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(Received for publication, September 25, 1995; and in revised form, February 20, 1996) From the
Retinoids regulate a broad range of biological processes through
two subfamilies of nuclear retinoid receptors, the retinoic acid
receptors (RARs) and the retinoid X receptors (RXRs). Recently, we
reported a novel type of retinoic acid antagonist (SR11335) and showed
that this compound can inhibit retinoic acid (RA)-induced activation of
a human immunodeficiency virus type 1 (HIV-1) promoter construct that
contains a special RA response element (RARE). We have now further
characterized the antagonism mediated by SR11335 and of newly
synthesized structurally related compounds. Two compounds, SR11330 and
SR11334, which are poor transactivators, also showed antagonist
activities, inhibiting all-trans-RA (tRA) and
9-cis-RA. The retinoids inhibited transcriptional activation
of RAR/RXR heterodimers effectively,while inhibition of RXR homodimers
was less efficient. Inhibition was observed on several RAREs, including
the TREpal,
Natural and synthetic vitamin A derivatives (retinoids) regulate
a large variety of essential biological functions including cellular
growth and differentiation (for reviews, see (1, 2, 3) ). The pleiotropic effects of
retinoids are mediated by specific nuclear receptors that belong to the
steroid/thyroid hormone receptor superfamily, a class of regulatory
proteins that can function as ligand-responsive transcription
factors(4) . The retinoid receptors so far identified fall into
two groups: the retinoic acid receptors (RAR Similar to steroid receptors, the retinoid receptors
interact as dimers with specific DNA sequences, the retinoic acid
response elements (RAREs), which are usually found in the promoter
region of responsive genes. However, in contrast to steroid receptors
that bind as homodimers to palindromic response elements, the RARs bind
effectively as RAR/RXR heterodimers to a structurally diverse set of
RAREs that contain a minimum of 2 hexanucleotide half-sites with the
consensus sequence
(5`-(A/G)GGTCA-3`)(5, 6, 7, 8) . The
RXRs can also form heterodimers with a number of other hormone
receptors such as thyroid hormone receptors, vitamin D Recently, retinoids
have been shown to enhance replication of several viruses such as the
human immunodeficiency virus type 1 (HIV-1) and human
cytomegalovirus(12, 16, 17) . The retinoid
response of the viruses appears to be controlled through elements in
the long terminal repeat region of the
viruses(12, 13) . Indeed, RAREs have been identified
in the long terminal repeat of HIV-1(18, 19) ,
cytomegalovirus(15) , and human hepatitis B virus(14) .
In the case of HIV-1, a distinct RARE that contains two consensus
half-sites arranged as a palindrome was located at nucleotides
-348 to -328 of the long terminal
repeat(18, 19) . Both RXR In general, the search for retinoid antagonists has met
with limited success, and little is known about common structural
features required for retinoid antagonists and their mechanisms of
action. Recently, we reported that a novel synthetic retinoid, SR11335,
repressed the retinoid-induced transcriptional activation of the
HIV-1-RARE(18) . These investigations were expanded to
characterize further the retinoid antagonist activity of SR11335 and
structurally related compounds as well as their mechanism of action.
Here, we report that the retinoid antagonists, SR11335 and SR11330, can
repress a broad spectrum of RAR/RXR heterodimer activities on a variety
of RAREs and inhibit tRA-induced differentiation of HL-60 cells.
Additionally, we studied the effects of the antagonists on the RAR/RXR
heterodimer binding to RAREs and on ligand-induced conformational
changes of retinoid receptors by limited proteolytic digestion
experiments.
Figure 2:
Transcriptional activation profiles.
Transcriptional activation activities of this series of retinoids was
determined with ER-RAR or ER-RXR hybrid receptors(21) . CV-1
cells were transiently transfected with 100 ng of ERE-tk-CAT reporter
plasmid and 50 ng of each hybrid receptor expression plasmid, i.e. ER-RAR
Figure 3:
Antagonistic effects of the synthetic
retinoids on tRA-induced activation of the HIV-1-RARE. CV-1 cells were
transiently transfected with 100 ng of HIV-1-RARE-tk-CAT (variant A)
reporter together with RAR
Figure 4:
Inhibition of retinoid receptor subtypes.
The TREpal-tk-CAT reporter was used to analyze receptor subtype
selective inhibition. A, RAR
Figure 5:
Antagonism is response element
independent. CV-1 cells were cotransfected with 5 ng of RAR
Figure 1:
Chemical structures of the
synthetic retinoids analyzed in the present
study.
The currently identified RAREs show considerable structural
differences (27, 28, 30, 41) . To
analyze whether SR11335 could differentially affect different RAREs, we
compared its antagonistic effects on the CRBPI-RARE, the apoAI-RARE,
both DR-2 type elements, and the
Figure 6:
Limited proteolytic digestions of retinoid
receptors in the presence of retinoid agonist and antagonist. In
vitro translated [
Figure 7:
Effects of the retinoid antagonists on
retinoid-induced differentiation (growth inhibition) of the human
promyelocytic leukemia cell line HL-60. HL-60 cells were grown in the
presence of 10
In the present study, we analyzed a related group of
retinoids for their antagonistic effects on retinoid-induced
transcriptional activation of nuclear retinoid receptors. Several of
these retinoids showed strong inhibition of RA-induced activation by
various combinations of RAR/RXR heterodimers, but were less effective
with RXR It has recently been shown that RAR/RXR
heterodimers interact with most RAREs with a defined polarity, such
that RAR usually interacts with the 3` half-site of the response
element(41) . In this configuration, RAR can control the
transcriptional activation of the heterodimer, preventing activation by
RXR-selective ligands(43) . By inhibiting the activation of
RARs, retinoid antagonists may also be able to inhibit activation of
the RXR component of the heterodimer. Such a mechanism could allow
particularly efficient antagonistic effects by RAR selective retinoid
antagonists. The potential value for limiting responses to retinoids by
RAR antagonists of this type is demonstrated by their ability to
antagonize effectively tRA-induced growth inhibition/differentiation of
HL-60 cells. Kaneko et al. (39) reported two
antagonists that inhibited tRA-induced differentiation of HL-60 cells.
These compounds have the basic ring structure of the RAR Our analyses indicate that the
antagonists identified here interact with the same binding pocket as
the agonists by blocking their access to the ligand binding domain.
Interaction of the antagonist with the ligand binding domain leads to
the same or a similar protease-resistant core as observed in the
presence of agonist. However, it is clear that the agonist must induce
an additional structural change allowing transcriptional activation not
induced (or inhibited) by the antagonist. Further studies are needed to
better understand the exact mechanism by which antagonists function at
the receptor level. The practical importance of retinoid antagonists
has recently become apparent by the observation that several viruses
are induced to replicate in the presence of retinoid agonists. Whether
antagonists can be designed that inhibit replication of viruses without
causing a general block of the retinoid agonist activity remains to be
explored.
Volume 271,
Number 20,
Issue of May 17, 1996 pp. 11897-11903
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
RARE, apoAI-RARE,and CRBPI-RARE. In addition, the
antagonists inhibited tRA-induced differentiation of HL-60 cells. The
antagonist did not interfere with DNA binding of the receptors. In
limited proteolytic digestion assays, SR11335 induced resistance of the
receptors to proteolysis, but the pattern of the degradation was not
altered from that induced by tRA, suggesting that these antagonists
induce their biological effects by competing with agonists for binding
to RARs, thereby preventing the induction of conformational changes of
the receptors necessary for transcriptional activation.
, -, and -
) (
)and the retinoid X receptors (RXR, -, and
-). The receptors contain a number of distinct subdomains, a
highly conserved cystine-rich domain necessary for sequence-specific
DNA binding and a hydrophobic ligand-binding domain (reviewed in (4) ).
receptors, and peroxisome proliferator-activated receptor (for
reviews, see (4) and (9) ). In addition, RXRs can also
act as homodimers in the presence of 9-cis-RA or RXR-selective
synthetic retinoids(10, 11) . homodimer and
RAR/RXR heterodimer are efficient activators of this
RARE(18) . Therefore, retinoid antagonists which can counteract
retinoid activity, could provide a means of repressing viral
replication by inhibiting the retinoid-dependent transcription and
replication of the viruses in vivo. Besides providing
important tools for deciphering mechanisms of retinoid action, the
development of retinoid antagonists could therefore also be of
importance because of their potential in the treatment of viral
infections.
Retinoids
All-trans-RA was purchased
from Sigma. 9-cis-RA was prepared by the method of Sakashita et al.(20) . The intermediate ethyl
4-(4-formyl-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoate
(FTTAB) was prepared by a Pd(0)-catalyzed Suzuki biaryl coupling (78%)
of
2-bromo-5,6,7,8-tetrahydro-4-hydroxymethyl-5,5,8,8-tetramethylanthracene
and 4-carbethoxyphenylboronic acid, followed by oxidation with
pyridinium chlorochromate, and then was used to synthesize SR11330,
SR11333, SR11334, SR11335, and SR11337. Treatment of FTTAB with
CFI and zinc in HCONMe
, followed by hydrolysis
(KOH, aqueous EtOH; diluted HCl), afforded SR11330. Oxidation of
SR11330 with CrO in aqueous
HSO
/acetone gave SR11334. Treatment of SR11334
with hydroxylamine hydrochloride and sodium acetate in EtOH, followed
by diluted HCl, produced SR11333. Reaction of SR11330 with NaH and MeI
in HCONMe, followed by hydrolysis (KOH, aqueous EtOH;
diluted HCl), afforded SR11335. An alternate synthesis of SR11335 has
also been reported(18) .
4-[5,6,7,8-Tetrahydro-4-amino-5,5,8,8-tetramethyl-2-anthracenyl]
benzoic acid (45) was acylated by treatment with
(CFCO)O in pyridine, followed by hydrolysis
(aqueous HCl), to give SR11336. FTTAB was oxidized under
Baeyer-Villiger conditions
(3-Cl-C
H-COH in
CHCl), followed by base hydrolysis of the
formate ester group (KOH, aqueous EtOH:diluted HCl) and acid hydrolysis
of the ethyl ester with HSO in aqueous acetic
acid to give SR11337. All retinoids were fully characterized
spectroscopically (IR, H NMR) and gave suitable elemental
analyses. Retinoid stock solutions (10 mM) were made in a
dimethyl sulfoxide:ethanol (1:1) mixture and were maintained at
-20 °C.Plasmids
The human and mouse retinoid receptor
expression plasmids pECE-hRAR, -hRAR, -hRAR, -hRXR,
-mRXR, and -mRXR and human estrogen receptor, pECE-hER, have
been described
previously(10, 22, 23, 24) . The
expression plasmids for chimeric receptors containing the DNA binding
domain of estrogen receptor and the ligand binding domain of the
retinoid receptor, i.e. pECE-ER-RAR, -ER-RAR,
-ER-RAR, and -ER-RXR, have been described
elsewhere(21) . Construction of the reporter plasmids,
HIV-1-RARE-tk-CAT, TREpal-tk-CAT, CRBPI-tk-CAT, RARE-tk-CAT,
apoAI-tk-CAT, and ERE-tk-CAT and have been described previously as well (18, 24, 25, 26, 27) .Transient Transfection
CV-1 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum. Cells were seeded at 5.0 
10
cells
per well in 24-well plates 16 to 24 h prior to transfection. A modified
calcium phosphate precipitation procedure was used for transient
transfection as described
previously(10, 11, 29) . Briefly, 100 ng of
reporter plasmid, 200 ng of -galactosidase expression vector
(pCH110, Pharmacia Biotech Inc.), and variable amounts of receptor
expression vector were mixed with carrier DNA (pBluescript, Stratagene)
to 1000 ng of total DNA per well. After the cells were grown in the
presence of retinoids for 24 h, chloramphenicol acetyltransferase (CAT)
and -galactosidase activity were assayed as described
previously(29) . CAT activity was normalized for transfection
efficiency by the corresponding -galactosidase activity. Depending
on the receptor and the reporter gene used, the fold inductions
observed in the experiments shown in Fig. 2Fig. 3Fig. 4Fig. 5varied between 3-
and 50-fold. To allow for better comparisons of the various
experiments, data are expressed in percent activation.
(
), ER-RAR (
), ER-RAR (
),
and ER-RXR (
). Transfected cells were grown in the presence
of the indicated concentration of retinoids and assayed for CAT
activity after 24 h as described under ``Materials and
Methods.'' 100% activity was the reporter gene activity measured
in the presence of 10
M tRA only
(ER-RAR, -, and -) or 10M 9-cis-RA (ER-RXR) after subtraction of constitutive
receptor activity. The data points represent means of two experiments
carried out in duplicate.
and RXR expression plasmids.
Transfected cells were treated with 10
M tRA in the presence of the indicated concentrations of SR11333
(), SR11334 (), SR11335 (), SR11336 (), SR11330
(
), and SR11337 (
). The activation obtained in the
presence of tRA only represents 100%. The data shown represent the
means of two experiments carried out in
duplicate.
and RXR expression
vectors were cotransfected with the reporter alone or in combination. B, RAR or RAR were cotransfected with RXR. As a
control, an ERE-tk-CAT vector was cotransfected with an ER expression
vector. C, RAR and RAR were also analyzed in the
presence of RXR and RXR. Transfected cells were treated with
10M tRA in the presence or absence of the
indicated concentration of SR11330 (), SR11335 (), or
Tamoxifen (). The activation obtained in the presence of
10M tRA alone represents 100%. When only
RXR was transfected, 10M 9-cis-RA was used, and 10M estradiol was used when ER was transfected. Results of a
representative experiment with duplicate measurements are
shown.
and
RXR expression plasmids and 100 ng of CRBPI-RARE-tk-CAT (),
apoA1-RARE-tk-CAT (), or RARE-tk-CAT () reporter.
Transfected cells were treated with 10M tRA in the presence or absence of the indicated concentrations of
SR11335. 100% activation represents the activity measured in the
presence of 10M tRA without
antagonist.
In Vitro Transcription and Translation
cDNAs of
RARs and RXR cloned in pBluescript were transcribed by using
either T7 or T3 RNA polymerase, and the transcripts were translated
with the rabbit reticulocyte lysate system (Promega) as described
previously(10, 11, 29) . The relative amounts
of translated proteins were estimated by separating the
[
S]methionine-labeled proteins on a
SDS-polyacrylamide gel, quantitating the amount of incorporated
radioactivity, and normalizing it relative to the content of methionine
residues in each protein. For limited proteolytic digestion
experiments, [S]methionine-labeled receptor
proteins were used.Gel Retardation Assays
Gel retardation assays were
performed essentially as described
previously(10, 11) . Briefly, oligonucleotides
corresponding to the sequence of the TREpal (5`-TGAGGTCATGAC-3`) was
synthesized with BglII adapter sequences at both ends, 
P-labeled, and used as probe. In vitro translated
receptor proteins were preincubated with vehicle (dimethyl
sulfoxide:ethanol = 1:1) and/or retinoids for 20 min at room
temperature in the dark. Final concentration of vehicle was 1%, and no
effect of vehicle on binding was observed at this concentration. This
mixture was incubated with the probe in a 20-µl reaction mixture
containing 10 mM HEPES, pH 7.9, 50 mM KCl, 1 mM dithiothreitol, 2.5 mM MgCl, 10% glycerol,
and 1 µg of poly(dI-dC) at 25 °C for 20 min. The reaction
mixtures were then loaded on a 5% nondenaturing polyacrylamide gel
containing 0.5 TBE (44.5 mM Tris borate, 44.5 mM boric acid, and 1 mM EDTA).Limited Proteolytic Digestion Analysis
Protease
digestion was carried out essentially as described
previously(30, 31, 32) .
[S]Methionine-labeled retinoid receptor was
treated with tRA, 9-cis-RA, or retinoid antagonist for 30 min
at room temperature. Retinoids were dissolved in a vehicle of dimethyl
sulfoxide:ethanol = 1:1 and final concentration of the vehicle
was 1%. To 8 µl of retinoid-treated receptor proteins, 2 µl of
proteases in 10 mM Tris buffer (pH 7.6) was added and
incubated for an additional 15 min at room temperature. The reaction
was stopped by addition of 30 µl of SDS-polyacrylamide gel
electrophoresis sample buffer, and then SDS-polyacrylamide gel
electrophoresis was carried out with 12% (w/v) polyacrylamide gels. The
gels were treated with a 25% isopropyl alcohol, 10% acetic acid aqueous
solution for 1 h and followed by Amplify (Amersham) for 20 min. After
drying with a vacuum drier for 50 min, the gels were autoradiographed.HL-60 Cultures and Differentiation
Measurements
The human promyelocytic leukemic cell line HL-60
cells were maintained in RPMI 1640 medium supplemented with 10%
charcoal-treated fetal calf serum. 1 10 cells
suspended in 100 µl of RPMI 1640 medium supplemented with 10%
charcoal-treated fetal calf serum were plated in each well of 96-well
plates and were grown in the presence or absence of variable
concentrations of retinoids. After 3 days of incubation, 50 µl of
fresh medium containing assigned amounts of retinoids were added, and
incubation was continued for another 3 days. At the end of retinoid
treatment, the cell proliferation was measured by the capacity of the
cells to reduce nitro blue tetrazolium (Promega) according to the
manufacturer's instruction. The inhibition of cell proliferation
by retinoids was used as a measurement of differentiation.
Retinoids with Low Transcriptional Activation
Activities Show Antagonistic Effects
We conducted
structure-activity studies on retinoids having low transcriptional
activation activity, to identify the structural features required for
retinoid antagonists.
4-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoic acid
(TTAB, SR3691), a retinoid having potent transcriptional activation
activities for all three RARs (21) with EC
values
of 10 nM and comparable binding affinities (45) was
selected as the starting structural template for these studies. The
hydrogen at the 4-position of the tetrahydroanthracenyl ring of TTAB
was then replaced by a variety of groups of different size,
electronegativity, and polarity, including the following:
2,2,2-trifluoro-1-hydroxyethyl (SR11330),
2,2,2-trifluoro-1-(hydroxyimino)ethyl (SR11333), 2,2,2-trifluoracetyl
(SR11334), 2,2,2-trifluoro-1-methoxyethyl (SR11335),
2,2,2-trifluoroacetamido (SR11336), and hydroxyl groups (SR11337) (Fig. 1). The transcriptional activities of these analogs were
determined by using ER/RAR hybrid receptors with the reporter construct
ERE-tk-CAT(21) . In this series, SR11330 and SR11335 showed the
lowest transcriptional activation activities, which were less than 10%
of those of tRA at 10
M, whereas SR11334
showed moderate activity and the other analogs had significant
activity, particularly with RAR and RAR (Fig. 2). The
antagonist activities of these analogs on the RAR/RXR
heterodimer were tested first on the HIV-1-RARE in competition
experiments with tRA. Of the substituents examined in this series, the
2,2,2-trifluoro-1-methoxyethyl group conferred the most potent
antagonist activity, followed by analogs having the
2,2,2-trifluoro-1-hydroxyethyl (SR11330) and trifluoracetyl (SR11334)
substituents (Fig. 3). Induction observed in the presence of
9-cis-RA was inhibited by the compounds to a similar extent
(data not shown).
Antagonist Activity by RAR Inhibition
To
characterize the mechanism of inhibition of the retinoid response by
the antagonists, we studied whether the inhibition was restricted to
certain combinations of retinoid receptors. SR11335 inhibited the
RAR/RXR heterodimer activity up to 80% (at 10M). Inhibition was also obtained by SR11330 with less
efficiency. When only RAR was cotransfected, the inhibition
observed was close to that of the RAR/RXR heterodimer
inhibition. In contrast, only about 30% inhibition was observed for the
RXR homodimer activity (Fig. 4A). This suggested
that the inhibition of the tRA-induced RAR/RXR heterodimer
activity is mediated mainly through inhibition of the RAR.
Heterodimers with other RAR subtypes, i.e. RAR/RXR
and RAR/RXR, were also repressed by the antagonists, but
inhibition of the RAR/RXR heterodimer was less effective (Fig. 4B). To document that the inhibition observed
with the antagonist was specific for the retinoid receptors, we also
investigated the effect of the antagonists on estrogen receptor (ER).
No significant effect on ER activation by estradiol was observed,
whereas tamoxifen (10M) had a profound
effect (Fig. 4B). GR activity was not affected either
(data not shown). RARs also form heterodimers with the two other RXR
subtypes, i.e. RXR and
RXR(7, 8, 34) . These heterodimers were
similarly inhibited by the antagonists (Fig. 4C). RARE, a DR-5. As shown in Fig. 5, the inhibition obtained was almost identical for those
three RAREs and was also very similar to the inhibition seen with the
HIV-1 RARE.Antagonist Effects on DNA Binding
To obtain
insight into the possible mechanisms by which these antagonists
function, we investigated whether SR11335 and SR11330 could inhibit DNA
binding of the retinoid receptors, possibly by inducing a receptor
conformation that prevents DNA interaction. DNA binding was analyzed by
gel retardation assay with in vitro translated receptors and
radiolabeled response elements. SR11330 and SR11335 did not induce any
noticeable changes in the binding of the receptors to the TREpal in the
presence or absence (data not shown) of tRA. The same results were
obtained with the RARE and the HIV-1-RARE (data not shown).Antagonist and Agonist Induce Overlapping Conformational
Changes in the Receptors at Different Concentrations
Recently,
it has been reported that hormone binding to nuclear receptor induces
conformational changes in the receptor, rendering the ligand binding
domain resistant to protease digestion(35, 36) . In
addition, anti-hormones can induce conformational changes, distinct
from those induced by the hormone(31, 32) . We
investigated whether the antagonists induced a distinct conformational
change in the RARs. Limited proteolytic digestions with in vitro translated, [S]methionine-labeled receptors
were employed for those analyses. Labeled receptor was incubated alone
or with tRA or SR11335, then digested for 15 min with a variety of
proteases. Under these conditions, RAR alone was almost completely
digested into fragments smaller than 14 kDa by trypsin. The receptor
was only partially digested in the presence of tRA, resulting in the
accumulation of a 30-kDa resistant fragment (Fig. 6A),
as has been observed by others(31) . The retinoid antagonist,
SR11335, generated the same size of trypsin digestion-resistant
fragment. However, the concentration of SR11335 required to induce the
same degree of resistance was about 200-fold higher than that of tRA.
Similar results were obtained with other proteases such as elastase and
chymotrypsin. tRA also protected RAR and RAR from trypsin
digestion and generated 33-kDa and 35-kDa fragments, respectively. In
the presence of SR11335, a higher concentration (2
10M) was required to obtain the same
degree of protection as seen with tRA (Fig. 6B). When
only 10- or 100-fold higher concentrations of SR11335 were used (2
10M, 2 10
M), little or no protection was seen (not shown). The
observation that only relatively high concentrations of the antagonist
were able to induce protected fragments is consistent with the
apparently low affinity of these compounds for the RARs (the possible
presence of retinoid agonist in the protein extracts could have only
enhanced the activities of the antagonists in these assays). RXR
also generated a 19-kDa fragment that was resistant to chymotrypsin
digestion in the presence of 9-cis-RA. However, SR11335 did
not protect RXR from the chymotrypsin digestion (Fig. 6C). These results are consistent with the weak
effect of this retinoid in antagonizing activation by RXR
homodimers.
S]methionine-labeled
RAR (A), RAR (B), or RXR (C)
was preincubated with vehicle alone or together with 10M tRA or 2 10M SR11335. The proteases used in the experiments were trypsin (A and B) and chymotrypsin (C). The migration of
molecular mass markers is indicated. The protein fragments resistant to
protease digestion in the presence of the retinoids are indicated by arrows.
Antagonists Inhibit RA-induced Differentiation in HL-60
Cells
To evaluate the antagonistic effects of SR11335 in
vivo, the human promyelocytic leukemia cell line, HL-60, was
chosen since retinoid-induced differentiation has been well studied and
characterized in these cells. HL-60 cells have been known to
differentiate into granulocytes upon treatment with retinoids, and the
retinoid effects are assumed to be mediated by RAR or together
with RAR since these receptors are expressed in this cell
line(37, 38) . HL-60 cells were incubated with the
antagonists in the presence of tRA for 6 days, before measuring
differentiation as a function of cell proliferation inhibition. As
shown in Fig. 8, tRA induced differentiation in a dose-dependent
manner. A concentration as low as 2.5 10M SR11335 effectively reversed the growth inhibition (or
differentiation) induced by 10
M and
10M tRA. In the presence of
10M tRA, the efficiency of repression of
differentiation was less. The repression of differentiation was also
obtained using two other retinoid antagonists, SR11330 and SR11334,
although the effects were weaker (Fig. 7), demonstrating a good
correlation between in vitro and in vivo results.
M tRA () or
10M tRA plus 2.5 10M (), 1.0 10M (), or 2.5 10M () concentrations of the indicated retinoids. Cell
proliferation was determined as a measure of differentiation as
described under ``Materials and
Methods.''
homodimers. It is likely that the inhibition observed on
RXR homodimers represented inhibition of heterodimers formed
between transfected RXR and endogenous RARs, since it has been
shown that in the presence of both receptor species the formation of
heterodimers is predominant in vivo. Inhibition was observed
on all the retinoid response elements tested. Our transactivation
studies, therefore, suggest that the antagonists function by competing
with RAR agonists for RAR occupancy. This is consistent with the
observation that antagonists induce a similar conformational change as
the agonists determined by limited protease digestion, and, like the
agonist tRA, do not significantly affect DNA binding of the receptor
heterodimers. However, the structural changes determined by the limited
protease digestion pattern represent only a crude assession of the
ligand-induced changes in the receptor, since the conformation induced
by agonists for receptor-mediated transcription activation must be
distinct from the conformation induced by antagonists. The antagonist
SR11335 did not induce a protease-resistant RXR fragment while
9-cis-RA did. This observation is also consistent with the
transcriptional activation data, confirming that this antagonist
functions through RARs.-selective
retinoidal benzamide
4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenylcarboxamido)benzoic
acid (Am580), which is a potent inducer of HL-60 cell differentiation,
but have a 3-diamantyl-4-methoxyphenyl group in place of the
tetrahydrotetramethylnaphthalene ring of Am580. The bulky diamantyl
group, which has a larger steric volume than the corresponding
7-methylene and 8,8-dimethylmethylene groups of Am580, permits binding
to RAR but inhibits gene transcription. Apfel et al. (40) reported that (E)-6-[2-(4-carboxyphenyl)propenyl]-7-(n-heptyloxy)-3,4-dihydro-4,4-dimethyl-2H-benzothiopyran
1,1-dioxide (Ro41-5253) is a potent and selective antagonist of
RAR. This antagonist has the basic ring structure of Am580 and the
potent RAR-selective retinoid (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)propenyl]benzoic
acid (TTNPB, Ro13-7410), but has a sulfone group at the position
corresponding to the 5-position of the tetrahydronaphthalene ring and a
heptyloxy group on this ring ortho to the bridge linking the aromatic
rings. These studies indicate that steric bulk can be placed in regions
corresponding to the hydrophobic head and tetraene side chain of the
retinoid skeleton without loss of receptor binding activity. Our
studies further define the constraints of binding and antagonism in the
region of the tetraene chain using TTAB as the template for structural
modification. The common structural feature of antagonists SR11335 and
SR11330 are the 2,2,2-trifluoroethyl group with a polar substituent at
its 1-position. The 1-methoxy group of SR11335 produces more potent
antagonism than the 1-hydroxyl group of SR11330, although neither
compound binds as efficiently to RAR as Ro41-5253 does (data not
shown). The trifluoro substituents appear to be essential for binding
but not receptor activation because the 1-hydroxyethyl analog (SR11326)
of SR11330 partially retains transcriptional activity. Replacement of
the 1-methylene by a carbonyl group (SR11334) also confers partial
agonist activity. Smaller hydrophobic groups at this ring position such
as methyl do not decrease RAR transcriptional activity, whereas
larger groups such as t-butyl are devoid of activity and poor
binders to RAR. Analogs having smaller polar groups such as
hydroxyl (SR11337), amino, and hydroxymethyl are poor activators of
RAR, with activities comparable with that of the 1-hydroxyethyl
analog. RAR and RAR are more tolerant of substitutional
modification at this position.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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