooled. The experiments were performed at least twice using
independent DNA preparations of each construct. Analysis of CAT
activity was carried out as described (52, 53). Acetylation of
[14C]chloramphenicol was determined by TLC and quantified
by radioactivity counting (AMBIS, San Diego, CA). The CAT activity was
normalized for variation in transfection efficiency using the
ß-galactosidase activity measured for each sample as a standard.
DNA Binding Experiments
Nuclear proteins were isolated from rat brown adipose tissue as
reported previously (5). Protein extracts from differentiated and
nondifferentiated brown adipocyte nuclei were prepared as described
(54). Protein concentration was determined by the micromethod of
Bio-Rad (Richmond, CA) using BSA as standard. Partially purified
bacterially expressed CREB was a kind gift from Dr. R. Hanson (45).
C/EBPß is an 11-kDa polypeptide form of C/EBPß (95% pure), kindly
provided by Dr. S. McKnight (55). Recombinant purified c-Jun was from
Promega.
For the gel retardation assays, the UCP-CRE or mut-UCP-CRE
oligonucleotides were end-labeled using [
-32P]dCTP and
Klenow enzyme. The DNA probe (10,00020,000 cpm) was incubated for 30
min at 25 C with 5 µg of brown adipose tissue nuclear protein extract
or purified CREB, C/EBPß, or c-Jun proteins. Reactions were carried
out in a final volume of 20 µl containing 20 mM HEPES (pH
7.6), 0.1 mM EDTA, 1 mM dithiothreitol, 50
mM NaCl, 10% glycerol, and 2 µg (nuclear extracts) or
0.5 µg (purified proteins) of poly(deoxyinosinic-deoxycytidylic)acid.
Samples were analyzed by electrophoresis at 4 C for 6080 min in
nondenaturing 5% polyacrylamide gels in 0.5x TBE (44.5 mM
Tris, 44.5 mM borate, 1 mM EDTA). Gels were
analyzed by autoradiography. In the competition experiments, 100-fold
molar excess of unlabeled oligonucleotide was included in each
respective binding reaction. When indicated, 0.2 µl or 1 µl of
rabbit antiserum against Jun proteins, kindly provided by Dr. R. Bravo
(56), or 1 µl of an antiserum against CREB (Santa Cruz Biochemicals,
Santa Cruz, CA), or equivalent amounts of preimmune (control) serum
were incubated with the brown adipose tissue nuclear extracts for
2 h at 4 C before incubation with the labeled probe.
RNA Isolation and Northern Blot Analysis
Total RNA was extracted from cultured brown adipocytes by a
single-step method using guanidine hydrochloride (57). For Northern
blot analysis, 15 µg of total RNA were denatured, electrophoresed on
1.5% formaldehyde/agarose gels, and transferred to nylon membranes
(Hybond N, Amersham). Ethidium bromide (0.2 µg/ml) was added to RNA
samples to check equal loading of gels and transfer efficiency (58).
Hybridization and washing were carried out as reported (43). Blots were
hybridized to DNA probes corresponding to the full-length cDNA for rat
UCP-1 (59) or rat c-Jun (50). The cDNA probes were labeled with
[
-32P]dCTP using the random oligonucleotide-primer
method. Autoradiographs were quantified by densitometric scanning (LKB
Instruments, Rockville, MD).
Immunoblot Analysis
Samples containing equal amounts of nuclear protein extracts
from differentiated or nondifferentiated brown adipocytes were
electrophoresed on 0.1% SDS/12% polyacrylamide gels. Proteins were
transferred to polyvylidene difluoride membranes (Millipore, Bedford,
MA) and probed with the antisera against CREB (Santa Cruz Biochemicals)
or Jun (56). Immunoreactive material was detected by the enhanced
chemiluminescence (ECL) detection system (Amersham). The sizes of the
proteins detected were estimated by using protein molecular mass
standards (Bio-Rad).
Statistical Analysis
Where appropriate, statistical analysis was performed by
Students t test and significance is indicated in the
text.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. D. Ricquier, T. Curran, R. Hanson, S.
McKnight, M. Muramatsu, and R. Bravo for the generous gifts of
plasmids, purified proteins, and/or antisera.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Marta Giralt, Departament de Bioquímica i Biologia Molecular,Universitat de Barcelona, Avda Diagonal 645, 08028-Barcelona, Spain. E-mail:
giralt{at}porthos.bio.ub.es
This work was supported by Grant PB9509695 from DGICyT,
Ministerio de Educación y Cultura, Spain, and by Grant
1995SGR-00096 from Generalitat de Catalunya.
1 These authors made equal contributions to this work. 
Received for publication October 21, 1997.
Revision received March 24, 1998.
Accepted for publication March 26, 1998.
 |
REFERENCES
|
---|
-
Nicholls DG, Cunningham SA, Rial E 1986 The bioenergetic
mechanisms of brown adipose tissue mitochondria. In: Trayhurn P,
Nicholls DG (eds) Brown Adipose Tissue. Edward Arnold Publishers,
London, pp 5285
-
Ricquier D, Bouillaud F, Toumelin P, Mory G, Bazin R, Arch J,
Pénicaud L 1986 Expression of uncoupling protein mRNA in
thermogenic or weakly thermogenic brown adipose tissue. J Biol
Chem 261:1390513910[Abstract/Free Full Text]
-
Cassard-Doulcier AM, Gelly C, Fox N, Schrementi J, Raimbault
S, Klaus S, Forest C, Bouillaud F, Ricquier D 1993 Tissue-specific and
beta-adrenergic regulation of the mitochondrial uncoupling protein
gene: control by cis-acting elements in the 5'-flanking region. Mol
Endocrinol 7:497506[Abstract]
-
Kozak UC, Kopecky J, Teisinger J, Enerback S, Boyer B, Kozak
LP 1994 An upstream enhancer regulating brown-fat-specific expression
of the mitochondrial uncoupling protein gene. Mol Cell Biol 14:5967[Abstract]
-
Yubero P, Manchado C, Cassard-Doulcier AM, Mampel T,
Viñas O, Iglesias R, Giralt M, Villarroya F 1994 CCAAT/enhancer
binding proteins alpha and beta are transcriptional activators of the
brown fat uncoupling protein gene promoter. Biochem Biophys Res Commun 198:653659[CrossRef][Medline]
-
Alvarez R, De Andrés J, Yubero P, Viñas O, Mampel
T, Iglesias P, Giralt M, Villarroya F 1995 A novel regulatory pathway
of brown fat thermogenesis. Retinoic acid is a transcriptional
activator of the mitochondrial uncoupling protein gene. J Biol
Chem 270:56665673[Abstract/Free Full Text]
-
Larose M, Cassard-Doulcier AM, Fleury C, Serra F, Champigny
O, Bouillaud F, Ricquier D 1996 Essential cis-acting
elements in rat uncoupling protein gene are in an enhancer containing a
complex retinoic acid response domain. J Biol Chem 271:3153331542[Abstract/Free Full Text]
-
Rabelo R, Schifman A, Rubio A, Sheng XY, Silva JE 1995 Delineation of thyroid hormone-responsive sequences within a critical
enhancer in the rat uncoupling protein gene. Endocrinology 136:10031013[Abstract]
-
Sears IB, MacGinnitie MA, Kovacs LG, Graves RA 1996 Differentiation dependent expression of the brown adipocyte uncoupling
protein gene: regulation by peroxisome proliferator-activated receptor
gamma. Mol Cell Biol 16:34103419[Abstract]
-
Cummings DE, Brandon EP, Planas JV, Motamed K, Idzerda RJ,
McKnight GS 1996 Genetically lean mice result from targeted disruption
of the RIIß subunit of protein kinase A. Nature 382:622626[CrossRef][Medline]
-
Rehnmark S, Nechad M, Herron D, Cannon B, Nedergaard J 1990 Alpha and beta-adrenergic induction of the expression of the uncoupling
protein thermogenin in brown adipocytes differentiated in culture.
J Biol Chem 265:1646416471[Abstract/Free Full Text]
-
Bronnikov G, Houstek J, Nedergaard J 1992 Beta-adrenergic,
cAMP-mediated stimulation of proliferation of brown fat cells in
primary culture mediation via beta 1 but not beta 3 adrenoreceptors.
J Biol Chem 267:20062013[Abstract/Free Full Text]
-
Cannon B, Jacobsson A, Rehnmark S, Nedergaard J 1996 Signal
transduction in brown adipose tissue recruitment: noradrenaline and
beyond. Int J Obes 20[Suppl 3]:S36S42
-
Roesler WJ, Vandenbark GR, Hanson RW 1988 Cyclic AMP and the
induction of eukaryotic gene transcription. J Biol Chem 263:90639066[Free Full Text]
-
Pestell RG, Jameson JL 1995 Transcriptional regulation of
endocrine genes by second-messenger signaling pathways. In: Weintraub
BD (ed) Molecular Endocrinology: Basic Concepts and Clinical
Correlations. Raven Press, New York, pp 5976
-
Brindle PK, Montminy MR 1992 The CREB family of transcription
activators. Curr Opin Genet Dev 2:199204[Medline]
-
Degroot RP, Sassonecorsi P 1993 Hormonal control of gene
expression: multiplicity and versatility of cyclic adenosine
3',5'-monophosphate-responsive nuclear regulators. Mol Endocrinol 7:145153[Medline]
-
Lamb P, Mcknight SL 1991 Diversity and specificity in
transcriptional regulation: the benefits of heterotypic dimerization.
Trends Biochem Sci 16:417422[CrossRef][Medline]
-
Habener JF 1990 Cyclic AMP response element binding proteins:
a cornucopia of transcription factors. Mol Endocrinol 4:10871094[Medline]
-
Gurney AL, Park EA, Giralt M, Liu JS, Hanson RW 1992 Opposing
actions of Fos and Jun on transcription of the phosphoenolpyruvate
carboxykinase (GTP) gene. Dominant negative regulation by Fos. J
Biol Chem 267:1813318139[Abstract/Free Full Text]
-
Park EA, Gurney AL, Nizielski SE, Hakimi P, Cao Z, Moorman A,
Hanson RW 1993 Relative roles of CCAAT/enhancer-binding protein-beta
and cAMP regulatory element-binding protein in controlling
transcription of the gene for phosphoenolpyruvate carboxykinase (GTP).
J Biol Chem 268:613619[Abstract/Free Full Text]
-
Riabowol K, Schiff J, Gilman MZ 1992 Transcription factor AP-1
activity is required for initiation of DNA synthesis and is lost during
cellular aging. Proc Natl Acad Sci USA 89:157161[Abstract]
-
Hunter T, Karin M 1992 The regulation of transcription by
phosphorylation. Cell 70:375387[Medline]
-
Angel P, Karin M 1991 The role of Jun, Fos and the AP-1
complex in cell-proliferation and transformation. Biochim Biophys Acta 1072:129157[CrossRef][Medline]
-
Hai T, Curran T 1991 Cross-family dimerization of
transcription factors Fos/Jun and ATF/CREB alters DNA binding
specificity. Proc Natl Acad Sci USA 88:37203724[Abstract]
-
Benbrook DM, Jones NC 1994 Different binding specificities and
transactivation of variant CREs by CREB complexes. Nucleic Acids Res 22:14631469[Abstract]
-
Inagaki N, Maekawa T, Sudo T, Ishii S, Seino Y, Imura H 1992 c-Jun represses the human insulin promoter activity that depends on
multiple cAMP response elements. Proc Natl Acad Sci USA 89:10451049[Abstract]
-
Pestell RG, Hollenberg AN, Albanese C, Jameson JL 1994 c-Jun
represses transcription of the human chorionic gonadotropin alpha and
beta genes through distinct types of CREs. J Biol Chem 269:3109031096[Abstract/Free Full Text]
-
Yubero P, Viñas O, Iglesias R, Mampel T, Villarroya F,
Giralt M 1994 Identification of tissue-specific protein binding domains
in the 5'-proximal regulatory region of the rat mitochondrial brown fat
uncoupling protein gene. Biochem Biophys Res Commun 204:867873[CrossRef][Medline]
-
Cassard-Doulcier AM, Larose M, Matamala JC, Champigny O,
Bouillaud F, Ricquier D 1994 In vitro interactions between
nuclear proteins and uncoupling protein gene promoter reveal several
putative transactivating factors including Ets1, retinoid X receptor,
thyroid hormone receptor, and a CACCC box-binding protein. J Biol
Chem 269:2433524342[Abstract/Free Full Text]
-
Bakker O, Parker MG 1991 CCAAT/enhancer binding protein is
able to bind to ATF/CRE elements. Nucleic Acids Res 19:12131217[Abstract]
-
Kushner PJ, Baxter JD, Duncan KG, Lopez GN, Schaufele F, Uht
RM, Webb P, West BL 1994 Eukaryotic regulatory elements lurking in
plasmid DNA: the activator protein-1 site in pUC. Mol Endocrinol 8:405407[Medline]
-
Ryseck RP, Hirai SI, Yaniv M, Bravo R 1988 Transcriptional
activation of c-jun during the G0/G1 transition in mouse fibroblasts.
Nature 334:535537[CrossRef][Medline]
-
Boyer BB, Kozak LP 1991 The mitochondrial uncoupling protein
gene in brown fat: correlation between DNAse-I hypersensitivity and
expression in transgenic mice. Mol Cell Biol 11:41474156[Medline]
-
Manchado C, Yubero P, Viñas O, Iglesias R, Villarroya F,
Mampel T, Giralt M 1994 CCAAT/enhancer-binding proteins alpha and beta
in brown adipose tissue: evidence for a tissue-specific pattern of
expression during development. Biochem J 302:695700[Medline]
-
Vallejo M, Ron D, Miller CP, Habener JF 1993 C/ATF, a member
of the activating transcription factor family of DNA-binding proteins,
dimerizes with CCAAT/enhancer-binding proteins and directs their
binding to cAMP response elements. Proc Natl Acad Sci USA 90:46794683[Abstract]
-
Thonberg H, Cannon B 1997 Norepinephrine-induced CREB
phosphorylation in brown fat cultures. Int J Obes 21[Suppl 2]:S49
-
Ellis MJC, Lindon AC, Flint KJ, Jones NC, Goodbourn S 1995 Activating transcription factor-1 is a specific antagonist of the
cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding
protein-1-mediated response to cAMP. Mol Endocrinol 9:255265[Abstract]
-
Lamph WW, Dwarki VJ, Ofir R, Montminy M, Verma I 1990 Negative
and positive regulation by transcription factor cAMP responsive
element-binding protein is modulated by phosphorylation. Proc Natl Acad
Sci USA 87:43204324[Abstract]
-
Cornelius P, McDougald OA, Lane MD 1994 Regulation of
adipocyte development. Annu Rev Nutr 14:99129[CrossRef][Medline]
-
Stone RL, Bernlohr DA 1990 The molecular basis for inhibition
of adipose conversion of murine 3T3L1 cells by retinoic acid.
Differentiation 45:119127[Medline]
-
Stephens JM, Butts M, Stone R, Pekala PH, Bernlohr DA 1993 Regulation of transcription factor messenger RNA accumulation during
3T3L1 preadipocyte differentiation by antagonists of adipogenesis.
Mol Cell Biochem 123:6371[Medline]
-
Giralt M, Martin I, Iglesias R, Viñas O, Villarroya F,
Mampel T 1990 Ontogeny and perinatal modulation of gene expression in
rat brown adipose tissue. Unaltered iodothyronine 5'-deiodinase
activity is necessary for the response to environmental temperature at
birth. Eur J Biochem 193:297302[Abstract]
-
Klaus S, Cassard-Doulcier AM, Ricquier D 1991 Development of
Phodopus-sungorus brown preadipocytes in primary cell culture. Effect
of an atypical beta-adrenergic agonist, insulin, and triiodothyronine
on differentiation, mitochondrial development, and expression of the
uncoupling protein UCP. J Cell Biol 115:17831790[Abstract]
-
Park EA, Roesler WJ, Liu J, Klemm DJ, Gurney AL, Thatcher JD,
Shuman J, Friedman A, Hanson RW 1990 The role of the CCAAT
enhancer-binding protein in the transcriptional regulation of the gene
for phosphoenolpyruvate carboxykinase (GTP). Mol Cell Biol 10:62646272[Medline]
-
Montminy MR, Sevarino KA, Wagner JA, Mandel G, Goodman RH 1986 Identification of a cyclic-AMP-responsive element within the rat
somatostatin gene. Proc Natl Acad Sci USA 83:66826686[Abstract]
-
Wasylyk C, Gutman AM, Nicholson R, Wasylyk B 1991 The c-Ets
oncoprotein activates the stromelysin promoter through the same
elements as several non-nuclear oncoproteins. EMBO J 10:11271134[Abstract]
-
Luckow B, Schütz G 1987 CAT constructions with multiple
unique restriction sites for the functional analysis of eukaryotic
promoters and regulatory elements. Nucleic Acids Res 15:5490[Medline]
-
Muramatsu M, Kaibuchi K, Arai K 1989 A protein kinase C cDNA
without the regulatory domain is active after transfection in
vivo in the absence of phorbol ester. Mol Cell Biol 9:831836[Medline]
-
Gentz R, Rauscher II FJI, Abate C, Curran T 1989 Parallel
association of Fos and Jun leucine zipper juxtaposes DNA binding
domains. Science 243:16951699[Medline]
-
Sonnenberg JL, Rauscher II FJI, Morgan JL, Curran T 1989 Regulation of proenkephalin by Fos and Jun. Science 246:16221625[Medline]
-
Gorman CM, Moffat LF, Howard BH 1982 Recombinant genomes which
express chloramphenicol acetyl transferase in mammalian cells. Mol Cell
Biol 2:10441051[Medline]
-
Pothier F, Ouellet M, Julien JP, Guerin SL 1992 An improved
CAT assay for promoter analysis in either transgenic mice or tissue
culture cells. DNA Cell Biol 11:8390[Medline]
-
Swick AG, Blake MC, Kahn JW, Azizkhan JC 1989 Functional
analysis of GC element binding and transcription in the hamster
dihydrofolate reductase gene promoter. Nucleic Acids Res 17:92919304[Abstract]
-
Cao ZD, Umek RM, Mcknight SL 1991 Regulated expression of
three C/EBP isoforms during adipose conversion of 3T3L1 cells. Gene
Dev 5:15381552[Abstract]
-
Kovary K, Bravo R 1991 The Jun and Fos protein families are
both required for cell cycle progression in fibroblasts. Mol Cell Biol 11:44664472[Medline]
-
Chomczynski P, Sacchi N 1987 Single-step method of RNA
isolation by guanidium thiocyanate-phenol-chloroform extraction. Anal
Biochem 162:156159[CrossRef][Medline]
-
Pappu HR, Hiruki C 1989 Improved RNA visualization in
formaldehyde-agarose gels. Focus 11:67
-
Bouillaud F, Ricquier D, Thibault J, Weissenbach J 1985 Molecular approach to thermogenesis in brown adipose tissue: cDNA
cloning of the mitochondrial uncoupling protein. Proc Natl Acad Sci USA 82:445448[Abstract]