(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, 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.
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, -
, 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) ).
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 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) .
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 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.
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 (
), 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 10
M 9-cis-RA (ER-RXR
) after subtraction of constitutive
receptor activity. The data points represent means of two experiments
carried out in duplicate.
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 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.
Figure 4:
Inhibition of retinoid receptor subtypes.
The TREpal-tk-CAT reporter was used to analyze receptor subtype
selective inhibition. A, RAR 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
10
M tRA in the presence or absence of the
indicated concentration of SR11330 (
), SR11335 (
), or
Tamoxifen (
). The activation obtained in the presence of
10
M tRA alone represents 100%. When only
RXR
was transfected, 10
M 9-cis-RA was used, and 10
M estradiol was used when ER was transfected. Results of a
representative experiment with duplicate measurements are
shown.
Figure 5:
Antagonism is response element
independent. CV-1 cells were cotransfected with 5 ng of RAR 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 10
M tRA in the presence or absence of the indicated concentrations of
SR11335. 100% activation represents the activity measured in the
presence of 10
M tRA without
antagonist.
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 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.
Figure 6:
Limited proteolytic digestions of retinoid
receptors in the presence of retinoid agonist and antagonist. In
vitro translated [S]methionine-labeled
RAR
(A), RAR
(B), or RXR
(C)
was preincubated with vehicle alone or together with 10
M tRA or 2
10
M 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.
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 10M tRA (
) or
10
M tRA plus 2.5
10
M (
), 1.0
10
M (
), or 2.5
10
M (
) concentrations of the indicated retinoids. Cell
proliferation was determined as a measure of differentiation as
described under ``Materials and
Methods.''
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 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.
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-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.
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.