From the Laboratorio de Biología Molecular, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II Piso 4, Ciudad Universitaria, 1428 Buenos Aires, Argentina
Received for publication, May 22, 2002, and in revised form, October 18, 2002
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ABSTRACT |
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Activation protein-1
(AP-1) transcription factors are early response genes involved in a
diverse set of transcriptional regulatory processes. The phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA) is often used to
induce AP-1 activity. The purpose of this work was to explore the
molecular mechanisms involved in the TPA regulation of ubiquitous
5-aminolevulinate synthase (ALAS) gene expression, the first and
rate-controlling step of the heme biosynthesis. Previous analysis of
the 5'-flanking sequence of ALAS revealed the existence of two
cAMP-response elements (CRE) required for basal and cAMP-stimulated
expression. The fragment Activation protein-1
(AP-1)1 transcription factors
are early response genes involved in a diverse set of
transcriptional regulatory processes. AP-1 is a dimeric complex
composed of members of the Fos and Jun family proteins (1). This
complex binds the consensus DNA sequence TGA(G/C)TCA, termed
12-O-tetradecanoylphorbol-13-acetate (TPA)-response element
(TRE) or AP-1, sites found in a variety of promoters of genes, such as
growth factors, chemokines, and cytokines (2). The Fos family contains
four proteins (c-Fos, Fos-B, Fra-1, and Fra-2), whereas the Jun family
is composed of three (c-Jun, JunB, and JunD). Fos and Jun are members
of the basic leucine zipper group of proteins, and this basic motif
mediates the formation of homo- and heterodimers. c-Jun is the major
component of the AP-1 complex, and c-Fos is its best known partner (3, 4). AP-1 is activated by mitogens, oncoproteins, cytokines, and stress
agents such as ultraviolet light. The phorbol ester TPA is often used
to induce AP-1 activity. The activation of this protein may be mediated
both by transcriptionally independent and dependent mechanisms, which
involve post-translational modifications of its components or increases
in the expression of their corresponding genes, respectively (5,
6).
Transcription coactivators bridge transcription factors and the
components of the basal transcriptional apparatus (7). One important
class of coactivators includes the cAMP-response element protein
(CREB)-binding protein (CBP) and the highly related p300 protein, which
were originally identified for their ability to interact strongly with
CREB (8). Subsequently, CBP and p300 were identified as essential
cofactors for a number of nuclear transcription factors, including AP-1
complex (9), several components of the basal transcriptional machinery
(TBP and TFIIB) (10), other histone acetyltransferases (SRC-1,
ACTR and P/CAF) (11), developmental proteins (GATA-1, MEF-2, Pit-1)
(12), viral oncoproteins (E1A, large T antigen, and Tax) (13), and nuclear receptors (14).
These coactivator proteins are important not only due to their role in
positive transcriptional regulation from DNA binding sites but also due
to their role as mediators of cross-talk between different signal
transduction pathways (15). Although negative binding elements have
been described, repression is mainly conducted by interference with
other transcription factors, of which AP-1 is one of the most
representative (16). CBP has been reported to play a significant role
in the negative cross-talk between members of the nuclear receptor
family, including glucocorticoid receptor, retinoic acid receptor,
thyroid hormone receptor, and AP-1 activity, without inhibition of DNA
binding (17-19). Several independent approaches revealed that CBP is
necessary for the activation of both AP-1 and of nuclear hormone
receptors (NHR) as well. As has been suggested, competition for
limiting amounts of CBP may account for many of the inhibitory effects
of NHR on AP-1 activation (17, 20). Thus, on genes containing NHR
binding sites but lacking AP-1 binding sites, positive regulation by
liganded NHR is inhibited by activation of AP-1. Conversely, liganded
NHR can inhibit AP-1-mediated transcription.
In this paper, we present a distinct mechanism of negative cross-talk
between CREB and AP-1 that involves competition by CBP on the
transcriptional activity of 5-aminolevulinate synthase (ALAS) gene
promoter. ALAS is a mitochondrial matrix enzyme that catalyzes the
first and rate-limiting step of heme biosynthesis (21). There are two
related ALAS isozymes that are encoded by two separate genes located on
different chromosomes. The erythroid cell-specific enzyme or ALAS-2 is
developmentally regulated, and it markedly increases during
erythropoiesis to meet the demand for heme during hemoglobin
production. The second enzyme, ubiquitous or liver type ALAS (ALAS-1),
is probably expressed in all tissues to provide heme for cytochromes
and other hemoproteins (21, 22).
Expression of ALAS in the liver was found to be subject to
feedback regulation by heme (21). In addition to this major mechanism of regulation, we have demonstrated that cAMP induces and phorbol esters repress the expression of liver ALAS through protein kinase A
(PKA) and protein kinase C (PKC) activation, respectively (23, 24).
Studies carried out on the 5'-regulatory region of ALAS gene showed the
presence of two functional CRE-like sites that are necessary not only
for the cAMP-mediated induction but also for basal expression. These
sites are bounded by CREB and recruit coactivator CBP (25).
The purpose of this study was to examine the molecular mechanism
underlying TPA-inhibited expression of ALAS gene. Promoter deletion
analysis were performed on the ALAS gene, which, as we have already
demonstrated, is repressed by TPA (23). We found that an AP-1 binding
site (TRE-ALAS) was crucial for the inhibition of the ALAS promoter
despite its widely reported positive responsiveness to TPA in several
promoters (26, 27). The nuclear heterodimeric complexes that bind to
TRE-ALAS would be composed of c-Fos and c-Jun or c-Fos and JunD. In
addition, our data indicate that overexpression of CBP relieved TPA
repression, suggesting that sequestration of CBP prevents the
downstream formation of the CREB·CBP complex necessary for
basal transcription of ALAS gene. This competition for limiting the
intracellular amount of a common coactivator could explain the bizarre
inhibitory effect of TPA on ALAS gene expression. Finally, we observed
different responses to TPA depending on the relative position of TRE
and CRE sites on the ALAS promoter. Therefore, AP-1 complex on TRE-ALAS
would interfere in a disposition-dependent manner with the
transcription machinery through CBP sequestration during the inhibition
of ALAS gene expression.
Reagents--
Eagle's minimum essential medium, guanidine
isothiocyanate, TPA, 4 Expression Vectors--
The following expression vectors were
used as indicated in each experiment. Plasmid pALAS/CAT contains the
5'-flanking region (
To perform transfection assays, plasmids were purified using the Wizard
Plus Maxipreps (Promega Co). DNA concentration was estimated spectrophotometrically.
Cell Culture and Treatments--
The human hepatoma cell line
HepG2 was grown as monolayer cultures in minimum essential medium
supplemented with 10% (v/v) heat-inactivated fetal calf serum and 1%
penicillin-streptomycin, 100 µM nonessential amino acids,
and 2 mM glutamine. Cells were cultured in a humidified
atmosphere with 5% CO2 at 37 °C. For RNA analysis,
phenobarbital and TPA were added to HepG2 cells at 75% confluence in
100-mm tissue culture plates for the times and concentrations detailed
in the figure legends. Phenobarbital was dissolved in 0.1 ml of the
corresponding medium. Phorbol esters and calphostin C were dissolved in
a small volume (less than 0.5% of the total volume of culture media)
of Me2SO.
RNA Extraction and Northern Blot Analysis--
Total cellular
RNA was isolated from cultured HepG2 cells according to Chomczinsky and
Sacchi (30). The yield and purity of RNA samples were assessed by the
ratio of absorbance at 260 and 280 nm. For Northern blot analysis, 20 µg of total RNA were denatured, electrophoresed in 1%
glyoxal-agarose gels, and transferred to nylon membranes (Hybond N,
Amersham Biosciences). The membranes were sequentially hybridized with
32P-labeled probes to human liver ALAS and Transient Transfection Experiments--
HepG2 transient
transfections were performed according to the standard calcium
phosphate precipitation method as previously described (25). In brief,
4 µg of pALAS/CAT or its derivatives and 6 µg of pCEFL
Then cells were collected, and CAT activity was measured in cell
extracts as described previously (25) according to the Seed and Sheen
phase-extraction method (33). The Electrophoretic Mobility Shift and Supershift
Assays--
Nuclear extracts were prepared from TPA-stimulated and
unstimulated HepG2 cells as described by Andrews and Faller (35). Double-strand DNA probes and cold competitors used were TRE-ALAS (AP-1
site located at Immunoprecipitation and Western Blot Analysis--
HepG2 cells
were plated on 100-mm dishes (2 × 106) and
transfected with 2.5 µg of CBP expression vector, where CBP was
tagged with HA, or the backbone vector using Escort transfection
reagent according to the recommendations of the manufacturer (Sigma). Two days after transfection, total cell lysates were prepared in
radioimmune precipitation assay buffer (1× phosphate-buffered saline,
1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 µg/ml
phenylmethylsulfonyl fluoride, 60 µg/ml aprotinin, and 1 mM sodium orthovanadate). The lysates were centrifuged at
10,000 × g for 10 min to remove cell debris. Cleared
lysates (100 µg) were immunoprecipitated with monoclonal anti-HA
antibody (Santa Cruz). The immune complexes were recovered on protein
A/G-agarose beads (Santa Cruz) for 1 h at 4 °C and then washed
4 times with phosphate-buffered saline. The precipitated proteins were
resuspended in sample buffer containing 2% SDS and 30 mM
TPA Inhibits ALAS Gene Expression in HepG2 Cells--
We first
studied the effect of TPA on the expression of ALAS gene in HepG2 cells
either in basal or induced conditions. These cells were
incubated with 1 µM TPA for up to 24 h in the
presence or the absence of 0.6 mM phenobarbital, a well
known inducer of ALAS gene expression (36). Northern blot analysis
showed that in both cases there was a time-dependent
decrease in mRNA levels for ALAS, suggesting that TPA inhibited
ALAS gene expression in HepG2 cells (Fig.
1A). The TPA
concentration-response relationship for ALAS mRNA inhibition
revealed a dose-dependent effect. TPA was effective over a
concentration range of 10
In many cell types, prolonged treatment with phorbol esters resulted in
almost complete depletion of cellular PKC. Because PKC activation led
to inhibition of ALAS gene expression, HepG2 cells transiently
transfected with ALAS/CAT were pretreated with 1 µM TPA
for 12 h to determine whether PKC diminution by translocation to
the cell membrane prevented this inhibition. We observed that prolonged
PKC stimulation resulted in the blockage of ALAS inhibition by TPA both
in basal and phenobarbital-stimulated conditions (data not shown).
Furthermore, incubation of ALAS/CAT-transfected HepG2 cells with 1 µM calphostin C, a PKC inhibitor, resulted in the blockage of the inhibitory effect of 1 µM TPA on ALAS
promoter activity (Fig. 2). These results
suggest that PKC is involved in the inhibitory effect of ALAS gene
expression.
ALAS Gene Expression Is Inhibited by TPA due to a Major Responsive
Site in the Proximal 5'-Noncoding Region--
To identify
cis-acting response elements in the 5'-flanking region of
the rat ALAS gene that are TPA-responsive, a series of progressively
longer deletion mutants of ALAS/CAT were constructed. As in the case of
ALAS/CAT, these deletion promoter mutants were transiently transfected
into HepG2 cells and assayed for CAT activity in the absence and
presence of 1 µM TPA. As shown in Fig.
3, progressive deletion of sequences from
Characterization of AP-1 Binding Site within the ALAS
Promoter--
In silico analysis of the 200-bp region
( Heterodimers c-Fos/c-Jun and c-Fos/JunD
Inhibit ALAS Promoter Activity--
The AP-1 protein, which is a
complex consisting of proteins from Jun and Fos families, binds to TREs
and modifies TPA-regulatable genes. To determine the effects of these
AP-1-related proteins on ALAS transcription, several expression vectors
of the Jun and Fos protein families were transfected into HepG2 cells.
Co-transfection of p-354ALAS/CAT with c-Fos and c-Jun or c-Fos and JunD
expression vectors resulted in a reduced expression of the fusion gene
similar to the level achieved when p-354ALAS/CAT-transfected cells were incubated with TPA (Fig. 5). None of the
other Fos/Jun combinations or Jun dimers modified CAT activity. No
changes in promoter activity were observed in similar co-transfection
experiments performed with p-156ALAS/CAT, a reporter vector that does
not contain the TRE-ALAS site (data not shown), thus discarding
unspecific effects of Fos/Jun-overexpressed proteins. We next performed
supershift analysis to determine the binding of these proteins to the
TRE-ALAS site. As shown in Fig. 4C, incubating HepG2 nuclear
extracts with either anti-c-Fos, anti-c-Jun, or anti-JunD antibodies
caused a supershifted band.
Because TRE, present in the promoter regions of several eukaryotic
genes, is known to increase the transcription of these genes in
response to TPA, in contrast to what happens with ALAS promoter, we
challenged the ability of TPA and Fos/Jun expression vectors under our
experimental conditions to induce CAT activity of a reporter vector
containing two TRE sites with a minimum thymidine kinase promoter
(pTRE-tk-CAT). Results in Table I clearly
show that any Fos/Jun or Jun/Jun heterodimer was able to induce CAT expression as TPA did.
To confirm the functional contribution of AP-1 factors to the
regulation of ALAS transcription by TPA, a dominant negative variant of
c-Fos (A-Fos) was used in co-transfection experiments. This factor
contains, instead of the DNA binding domain, an acidic domain
complementary in charge distribution to the basic region of the
targeted factor. As a result, when A-Fos dimerizes with a wild type
factor to form a coiled coil through the leucine zipper region, the
respective acidic and basic regions continue the formation of a very
stable factor and prevent it from binding to DNA (28). As seen in Fig.
6, the overexpression of A-Fos
significantly diminished the inhibitory effect caused by TPA or by the
overexpression of c-Fos/c-Jun or c-Fos/JunD on ALAS promoter activity.
Similarly, overexpressed A-Fos blocked the induction effect obtained on
pTRE-tk-CAT expression by TPA or by the mentioned heterodimers Fos/Jun
(Table II).
All these results strongly indicate that the nuclear proteins
interacting with the TRE-ALAS region are most likely composed of c-Fos
and c-Jun or c-Fos and JunD and that these AP-1 complexes are
responsible for the TPA-mediated inhibition of ALAS transcriptional activity.
Overexpression of CBP Counteracts the TPA Inhibitory Effect on ALAS
Promoter Activity--
In previous work we demonstrated the presence
of two CRE-like sites on the regulatory region of ALAS gene, located
downstream TRE-ALAS site, necessary not only to confer cAMP/PKA
responsiveness but for also ALAS basal expression (25). These findings
allow us to hypothesize that the competition for a common co-activator like CBP that prevents the downstream formation of the CREB·CBP complex necessary for basal transcription of ALAS gene could explain the bizarre inhibitory effect of TPA. To evaluate this hypothesis, HepG2 cells were co-transfected with p-354ALAS/CAT and CBP expression vector and treated or not with TPA. Overexpression of CBP severely curtailed the TPA-mediated inhibition of ALAS promoter activity (Fig.
7). Consistent with this result, when the
inhibition of ALAS transcription was achieved by co-transfection of
c-Fos and c-Jun or c-Fos and JunD expression vectors, CBP
overexpression partially reversed this effect. Furthermore, the
addition of TPA blocked the CBP-mediated reversion (Fig. 7). Similar
experiments were performed with an expression vector encoding
co-activator p300. Again, TPA-mediated inhibition of ALAS promoter
activity was avoided by the overexpression of the transcriptional
integrator (data not shown).
CBP Interacts with Endogenous CREB in Vivo--
To challenge our
hypothesis, we next asked whether CBP associates with CREB in HepG2
cells in basal conditions. To address this, an expression vector for
CBP, harboring a HA tag, was transfected into the HepG2 cells.
Immunoprecipitation of tagged CBP followed by Western blot analysis of
the precipitants for the presence of phospho-CREB indicated an
interaction between the proteins (Fig.
8A, upper panel).
As expected the CREB-CBP interaction was increased in HepG2 cells
previously treated with 100 µM 8-CPT-cAMP. Likewise,
incubating cells with 100 µM H-7, a protein kinase
inhibitor, partially blocked the protein association. No binding to
CREB was observed when HA-tagged empty vector was used as the control. None of the treatments had any effect on endogenous levels of CREB in
HepG2 cells (Fig. 8A, lower panel). These results
strongly suggest that the presence of a basal level of phosphorylated
CREB is able to recruit CBP in HepG2 cells despite the absence of
exogenous cAMP stimulus.
It was of interest to determine whether ALAS promoter activity in HepG2
cells transfected with p-354ALAS/CAT correlates with CREB-CBP
interaction in the conditions tested in the immunoprecipitation assay.
As shown in Fig. 8B, cAMP-treated cells displayed the
highest CAT activity, whereas the presence of the inhibitor H-7
diminished it below the basal levels, suggesting that the extent of
CREB and CBP association is critical for ALAS promoter activity.
Relative Position of CRE-ALAS and TRE-ALAS in a Heterologous
Promoter Drives TPA Responsiveness--
To strengthen the previous
analysis we constructed three expression vectors cloning two different
regions of ALAS promoter in the heterologous reporter vector pBLCAT2.
As shown in Fig. 9A, vector
pASCAT comprises the region between
In a previous work we demonstrate that the effect of TPA in reducing
ALAS expression was dominant over the stimulatory action of cAMP (23).
To assess the possibility that the stimulatory and inhibitory effects
of cAMP and TPA, respectively, would be a consequence of the relative
positions of the sites for its specific binding factors, we tested the
action of 100 µM 8-CPT-cAMP and 1 µM TPA in
cotransfection experiments with pAECATd or pAECATi. As shown in Fig.
10, TPA inhibited the transcription
activity of the ALAS promoter in the right orientation, whereas the
phorbol ester induced ALAS/CAT expression when the promoter was located in the inverted position. Conversely, cAMP induced the expression of
the fusion gene when the promoter was in the right orientation but did
not display any significant stimulation when the regulatory region was
located in the inverted position. We then tested the effect of mutation
of the TRE-ALAS in these constructions. TPA inhibitory or stimulatory
effects were impaired when TRE-ALAS was mutated in pAECATdm or
pAECATim, respectively, whereas the effect of cAMP was not modified.
Coincident with the above hypothesis, when cAMP and TPA were jointly
added to HepG2 cells, the dominant inhibitory effect of TPA was
observed when the promoter was located in the right position. In this
case, mutation of TRE-ALAS in the direct orientation promoter
alleviated the TPA dominant inhibition and allowed cAMP to induce CAT
activity, whereas mutation in the inverted orientation promoter did not
modify reporter activity, suggesting that increasing the distance
between the CRE and the transcription initiation sites impairs the
ability of cAMP to induce ALAS promoter activity. To support these
findings, a similar experiment was performed in HepG2 cells
cotransfected with the expression vector for CBP co-activator. As shown
in the same figure, overexpression of CBP either blocked the inhibitory
effects or enhanced the stimulatory action observed in the absence of
the coactivator.
These results strongly suggest that the relative position of TRE with
respect to CREs could drive the response to TPA and also reinforce the
idea that competition by co-activator CBP could be involved in
TPA-mediated inhibition of ALAS transcriptional activity.
The aim of this work was to determine the molecular mechanism
involved in the TPA-mediated inhibition of the rat ubiquitous ALAS gene
in human hepatoma HepG2 cells. Previous analysis of the 5'-flanking
sequence of ALAS gene revealed the existence of binding sites for
nuclear respiratory factor I (39) and for transcription factor CREB
(25). We describe for the first time a DNA sequence (TRE-ALAS) that
binds AP-1 proteins and is necessary for the inhibitory effect of
phorbol esters on ALAS gene expression. Based on Northern blot analysis
and transient transfection experiments with a promoter-reporter fusion
gene containing a 870-bp fragment of the 5' region of the ALAS gene, we
conclude that the effect of TPA occurs at the transcriptional level. We
have previously demonstrated that phorbol esters do not modify the ALAS
mRNA turnover (23).
The well documented interaction of phorbol esters with PKC (40)
suggests that protein phosphorylation is involved in the mechanism by
which TPA suppresses ALAS mRNA transcription. The blockage of TPA
inhibitory effect caused by a specific inhibitor of PKC such as
calphostin C (41), the desensitization developed after pretreatment of
HepG2 cells with TPA, and the fact that 4 Deletion analysis of the 5'-regulatory region of the ALAS gene allowed
us to identify a major TPA-responsive region located at Additional evidence for functional TRE in the ALAS gene is as follows.
First, CAT activity was reduced when a reporter gene containing the TRE
region was cotransfected with a combination of vectors that expressed
c-Fos and c-Jun or c-Fos and JunD, proteins that belong to the AP-1
family. Second, cotransfection with the dominant negative A-Fos
abolished the response of the ALAS promoter to the inhibitory effect of
either TPA or c-Fos/c-Jun or c-Fos/JunD. The suggested effect is the
titration of c-Jun and JunD factors (28). Finally, TRE-ALAS conferred
sensitivity to the thymidine kinase promoter on the action of TPA when
this heterologous plasmid was transfected to HepG2 cells.
The results of gel-shift experiments merit some comments. Incubation of
HepG2 nuclear extracts with an oligonucleotide containing the TRE-ALAS
sequence of ALAS determined a complex that was highly increased with
extracts from TPA-treated HepG2 cells. This complex was competed with
the unlabeled probe in a simultaneous incubation but not with a mutated
version. Likewise, competition with an unlabeled consensus AP-1 probe
indirectly confirmed the TRE-ALAS identity. It has been reported that
all Jun·Jun and Jun·Fos complexes have the same binding specificity
but exhibit different binding affinities (4). Thus, Jun and Fos
proteins have the ability to interact among themselves and activate
different targets that contain AP-1-response elements in their
promoters. In addition to transfection experiments, supershift assays
confirm that nuclear proteins that bind to the TRE-ALAS are most likely
composed of c-Fos and c-Jun or c-Fos and JunD. It has been reported
that c-Fos, c-Jun, and JunD are expressed in HepG2 cells (43).
Therefore, our finding is compatible with the expression pattern of the
AP-1 protein family in this cell line. The binding of c-Jun and c-Fos to the TRE-ALAS site after treatment of HepG2 cells with TPA is not
surprising given that these nuclear proteins can be induced rapidly and
transiently and display enhanced AP-1 binding activity upon treatment
of cultured cells with an array of compounds, including TPA (44).
Conversely, JunD appears to be constitutively expressed as relatively
high levels in cultured cells, including hepatomas, with only modest
increases in mRNA levels after treatment with TPA or growth factors
(45). Formation of heterodimeric and homodimeric complexes between Fos
and Jun family members and their association with DNA depends on their
zipper domains. Thus, the Fos zipper avidly binds to the Jun zipper but
does not bind to itself. Members of the Jun family can weakly
homodimerize or form stronger heterodimeric complexes with Fos family
members (46). This could be the reason for the presence of Fos in the
two forms of AP-1 that bind TRE-ALAS site. Because TRE-ALAS does not
exactly match with the consensus TRE site, the stronger AP-1 factors
could establish the most stable complexes on the DNA and would be the
most effective inhibitors.
The inhibitory effect of TRE-ALAS on the transcriptional activity of
the ALAS promoter is rather interesting since the presence of the same
sequence in a heterologous promoter enhanced TPA transcription. Promoter regions of eukaryotic genes are generally composed of multiple
binding sites for transcriptional activators and repressors that act in
combination to regulate the expression of a linked gene (47). Previous
analysis of the 5'-flanking sequence of ALAS revealed the existence of
two CRE sites. This elements are required for basal and cAMP-stimulated
expression of the ALAS gene in hepatoma human cells (25). Our results
show that TPA inhibitory effect can be blocked by CBP overexpression.
Thus, our hypothesis to explain TPA inhibitory effect is based on the competition between AP-1 and CREB factors for limiting amounts of CBP.
There are several evidences on the interaction of CBP with CREB and
Fos/Jun proteins (8, 48) as well as on the competition mechanisms that
regulate the action of different transcription factors, which involve
CBP and other coactivators. Fronsdal et al. (49) provide
evidence that the transcriptional interference between androgen
receptors and AP-1 may be mediated through competition for limiting
amounts of CBP. Similarly, DiSepio et al. (19) suggest that
the mutual transrepression of the retinoic acid-receptor and AP-1 might
be due to the competition for limiting coactivators, including CBP. In
this regard, our demonstration that CBP interacts with CREB under basal
conditions in HepG2 cells and that the extent of this association is
correlated with the ALAS promoter activity provides a strong support
for our hypothesis.
Even though the competition between different transcription factors for
CBP is well known, there is no conclusive evidence that coactivator
levels are limiting, as has been proposed (17, 50). Karin and Chang
(51) suggest that, because the amounts of nuclear CBP/p300 seem to
exceed those of AP-1 or glucocorticoid receptor and CBP/p300 is
also a common target for many other sequence-specific transactivators,
which do not transrepress AP-1 activity, it is unlikely that simple
competition for a limiting amount of CBP/p300 can explain the
transrepression of AP-1 activity by glucocorticoid receptor. Despite
this, our results present a cross-talk between different signal
pathways that regulate gene expression, like cAMP and TPA, and that
occur at the level of a coactivator.
Finally, our work highlights the importance of the natural promoter
context when studying AP-1-mediated gene expression. It seems likely
that a completely orientation-dependent silencer or
enhancer acts by presenting its specific binding factor in a particular
position or direction relative to other regulatory sequences or factors
(52). We suggest that the contribution of the AP-1 binding factors to
the promoter activity might be determined mainly by the promoter
context (e.g. surrounding binding sites for other
transcription factors) rather than by the element itself. Results
depicted in Figs. 9 and 10, in which the TRE-ALAS site was placed in
different contexts with respect to CRE sites, thus acting as a
transcriptional enhancer, support this view.
In addition to the feedback regulation by heme (21) and in addition to
the transcription inhibitory effect described in this paper, ALAS is
also repressed by insulin (53). Recently, the postreceptor-signaling
mechanisms for insulin regulation of ALAS gene expression has begun to
be uncovered. We suggest that both pathways, phosphatidylinositol
3-kinase/PKB and Ras/mitogen-activated protein
kinase/p90RSK are jointly required for insulin-mediated
inhibition of ALAS gene expression in rat hepatocytes and human
hepatoma cells (54). Also, the activation of PKC is indispensable for
the transduction of the insulin effect to the ALAS promoter, although
the connection with the mentioned pathways remains unclear (53). In a
number of instances investigators have described that phorbol esters mimic the action of insulin (55). On the other hand, there is evidence
that insulin increases diacylglycerol concentrations and PKC activity
(56). Because PKC is a primary target of phorbol esters, one could
speculate that this protein kinase acts as a common factor linking
insulin and TPA signaling. However, there are several reports
demonstrating that genes that are acutely inhibited by insulin present
different responses to this hormone after inhibition or down-regulation
of PKC (57-59). In the particular case of ALAS, beyond the involvement
of PKC in both insulin and TPA signaling, the phorbol ester inhibits
transcription through cis-acting elements, the TRE site, and
trans-acting factors, AP-1 proteins, that differ from those
involved in insulin regulation of ALAS promoter activity, which would
include hepatocyte nuclear factor-3 and NF-1 sites (this paper and Ref.
54).
There are many signals that activate PKC in hepatocytes. Various
prostaglandins, metabolites of arachidonic acid, and other lipid
mediators produced by phospholipases C and D are thought to play
important roles in hepatocyte proliferation through PKC activation
(60). The proliferative action of transforming growth factor- The inhibition of ALAS expression can have important physiologic
outcomes. Its imbalanced repression results in a deficit of vital
hemeproteins, such as hemoglobin, enzymes taking part in cellular
respiration, reduction of sulfite and nitrite, neutralization of
reactive oxygen species, and the oxidative metabolism of lipophilic xenobiotics, as a consequence of an impaired synthesis of heme (21,
63). The inhibitory effect of TPA-mediated PKC activation on the ALAS
transcription could explain the apoptotic cellular response driven by
phorbol ester treatment (64, 65). One of the reasons could be the
increase of free radicals produced by TPA and the decrease in the
amount of peroxisomal hemeproteins involved in neutralizing reactive
oxygen species. A similar mechanism could be utilized by prostaglandin
A2 to induce apoptosis in human hepatocarcinoma Hep3B and
HepG2 cells (66).
In summary, our results demonstrate the presence of a functional TRE
site on the ALAS promoter that, upon TPA stimulation, interacts with
c-Fos/c-Jun or c-Fos/JunD heterodimers. We propose that the decrease in
ALAS basal activity observed in the presence of TPA may reflect a
reduction in the capability of this promoter to assemble the productive
pre-initiation complex involving the interaction of CREB-CBP with the
CRE sites through sequestration of the coactivator (Fig.
11). We also demonstrate that the
transcriptional properties of this AP-1 site would depend on a
spatial-disposition-dependent manner with respect to CRE
sites and/or the transcription initiation site.
833 to +42 in the 5'-flanking region of rat
ALAS gene was subcloned into a chloramphenicol acetyltransferase (CAT)
reporter vector. The expression vector pALAS/CAT produced a significant
CAT activity in transiently transfected HepG2 human hepatoma cells,
which was repressed by TPA. Sequence and deletion analysis detected a
TPA response element (TRE), located between
261 and
255 (TRE-ALAS), that was critical for TPA regulation. We demonstrated that c-Fos, c-Jun, and JunD are involved in TPA inhibitory effect due to their ability to bind TRE-ALAS, evidenced by supershift analysis and their
capacity to repress promoter activity in transfection assays. Repression of ALAS promoter activity by TPA treatment or Fos/Jun overexpression was largely relieved when CRE protein-binding protein or
p300 was ectopically expressed. When the TRE site was placed in a
different context with respect to CRE sites, it appeared to act as a
transcriptional enhancer. We propose that the decrease in ALAS basal
activity observed in the presence of TPA may reflect a lower ability of
this promoter to assemble the productive pre-initiation complex due to
CRE protein-binding protein sequestration. We also suggest that the
transcriptional properties of this AP-1 site would depend on a
spatial-disposition-dependent manner with respect to the
CRE sites and to the transcription initiation site.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phorbol-12,13-diacetate (4
PDA), agarose,
calphostin C, chloramphenicol, butyryl coenzyme A, 8-CPT-cAMP, and
o-nitrophenyl-
-D-galactopyranoside were
purchased from Sigma.
[ring-3,5-3H]Chloramphenicol (specific
activity 1.1-2.2 GBq/mmol), [
-32P]ATP (specific
activity 222 TBq/mmol), and [
-32P]dCTP (specific
activity 111 TBq/mmol) were purchased from PerkinElmer Life Sciences.
Random primers kit, restriction endonucleases, and DNA modifying
enzymes were from New England Biolabs, Inc. All other chemicals were of
analytical grade. Oligodeoxynucleotides were chemically synthesized by
Bio-Synthesis Inc. (Lewisville, TX).
833 to +42 bp) of rat ubiquitous ALAS gene cloned
upstream the CAT reporter gene in vector pBLCAT6. Deletion mutant
plasmids p-459ALAS/CAT, p-354ALAS/CAT, p-156ALAS/CAT, and p-75ALAS/CAT were described previously (25). Vectors pSG5 encoding wild type forms
of c-Fos, c-Jun, JunB or JunD, and a pRc/RSV vector encoding the
cDNA for the wild type version of CBP were kindly provided by Dr.
P. Sassone-Corsi (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France). Vector pCEFLp300 encoding cDNA for the wild type version of p300 was a gift from Dr.
J. Silvio Gutkind (NIDCR, National Institutes of Health, Bethesda, MD).
The pCMV/A-Fos (designated pA-Fos) is a cytomegalovirus-driven expression vector in which the normal basic region critical for DNA
binding at the N terminus of the Fos leucine zipper was replaced for an
acidic sequence (a generous gift from Dr. Charles Vinson, NCI, National
Institutes of Health, Bethesda, MD) (28). The plasmid pTRE-tk-CAT
contains two TRE consensus sequence upstream of the HSV thymidine
kinase promoter (29). The heterologous pASCAT vector, in which a copy
of the
354 to
156-bp sequence of ALAS gene was placed upstream of
the thymidine kinase promoter, was generated by cloning the
AflII/StuI fragment of pACAT into the
SalI site of pBLCAT2. Similarly, pAECATd or pAECATi vectors containing a copy of the
354 to
38-bp sequence of ALAS gene in the
right or inverted position, respectively, upstream of the thymidine
kinase promoter were generated by cloning the
AflII/BstEII fragment of pACAT into the
SalI site of pBLCAT2. The mutations were generated by
polymerase chain reaction-based site-directed mutagenesis (Stratagene,
La Jolla, CA). In p-354ALAS/CATm, pAECATdm, and pAECATim vectors the
TRE-ALAS site was mutated from wild-type TGACGCA (coding strand) to
TGACGTG (coding strand). The fidelity of all mutated vectors was
checked by DNA sequence. Plasmid pCEFL containing the
-galactosidase
gene was also used.
-tubulin. To
detect ALAS mRNA, a 26-mer oligodeoxynucleotide was
synthesized complementary to bases +328 to +353 of human ubiquitous
ALAS mRNA (31). The oligodeoxynucleotide was purified and
5'-end-labeled using [
-32P]ATP and T4
polynucleotide kinase. The resulting probe had a specific activity of
about 5-6 × 103 cpm/fmol. Hybridization was carried
out overnight at 70 °C in the same prehybridization solution by
adding the 32P-labeled oligodeoxynucleotide (3.0 × 105 cpm/cm2) as previously described (24). To
detect
-tubulin mRNA, chicken
-tubulin cDNA (a generous
gift of Dr. J. Messina, Florida) was labeled by random priming using
[
-32P]dCTP and Klenow to a specific activity of about
6 × 108 cpm/µg. Membranes were stripped,
prehybridized, hybridized, and washed in standard conditions described
by Sambrook et al. (32). Autoradiographs were obtained by
exposing these blots to Kodak XAR-5 film with an intensifying screen
for 3-5 days at
70 °C.
gal were
cotransfected into 5 × 105 cells plated on 35-mm
Petri dishes. The
-galactosidase plasmid was used as the internal
standard to normalize transfection efficacy. The use of other
cotransfected plasmids is indicated in each experiment. The amount of
Fos/Jun, CBP, and p300 expression vectors that have been used in the
different experiments was previously determined through dose-response
curves. Different quantities of the plasmids mentioned were
co-transfected into HepG2 cells together with p-354ALAS/CAT, and CAT
expression was measured. The minimum amount of each plasmid that
produced the maximum effect was used in later experiments. Final DNA
concentration was adjusted to 30 µg/35-mm dish with nonspecific DNA
carrier. Control transfections with carrier alone and carrier plus
vector pBLCAT6 or pBLCAT2 were done in parallel. Sixteen hours later,
the medium was replaced with 3 ml of serum-free medium containing the
reagents indicated in each experiment and incubated for 24 h.
-galactosidase activity was
determined spectrophotometrically in the transfected cell extracts
(32). CAT activity was expressed as the amount of radiolabeled
chloramphenicol acetylated by 1 mg of protein in 1 min and normalized
with
-galactosidase activity.
-Galactosidase activity was not
modified by any of the treatments used. The protein concentration of
the cell extracts was determined by the Bradford assay (34).
261 bp), 5'-AGGAGTCTGACGCACAGGGCT-3', and mutant,
5'-AGGAGTCTGACGTGCAGGGCT-3', called TRE-ALASmut, in which the underlined bases have been mutated to disrupt the specific binding of AP-1-binding proteins. A positive control
oligodeoxynucleotide containing a consensus AP-1 binding site,
5'-CGCTTGATGAGTCAGCCGGAA-3', and an oligodeoxynucleotide containing a
consensus CRE site corresponding to the
58 to
31 bp of rat
somatostatin gene promoter 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3', were also
used. Oligodeoxynucleotides were 5'-end-labeled with T4
polynucleotide kinase and 222 TBq/mmol [
-32P]dATP at
37 °C for 60 min. Binding reactions were performed mixing 10 µg of
the nuclear extract with 2 µg of poly(dI-dC) and 150,000 dpm of the
labeled probe in TM buffer (50 mM Tris-HCl, pH 7.9, 12.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 20% (v/v) glycerol) to a final volume
of 20 µl and incubated at room temperature for 30 min. When noted,
the nuclear extract was incubated for 20 min at room temperature in a
binding mixture with the indicated molar excess of unlabeled competitor
DNA before the addition of labeled probe. After the binding reaction,
electrophoresis was carried out through a 5% non-denaturating
polyacrylamide gel containing 0.25× TBE (1× TBE: 50 mM
Tris borate, pH 8.3, 1 mM EDTA). The gel was then dried,
and autoradiography was performed. The supershift analysis was
performed by incubating the nuclear extract with 3 µl of specific
antibody at 4 °C for 4 h before the band shift assays already
described. Antibodies against c-Fos, c-Jun, and JunD were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA).
-mercaptoethanol, boiled for 3 min, fractionated by SDS-PAGE on a
10% gel, and thereafter blotted to a nitrocellulose membrane. The
membrane was then immunoblotted with polyclonal anti-rabbit
anti-phospho-CREB (Cell Signaling Technology). The
antibody was detected using horseradish peroxidase-linked goat
anti-rabbit IgG (Sigma) and visualized by the Pierce Super Signal Ultra
Chemiluminescence signaling systems and a Bio-Imaging Analyzer
Fujifilm LAS-1000. For direct immunoblotting of cell proteins, total
cell lysates of parallel samples were fractionated by electrophoresis
in SDS-PAGE on a 10% gel, and the proteins were transferred to a
nitrocellulose membrane. Protein CREB was detected using the rabbit
polyclonal antibody anti-CREB (Cell Signaling). The immune complexes
were visualized by chemiluminescence as describe above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
8 to 10
5
M, and maximum inhibition was reached at 10
6
M and over (Fig. 1B). These results agree with
previous observations made in primary cultures of rat hepatocytes (23).
To determine whether sequences in the 5'-flanking region of the ALAS
gene could confer TPA responsiveness, we fused about 870 bp of this
region to the bacterial reporter gene for CAT. HepG2 cells were
transiently transfected with this ALAS/CAT vector and then incubated
with different amounts of TPA before harvesting and analysis of CAT activity (Fig. 1C). TPA caused a dose-dependent
inhibition in ALAS/CAT expression with a maximum decrease of almost
5-fold in untreated cells and 8-fold in cells exposed to 0.6 mM phenobarbital. The action of 4
PDA, a TPA analog that
is ineffective in tumor promotion and PKC activation, was tested to
exclude the possibility of a general inhibitory effect due to
incubation with phorbol esters. The addition of 1 µM
4
PDA failed to reduce ALAS/CAT expression (data not shown).
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Fig. 1.
Effect of TPA on ALAS mRNA and promoter
activity. A, HepG2 cells were incubated in
serum-free medium with 1 µM TPA in the presence or
absence of 0.6 mM phenobarbital (PB) for the
times indicated. B, HepG2 cells were incubated in serum-free
medium with different concentrations of TPA in the presence or absence
of 0.6 mM phenobarbital for 8 h. In A and
B total RNA was extracted from the cells and subjected to
Northern blot analysis using a 32P-labeled probe specific
for human ALAS mRNA and reprobed for -tubulin mRNA as
described under "Experimental Procedures." Each figure shows a
representative autoradiograph of three independent experiments with
similar results. C, HepG2 cells transiently transfected with
4 µg of p-ALAS/CAT plasmid were incubated in serum-free medium with
different concentrations of TPA in the presence (
) or absence (
)
of 0.6 mM phenobarbital. After 24 h, cells were
harvested, and CAT activity was determined as described under
"Experimental Procedures." Results are expressed as relative CAT
activity with respect to samples non-treated with TPA, which were set
to 100. Each point represents the mean ± S.E. of four different
experiments performed in duplicate. Student's t test was
used to compare samples treated with TPA with non-treated samples (*,
p < 0.05, indicates the minimum TPA
concentration that causes a significant reduction in CAT
activity).
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Fig. 2.
Calphostin C blocks the TPA inhibitory effect
on ALAS promoter activity. HepG2 cells transiently transfected
with 4 µg of p-ALAS/CAT plasmid were incubated in serum-free medium
with the indicated additions. After 24 h, cells were harvested,
and CAT activity was determined as described under "Experimental
Procedures." Results are expressed as relative CAT activity with
respect to basal value without any addition, which was set to 100. Bars represent the mean ± S.E. of three independent
experiments performed in duplicate. Student's t test was
used to compare samples containing TPA with the respective basal value
(*, p < 0.05) or samples containing TPA plus
calphostin C with samples containing TPA alone (**, p < 0.05). PB, 0.6 mM phenobarbital;
Cpn, calphostin C.
833 to
354 bp did not significantly impair TPA-mediated inhibition
of promoter activity. Further deletion of the promoter from
354 to
156 bp almost completely blocked the TPA-inhibited ALAS/CAT
expression, suggesting that an essential TPA-responsive element is
contained within this region. There were only slight differences in the
basal levels of promoter activity in the deletion mutants tested (Fig.
3, inset).
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Fig. 3.
Deletion analysis reveals a region important
for TPA-mediated inhibition of ALAS promoter activity. HepG2 cells
transiently transfected with 4 µg/plate of p-ALAS/CAT or equivalent
amounts of deletion mutants containing the 5'-flanking region of ALAS
gene illustrated on the left were incubated in serum-free
medium alone (white bars) or with the addition of 1 µM TPA (black bars). After 24 h, cells
were harvested, and CAT activity was determined as described. Results
are expressed as relative CAT activity with respect to basal value for
each construction, which was set to 100. Bars represent
mean ± S.E. of four independent experiments performed in
duplicate. Student's t test was used to compare TPA-treated
and non-treated samples (*, p < 0.05).
Inset, relative basal CAT activity of each promoter mutant
with respect to the basal activity of p-ALAS/CAT, which was set to 100. Bars represent the mean ± S.E. of four independent
experiments performed in duplicate.
354 to
156 bp) included in our 5'-deletion analysis in the
TPA-mediated inhibition of ALAS promoter activity revealed a potential
TRE/AP-1 regulatory element (TRE-ALAS) corresponding to
nucleotides
261 to
255 bp (core similarity 1.000; matrix
similarity 0.925) (37). Consistent with 5' deletion analysis,
mutation of the TRE-ALAS site impaired the ability of TPA to decrease
promoter activity, revealing a role for this element in the regulation
of ALAS gene expression (Fig. 3). However, basal promoter activity was
unaffected by the TRE-ALAS mutation (Fig. 3, inset). To
determine whether AP-1 proteins can bind the TRE-ALAS motif, we
performed electrophoretic mobility shift assays with oligonucleotides
containing this sequence and nuclear extracts from HepG2 cells treated
or not treated with 1 µM TPA. As shown in Fig.
4, a probe containing the TRE-ALAS motif
(TGACGCA) formed a shifted complex that increased several times with
the addition of TPA-treated extracts. This complex was competed in a
dose-dependent manner by the same unlabeled oligonucleotide
but not by unlabeled oligonucleotide containing a mutated TRE-ALAS
binding site (TGACGTG). Moreover, the DNA-protein complex
was competed by unlabeled oligonucleotides containing the consensus
AP-1 sequence but not by the consensus CRE sequence. Likewise, a
shifted complex formed by a probe containing the AP-1 consensus motif
was competed in a dose-dependent manner with unlabeled oligonucleotides containing the TRE-ALAS sequence (data not shown). Taken together, these data clearly show that the TRE-ALAS element is an
AP-1 binding site.
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Fig. 4.
TRE-ALAS site forms a complex with
c-Fos/c-Jun or c-Fos/JunD. A, 10 µg of protein
prepared from extracts of HepG2 cells previously treated or not treated
with 1 µM TPA for 6 h were incubated with a
32P-labeled probe representing the TRE-ALAS site.
B, 10 µg of protein prepared from extracts of HepG2 cells
previously treated with 1 µM TPA for 6 h were
incubated with 32P-labeled probes representing the TRE-ALAS
or the mutated TRE-ALAS (TRE-ALASmut) in the presence or absence of
increased quantities of unlabeled competitor oligonucleotides as
indicated. The competitor oligonucleotides were either the unlabeled
TRE-ALAS, mutated TRE-ALAS, AP-1, or CRE consensus sequences.
C, 10 µg of nuclear extract prepared from the HepG2 cells
treated with 1 µM TPA for 6 h were subjected to
supershift assays. Nuclear proteins were preincubated with 3 µl of
each antibody against c-Fos, c-Jun, or JunD proteins, and then
32P-labeled TRE-ALAS or AP-1 probe was added. Controls were
incubated with preimmune immunoglobulins (pi). In
A, B, and C, protein-DNA complexes
were resolved by electrophoresis on a 5% non-denaturing polyacrylamide
gel in 0.25× TBE buffer with 120 V at room temperature.
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Fig. 5.
Heterodimers of Fos and Jun proteins inhibit
ALAS promoter activity. HepG2 cells were transiently transfected
with 4 µg/plate of p-354ALAS/CAT and cotransfected or not with 3 µg/plate of each of the indicated expression vectors for Fos and Jun
proteins. Non-cotransfected sample was incubated in serum-free medium
containing 1 µM TPA during 24 h. Results are
expressed as relative CAT activity with respect to p-354ALAS/CAT
without any treatment, which was set to 100. Bars represent
the mean ± S.E. of four independent experiments performed in
duplicate. Student's t test was used to compare samples
cotransfected with some Fos/Jun expression vector and non-cotransfected
and non-treated sample. *, p < 0.05.
TPA or Fos and Jun heterodimers stimulate the expression of a reporter
vector containing TRE
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Fig. 6.
Effect of a dominant negative variant of
c-Fos on the TPA or Fos/Jun-mediated inhibition of ALAS promoter
activity. HepG2 cells were transiently transfected with 4 µg/plate of p-354ALAS/CAT and cotransfected or not with 3 µg/plate
of each of the indicated expression vectors for Fos and Jun proteins.
Non-cotransfected samples were incubated in serum-free medium
containing 1 µM TPA during 24 h. Transfections may
include 6 µg/plate of expression vector for A-Fos or the empty vector
as indicated. Results are expressed as relative CAT activity with
respect to basal value of p-ALAS/CAT, which was set to 100. Bars represent the mean ± S.E. of three different
experiments performed in duplicate. Student's t test was
used to compare in each group samples cotransfected and
non-cotransfected with A-Fos. *, p < 0.05.
Effect of a dominant negative variant of c-Fos on the expression of a
reporter vector containing TRE
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Fig. 7.
CBP overexpression blocks the inhibitory
effect of TPA or Fos/Jun-mediated inhibition of ALAS transcriptional
activity. HepG2 cells were transiently transfected with 4 µg/plate of p-354ALAS/CAT and cotransfected or not cotransfected with
3 µg/plate of each of the indicated expression vectors for Fos and
Jun proteins. Transfections may include 4 µg/plate of expression
vector for CBP coactivator as indicated. Some cell cultures were
treated with 1 µM TPA for 24 h. Results are
expressed as relative CAT activity with respect to non-cotransfected
sample, which was set to 100. Bars represent the mean ± S.E. of three different experiments performed in duplicate.
Student's t test was used to reveal significant differences
in each group. *, p < 0.05, between samples
cotransfected and non-cotransfected with CBP; **, p < 0.05, between samples cotransfected with CBP plus TPA-treated and
samples cotransfected with CBP only.
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Fig. 8.
CBP interacts with CREB under basal
conditions in HepG2 cells. A, HA-tagged CBP protein was
expressed in HepG2 cells before treatment with 100 µM H-7
or 100 µM 8-CPT-cAMP (cAMP). Equal amounts of
proteins from cell lysates were immunoprecipitated (IP)
using a monoclonal anti-HA antibody. The immune complexes were
subjected to 10% SDS-PAGE, transferred to nitrocellulose membranes,
and analyzed by Western blot with an anti-phospho (Ser-133)-CREB
antibody (upper panel). Parallel samples of HepG2 cells used
in immunoprecipitation assay were electrophoresed, transferred to
membranes, and probed with an anti-CREB antibody that recognizes total
CREB (lower panel). Immunoprecipitation and direct
immunoblotting experiments were performed twice and showed similar
results. B, HepG2 cells were transiently transfected with 4 µg/plate of p-354ALAS/CAT and treated with 100 µM H-7
or 100 µM 8-CPT-cAMP (cAMP). After 24 h,
cells were harvested, and CAT activity was determined as described.
Results are expressed as relative CAT activity with respect to non
treated sample, which was set to 100. Bars represent the
mean ± S.E. of three different experiments performed in
duplicate. Student's t test was used to compare treated
with non treated samples. *, p < 0.05.
354 and
156 bp containing the
critical TRE-ALAS for TPA-mediated inhibition, vector pAECATd comprises
the
354 to
38-bp region including TRE-ALAS and the two CRE-ALAS
sites, and vector pAECATi contains the same sequence but in the inverse
direction. In agreement with our hypothesis, when the TRE-ALAS region
was attached to the thymidine kinase promoter bound to the CAT gene
(pASCAT), it expressed high levels of CAT activity upon transfection in
HepG2 cells treated with 1 µM TPA. This induction was
slightly increased by CBP co-transfection (Fig. 9B). On the
contrary, transfection of the fusion gene containing TRE and CRE sites
(pAECATd) in TPA-treated cells resulted in a decreased CAT expression
that was blocked by CBP overexpression, in agreement with the results
obtained with the ALAS promoter (Fig. 9B). Interestingly,
TPA induced CAT activity when vector pAECATi, with TRE and CRE sites in
inverted position, was transfected.
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Fig. 9.
TRE-ALAS site linked to heterologous promoter
confers TPA responsiveness. A, maps of the
p-354ALAS/CAT and chimerical ALAS promoters indicating the elements
that are present. B, HepG2 cells were transiently
transfected with 6 µg/plate of p-354ALAS/CAT or the depicted
chimerical expression vectors and cotransfected or not with 4 µg/plate of expression vector for CBP coactivator as indicated. Some
cell cultures were treated with 1 µM TPA for 24 h.
For each ALAS/CAT expression vector, results are expressed as relative
CAT activity with respect to non-cotransfected and non-TPA-treated
sample, which was set to 100. Bars represent the mean ± S.E. of three different experiments performed in duplicate.
Student's t test was used to reveal significant
differences. *, p < 0.05, between samples treated with
TPA, samples cotransfected with CBP, or samples cotransfected with CBP
plus TPA-treated, non-treated, and non-cotransfected samples; **,
p < 0.05, between samples cotransfected with CBP plus
TPA-treated and samples treated with TPA; ***, p < 0.05, between samples cotransfected with CBP plus TPA-treated and
samples cotransfected with CBP). Inset, relative basal CAT
activity of each chimerical ALAS/CAT expression vector respect to empty
vector (pBLCAT2), which was set to 100. *, p < 0.05.
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Fig. 10.
Relative position of CRE and TRE sites in a
heterologous promoter drives TPA and cAMP responsiveness. HepG2
cells were transiently transfected with 6 µg/plate of pAECATd
(d) or pAECATi (i) or their mutated version
(pAECATdm (dm) or pAECATim (im)) and
cotransfected or not with 4 µg/plate of CBP expression vector as
indicated. Some cell cultures were treated with 1 µM TPA
and/or 100 µM 8-CPT-cAMP (cAMP) for 24 h.
For each ALAS/CAT expression vector, results are expressed as relative
CAT activity with respect to the wild type version of non-cotransfected
and non-treated sample, which was set to 100. Bars represent
the mean ± S.E. of three different experiments performed in
duplicate. Student's t test was used to reveal significant
differences. *, p < 0.05, between samples 5, 9, and 13 and sample 1; *, p < 0.05, between sample 7 and sample
3; *, p < 0.05, between sample 13 and sample 9; *,
p < 0.05, between sample 15 and sample 7; *,
p < 0.05, between sample 21 and sample 13; *,
p < 0.05, between sample 22 and sample 15. In all
cases, relative CAT activity of mutants was compared with their
respective wild type version.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PDA was ineffective in
reducing ALAS promoter activity support the hypothesis that PKC
activation mediates TPA inhibitory effect on ALAS gene expression.
354 to
156
bp. Transfection assays with plasmids containing sequences shorter than
156 bp of the ALAS promoter in HepG2 cells abrogate the response to
the influence of TPA. This sequence contains a TRE motif at
261 bp
that differs from the consensus sequence in only 1 nucleotide (42). The
mutation of this TRE-ALAS site abolished the response to TPA in the
p-354ALAS/CAT vector.
is
mediated by phospholipase C and PKC, among other effectors (61).
Moreover, mechanisms involving diacylglycerol and PKC seem to play a
role in the mitogenic effects of various agents that bind to G
protein-coupled receptors and activate cells in early G1,
such as norepinephrine, angiotensin II, and vasopressin (62).
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Fig. 11.
Model of positive and negative cross talk
modulated by the transcriptional coactivator CBP in ALAS promoter.
ALAS basal expression requires the interaction between CREB and the
promoter CRE sites, thus facilitating the incorporation of coactivator
CBP. The formation of this complex would have a stabilizing effect on
the transcription mechanism in basal conditions (25). TPA stimulates
the synthesis of proteins from the AP-1 family, which would occupy TRE
sites located upstream CRE sites. The subsequent activation of Fos/Jun
factors due to post-transductional changes would lead to CBP shift from
the CRE·CREB complex to further TRE·AP-1 complex, thus
destabilizing the transcription mechanism and causing the inhibition of
ALAS gene expression.
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ACKNOWLEDGEMENT |
---|
We thank Laura Gutiérrez for help with manuscript preparation and language advice.
![]() |
FOOTNOTES |
---|
* This work was supported by research grants from the Universidad de Buenos Aires and Consejo Nacional de Investigaciones Científicas y Técnicas.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A Research Fellow of the Consejo Nacional de Investigaciones
Científicas y Técnicas.
§ To whom correspondence should be addressed. Tel.: 54-11-4821-4893; Fax: 54-11-4576-3342; E-mail: ecanepa@qb.fcen.uba.ar.
Published, JBC Papers in Press, November 13, 2002, DOI 10.1074/jbc.M205057200
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ABBREVIATIONS |
---|
The abbreviations used are:
AP-1, activation
protein-1;
4PDA, 4
-phorbol 12,13-diacetate;
ALAS, 5-aminolevulinate synthase;
CAT, chloramphenicol acetyltransferase;
CREB, cAMP-responsive element protein;
CBP, CREB-binding protein;
CRE, cyclic AMP-responsive element;
8-CPT-cAMP, 8-(4-chlorophenylthio)-cAMP;
HA, hemagglutinin;
NHR, nuclear hormone receptors;
PKA and PKC, protein
kinase A and C, respectively;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
TRE, TPA-responsive
element.
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REFERENCES |
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