Ligand-Activated Retinoic Acid Receptor Inhibits AP-1 Transactivation by Disrupting c-Jun/c-Fos Dimerization
Xiao-Feng Zhou,
Xi-Qiang Shen and
Lirim Shemshedini
Department of Biology University of Toledo Toledo, Ohio
43606
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ABSTRACT
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In the presence of retinoic acid (RA), the
retinoid receptors, retinoic acid receptor (RAR) and retinoid X
receptor (RXR), are able to up-regulate transcription directly by
binding to RA-responsive elements on the promoters of responsive genes.
Liganded RARs and RXRs are also capable of down-regulating
transcription, but, by contrast, this is an indirect effect, mediated
by the interaction of these nuclear receptors not with DNA but the
transcription factor activating protein-1 (AP-1). AP-1 is a dimeric
complex of the protooncoproteins c-Jun and c-Fos and directly regulates
transcription of genes important for cellular growth. Previous in
vitro results have suggested that RARs can block AP-1 DNA
binding. Using a mammalian two-hybrid system, we report here that human
RAR
(hRAR
) can disrupt in a RA-dependent manner the homo- and
heterodimerization properties of c-Jun and c-Fos. This inhibition of
dimerization is cell specific, occurring only in those cells that
exhibit RA-induced repression of AP-1 transcriptional activity.
Furthermore, this mechanism appears to be specific for the RARs, since
another potent inhibitor of AP-1 activity, the glucocorticoid receptor,
does not affect AP-1 dimerization. Our data argue for a novel mechanism
by which RARs can repress AP-1 DNA binding, in which liganded RARs are
able to interfere with c-Jun/c-Jun homodimerization and c-Jun/c-Fos
heterodimerization and, in this way, may prevent the formation of AP-1
complexes capable of DNA binding.
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INTRODUCTION
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Retinoic acid (RA), the most biologically active natural
metabolite of vitamin A (retinol), exerts profound effects on
vertebrate development, cellular differentiation, and homeostasis
(reviewed in Ref. 1). RA is involved in epithelial differentiation (2),
has a central role as a tissue-specific morphogen during embryogenesis
(3), and represses malignant transformation of epithelial cells both
in vitro (4) and in vivo (5). These diverse and
pleiotropic effects of RA are mediated through the binding of RA to a
family of nuclear receptors, which, together with the receptors for
steroid and thyroid hormones and vitamin D, form the nuclear receptor
superfamily (reviewed in Refs. 6, 7, 8, 9, 10). Nuclear receptors comprise the
largest family of transcription factors, which, upon binding of their
cognate ligands, modulate transcription initiated from promoters of
target genes by interacting with specific cis-acting, DNA
response elements.
In contrast to this positive effect of nuclear receptors on
transcription, which requires receptor-DNA interactions, the retinoid
receptors and other nuclear receptors can negatively affect gene
expression without binding to DNA, via their ability to functionally
interact with the transcription factor AP-1 (activating protein-1)
(reviewed in Refs. 11, 12, 13, 14). AP-1 consists of homodimers and
heterodimers of Jun (c-Jun, v-Jun, JunB, and JunD), Fos (c-Fos, v-Fos,
FosB, Fra1, and Fra2), or activating transcription factor (ATF2,
ATF3/LRF1, B-ATF) bZIP (basic region leucine zipper) proteins (15, 16, 17).
The transcription of the c-jun and c-fos genes,
encoding the major components of AP-1, is rapidly induced upon
stimulation of cellular proliferation (37, 38). Like nuclear receptors,
AP-1 activates transcription of target genes by binding to specific
promoter elements, called TREs (TPA-responsive elements) (17, 18). TPA
(12-O-tetra-decanoyl-phorbol-13-acetate) is a tumor promoter
that induces the expression of the c-fos and
c-jun genes (19, 20) and thereby indirectly stimulates the
expression of AP-1 target genes.
Both positive and negative regulatory interactions between nuclear
receptors and c-Jun/c-Fos have been reported (reviewed in Refs.
11, 12, 13, 14). The first results showed an inhibition of glucocorticoid
receptor (GR)-induced transcription by either c-Fos or c-Jun (21, 22, 23, 24).
We (25) and others (26, 27, 28, 29, 30, 31) have shown that this type of interference
is not restricted to the GR, but seems to be a common characteristic of
nuclear receptors, including the receptors for the hormones
progesterone (PR), estrogen (ER), androgen (AR), and thyroid (TRs), and
the RA (RARs, RXRs). Conversely, the activation of the collagenase and
stromelysin genes by AP-1 is repressed in a ligand-dependent manner by
several receptors, including GR (21, 22, 23, 24), PR (25), AR (25), ER (25), TR
(28), and RARs/RXRs (26, 27, 29). By contrast, coexpression of c-Jun,
c-Fos, and ER causes synergistic activation of the ovalbumin gene (32).
GR has been shown to potentiate c-Jun-activated transcription from the
proliferin- regulatory element (33). Similarly, transfected c-Jun
enhances AR-induced transactivation, but it does so independently of
promoter or cell type specificity (25, 34, 35).
In contrast to the interaction between AP-1 and AR, PR, or GR, which is
nonmutual and can be either negative or positive (25, 32, 33), the
interaction between AP-1 and the retinoid receptors is mutual and
solely inhibitory. c-Jun and c-Fos, either individually or together,
have been shown to repress the transcriptional activity of RAR and/or
RXR (27). Conversely, both RAR/RXR heterodimers or homodimers of either
can inhibit AP-1 transactivation of several AP-1-responsive
promoters (27, 29). Indeed, the RAR/RXR antagonism of AP-1 has been
directly implicated in the regulation of collagenase (27, 29) and stromelysin (26), two genes that play key roles in
tumor potential and invasiveness.
While the molecular bases of these diverse regulatory interactions
between nuclear receptors and AP-1 are not known, recent studies
provide several attractive models. Based on the demonstration that
CREB-binding protein (CBP) and the related p300 can act as
transcriptional coactivators for both nuclear receptors (36, 37, 38) and
AP-1 (39, 40), it has been proposed that the nuclear receptor-AP-1
antagonism depends on competition for limiting amounts of these two
coactivator proteins (36). While this model may explain some of the
observations made, it is not able to explain all of the cell, promoter,
and receptor specificity that has been observed in the nuclear
receptor-AP-1 interactions (25). More recently, it has been suggested
that GR, RARs, and TRs can block AP-1 activity by inhibiting the
activity of Jun amino-terminal kinase (JNK) (41), which enhances c-Jun
transcriptional activity by phosphorylating Ser63/73 (42, 43). However,
this model also is unable to account for the diverse nature of the
interactions between nuclear receptors and AP-1. Others have argued,
based on in vitro results, that AP-1 and receptors mutually
inhibit each others DNA-binding ability (21, 24, 33). However, Konig
et al. (44) have provided strong evidence that in
vivo DNA binding, at least for GR and AP-1, is not affected, since
the in vivo footprint of either of these transcriptional
activators in the presence of the other did not change. Thus, the
nuclear receptor-AP-1 antagonism may depend on multiple mechanisms with
the involvement of cell- and receptor-specific factors.
Using a mammalian two-hybrid system, we provide evidence here that RAR,
but not GR, is able to disrupt in vivo c-Jun/c-Fos
dimerization in a ligand-dependent manner. This effect is not only
receptor specific, but also cell specific, paralleling what has been
reported previously with RA-induced inhibition of AP-1 transcriptional
activity (27). Our results suggest that c-Jun/c-Fos dimerization may be
a third target of nuclear receptor-mediated repression of AP-1 that may
be specific for the transrepression activity of RARs and may partially
explain the receptor- and cell-specific nature of this repression.
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RESULTS
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RA-Bound human RAR
(hRAR
) Inhibits AP-1 Activity in a
Cell-Specific Manner
The interaction between nuclear receptors and AP-1 has been shown
previously to be dependent on cell type (Refs. 25, 27 ; reviewed in
Ref. 11). To test for cell specificity in RARs ability to inhibit
AP-1 transcriptional activity, cells were transiently transfected with
expression plasmids for c-Jun and hRAR
and the AP-1-inducible
reporter TRE-tk-CAT (34). In keeping with previously
published data (27), hRAR
inhibited in a ligand- and dose-dependent
manner exogenous c-Jun activity in HeLa cells (Fig. 1A
), but not in Cos cells (Fig. 1B
). The
same difference in activity was observed on endogenous AP-1 activity
(data not shown).

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Figure 1. Ligand-Bound RAR Inhibits AP-1 Activity in HeLa
Cells, but Not Cos Cells
HeLa (A) and Cos (B) cells were transfected with 1 µg of the
TRE-tk-CAT reporter plasmid together with 1 µg of c-Jun and 1, 3, or
5 µg of hRAR expression plasmids. Cells receiving hRAR were
treated with 10-7 M AT-RA as indicated. Note
that CAT activity is represented relative to activity of first
condition, which was set to 1.
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Mammalian Two-Hybrid System Can Be Used to Measure c-Jun/c-Fos
Dimerization in Vivo
The yeast two-hybrid system has been used previously to measure
in vivo protein-protein interactions (45). In the current
study, we have used a similar system to measure c-Jun/c-Fos
heterodimerization and c-Jun/c-Jun homodimerization in cultured
mammalian cells. In our system, two fusion proteins are expressed, one
containing the GAL4 DNA-binding domain (DBD) fused to either
full-length c-Jun or only its bZIP region and the other containing the
VP16 transactivation domain fused to either full-length c-Fos or only
its bZIP region (Fig. 2A
). HeLa cells
were transfected with expression plasmids for these different fusion
proteins, and dimerization was monitored with the GAL4-inducible
reporter 17M-tk-CAT. While GAL-cJun had a weak activity and VP16-cFos
had no measurable activity, these two fusion proteins together resulted
in a 14-fold stimulation in transcription (Fig. 2B
), demonstrating a
strong in vivo interaction between transfected c-Jun and
c-Fos. If either c-Fos or c-Jun or both were absent from the fusion
proteins, no interaction was detected (Fig. 2B
). Since earlier work
(46) has shown that the respective bZIP regions of these two
protooncoproteins are sufficient for heterodimerization, we tested
these same regions in our system. Indeed, GAL-cFos(137216)
and VP16-cJun(237331) exhibited a dimerization capacity that is
comparable to that observed with the full-length proteins (Fig. 2B
). As
further evidence, we measured c-Jun/c-Jun homodimerization by
coexpressing GAL-cJun and VP16-cJun(237331). In agreement with
previous work (47), c-Jun/c-Jun homodimerization was significantly
weaker than c-Jun/c-Fos heterodimerization, even with higher
(
3-fold) amounts of c-Jun fusion proteins expressed (Fig. 2B
).
Identical results have been obtained in Cos cells (data not shown).
These results together indicate that our mammalian two-hybrid system is
faithfully measuring the ability of c-Jun and c-Fos to dimerize
in vivo.

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Figure 2. c-Jun/c-Fos Heterodimerization Is More Efficient
Than Is c-Jun-c-Jun Homodimerization in Vivo
A, A schematic representation showing full-length c-Jun and c-Fos and
truncations of these two proteins, which were used as fusion proteins
with GAL4 or VP16 in the mammalian two-hybrid screen.
Numbers represent amino acid residues. B, HeLa cells
were transfected with 1 µg of the 17 M-tk-CAT reporter
plasmid together with 1 or 3 µg each of expression plasmids for
GAL-cJun or VP16-cJun or 1 µg each of expression plasmids for
VP16-cFos, GAL-cFos(137216), VP16-cJun(237331), GAL-DBD, or
VP16. Note that CAT activity is represented relative to activity of
GAL-cJun, which was set to 1.
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hRAR
Disrupts c-Jun/c-Fos Dimerization in Vivo in a
Ligand-Dependent Manner
To determine whether RAR can affect the in vivo
dimerization between c-Jun and c-Fos, HeLa cells were transfected with
an expression plasmid for hRAR
and treated with 10-7
M all-trans-retinoic acid (AT-RA). hRAR
was
able to severely block dimerization between full-length c-Jun and c-Fos
(Fig. 3A
). This negative effect occurred
only in the presence of AT-RA, since there was no effect by hRAR
in
the absence of AT-RA (Fig. 3A
). A similar ligand-dependent
RAR
-induced inhibition was observed on c-Jun/c-Jun
homodimerization (Fig. 3B
). Since the bZIP regions of c-Jun and c-Fos
are sufficient for dimerization (see Fig. 2B
), we wanted to determine
whether these regions are also sufficient for the negative effect of
hRAR
on AP-1 dimerization. Liganded hRAR
was also able to repress
the dimerization between GAL-cFos(137216) and VP16-cJun (Fig. 4A
) and that between GAL-cFos(137216) and
VP16-cJun(237331) (Fig. 4B
). Note that hRAR
, with or without
AT-RA, did not affect the activity of GAL-VP16 (Fig. 3C
), excluding a
possible hRAR
interference of either GAL(DBD) or VP16 function.
These results clearly show that hRAR
is able to block in a
ligand-dependent manner both hetero- and homodimerization of AP-1, and
thereby blocking in vivo DNA binding, by possibly targeting
the respective bZIP regions of c-Jun and c-Fos.

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Figure 3. Ligand-Bound RAR Disrupts Both AP-1
Homodimerization and Heterodimerization
HeLa cells were transfected with 1 µg of the 17M-tk-CAT reporter
plasmid and 1 µg of hRAR expression plasmid together with 1 µg
each of expression plasmids for GAL-cJun and VP-cFos for
heterodimerization (A), 1 µg each of expression plasmids for GAL-cJun
and VP16-cJun for homodimerization (B), or 1 µg GAL-cJun and 0.5 µg
of GAL-VP16 expression plasmids (C). Cells receiving hRAR were
treated with 10-7 M AT-RA as indicated. Note
that CAT activity is represented relative to activity in the absence of
activator, which was set to 1.
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Figure 4. RAR Inhibition of c-Jun/c-Fos Dimerization Is
Targeted to the bZIP Regions of These Protooncoproteins
HeLa cells were transfected with 1 µg of the 17M-tk-CAT reporter
plasmid and 1 µg of hRAR expression plasmid together with 1 µg
each of expression plasmids for GAL-cFos(137216) and either VP16-cJun
(A) or VP16-cJun(237331) (B). Cells receiving hRAR were treated
with 10-7 M AT-RA as indicated. Note that CAT
activity is represented relative to activity of GAL-cFos(137216),
which was set to 1.
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hRAR
Disruption of c-Jun/c-Fos Dimerization Is Cell Specific
The cell-specific nature of nuclear receptor-induced inhibition of
AP-1 transcriptional activity prompted us to study hRAR
-induced
inhibition of AP-1 dimerization in several different cells. As observed
before (see Fig. 4B
), liganded hRAR
is able to block the
dimerization between the bZIP regions of c-Jun and c-Fos in HeLa cells
(Fig. 5A
); note that the ligand-dependent
activity observed in the absence of transfected hRAR
is likely due
to endogenous receptor. Importantly, however, when the same experiment
was repeated in Cos cells, there was no detectable effect of hRAR
,
either in the absence or presence of AT-RA, on c-Jun/c-Fos dimerization
(Fig. 5B
). This lack of RAR activity in Cos cells on AP-1 dimerization
correlates with the lack of RAR activity on AP-1 transcriptional
activity in these same cells (see Fig. 1B
). To further examine the cell
specificity, we used the yeast two-hybrid system to test the activity
of hRAR
on AP-1 dimerization. In this system, we analyzed the
interaction between c-Jun(237331) fused to the B42 (acid blob)
transcriptional activation domain and the LexA DBD joined to either
c-Fos(137216) for heterodimerization or full-length c-Jun for
homodimerization. B42-cJun(237331) interacted strongly with both
LexA-cFos(137216) and LexA-cJun, but cotransformed hRAR
, either in
the absence or presence of AT-RA, had no significant effect (Fig. 6A
). Accordingly, liganded hRAR
was
unable to repress the transcriptional activity of LexA-cJun (Fig. 6B
).
These results together suggest that the ability of hRAR
to repress
AP-1 dimerization is dependent on cell-specific factors that may not be
found in either Cos or yeast cells.

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Figure 5. RAR Disrupts c-Jun/c-Fos Dimerization in a
Cell-Specific Manner
HeLa (A) and Cos (B) cells were transfected with 1 µg of the
TRE-tk-CAT reporter plasmid together with 1 µg each of
expression plasmids for GAL-cFos(137216), VP16-cJun(237331), or
hRAR . Cells receiving hRAR were treated with 10-7
M AT-RA as indicated. Note that CAT activity is represented
relative to activity of GAL-cFos(137231), which was set to 1.
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hRAR
Disruption of AP-1 in Vitro DNA Binding Is Also
Cell Specific
Our transfection results above show that liganded hRAR
can
block AP-1 dimerization in a cell-specific manner (see Fig. 5
).
Disruption of AP-1 dimerization should lead to abolishment of AP-1
sequence-specific DNA binding. To analyze this, we measured the
in vitro DNA-binding ability of endogenous AP-1 from either
HeLa or Cos cells, which had been transfected with hRAR
, in the
absence or presence of RA. Extracts from both cells exhibited
significant AP-1 DNA-binding activity (Fig. 7
, lanes 1 and 3). This AP-1 acitivity
was confirmed by both addition of antibody, which disrupts c-Jun DNA
binding, (compare lanes 5 and 6) and competition with unlabeled DNA
(compare lanes 7 and 8). Importantly, RA-bound hRAR
was able to
repress AP-1 DNA binding in HeLa cells (compare lanes 1 and 2), but not
Cos cells (compare lanes 3 and 4), paralleling what was observed with
the mammalian two-hybrid system (see Fig. 5
).

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Figure 7. RAR Disrupts AP-1 in Vitro DNA
Binding in a Cell-Specific Manner
HeLa and Cos cells were transfected with 10 µg of hRAR . These
cells were treated with 10-7 M AT-RA as
indicated. Nuclear extracts were tested for AP-1 DNA binding using a
gel mobility shift assay. Antibody disruption analysis was done with
either an anti-c-Jun or antiandrogen receptor (AR) antibody. Note that
the anti-c-Jun antibody is directed against the bZIP region and thus
disrupts c-Jun DNA binding; therefore, no supershift is detectable. DNA
competition was carried out with a 50-fold excess of unlabeled DNA
elements (TRE, ARE). The arrow points to the AP-1-TRE
complex, and FP represents the free probe. The protein amount (in
micrograms) used in each reaction is the following: lane 1, 11.5; lane
2, 11.8; lane 3, 12.8; lane 4, 14.1; lane 5, 11.4; lane 6, 11.5; lane
7, 11.5; lane 8, 11.5.
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hRAR
Disruption of c-Jun/c-Fos Dimerization Is Receptor
Specific
Several nuclear receptors, including that for glucocorticoids
(GR), have been shown to block AP-1 transcriptional activity in a
ligand-dependent manner. Interestingly, however, GR has been shown to
have no effect on in vivo AP-1 DNA binding, as measured by
in vivo footprint analysis (44). Therefore, we tested the
activity of GR in our mammalian dimerization assay in HeLa cells, which
have previously been shown to exhibit dexamethasone (Dex)-induced
repression of AP-1 transcriptional activity (Refs. 20, 21, 22, 23, 24 and data not
shown). Importantly, GR, with or without Dex, had no significant
influence on dimerization between GAL-cFos(137216) and
VP16-cJun(237331) in HeLa cells (Fig. 8
) Thus, inhibition of AP-1 dimerization
in vivo is not only cell specific, but also receptor
specific.

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Figure 8. GR Is Unable to Disrupt c-Fos/c-Jun Dimerization
HeLa cells were transfected with 1 µg of the 17M-tk-CAT reporter
plasmid and 1 µg each of expression plasmids GAL-cFos(137216)
and/or VP16-cJun(237331), and 1, 3, or 5 µg of hGR expression
plasmid. Cells receiving hGR were treated with 10-7
M Dex as indicated. Note that CAT activity is represented
relative to activity of GAL-cFos(137216), which was set to 1.
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DISCUSSION
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Previous in vitro DNA binding studies suggested that
RARs are able to antagonize AP-1 activity by blocking its DNA-binding
ability (21, 23, 24, 27, 28, 29). We show in this paper that this
interference is cell specific, occurring in HeLa but not Cos cells,
and, significantly, provide the first in vivo evidence
supporting this mechanism of transcriptional interference. Moreover,
these data provide a potential basis for some of the cell- and
receptor-specific effects that have been reported (25, 27). Our results
indicate that hRAR
is able to disrupt in a RA-dependent manner the
in vivo dimerization capacity of c-Jun with either itself or
c-Fos, which would preclude the formation of DNA-binding-competent AP-1
complexes. In support of previous work (27), this RA-dependent effect
on dimerization is cell specific, paralleling the previously observed
activity of RARs on AP-1 transcriptional activity.
How might RAR prevent the formation of AP-1 homo- and heterodimers?
AP-1 dimerization is mediated via the conserved bZIP regions found
within c-Jun, c-Fos, and their protein families (reviewed in Ref. 16).
Our transfection studies show that the bZIP regions of c-Jun and c-Fos
are sufficient for the ligand-dependent RAR inhibition of dimerization.
Interestingly, the bZIP regions of c-Jun and c-Fos have been previously
shown to be essential for the transcriptional interactions between
these protooncoproteins and nuclear receptors (23, 25). Thus, the bZIP
regions of c-Jun and c-Fos may provide a common surface through which
nuclear receptors can engage in a protein-protein interaction with
c-Jun and c-Fos.
Several studies (21, 24, 27, 33, 48), using chemical cross-linking and
coprecipitation approaches, have suggested that nuclear receptors can
physically associate with both c-Jun and c-Fos, and thus block their
ability as AP-1 to bind to DNA. However, these protein-protein
interactions appear to be weak or indirect, since we and others (22, 23, 25, 26, 34) have been unable to detect a stable interaction between
RAR and the AP-1 components. In fact, our current data support the
model proposed by Pfahl (11), that additional factors, which appear to
be expressed in a cell-specific manner, are essential for the
antagonism between receptors and AP-1. Our previous results showed that
the direction and magnitude of the nuclear receptor-AP-1 interaction is
dependent on the type of cell, promoter, receptor, and AP-1 component
(25). We now provide evidence that liganded hRAR
is able to inhibit
AP-1 dimerization in cell-specific manner, occurring in HeLa cells, in
which this receptor can inhibit AP-1 transcriptional activity, but not
in Cos cells, where it is has no effect on AP-1 transcriptional
activity. Further, liganded hRAR
does not disrupt c-Jun/c-Fos homo-
and heterodimerization reconstituted in yeast, and, accordingly, as
would be predicted, it is unable to repress LexA-cJun transcriptional
activity in yeast. Thus, it is possible that RARs, and other nuclear
receptors, can directly associate with c-Jun and/or c-Fos, but the
affinity of this direct interaction is not sufficient in
vivo to modulate transcription. Additional factors, expressed in a
tissue-specific manner, may be needed to stabilize the RAR-AP-1
interaction and thus prevent AP-1 dimerization (see Fig. 9
for a scheme). An example of a
nonreceptor, cell-specific factor mediating the interaction of a
nuclear receptor with another transcription factor comes from a study
on the transactivation of the RARß2 promoter. Berkenstam et
al. (49) have found that this promoter is synergistically
activated by RAR and the TATA box-binding protein (TBP) in embryonal
carcinoma (EC) cells but not Cos, and that this synergy can be restored
in Cos cells by ectopically expressing E1A (50). It is also possible
that RAR has a secondary effect, by inducing the expression of a
protein that is directly involved in inhibiting AP-1 dimerization.

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Figure 9. A Model of How Ligand-Bound RAR Can Inhibit AP-1
Transcriptional Activity
In the absence of RA-activated RAR, c-Jun and c-Fos are able to
dimerize to form AP-1, and this dimeric complex can bind to promoter
elements (AP-1 elements) of AP-1-responsive genes. When RA is bound to
RAR and the necessary cell-specific factor(s) (factor x) is present,
the c-Jun/c-Fos heterodimer does not form; thus, the AP-1 element is
vacant and the gene remains transcriptionally silent.
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In the case of nuclear receptor- and AP-1-mediated transcription, there
is compelling evidence that CBP and p300 can act as coactivators for
both pathways (36, 37, 38, 39, 40). It was recently reported that CBP, and perhaps
p300, can also facilitate the homodimerization of AR (51). Since CBP
and p300 can associate with both c-Jun and c-Fos, it is possible that
they are serving a similar role in AP-1 dimerization. However, our
mammalian two-hybrid assay detected no p300 influence on either
c-Jun/c-Fos dimerization or RA-induced disruption of dimerization (data
not shown). It has been suggested that RARs and other nuclear receptors
can compete with AP-1 for limiting amounts of CBP and p300 (36),
thereby resulting in transcriptional efficacy going to one activator at
the expense of the other. Since CBP and p300 are known to interact with
and mediate the activities of several nuclear receptors, including TR
(36, 52), GR (53), ER (54), PR (53, 55), and AR (51), then these same
receptors should be in a competitive and mutually inhibitory
interaction with the AP-1 components c-Jun and c-Fos. Although this is
generally the case, there are several exceptions. First, the AP-1
interaction with AR can be either negative or positive, depending on
the AP-1 component. Indeed, our laboratory has shown that c-Jun
strongly enhances AR-induced transcription and c-Fos can inhibit this
activity, independent of cell or promoter specificity (25, 34, 35). On
GR-inducible promoters, c-Jun generally blocks GR activity except in
several T cell lines (56). On the AP-1-inducible proliferin
promoter, which contains a composite response element, GR and
mineralocorticoid receptor (MR) can act either cooperatively or
antagonistically with AP-1, depending on the identity of receptor (GR,
MR) and AP-1 component (c-Jun/c-Jun or c-Jun/c-Fos) (33). In view of
this complexity, it is likely that additional modes of interaction
occur between nuclear receptors and AP-1. Recently, it was reported
that several nuclear receptors, including RARs, are able to repress JNK
activation of c-Jun, providing a second mechanism of nuclear
receptor-induced inhibition (41). Our data suggest that an additional
mode of action exists, in which RARs can disrupt the ability of c-Jun
to homodimerize with itself and heterodimerize with c-Fos. In contrast
to the other mechanisms, the last one closely parallels the
cell-specific nature of RAR-induced repression of AP-1 transcriptional
activity, appears to be specific for RARs, and may depend on
involvement of cell-specific factors. Thus, it appears that RARs can
use multiple mechanisms by which to blunt the transcriptional activity
of AP-1. It is possible that one of these proposed mechanisms is the
preferred choice in vivo or that these different modes of
actions may cooperate to ensure the appropriate expression of
AP-1-responsive genes.
RARs effect on AP-1 activity has been proposed to be responsible for
the clinical effects of retinoids as antineoplastic, antiinflammatory,
and immunosuppressive agents (26, 27, 29). In this regard, it is
noteworthy that selective retinoids have been reported that allow a
separation of the transactivation and transrepression activities of
RARs (57, 58). Future work will be needed to determine whether some of
these anti-AP-1-selective retinoids act by inducing RAR-mediated
disruption of c-Jun/c-Fos dimerization.
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MATERIALS AND METHODS
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Plasmids
For mammalian expression, hRAR
(59), hGR (59), GAL-VP16 (59),
and c-Jun (25) in pSG5 have been described. GAL-c-Jun and
GAL-c-Fos were also expressed from the mammalian expression plasmid
pGAL0 (60). GAL-cFos(137216) was constructed by PCR amplification of
c-Fos amino acids 137216 using the upstream oligo
5'-GATCGAATTCATGGAAGAGAAACGGAGA-3' and the downstream oligo
5'-GATCGGATCCTCACATCTCCTCTGGGAA-3' and inserting into pGAL0. VP16-cJun
was constructed by inserting full-length c-Jun into the
BamHI/BglII sites of VP16/pTL1, pTL1 (34)
containing the activation domain of VP16. VP16-cJun(237331) was
constructed by PCR amplification of c-Jun amino acids 237331 using
the upstream oligo 5'-GATCGAATTCCTCGAGATGGGCGAGACACCGCCC-3' and
the downstream oligo 5'-GATCGGATCCTCAAAATGTTTGCAA-3' and inserting into
VP16/pTL1.
For yeast expression, the expression plasmids pEG202 (45), pJG45
(45), and pYE10 (61) were used. LexA-cFos(NOREF>137231) was constructed by
digestion of cFos(137231) from pGAL0 and insertion into pEG202.
LexA-cJun was constructed by digestion of c-Jun from pTL1 with
BglII, extension with klenow, and cutting with
BamHI. This fragment was inserted into pEG202.
B42-cJun(237331) was constructed by inserting the PCR fragment
encoding c-Jun amino acids 237331 into pJG45. hRAR
was expressed
from the plasmid pYE10 (61).
For mammalian cells, the reporter plasmids have the gene for
chloramphenicol acetyl transferase (CAT) driven by the RA-inducible
RARE-tk, AP-1-inducible TRE-tk, or GAL4-inducible 17M-tk promoters
(34). Transfection efficiency was standardized by measuring the
ß-galactosidase (ß-gal) activity, originating from the
cotransfected plasmid pCH110 or CMV-LacZ (25). For yeast cells, the
reporter was pSH1834 (45), which has the LacZ gene under the control
of a LexA-inducible promoter.
Cell Transfections and CAT Assays
HeLa and Cos cells were grown and transfected as described
previously (34). hRAR
and GR were activated by the addition of
10-7 M of either AT-RA and Dex, respectively.
CAT assays were performed and standardized according to the measured
ß-gal activity as previously described (25). For all transfections,
we used different amounts of expression plasmid, 1 µg of reporter
plasmid (RARE-tk-CAT, 17 M-tk-CAT, or TRE-tk-CAT), 2 µg
of pCH110 for Cos cells, and 0.5 µg CMV-LacZ for HeLa cells, and
enough carrier DNA (Bluescript) to bring the final plasmid amount to 9
µg per dish. CAT assay results were quantified by densitometric
scanning of autoradiograms of at least three repeats for each
transfection, and each value represents the average of three to four
repetitions plus standard deviation.
Gel Mobility Shift Assay
HeLa and Cos cells were transfected with 10 µg of hRAR
and
2 µg of pCH110. Cells receiving ligand were treated with 100
nM RA 24 h before harvesting. Cells were harvested in
ice-cold PBS and spun at 5000 rpm for 5 min. Ten percent of the cells
were used to perform a ß-gal assay for quantification of transfection
efficiency. The remainder of the cells were resuspended in buffer I (10
mM Tris-HCl, pH 7.5; 10 mM NaCl; 5
mM MgCl2) and incubated at 4 C for 5 min.
Sucrose (0.3 M) was then added and cells were lysed with a
dounce homogenizer. Nuclei were pelleted by centrifuging lysed cells at
2500 rpm (600 x g) for 10 min. The nuclear pellet was
washed once with buffer II (buffer I containing 0.3 M
sucrose). Then, the nuclear pellet was resuspended in buffer III (50
mM Tris-HCl, pH 8; 150 mM NaCl; 5
mM EDTA; 0.1% Nonidet P-40) with protease inhibitors and
incubated with shaking at 4 C for 30 min. The lysed nuclei were
centrifuged at 15,000 rpm for 15 min, and the supernatant, constituting
the nuclear extract, was saved. The amount of extract used was
standardized according to ß-gal activity.
Gel mobility shift assays were performed with nuclear extracts
containing the same amount of ß-gal activity. These reactions were
performed in a final volume of 20 µl in DNA-binding buffer (10
mM Tris, pH 8; 0.1 mM EDTA; 4 mM
dithiothreitol) which also contained 1 µg of poly(dI-dC), 100
mM KCl, and 150,000 cpm of 32P-labeled probe
(5'-TCGAGTTGCATGAGTCAGACATCGATTGCA-3'). After the addition
of x ß-gal units of nuclear extract, the reactions were gently
vortexed and incubated for 15 min at 25 C. The samples were run on a
6% polyacrylamide gel for 1.5 h at room temperature, after which
the gel was dried and exposed to autoradiography. In some reactions, 1
µl of either anti-c-Jun (sc-44, Santa Cruz Biotechnology, Santa Cruz,
CA) or anti-hAR (sc-815, Santa Cruz Biotechnology) were added before
addition of probe. Other reactions received a 50-fold excess of either
unlabeled AP-1 element (given above) or androgen-response element (ARE)
(5'-GATCCAAAGTCAGAACACAGTGTTCT-GATCAAAGA-3').
Yeast Two-Hybrid System
Yeast two-hybrid analysis and LexA-cJun activity were measured
by quantifying ß-gal activity using
o-nitrophenyl-ß-D-galactoside as a
substrate as described (62). AT-RA was added to a final concentration
of 10-6 M.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Roger Brent, Barak Cohen, and Lauren Ha
for providing the materials for the yeast two-hybrid system, Gordon
Tomaselli for GAL-cFos and GAL-cJun, Pierre Chambon and Hinrich
Gronemeyer for the hRAR
plasmid, Athanasios Bubulya for helpful
discussions, Yun Zhou for technical support, and Scott Leisner for
critical reading of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. L. Shemshedini, Department of Biology, University of Toledo, Toledo, Ohio 43606. Email: lshemsh@uoft02.utoledo.edu.
This work was supported in part by American Heart Association Grant
NW-9516-YI and NIH Grant DK-51274 to L.S.
Received for publication July 9, 1998.
Revision received November 2, 1998.
Accepted for publication November 4, 1998.
 |
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