From the Center for Cardiovascular Research, Departments of
** Medicine, Pediatrics, and
Molecular Biology & Pharmacology, Washington University School of Medicine,
St. Louis, Missouri 63110
Received for publication, August 9, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Dietary vitamin A and its derivatives, retinoids,
regulate cardiac growth and development. To delineate mechanisms
involved in retinoid-mediated control of cardiac gene expression, the
regulatory effects of the retinoid X receptor Retinoids are compounds, derived from vitamin A, which exert
pleiotropic effects on cellular differentiation, morphogenesis, and
metabolism. The effects of retinoids on cardiac growth and development
are well recognized. Offspring of rodents and birds fed a vitamin
A-deficient diet display congenital defects involving many organ
systems including the cardiovascular system (1, 2). Vitamin A
replacement at different stages of embryonic development in these
animals actually alters the severity and type of heart defects seen at
birth indicating that retinoid signaling pathways play key roles in a
variety of developmental programs (1). Similarly, retinaldehyde
dehydrogenase-deficient mice die in utero at embryonic day 8 secondary to poor maturation of the ventricular myocardium, an effect
that can be rescued by supplementation of maternal vitamin A (3, 4). In
addition, retinoids can exert teratogenic effects. Human offspring born
of mothers who ingested isotretinoin, a vitamin A analog, during
pregnancy exhibited a high incidence of complex cardiac malformations
including transposition of the great vessels, tetralogy of Fallot,
hypoplastic aortic arch, and ventricular septal defects (5). Moreover,
in the developing chick, local application of high concentrations of retinoic acid disrupts the migration of the pre-cardiac mesoderm, an
effect that is dose-dependent and developmental
stage-specific (6).
Retinoids bind to ligand-activated nuclear receptor transcription
factors called retinoid receptors. The retinoid receptors comprise a
subfamily within the nuclear receptor superfamily including the
retinoic acid receptors
(RARs)1 and the
9-cis-retinoic acid or retinoid X receptors (RXRs). RXRs participate in nuclear receptor regulatory pathways in two distinct manners. First, RXRs serve as heterodimeric partners for other class II
nuclear receptors such as thyroid receptor, vitamin D receptor,
peroxisome proliferator-activated receptors (PPARs), or RARs.
Heterodimeric complexes containing RXR in the presence of ligand,
recognize and bind cognate DNA elements within the regulatory regions
of target genes. Alternatively, RXR can form homodimers in the presence
its own ligand, 9-cis-retinoic acid (9-cis-RA).
Thus, RXR has been termed a "master regulator" of retinoid
signaling pathways. Interestingly, the transcriptional regulatory
effects mediated by retinoids is influenced at multiple levels
including specificity of the ligand-receptor interaction, temporal and
spatial variations in receptor expression, selection of heterodimeric
partners (some of which are permissive for liganded RXR), and the
levels of specific co-activators and co-represssor molecules within
target tissues. More recently, RXRs and other members of the nuclear
receptor superfamily have been shown to interact with non-nuclear
receptor transcription factors providing yet another level of
complexity in the retinoid signaling pathway (7-9). Accordingly, it is
not surprising that the observed effects of retinoids on cardiac
morphogenesis involves multiple developmental stages and cell types.
The cardiac phenotype of RXR As an initial step in the characterization of mechanisms involved in
retinoid-mediated cardiac gene regulation, we sought to use an
experimental system in which retinoid signaling was isolated from the
myriad of signals concurrent during development. To this end, we
focused on the observations of others that retinoids inhibit cardiac
myocyte hypertrophic growth in vitro. In response to a
variety of mechanical or hormonal stimuli, the terminally differentiated cardiomyocyte undergoes hypertrophic growth, a physiological response characterized by an increase in cell size, an
increase in sarcomeric organization, and activation of the fetal
expression pattern of contractile and metabolic genes (reviewed in Ref.
13). The cellular regulatory pathways involved in the induction of
cardiac hypertrophy and development of heart failure are currently
being unraveled. Atrial natriuretic factor (ANF) gene expression is
increased during cardiac hypertrophic growth and has been used as a
consistent marker of the hypertrophy program. Several groups have shown
that RXR Cell Culture and Transfections--
Rat neonatal cardiomyocyte
cultures were prepared as described (16). Briefly, hearts were removed
from 1-day-old Sprague-Dawley rats, the atria and great vessels were
trimmed off, and tissue was finely minced followed by sequential
digestion with 0.5 mg/ml collagenase (WAKO, Richmond, VA). Ventricular
cardiomyocytes were separated from fibroblasts by differential plating
and were cultured in gelatin-coated 12-well tissue culture plates (0.4 hearts/well) in media containing Dulbecco's modified Eagle's medium,
10% horse serum, 5% fetal calf serum, 100 µM
bromodeoxyuridine, penicillin, streptomycin, and
L-glutamine. Cardiomyocytes were transfected with
promoter-reporter constructs and transcription factors using the
calcium-phosphate precipitation method (17) with 4 µg of reporter and
0.5 µg of receptor per well. After 24 h, media was changed to
serum-free media containing 10 µg/ml transferrin, 10 µg/ml insulin,
and 100 µM bromodeoxyuridine, with the addition of
phenylephrine (5 µM) and retinoid ligand daily for 3 days. CV-1 cells (African green monkey kidney fibroblasts, obtained from ATCC, Manassas, VA) were maintained in minimal essential medium
with 10% fetal bovine serum. Transfections of CV-1 cells were also
performed using the calcium-phosphate precipitation method in minimal
essential medium supplemented with fetal bovine serum that was
pre-stripped using activated charcoal, to remove retinoid ligand
normally present in serum. 9-cis-Retinoic acid (9-cis-RA) and TTNPB
((E)-4-(2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl)benzoic acid) were obtained from Sigma. Luciferase was assayed in
cardiomyocytes 48-72 h after transfection. Cells were washed with
phosphate-buffered saline and lysed using Promega reporter lysis
buffer. Luciferase activity in the cell lysates was assayed using BD
Pharmingen substrates A and B according to the manufacturer's
instructions in a analytical luminescence luminometer. Growth hormone
was assayed from supernatants 72 h after transfection of
cardiocytes or 48 h after transfection of CV-1s, using a kit from
Nichols Institute Diagnostics (San Juan Capistrano, CA). All reporter
activities were corrected for the activity of an SV40- Plasmids--
Serial truncations of the rat ANF gene promoter
were produced by PCR amplification followed by cloning into pGL2 basic
(Promega) using MluI and XhoI sites to produce
The pMT2 mGATA-4, GATA-RE.growth hormone, and GST-mGATA-4 have been
described (18). GATA-4 mutants used in GST "pull-down" experiments
were cloned by PCR amplification using pSG5mGATA-4 as template into the
EcoRI/SalI sites in pSG5 using the following primers: for GATA, aa 1-440,
5'-agaatcgaattccttggggcgatgtaccaaagcctgccatgg-3' and
5'-gttacagtcgacttacgcggtgattatgacccctag-3'; aa 1-186,
5'-agaatcgaattccttggggcgatgtaccaaagcctgccatgg-3' and
5'-ataaaagtcgacttatgggctgtcgaaggggccggcaga-3'; aa 204-300, 5'-agcgctgaattccttggggcgatgtttgatgacttctcagaaggcaga-3' and
5'-agactggtcgacttacatggagcttcatgtagaggccgc-3'; aa 244-440,
5'-attacagaattccttggggcgatgaacggcatcaaccggccc-3' and 5'-gttacagtcgacttacgcggtgattatgacccctag-3'. The point mutation in ZF2
of GATA-4 (C290293S( GST Pull-downs--
GST fusion proteins were produced using pGEX
vectors (Amersham Biosciences) in DE3 bacteria according to the
manufacturer's instructions with
isopropyl-1-thio- Statistical Analyses--
Significant differences
(p < 0.05) were determined using unpaired, two-tailed
Student t tests.
RXR
To evaluate the role of the proximal GATA-response element
(GATA-RE) in the retinoid-mediated repression of the ANF gene
promoter, the site was mutated in the context of Retinoid-mediated Repression of the ANF Promoter Is
Ligand-dependent and RXR-specific--
RXR is capable of
heterodimerizing with other nuclear receptors including members of the
RAR, vitamin D receptor, and PPAR families. To define the receptor
specificity of the RXR
The RXR specificity of the repressive response was further evaluated by
dose-response studies using RXR- and RAR-specific ligands in the
cardiomyocyte culture system. The RXR ligand, 9-cis-RA, repressed RXR RXR
To further characterize the specificity of the interaction between
RXR
To map the RXR Liganded RXR
The functional data shown in Fig. 6 strongly suggested that liganded
RXR The importance of retinoid signaling in various stages of cardiac
growth and development has been recognized for more than 50 years, yet
the specific molecular targets within the heart are largely unknown. We
sought to characterize a retinoid signaling/transcription factor
interface relevant to cardiogenic gene regulatory programs. To this
end, we focused on the transcriptional regulatory region of the ANF
gene that has been well characterized. ANF gene expression has been
established as a reliable marker of cardiac hypertrophy in rodents and
humans (see review by Levin (35)). In this report, we provide evidence
that retinoid signaling via RXR In previously published studies (36, 37), both RXR and vitamin D
receptor ligands were shown to suppress basal and
endothelin-1-activated expression of the ANF gene in rat atrial
myocytes. In addition, retinoid or vitamin D ligands were shown to
inhibit ET-1-induced cardiomyocyte hypertrophy (15). In these studies,
high concentrations of all-trans-retinoic acid
(i.e. enough to allow substantial isomerization of
all-trans-RA to 9-cis-RA) were necessary, leading
to the conclusion that liganded RXR rather than RAR is the dominant
retinoid receptor conferring this repression. Our results are
consistent with this conclusion. Additional information was gleaned by
the studies of Zhou and colleagues (14) who investigated the effects of retinoids on ANF gene expression and hypertrophic growth on rat neonatal ventricular cardiomyocytes in culture. Hypertrophic growth and
ANF expression were inhibited by treatment with
all-trans-retinoic acid. Furthermore, a dominant negative
retinoic acid receptor mutant (hRAR Analysis of the ANF promoter region revealed that it does not contain
cognate retinoid receptor response element DNA recognition sites
suggesting that RXR Several lines of evidence shown here indicate that the repression of
GATA-4 conferred by RXR The precise mechanism by which FOG-2 mediates repression has not been
completely characterized. Indeed, depending upon the cell context and
the target, FOG-2 is capable of activating or repressing GATA function
(44, 47). Recently, FOG-2 was shown to interact with the orphan nuclear
receptor COUP-TF (48). Given that COUP-TF confers repression to most of
its targets via recruitment of co-repressors containing histone
deacetylase activity, it is possible that COUP-TF participates in the
RXR·FOG-2 complex to deactivate GATA-4. In addition, given
that RXR binds GATA-4 via the same region (ZF2) as Nkx2.5 and SRF, it
is possible that the RXR The results of previous studies have shown that retinoids antagonize
the hypertrophic growth of cardiac myocytes (14, 15). The results
presented here focused on ANF gene expression, a single, albeit
representative component of the cardiac hypertrophic growth program. The link between the RXR (RXR
) on atrial
naturietic factor (ANF) gene transcription was investigated. The
transcriptional activity of an ANF promoter-reporter in rat neonatal
ventricular myocytes was repressed by RXR
in the presence of
9-cis-RA and by the constitutively active mutant
RXR
F318A indicating that liganded RXR confers the regulatory effect.
The RXR
-mediated repression mapped to the proximal 147 bp of the rat
ANF promoter, a region lacking a consensus retinoid response element
but containing several known cardiogenic cis elements
including a well characterized GATA response element.
Glutathione S-transferase "pull-down" assays revealed
that RXR
interacts directly with GATA-4, in a ligand-independent manner, via the DNA binding domain of RXR
and the second zinc finger
of GATA-4. Liganded RXR
repressed the activity of a heterologous promoter-reporter construct containing GATA-response element
recognition sites in cardiac myocytes but not in several other cell
types, suggesting that additional cardiac-enriched factors participate in the repression complex. Co-transfection of liganded RXR
and the
known cardiac-enriched GATA-4 repressor, FOG-2, resulted in additive
repression of GATA-4 activity in ventricular myocytes. In addition,
RXR
was found to bind FOG-2, in a
9-cis-RA-dependent manner. These data reveal a
novel mechanism by which retinoids regulate cardiogenic gene expression
through direct interaction with GATA-4 and its co-repressor,
FOG-2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-null mice provides additional evidence
for the importance of retinoid signaling in the development, growth,
and maturation of the heart. The ventricular myocardium does not
develop normally in RXR
-null mice leading to heart failure at
embryonic day 15 (10, 11). The incidence of conotruncal defects in
RXR
/
mice is greater than 50% (12). Furthermore, RXR
heterozygous animals display a wide array of defects involving the
conotruncal region, atrioventricular canal, and ventricular myocardium.
Taken together, these data indicate that retinoid signaling via RXR
serves a critical role in cardiac growth and development programs.
However, the molecular mechanisms by which RXR exerts regulatory
effects within the complex process of cardiac morphogenesis remains unclear.
, in the presence of 9-cis-RA, represses the
hypertrophic growth of cardiomyocytes in culture (14, 15). We
hypothesized that delineation of cis-elements and
trans-acting factors mediating the effects of
9-cis-RA on ANF gene transcription would provide information
about the mechanisms involved in the regulation of cardiogenic gene
transcription by retinoids. In this report, we demonstrate that
liganded RXR
regulates ANF gene transcription through a mechanism
uncharacteristic of previously described retinoid signaling pathways.
Our results indicate that RXR
represses ANF promoter activity in
cardiomyocytes by interacting directly with the transcription factor
GATA-4 and its co-repressor FOG-2. These results identify a mechanism
whereby retinoid signaling modulates gene expression in the heart and unveils a new permutation in the retinoid signaling pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase plasmid.
633ANF.luc,
316ANF.luc,
147ANF.luc, and
107ANF.luc using the
following 5' primers: 5'-gatcgcacgcgttttagacctgtatcatgttgg-3' (for
633), 5'-tagataacgcgtccagggagaaggaaatcctga-3' (for
316), 5'-agttatacgcgtgggctgtgacaagcttcg-3' (for
147), and
5'-atattaacgcgtgcatcttctcctggccgc-3' (for
107) and the same 3' primer
5'-agatcactcgagatctgatgtttgctgtctcgg-3'. Point mutations of the rat ANF
promoter were performed as follows: the GATA response element at
122
was mutated in both the
316ANF.luc and the
147ANF.luc constructs
from TGATAA to TACTAA by site-directed mutagenesis using the
following primers:
5'-GCTGTGACAAGCTTCGCTGGACTACTAACTTTAAAAGCGCATCTTCTCCTGGC-3' and
5'-GCCAGGAGAAGATGCGCTTTTAAAGTTAGTAGTCCAGCGAAGCTTGTCACAGC-3'. pSG5mRXR
F318A was produced by site-directed
mutagenesis of pSG5 mRXR
(Pierre Chambon) using the QuikChange kit
(Stratagene) according to the manufacturer's instructions with the
following oligonucleotide primer pairs:
5'-gctgctgatcgcctccgcctcccaccgctccatagc-3' and
5'-gctatggagcggtgggaggcggaggcgatcagcagc-3'. RXR truncation mutants were
made by PCR into the EcoRI and HindIII sites of
pSG5, using the following primers: pSG5mRXR
aa 132-467, 5'-catgtagaattccatgagttagtcgtagacatgtgtgctatctgtggggaccgctcc-3' and
5'-gattctaagcttctggagctgagcagctgtgtccaggca-3'; pSG5mRXR
aa 132-238,
5'-catgtagaattccatgagttagtcgtagacatgtgtgctatctgtggggaccgctcc-3' and
5'-aaagcttctatacaggcatgtcctcgttggcact-3'; pSG5mRXR
AF-2, 5'-cctgtcgaattgatggacaccaaagatttc-3' and
5'-ctcgagaggcttctagatgagcttgaagacaggtgctcctggca-3'; pCR2.1mRXR
203-467 was constructed by using
5'-atgaagcgggaagcgggaagctgtg-3' and
5'-gattctaagcttctggagctgagcagctgtgtccaggca-3'. GST-RXR was produced using the pGEX 2TKG vector (a gift from Ellen Li, Washington University) and the EcoRI/HindIII fragment from
pSG5.mRXR
. GST-mRXR
F318A was made using site-directed
mutagenesis, with the same primers as the pSG5mRXR
F318A mutant.
ZF2)) was made by site-directed
mutagenesis as above using oligonucleotide primer pairs
5'-ggtgagccctgtaagtaatgccagcggcctctacatgaagc-3' and
5'-gcctcatgtagaggccgctggcattacttacaggctcacc-3' Plasmids were verified
by sequence analysis using ABI prism automated sequencing technology at
the institutional core facility.
-D-galactopyranoside induction of
bacterial protein expression with some modifications: insoluble proteins were solubilized in 7 M urea/phosphate-buffered
saline followed by dialysis. Proteins were verified by Coomassie
staining of SDS-PAGE gels and by Western blot using an anti-GST
antibody. Bacterial lysate containing 2 µg of the GST fusion protein
or control GST protein were bound to 15 µl of glutathione-Sepharose in bead binding buffer (20 mM Tris, pH 7.4, 60 mM sodium chloride, 1 mM dithiothreitol, 15%
glycerol, 0.1% Nonidet P-40 (Calbiochem)) at room temperature and then
washed in bead binding buffer. Five µl of in vitro
translated protein (TNT T7 Quick-coupled kit according to
the manufacturer's instructions) was incubated for 1 h at room temperature, washed with bead binding buffer, and resolved on SDS-PAGE,
then visualized by autoradiography. In binding studies involving
retinoid ligand, ligand was included in the wash buffers as well as the
protein incubation period.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Represses Basal and
1-Adrenergic
Agonist-mediated Activation of the ANF Promoter via Elements within the
Proximal Promoter Region--
The traditional mechanism of
retinoid-mediated transcriptional control occurs via the action of
liganded retinoid receptors bound to DNA recognition sequences within
regulatory regions of target genes. Analysis of the rat ANF promoter
sequence failed to reveal retinoid receptor recognition sequences
(AGGTnA). Thus, we hypothesized that the known repression of ANF gene
transcription by retinoids occurs through an indirect mechanism such as
the modulation of trans-acting factors by RXR. The
cis-acting regulatory elements necessary for RXR-mediated
repression of ANF gene transcription were localized by assaying a 5'
deletion series of the rat ANF promoter fused to a luciferase reporter
(ANF.Luc) in rat neonatal cardiomyocytes. The experiments were
performed in the absence and presence of the
1-adrenergic agonist, phenylephrine, to allow evaluation
of effects on basal activity and hypertrophic response. Cotransfection
of an expression plasmid for RXR
markedly repressed the activity of
633ANF.luc in a 9-cis-RA-dependent manner in the presence or absence of phenylephrine (Fig.
1A). Similar
RXR
-dependent repression was observed for the
316ANF.luc and
147ANF.luc constructs (Fig. 1A). Given
that the basal activity of the smallest construct (
107ANF.luc) was
very low it was not possible to determine with certainty whether it
localized the cis-acting element mediating the repression by
retinoids. These results localized the cis-acting elements
involved in the RXR-mediated repression of ANF gene transcription to a
region within 147 nucleotides of the transcription start site.
Importantly, this region contains response elements for several
transcription factors known to be critical for the transcriptional control of ANF and other cardiac genes; GATA-4, a zinc finger transcription factor important in cardiogenesis and the cardiac hypertrophic program (19-21); serum response factor (SRF), a
transcription factor critical for basal and
1-adrenergic
agonist-induced activation of ANF gene expression (22, 23); and Nkx2.5,
a cardiac-enriched homeobox transcription factor necessary for proper
cardiac development (24) and a known co-activator of both GATA-4 and
SRF (25-30).
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Fig. 1.
Repression of ANF promoter activity by
retinoids. Ventricular myocytes were prepared from neonatal rats.
A, the graph represents the activity of a 5'
deletion series of the rat ANF gene promoter fused to a luciferase
reporter (shown on the left) assayed after co-transfection
with RXR under the conditions denoted in the key. Transfections were
performed at the time of plating the cardiomyocytes, in medium
supplemented with serum. Phenylephrine (PE, 5 µM) and/or 9-cis-RA (1 µM) were
added after changing to serum-free medium 12 h after transfection.
Luciferase activity (relative luciferase units, RLU) was
assayed 72 h later. B, the activity of ANF deletion
mutants (shown at left), in which the proximal GATA-response
element was mutated and assayed in cardiomyocytes as above. The
ordinate, indicating RLU with expanded scale, also
demonstrates differences in basal activity of the constructs. The
values represent mean (+S.E.) RLU of triplicate samples from at least
three independent experiments.
316ANF.luc
(
316
GATA.ANF.luc) and
147ANF.luc (
147
GATA.ANF.luc) for use
in cotransfection assays. The basal activity of both
316
GATA.ANF.luc and
147
GATA.ANF.luc, although still active,
was significantly diminished compared with the corresponding wild-type
constructs (Fig. 1B). The retinoid-mediated repression
observed with
316
GATA.ANF.luc was significantly less than that of
316ANF.luc but was not completely obliterated (30% compared with
94%, Fig. 1B). Similarly, the repression of the
147
GATARE ANF.luc construct was also significantly less than that
of the wild-type construct (42 compared with 88%, Fig. 1B). Taken together, these data indicate that the GATA-response element is
necessary for the full repression conferred by retinoids in cardiomyocytes, but also suggest that other elements may play a role.
Furthermore, the importance of the interaction between adjacent
elements within the native promoter cannot be underestimated, especially in light of the known cooperativity between GATA, Nkx2.5, and SRF.
-mediated repression of the ANF promoter, the
requirement of its activation domain and the effects of RXR
homodimerization were analyzed using two mutants, mRXR
F318A and
RXR
AF-2. RXR
F318A is a constitutively active mutant (31). The
conformational change induced by the phenylalanine to alanine
substitution allows the RXR
F318A mutant to exist in the dimer but
not the tetramer form and to possess constitutive
trans-activation properties in the absence of exogenous ligand (32, 33). RXR
AF-2, which lacks the COOH-terminal 19 amino
acids, is unable to recruit co-activator molecules (31, 34). We
confirmed the predicted activity of the RXR
mutants in CV-1 cells
using a known RXR-response element (RXR-RE) derived from the cellular
retinoid-binding protein II gene (Fig.
2A). CV-1 cells were used for
these experiments because they are largely devoid of endogenous
retinoid receptor activity. As expected, wild-type RXR
conferred
marked activation of the RXR-RE only in the presence of the ligand
9-cis-RA (Fig. 2A). In contrast, the mRXR
F318A
mutant activated the RXR-RE in the absence of ligand. As expected,
RXR
AF-2 was inactive with the RXR-RE. We then assayed the effect of
these mutants on ANF promoter-reporter activity in ventricular
cardiomyocytes (Fig. 2B). ANF promoter activity was
profoundly repressed with the F318A mutant in the absence of ligand. In
contrast, the RXR
AF-2 mutant did not confer repression. These latter
results indicated that as predicted by the 9-cis-RA dependence, an intact AF-2 domain is required for the repressive effects of RXR
on the ANF promoter. Moreover, these data demonstrate that the repressive effect is mediated largely by RXR
rather than
one of its nuclear receptor partners.
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Fig. 2.
Liganded RXR
represses the ANF gene promoter. Promoter-reporter
constructs and nuclear receptor expression plasmids were co-transfected
into cardiomyocytes. Retinoid concentration was 10
6
M unless stated otherwise. A, the
trans-activating activity of wild-type (WT) RXR
and
RXRF318A and RXR
AF-2 mutants was established using a luciferase
reporter containing an RXR-response element from the cellular
retinoid-binding protein II (CRBP) promoter in the absence
(open bar, left) or presence (filled
bar, right) of 9-cis-RA. B, the
effect of the RXR
mutants on
638ANF.luc. C and
D, the effects of co-transfection with RXR
, and/or RAR
on ANF promoter-reporter activity assayed in the presence of increasing
concentrations of the RXR-selective ligand, 9-cis-RA
(C) or the RAR-selective ligand, TTNPB (D). The
values represent mean (±S.E.) relative luciferase units
(RLU) of triplicate assays from at least three separate
experiments.
633ANF.luc in a dose-dependent manner only in
the presence of RXR
(Fig. 2C). Addition of RAR
had no
affect on the 9-cis-RA response (Fig. 2B).
Specifically, 9-cis-RA did not mediate a repressive response
in the presence of RAR
alone, and the effects observed with addition
of both RXR
and RAR
were similar to that of RXR
alone.
Moreover, a dose-response analysis using the RAR-specific ligand TTNPB
revealed that at no concentration did liganded RAR alone confer
repression (Fig. 2D). In the presence of RXR
, however, repression was observed at a concentration 100-fold greater than the
effective 9-cis-RA concentration (Fig. 2D).
Similar studies to analyze "permissive" RXR heterodimers
demonstrated that PPAR
/RXR
heterodimers, at high concentrations
of the PPAR
ligand ETYA (10
5 M), conferred
only 40% repression of ANF promoter activity in cardiomyocytes (data
not shown). Furthermore, VDR/RXR
heterodimers demonstrated a ligand
dose-dependent repressive effect on ANF promoter activity,
with 30 and 60% repression at 10
7 and 10
6
M vitamin D, respectively (data not shown). Accordingly,
the effects of ligands specific for permissive heterodimeric partners of RXR did not approach the potency seen with RXR ligands. Taken together these results indicate that liganded RXR is the dominant receptor conferring repression of the ANF promoter.
Antagonizes GATA-4 in a Ligand-dependent,
Cardiomyocyte-specific Manner--
The data shown in Fig. 1
demonstrated that the GATA-response element present in the proximal
region of the ANF gene promoter was involved in the retinoid-mediated
repression. However, a role for SRF or Nkx2.5 was not excluded. To
explore this further, the effect of RXR
on GATA-4, SRF, and Nkx2.5
was explored using independent reporters containing response elements
for each of the factors. RXR
/9-cis-RA had no affect in
cardiomyocytes on the activity of reporters containing heterologous
Nkx2.5 or SRF-response elements, respectively (data not shown). In
striking contrast, the activity of a GATA-4 responsive construct
(GATA-RE6.GH) was markedly repressed by
RXR
/9-cis-RA. Specifically, the high level activity of
GATA-RE6.GH in cardiomyocytes was abolished by RXR
in a
9-cis-RA-dependent manner (Fig.
3A). Interestingly, the
RXR
-mediated repression of GATA-RE6.GH was not observed
in the noncardiomyocyte cell line CV-1, even in the presence of
overexpressed GATA-4 (Fig. 3B). Furthermore, addition of
Nkx-2.5, SRF, or the known co-repressing RXR interacting partner SHP,
alone or in combination, did not recapitulate the RXR
-mediated
repression in CV-1 cells (data not shown). These results together with
the data shown in Fig. 1B demonstrate that the GATA-response
element is sufficient for retinoid-mediated repression and necessary
for the full repressive effect on the ANF gene promoter.
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Fig. 3.
Inhibition of GATA-4-mediated transactivation
by liganded RXR . A, the activity of a heterologous
promoter-reporter construct comprised of 6 copies of a GATA-response
element (GATA-RE) upstream of a growth hormone
(GH) reporter was assayed in ventricular cardiomyocytes in
the presence or absence of co-transfected RXR
and its ligand
9-cis-RA as indicated. B, the effect of RXR
on
GATA activity in CV-1 cells. For these experiments, expression vectors
for GATA-4 and RXR
were co-transfected in the presence or absence of
9-cis-RA as indicated. Values represent mean (+S.E.) growth
hormone levels corrected for
-galactosidase activity in arbitrary
units (AU) as determined from triplicate samples from at
least three independent experiments.
Interacts Directly with GATA-4 in a Ligand-independent
Manner--
The data shown above suggested that liganded RXR
inhibits GATA-4 activity through a cardiomyocyte-specific mechanism,
possibly via the effects of a cell-specific repressor recruited by
RXR
. Alternatively, GATA-4 expression could be down-regulated by a retinoid signaling pathway. However, the amount of cardiomyocyte GATA-4
protein detected by Western blot analysis was not affected by
overexpression of RXR
in the presence of 9-cis-RA (data
not shown). To determine whether RXR interacts directly with GATA-4, GST pull-down assays were performed. GATA-4 bound to GST-RXR
in an interaction that, surprisingly, did not require ligand (Fig. 4). Indeed, addition of
9-cis-RA had no effect on the binding. The specificity of
this interaction was confirmed by the lack of binding with GST alone
(Fig. 4). The interaction of RXR
with PPAR
is also shown as a
control. These data demonstrate a direct interaction between RXR
and
GATA-4 but does not explain the precise role of ligand in the
RXR-mediated repression of the ANF-promoter.
View larger version (20K):
[in a new window]
Fig. 4.
Ligand-independent interaction between GATA-4
and RXR . GST pull-down assays were performed using immobilized
GST control protein or GST-RXR fusion protein with in vitro
translated, 35S-labeled GATA-4 or PPAR
, in the presence
or absence of 9-cis-RA as indicated at the top.
Following incubation, beads were boiled in SDS-PAGE reducing buffer,
resolved on 10% SDS-PAGE gel, and visualized by autoradiography.
Numbers at the left reflect the molecular mass of
protein standards. KD, kilodaltons.
and GATA-4 and to map the relevant domains within RXR
, a
series of RXR
truncation and point mutants were used in GST
pull-down studies. The results demonstrated that the C domain of RXR,
which contains the DNA binding site, is critical for the interaction
with GATA-4 (Fig. 5A).
Specifically, an 35S-labeled RXR
amino-terminal deletion
mutant protein containing the C domain (aa 132-467), bound GST-GATA-4
while an amino-terminal deletion mutant lacking the C domain (203-467)
did not bind (Fig. 5A). An RXR
fragment containing only
the C domain (132-210) also bound GATA-4 (Fig. 5A). In
addition, the RXR
AF-2 mutant (1-449) also bound GATA-4. Of note,
this latter mutant did not confer repression on the ANF promoter
providing additional evidence that the RXR
/GATA interaction
alone is not sufficient to confer the observed repression.
Collectively, these data suggest that GATA-4 interacts with the DNA
binding domain of RXR
and that neither the AB domain nor the DEF
domains are necessary for this interaction. Furthermore, this
interaction is likely to occur on a GATA-RE in the absence of direct
DNA binding of RXR
because a critical DNA interaction site would
likely be obscured by the interaction with GATA-4.
View larger version (19K):
[in a new window]
Fig. 5.
Mapping of
RXR /GATA-4 interaction domains. GST
pull-down assays were carried out as above using truncation or point
mutants of RXR
and GATA-4. A, representative
autoradiographs of 35S-labeled proteins resolved on
SDS-PAGE. B, wild-type or mutant GATA-4 proteins were
synthesized as above and interactions with GST control protein-bound
Sepharose or GST-RXR
fusion protein were assayed; representative
autoradiographs are presented. Numbers below each
autoradiograph represent quantitation (% total input) of bound GATA-4
proteins, using 10% of the input (left) as an internal
standard.
interaction regions within GATA-4, GST pull-down
assays were performed using a series of GATA-4 truncation and point
mutants. A GATA-4 carboxyl-terminal deletion mutant lacking the ZF1 and
ZF2 regions did not bind RXR (Fig. 5B). A truncation mutant
containing only the DNA binding domain of GATA-4-(204-300) bound
GST-RXR, but to a lesser extent than the wild-type GATA-4 protein. A
robust interaction was noted between the GATA-4 mutant containing the
COOH-terminal half of the protein including ZF2 but not ZF1 (aa
244-440). Mutation of ZF2 significantly decreased but did not abolish
the interaction, indicating that in addition to ZF2, another region in
the COOH-terminal half of GATA-4 likely contributes to the interaction
with RXR. These results indicate that RXR
requires the ZF2 region of
GATA-4 for binding. In addition, a domain(s) in the region
carboxyl-terminal to ZF2 may also participate in the RXR
/GATA-4 interaction.
Recruits FOG-2: A Mechanism Whereby Retinoids
Repress GATA-4 Transactivation in Cardiomyocytes--
Given the likely
participation of a cardiac-specific co-repressor molecule in the
cell-type specific modulation of GATA-4 by RXR
, we assayed the
effect of the known cardiac-specific, GATA-4 co-repressor, FOG-2 on
RXR
-mediated repression of GATA-4. Transient transfection assays
were performed in cardiomyocytes to assess the effect of FOG-2 and RXR
on ANF promoter activity. As expected the addition of RXR
, in the
presence of 9-cis-RA, resulted in repression of
633ANF.luc
(Fig. 6A). The addition of
FOG-2, alone, confirmed its known repressive effect on
GATA-4-dependent transcription. The combination of RXR
and FOG-2, in the presence of 9-cis-RA, resulted in a
significantly greater degree of repression than either alone
(p = 0.002; Fig. 6A). The cooperativity
between RXR
and FOG-2 was further evaluated using titrations of both retinoid ligand and FOG-2. We first determined the minimal amount of
exogenous RXR
necessary to achieve maximal repression by titrating the amount of RXR
expression vector. Full repression of
633ANF.luc was noted with the addition of 350 ng (per well) of pSG5.mRXR
at
10
8 or 10
7 M
9-cis-RA (Fig. 6B). Utilizing these same
conditions, we assayed the effect of adding increasing amounts of FOG-2
on ANF promoter activity at two different ligand concentrations. As
expected, FOG-2 mediated a dose-dependent repression of
633ANF.Luc activity in the absence of ligand (Fig. 6C, open
bars), consistent with the known role of FOG-2 as a GATA
repressor. Addition of retinoid ligand and increasing amounts of FOG-2
expression vector incrementally repressed the activity of
633ANF.Luc
amounts at the lower ligand concentration of 10
8
M. Thus, the ligand-dependent repression
conferred by RXR
is further enhanced by FOG-2, in a
dose-dependent manner.
View larger version (20K):
[in a new window]
Fig. 6.
Functional cooperativity between RXR and
FOG-2 in the repression of the ANF gene promoter in cardiomyocytes.
A,
633ANF.luc and expression plasmids for RXR
and/or
FOG-2 were co-transfected into cardiomyocytes in the presence or
absence of 9-cis-RA as indicated. The results are shown with
a disrupted ordinate to demonstrate the broad range of activities.
Statistically significant differences between results of vector alone
and other conditions (*, p < 0.0001), and between
FOG-2 co-transfected and ligand treated/FOG-2-RXR transfected
conditions (**, p = 0.002) are denoted. B,
titration of the effect of different amounts of RXR
expression
vector (amounts in nanogram are shown at the bottom)
co-transfected into cardiomyocytes in the presence (10
8
or 10
7 M) or absence of 9-cis-RA.
C, addition of increasing amounts of FOG-2 expression vector
(denoted at the bottom in nanograms), in the presence of a
constant amount of RXR
expression vector (350 ng/well) was assayed
at the ligand concentrations shown. Note, ordinate scale is
relative luciferase units (RLU) (×103) in
B and C. Data presented are mean (±S.E.) of
triplicate wells from at least three separate experiments.
mediates repression of GATA-4 by cooperating directly or
indirectly with FOG-2. To distinguish between these possibilities, GST
pull-down assays were performed to determine whether RXR
and FOG-2
interact. The known ligand-enhanced interaction between the coactivator
SRC-1 and RXR
was included as a positive control for the effect of
ligand. As expected, addition of 9-cis-RA or substitution
with the constitutively active RXR
F318A resulted in a stronger
RXR
/SRC-1 interaction than observed with RXR
alone (Fig.
7). Pull-down experiments with
35S-FOG-2 and RXR
demonstrated that FOG-2 bound
GST-RXR
only in the presence of 9-cis-RA. In addition,
FOG-2 bound the constitutively active form of RXR
(RXR
F318A) in
the absence of ligand. These results reveal a strong,
ligand-dependent direct interaction between RXR
and
FOG-2 and strongly suggest that RXR
recruits the co-repressor FOG-2
to mediate deactivation of GATA-4.
View larger version (26K):
[in a new window]
Fig. 7.
Ligand-dependent interaction
between RXR and FOG-2. GST pull-down
assays were performed using GST control, GST-RXR, and GST-RXR F318A
proteins with in vitro translated, 35S-labeled
SRC-1 or FOG-2, in the absence or presence of 9-cis-RA
(10
7 M). SRC-1 is shown as a control for the
effect of ligand.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
converges on GATA-4-mediated
transcriptional control of the ANF gene in cardiomyocytes. These
results extend the findings of others that RXR antagonizes the cardiac
gene hypertrophic growth response.
403) abrogated the repression.
These latter results suggested that RXR/RAR heterodimers conferred the
repressive effect. However, the hRAR
403 mutant likely alters most if
not all RXR signaling pathways because of its potential to
heterodimerize and "squelch" RXR. Our data demonstrate a key role
for liganded RXR in this pathway. The constitutively active RXR
mutant RXR
F318A confers repression of the ANF promoter in the
absence of ligand. Moreover, we found that the RXR ligand
9-cis-RA is significantly more effective than the
RAR-selective ligand TTNPB in mediating the repressive effect. In fact,
TTNPB only repressed at concentrations known to also activate RXR (38,
39). In addition, repression was not seen with the RXR
AF-2
mutant, which should maintain trans-repressive properties if the
heterodimeric partner was the operative factor (40). Taken together we
conclude that liganded RXR
is critical for the observed repressive
affect on ANF gene expression. These data do not eliminate the
possibility that RXR
heterodimeric partners such as vitamin D or RAR
participate in this pathway to regulate other genes involved in the
hypertrophic growth program.
alters ANF gene transcription through an
indirect mechanism. Our results are consistent with this conclusion. We
found that RXR
represses ANF gene transcription via direct interaction with GATA-4. Through its DNA binding domain, RXR
interacts with the zinc finger region (ZF2) of GATA-4 as well as
additional site(s) within the COOH terminus of the protein. There is
significant precedence for interaction of GATA factors with other
transcription factors. In cardiac tissues, GATA-4 interacts with
Nkx2.5, NFAT, and MEF-2 through ZF2 (25, 41, 42), and with FOG-2
through ZF1 (43, 44). p300 requires other sites in the
NH2-terminal and carboxyl-terminal regions of GATA for its
interaction (45). Interestingly, GATA-4 interacts with RXR
in a
manner similar to the GATA-SRF interaction, involving a portion of the
COOH terminus of GATA-4 in addition to ZF2 (46). This suggests that
competition among activating and repressing factors for binding to
GATA-4 could modulate its activity. It should also be noted that our
results with the mutated ANF promoter suggest that other elements
participate in the RXR
-mediated repression. Nkx2.5 and SRF are
candidates for this role although our results suggest that they cannot
confer this repression alone.
involves an additional corepressor. First,
regulation of GATA-4 by RXR
occurred in a cardiac myocyte-restricted manner. Second, although the RXR
-mediated repression of the ANF promoter and a GATA-RE reporter required 9-cis-RA, the
RXR
/GATA-4 interaction is ligand independent. Third, the
RXR
/GATA-4 interaction does not require the AF-2 region of RXR
,
yet the observed repression effect required an intact AF-2. These
results led us to explore the possibility that FOG-2, the known
cardiac-enriched repressor of GATA-4 was recruited by RXR
. Our data
demonstrate that FOG-2 interacts with RXR
in a
ligand-dependent manner. Interestingly, the interaction
domains on GATA-4 are distinct for RXR
and FOG-2 and, thus, it is
possible that GATA-4 can bind both proteins simultaneously. We propose
that RXR
serves as a high affinity recruiter of FOG-2 to the GATA-4
interface. The ligand dependence of this interaction provides a
mechanism for dynamic modulation of the repressive effect in a cell-
and developmental stage-specific manner. In addition, these data
suggest a novel signaling pathway through which retinoids modulate key
cardiac transcription factors.
·FOG-2 complex displaces these
coactivating transcription factors.
·ATA-4·FOG-2 complex and the broad hypertrophic program is suggested but not definitively
established by this work. However, it is tempting to speculate that the
RXR
-mediated mechanism described here serves to block multiple
pathways involved in the hypertrophic response. It will also be of
interest to determine whether additional nuclear receptor or
non-nuclear receptor RXR interacting proteins are involved in the
proposed retinoid-mediated effects on the cardiac hypertrophic growth program.
![]() |
ACKNOWLEDGEMENT |
---|
Special thanks to Mary Wingate for assistance with manuscript preparation.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants RO1 HL58493, P50 HL61006, P30 DK52574, and P30 DK56341.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by an NHLBI, National Institutes of Health Grant KO8 HL03829.
¶ Current address: Depts. of Pediatrics and Medicine, Emory University School of Medicine, 1638 Pierce Dr., WMB 319, Atlanta, GA 30322.
To whom correspondence should be addressed: Center for
Cardiovascular Research, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8086, St. Louis, MI 63110. Tel.: 314-362-8908; Fax: 314-362-0186; E-mail: dkelly@im.wustl.edu.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M208173200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RAR, retinoic acid receptor; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptors; ANF, atrial naturietic factor; GST, glutathione S-transferase; SRF, serum response factor; RE, receptor element; aa, amino acid(s); ZF, zinc finger.
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