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INTRODUCTION |
Retinoids, the natural and synthetic vitamin A derivatives, are
known to regulate many biological processes, including growth, differentiation, and development (1-3). They are currently used in the
treatment of epithelial cancer and promyelocytic leukemia and are being
evaluated as preventive and therapeutic agents for a variety of other
human cancers (4). One of the major drawbacks of retinoid therapy has
been the wide range of undesirable side effects. Development of
anticancer-specific retinoids with improved clinical value is largely
dependent on the understanding of the mechanistic basis of the
pleiotropic activities induced by retinoids and their receptors. The
effects of retinoids are mainly mediated by two classes of nuclear
retinoid receptors: the retinoic acid receptors
(RARs)1 (5-9) and the
retinoid X receptors (RXRs) (10-14), and both receptors are members of
the steroid-thyroid hormone receptor superfamily and are encoded by
three distinct genes,
,
, and
(5-14).
All-trans-retinoic acid (RA) acts as a ligand for RARs,
while 9-cis-RA is a ligand for both RARs and RXRs. RARs and
RXRs modulate the expression of their target genes by interacting as
either homodimers or heterodimers with RA response elements (RAREs)
(11, 14-17). A RARE (
RARE) in the RAR
gene promoter mediates
RA-induced RAR
gene expression in many different cell types (18,
19). Up-regulation of the RAR
gene by RA plays a critical role in
amplifying the RA response and is required for RA-induced growth
inhibition and apoptosis in human breast cancer (20) and lung cancer
cells (21).
In addition to the regulation of RARE-containing genes, retinoid
receptors can inhibit the effect of the tumor promoter TPA by
repressing the transcriptional activity of AP-1 (22). Inhibition of
AP-1 activity by retinoid receptors may involve either direct protein-protein interaction between retinoid receptor and components of
AP-1, such as c-Jun (23), or competition for a common coactivator CBP
(24). Recent studies have suggested that different receptor conformational changes may account for gene regulation on RAREs and
inhibition of AP-1 activity and that they can be dissociated as
anti-AP-1-specific retinoids have been described (25-27). Interaction between membrane and nuclear receptor signaling pathways mediated by
RAR/AP-1 interaction may represent an important mechanism underlying the potent antineoplastic effects of retinoids. Because many of the
AP-1 responsive genes, such as collagenases and stromelysins, are
involved in cancer cell proliferation and transformation (28), retinoids that specifically inhibit AP-1 activity may be
therapeutically desirable because they may have reduced side effects
associated with gene activation, but retain their anticancer activity.
This is demonstrated by recent studies showing that retinoids that inhibit AP-1 activity but cannot induce transactivation of
RARE-containing genes were able to inhibit TPA-induced transformation
and the clonal growth of mouse epidermal JB6 cells (29). In addition, a
group of anti-AP-1-specific retinoids inhibited the proliferation of
lung and breast cancer cells but had impaired ability to induce differentiation of F-9 cells (25).
Each subtype of RARs has been implicated in the regulation of cancer
development and the anticancer activities of retinoids. Translocation
of the RAR
gene is responsible for the development of acute
promyelocytic leukemia (30), whereas RAR
may play a role in
mediating growth inhibition and apoptosis by certain retinoids (31).
Recently, evidence is emerging showing that RAR
may play a critical
role in the regulation of cancer cell growth. RAR
is located in
chromosome 3p24, a region that is often deleted or mutated in a variety
of cancer (32), and RAR
was found to be integrated by hepatitis B
virus in human liver cancer (33). It is not expressed in many different
types of cancer cell lines (20, 34-38), and re-expression of RAR
in
RAR
-negative cancer cells restored the ability of RA to induce
growth inhibition and apoptosis (20). Despite these studies, further
investigation is needed to dissect function of each retinoid receptor
in cancer cells. Several approaches have been used often to determine
specific function of each receptor subtype, including loss of function, such as knock-out and antisense technique, and gain-of-function, such
as ectopic expression of a receptor subtype. RAR subtype-selective agonists and antagonists are being developed (39-43), and they have
been widely used to study the function of each receptor. However, the
degree of selectivity and receptor transactivation activity needs to be
improved. In addition, development of more and effective
RAR
-selective retinoids is important to further study RAR
function in cancer. Furthermore, receptor-selective retinoids allow
dissociation of desired and undesired effects of retinoids and are also
believed to be more specific and with less toxicity in cancer
prevention and treatment.
To further study RAR
function in cancer cells, we analyzed a class
of conformational restricted retinoids. Our data demonstrated that
LE135, LE540, and LE550 inhibited all-trans-RA-induced
transcriptional activation of RAR
but not RAR
, RAR
, or RXR
on a number of RAREs, whereas LE511 selectively induced transactivation
activity of RAR
. The RAR
-selective antagonist effect was further
demonstrated by their ability to inhibit
all-trans-RA-induced apoptosis of ZR-75-1 human breast
cancer cells. Interestingly, the antagonists LE135 and LE540 also exert
anti-AP-1 activity. They effectively repressed TPA-induced AP-1
activity in both HeLa and breast cancer cells when RAR
was
expressed. In contrast, LE550 induced AP-1 activity in HeLa but not in
breast cancer cells. Together our results demonstrate that a novel
class of RAR
-selective retinoids with variable biological functions
should represent useful tools for studying RAR
function.
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EXPERIMENTAL PROCEDURES |
Retinoids--
All-trans-RA was obtained from Sigma.
LE135 was prepared as described by Eyrolles et al. (44).
LE511, LE540, and LE550 were prepared as described by (45). Ro 41-5253 was kindly provided by Dr. Michael Klaus (46).
Cell Culture--
Monkey kidney CV-1 and HeLa cells were grown
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
calf serum, and ZR-75-1 breast cancer cells were maintained in RPMI
1640 medium supplemented with 10% fetal calf serum.
Plasmid Constructions--
The receptor expression plasmids
pECE-RAR
, -RAR
, -RAR
, and -RXR
and the construction of the
reporter plasmids
RARE-tk-CAT, TREpal-tk-CAT, CRBPI-RARE-tk-CAT, and
ApoAI-RARE-tk-CAT have been described previously (15, 16, 47, 48).
Transient Transfection Assay--
CV-1 cells were seeded at
5.0 × 105 cells/well in 24-well plates. A modified
calcium phosphate precipitation procedure was used for transient
transfection as described previously (20). 100 ng of reporter plasmid,
100 ng of
-galactosidase expression vector (pCH110, Amersham
Pharmacia Biotech), and various amounts of receptor were mixed with
carrier DNA (pBluescript, Stratagene) to 1,000 ng of total DNA/well.
After 16-24 h, transfected cells were treated with or without
10
7 M all-trans-RA in the absence
or presence of the indicated concentrations of retinoid antagonists.
For anti-AP-1 assay, a reporter construct containing the collagenase
promoter linked with the CAT gene, -73-Col-CAT (23), was used in HeLa
and ZR-75
1 cells. After transfection, cells were grown in a medium
containing 0.5% charcoal-treated fetal calf serum with retinoids
and/or TPA (100 ng/ml). Transfection efficiency was normalized to
-galactosidase activity. The data shown are the means of three
separate experiments.
Apoptosis Assay--
Nuclear morphological change analysis and
DNA fragmentation (TdT) assay were as described previously (20, 21).
For nuclear morphological analysis, ZR-75-1 cells were treated with or
without 10
7 M all-trans-RA in the
absence or presence of 10
6 M LE135, LE540, or
LE550 for 4 days, trypsinized, washed with phosphate-buffered saline,
fixed with 3.7% paraformaldehyde, and stained with 50 µg/ml
4,6-diamidino-2-phenylindole containing 100 µg/ml DNase-free RNase A
to visualize the nuclei. Stained cells were examined by fluorescent
microscopy. For DNA fragmentation (TdT) assay, ZR-75-1 cells were
treated with or without 10
7 M
all-trans-RA in the absence or presence of 10
6
M LE135, LE540, or LE550. After 3 days, cells were
trypsinized, washed with phosphate-buffered saline, fixed in 1%
formaldehyde in phosphate-buffered saline, washed with
phosphate-buffered saline, resuspended in 70% ice-cold ethanol, and
immediately stored at
20 °C overnight. Cells were then labeled
with biotin-16-dUTP by terminal transferase and stained with
avidin-fluorescein isothiocyanate (Roche Molecular Biochemicals). The
labeled cells were analyzed using a FACScater-Plus. Representative
histograms are shown.
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RESULTS |
Transactivation Activity of the Synthetic Retinoids--
Recently,
we designed several retinoid antagonists, LE135, LE511, LE540, and
LE550 (49, 50), based on the structure-activity relationship of
retinoids (Fig. 1). Transcriptional
activation of these retinoids on RAR
, RAR
, RAR
, or RXR
was
determined by transient transfection assay using the reporter construct
TREpal-tk-CAT (15) in CV-1 cells. As shown in Fig.
2, all-trans-RA strongly induced transcriptional activation of each RARs, whereas
9-cis-RA effectively promoted RXR homodimer activity.
However, LE135, LE540, and LE550, at 10
7 M
and 10
6 M, had very little effect on
transcriptional activation of RAR
, RAR
, RAR
, or RXR
. LE135
and LE540 even showed inhibitory effect on basal RAR
activity in a
dose-dependent manner. Interestingly, LE511, a LE135 analog
with bulky acyclic alkyl group, showed a strong induction of RAR
transactivation activity. Activation of RAR
could be observed when
10
7 M LE511 was used, and it is about 65% of
efficiency as compared with all-trans-RA. Together, these
results demonstrate that LE135, LE540, and LE550 are ineffective on
transcriptional activation of RARs and RXR, whereas LE511 is a
RAR
-selective agonist.

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Fig. 2.
Transcriptional activation profiles.
Transcriptional activation activities of retinoids were determined on
RAR , RAR , RAR , or RXR receptors. CV-1 cells were
transiently transfected with 100 ng of TREpal-tk-CAT reporter plasmid
and 100 ng of RAR , RAR , or RAR or 20 ng of RXR receptor
expression plasmid. Transfected cells were grown in the absence or
presence of the indicated concentrations of retinoids, and assayed for
CAT activity after 24 h. 100% activity was the reporter gene
activity measured in the presence of 10 6 M
all-trans-RA for RARs or 10 6 M
9-cis-RA for RXR after subtraction of constitutive
receptor activity.
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Antagonistic Effect of the Synthetic Retinoids on RA-induced RAR
Transcriptional Activation--
LE135 was previously shown to
selectively bind to RAR
(44). The observations that LE135 could not
induce RAR
transactivational activity and that it inhibited basal
RAR
activity (Fig. 2) suggested that binding of RAR
by LE135
might repress its transactivation function. We therefore analyzed the
effect of LE135 on all-trans-RA-induced transcriptional
activation of RAR
on
RARE-tk-CAT reporter (19). For comparison,
its effect on RAR
or RAR
was analyzed. As shown in Fig.
3a, 10
7
M all-trans-RA-induced RAR
activity was
strongly inhibited by LE135 in a concentration-dependent
manner, with more than 70% inhibition when 10
6
M LE135 was used. For comparison, RAR
-selective
antagonist Ro 41-5253 did not show any effect on RAR
activity. When
RAR
was analyzed, LE135 did not exhibit clear inhibitory effect on
all-trans-RA-induced RAR
activity, whereas Ro 41-5253 significantly repressed the reporter transcription. Both LE135 and Ro
41-5253 did not show any influence on all-trans-RA-induced
RAR
activity. Similar results were obtained on another reporter, the
TREpal-tk-CAT (Fig. 3b and data not shown). Because RAR
is likely to function as RAR
/RXR heterodimer in cells, we determined
whether LE135 could act as a RAR
/RXR heterodimer antagonist. As
shown in Fig. 3b, LE135 exhibited a similar degree of
inhibition on both all-trans-RA-induced RAR
and
RAR
/RXR heterodimer activity (Fig. 3b), whereas it had no
effect on 9-cis-RA-induced RXR homodimer activity. Similar results were obtained with LE540 (data not shown). Together, these data
demonstrate that LE135 and LE540 are effective antagonist of RAR
and
RAR
/RXR heterodimer.

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Fig. 3.
Antagonistic effects of retinoids on
RA-induced transcriptional activation of the RARs. a,
effect of LE135 on RAR , RAR , or RAR receptor on RARE.
RAR , RAR , or RAR expression vector (100 ng) was cotransfected
with the RARE-tk-CAT repoter into CV-1 cells. Transfected cells were
treated with 10 7 M all-trans-RA in
the absence or presence of the indicated concentration of LE135 or Ro
41-5253 (10 7 M). b, effect of
LE135 on RAR , RXR , or RAR /RXR heterodimer activity on the
TREpal. RAR (100 ng), RXR (25 ng), or RAR /RXR expression
vectors were cotransfected with the TREpal-tk-CAT reporter into CV-1
cells. Transfected cells were treated with 10 7
M all-trans-RA in the absence or presence of the
indicated concentrations of LE135. c, the antagonist effect
of retinoids is response element independent. CV-1 cells were
cotransfected with 100 ng of RAR expression plasmid and 100 ng of
RARE-tk-CAT, CRBPI-RARE-tk-CAT or ApoAI-RARE-tk-CAT reporter.
Transfected cells were treated with 10 7 M
all-trans-RA in the absence or presence of the
10 6 M LE135, LE540, or LE550.
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To further analyze the antagonist effect of LE135 and its analogs,
reporter constructs containing different RAREs, including
RARE,
CRBPI-RARE and ApoAI-RARE were used. As shown in Fig. 3c, LE135, LE540, and LE550 inhibited all-trans-RA-induced
RAR
transcriptional activation on these different RAREs. Similar
degrees of inhibition (about 60-70%) were observed with the
RARE
and the ApoAI-RARE, whereas a less degree of inhibition (50-60%) was
obtained with the CRBPI-RARE. Interestingly, LE540 showed a more
effective inhibition on all RAREs than its isomer LE550, consistent
with their antagonist effect on HL-60 cell differentiation (45). These
results demonstrate that LE135, LE540, and LE550 could inhibit
transactivation of RAR
and that the antagonistic effect of these
retinoids is response element independent.
Effect of the Retinoid Antagonists on RA-induced Growth Inhibition
and Apoptosis in Human Breast Cancer Cells--
We have previously
demonstrated that expression of RAR
is required for
all-trans-RA-induced apoptosis of human breast cancer cells
(20). We then analyzed whether inhibition of RAR
activity by
RAR
-selective antagonists could repress all-trans-RA
activity in ZR-75-1 human breast cancer cells. ZR-75-1 cells underwent extensive apoptosis when they were treated with all-trans-RA
as revealed by both morphological analysis (DAPI staining) (Fig. 4a) and DNA end-labeling assay
(TdT) (Fig. 4b). However, all-trans-RA-induced apoptosis was strongly prevented when 10
7 M
all-trans-RA was used together with 10
6
M of LE135, LE540, or LE550. Morphological analysis showed
that all-trans-RA-induced apoptosis was reduced from about
40 to 20% by LE135, LE540, or LE550 (Fig. 4a). Similar
results were obtained by TdT assay (Fig. 4b). These results
are in agreement with previous observation made by ectopic expression
of RAR
(20), and suggest that these RAR
-selective antagonists are
useful tools for studying RAR
function.

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Fig. 4.
Effect of the retinoid antagonists on
RA-induced apoptosis in ZR-75-1 human breast cancer cells.
a, morphological analysis of apoptotic breast cancer cells.
ZR-75-1 cells were treated with or without 10 7
M all-trans-RA in the absence or presence of
10 6 M LE135, LE540, or LE550 for 4 days and
nuclear morphology was analyzed by DAPI staining. b, DNA
fragmentation analysis. ZR-75-1 cells were treated with or without
10 7 M all-trans-RA in the absence
or presence of 10 6 M LE135, LE540, or LE550
for 3 days, and DNA fragmentation was determined by the TdT assay.
Representative histograms show relative apoptotic cell numbers.
FL, fluorescence.
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Effect of Retinoid Antagonists on TPA-induced AP-1
Activity--
Recently, it was reported that RA can inhibit AP-1
activity (22) and that receptor conformational change required for AP-1 inhibition is different from that required for receptor transcriptional activation and they can be dissociated (25-27). To determine whether LE135, LE540, and LE550 could also induce RAR conformational changes for inhibiting AP-1 activity, we investigated their effect on TPA-induced AP-1 activity. When a reporter containing the collagenase (23) linked with the CAT gene, -73Col-CAT (23), was transiently transfected into HeLa cells, treatment of the cells with TPA strongly induced the reporter activity, consistent with previous observations. Co-treatment of the cells with either all-trans-RA, LE135,
LE540, or LE550 did not show a clear effect on TPA-induced reporter
activity (Fig. 5a). However,
when RAR
was cotransfected, the TPA-induced reporter transcription
was slightly inhibited, which was further inhibited when cells were
treated with all-trans-RA but not with LE135, LE540, and
LE550. Interestingly, treatment with LE550 slightly enhanced reporter
transcription. When RAR
and RXR
were cotransfected, however,
LE135 and LE540 showed a strong inhibition of the TPA-induced reporter
activity. The enhancing effect of LE550 was also increased. These data
suggest that LE135 and LE540 could induce a conformational change of
RAR
required for inhibiting AP-1 activity only when RAR
is
heterodimerized with RXR
and that LE550 may induce another RAR
conformation that stimulates AP-1 transcriptional activity. To
determine whether these retinoids could inhibit AP-1 activity in breast
cancer cells, the -73Col-CAT was transiently transfected into ZR-75-1
cells (Fig. 5b). Treatment of the cells with TPA led to an
increase of reporter gene transcription for about 7-fold. Both LE135
and LE540 inhibited the TPA-induced activity in a
concentration-dependent manner when RAR
expression
vector was cotransfected. These data demonstrate that LE135 and LE540
could also inhibit AP-1 activity in breast cancer cells. The fact that
LE135 and LE540 could inhibit the TPA-induced reporter transcription
without RXR
cotransfection is consistent with observation that
RXR
is expressed in ZR-75-1 cells (20). Interestingly, we did not
observe any induction of the reporter activity when cells were treated
with LE550, suggesting that the AP-1-inducing effect of LE550 is
cell-type specific.

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Fig. 5.
Effect of retinoids on TPA-induced
collagenase promoter activity. a, effect of LE
compounds on TPA-induced collagenase promoter activity in HeLa cells.
The -73Col-CAT reporter (100 ng) was cotransfected without or with the
indicated retinoid receptor expression vector (100 ng) into HeLa cells.
After transfection, cells were cultured in Dulbecco's modified
Eagle's medium containing 0.5% FCS and treated with either
all-trans-RA or the indicated retinoids and/or TPA (100 ng/ml). 24 h later, the cells were harvested and CAT activities
were determined. The mean of CAT activity in three independent
experiments is shown. b, effect of LE compounds on
TPA-induced collagenase promoter activity in ZR-75-1 cells. The
-73Col-CAT reporter (250 ng) was transfected into ZR-75-1 cells and
assayed for its activity as described in panel a.
Empty bar, no TPA treatment; black bar, with TPA
treatment.
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DISCUSSION |
Previous studies on receptor-ligand interaction have suggested
requirements for being a potent retinoid antagonist: (i) strong binding
to the receptor mediated by the hydrophobic alkylated benzo group of a
retinoid contributes to its binding affinity to RARs and (ii)
inhibition of the conformational changes, such as proper folding of the
helix 12 where the binding site for co-activators (AF2 domain) exists.
This has established structure-activity relationships in both retinoid
agonists and antagonists. We describe here a class of related synthetic
retinoids, LE135, LE540, LE511, and LE550, which either specifically
activate or inhibit RAR
transactivation function. In assaying for
transactivation function of the retinoids on each subtype of RARs and
RXR
, LE511 selectively activated RAR
(Fig. 2), suggesting that it
is a RAR
-selective agonist. In contrast, other LE compounds, LE135,
LE540, and LE550, did not display any activation function on the
retinoid receptors tested (Fig. 2). Instead, LE135 and LE540 showed a
strong inhibition of RAR
basal transactivation activity. When they
were used together with all-trans-RA, LE135 or LE540
effectively inhibited all-trans-RA-induced RXR
/RAR
activity (Fig. 3). LE135 does not bind to RXRs and RAR
in receptor
binding assay (45). It binds with higher affinity to RAR
(Ki = 0.22 µM) than to RAR
(Ki = 1.4 µM) (45). Thus, LE135 and
its related analog LE540 act as RAR
-selective antagonists. These
results suggest that the tetramethyltetrahydronaphthalenyl group as
seen in LE135, LE540, and LE550 may be required for RAR
antagonist
effect, because its replacement with effective mono-tertiary butylphenyl group as seen in LE511 resulted in loss of RAR
antagonism effect (Fig. 3). The smaller hydrophobic
tert-butyl group of LE511 not only decreases the binding
affinity to RARs but also may change the binding occupation in the
ligand-RAR complex with less disturbance of the helix folding. This may
explain the critical agonistic activity of LE511. The existence of
another benzo (or naphtho) group impairs the transcriptional activating
activity due to the different conformational change in the
ligand-receptor complex. From the reported crystal structures of RXR
and RAR
(51, 52), proper folding of the helix 12 in the
ligand-binding domain of RARs is critical for receptor activation. The
benzo/naphtho group fused to the diazepine ring may disturb the proper
folding of the helix to elicit the antagonistic activity. The bulkier
naphtho group of LE540 is expected to be more effective than the benzo group of LE135 and that of LE550 with a different direction seems to
affect weakly the conformation around the helix as it could not inhibit
RAR
basal transactivation activity (Fig. 2).
The pleiotropic effects of retinoids are mainly mediated by RARs and
RXRs. Both types of retinoid receptors are encoded by three distinct
genes,
,
, and
. The fact that these receptors display
distinct patterns of expression during development and differentiation
suggests that each of them may have specific function, which is being
unraveled recently by a variety of technologies, such as homologous
recombination, antisense, and ectopic expression of a receptor of
interest. The complexity of retinoid responses also can be dissected
with the use of both receptor-selective agonist and antagonists,
activating or interfering specifically or preferentially with one given
receptor. We have previously reported (20) that
all-trans-RA-induced apoptosis of human breast cancer cells
requires RAR
expression. This was based on our observation that
stable expression of RAR
in RAR
-negative cells induced apoptosis,
whereas expression of RAR
antisense RNA in RAR
-positive cells
abolished apoptotic effect of all-trans-RA (20). Here, we
used RAR
-selective antagonists to study the involvement of RAR
in
all-trans-RA-induced growth inhibition and apoptosis of ZR-75-1 human breast cancer cells. When RAR
-selective antagonists LE135, LE540, or LE550 was used together with all-trans-RA,
the effect of all-trans-RA on apoptosis in ZR-75-1 cells was
largely reduced (Fig. 4). This result further supports the role of
RAR
in all-trans-RA-induced growth inhibition and
apoptosis of human breast cancer cells. It also demonstrates that LE135
and its analogs are valuable tools for studying RAR
function.
In this study, we also show that RAR
antagonists could act as
effective anti-AP-1 retinoids (Fig. 5). These retinoids, which did not
show any transactivation function on RARs and RXRs, could repress AP-1
activity in the presence of RAR
/RXR heterodimer. Therefore, they are
anti-AP-1 specific retinoids. Interestingly, they could not affect AP-1
activity in the presence of RAR
alone but required RXR for effective
inhibition of AP-1 activity. This suggests that binding of retinoids to
RAR
alone is not sufficient to induce a specific anti-AP-1
conformational change. Interestingly, LE550 could induce AP-1 activity
in HeLa cells but not in ZR-75-1 cells (Fig. 5). The mechanism by which
LE550 induces AP-1 activity is unclear. It is likely that LE550 induces
a different conformational change of RAR
. This is supported by our
observation that LE540 and LE135 inhibited basal RAR
activity,
whereas LE550 could not (Fig. 2). This is interesting since LE540 and
LE550 are regio-isoforms, in which the bulkier naphtho group of both
compounds is arranged in different directions, which may be critical
for AP-1 interaction (Fig. 5). Previous studies have demonstrated that
AP-1 can either inhibit or stimulate nuclear receptor activity,
depending on cell type, promoter, and nuclear receptor (53). Our
observation that LE550 could induce AP-1 activity in HeLa cells
suggests that the retinoid receptor could also stimulate AP-1 activity
in response to appropriate ligand. The fact that induction of AP-1
activity by LE550 was only observed in HeLa cells but not in ZR-75-1
cells implies that inhibition or induction of AP-1 activity by retinoid receptor is also cell-type specific. LE550 may induce RAR
in a
conformation that allows a positive effect on AP-1 transcription, probably through transcriptional mediators specifically expressed in
HeLa cells. Such a compound may be a valuable tool for studying mechanisms underlying AP-1/nuclear receptor interaction and for dissecting complexity of AP-1/nuclear receptor interaction.