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Departament de Bioquímica i Biologia Molecular Universitat de Barcelona 08028-Barcelona, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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.
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-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
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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.
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). 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).
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). 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.
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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 260–266 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.
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). 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).
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, 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).
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, 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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-JunL3 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 260–266 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-Ham’s 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 80–90% 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 (10,000–20,000 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 60–80 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
Student’s t test and significance is indicated in the
text.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by Grant PB95–09695 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.
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