From the Diabetes Center/Metabolic Research Unit and
the § Department of Medicine, University of California,
San Francisco, California 94143-0540
Received for publication, July 29, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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Brain natriuretic peptide (BNP) gene expression
is a well documented marker of hypertrophy in the cardiac myocyte.
Triiodothyronine (T3), the bioactive form of thyroid
hormone, triggers a unique form of hypertrophy in cardiac myocytes that
accompanies the selective activation or suppression of specific gene
targets. In this study, we show that the BNP gene is a target of
T3 action. BNP secretion was increased 6-fold, BNP mRNA
levels 3-fold, and BNP promoter activity 3-5-fold following
T3 treatment. This was accompanied by an increase in
myocyte size, sarcomeric organization, and protein synthesis. Of note,
several of the responses to T3 synergized with those to the
conventional hypertrophic agonist endothelin. The response to the
liganded thyroid hormone receptor (TR) was mediated by an unusual
thyroid hormone response element located between The heart is highly sensitive to the effects of thyroid
hormone. Triiodothyronine
(T3),1 the most
active form of thyroid hormone, increases heart rate, cardiac
contractility (direct and indirect effects), and cardiac output.
Pathological states associated with either hypo- or hyperthyroidism display abnormalities in cardiac function that frequently contribute to
the morbidity and mortality associated with these disorders (1).
T3 has also been shown to activate growth in cardiac
myocytes in vitro (2, 3) and in vivo (4, 5),
through a combination of direct and indirect effects, leading to
increased cell size, protein synthesis, and changes in gene expression.
Thyroid hormone regulates a number of genes in the heart, including
those encoding sarcoplasmic endoplasmic reticulum
Ca2+-ATPase (SERCA) (6, 7), atrial natriuretic peptide (8), Brain natriuretic peptide (BNP) is a cardiac hormone that is linked
closely with hypertrophy both in animal models (19) and humans (20,
21). Thyroid hormone has been shown to stimulate secretion of BNP
in vitro and in vivo (22). However, there is no
evidence to indicate that this is accompanied by changes in BNP gene
expression in the cardiac myocyte, nor have these changes been linked
to the hypertrophic phenotype.
Thyroid hormone exerts its effects in target cells through interaction
with high affinity nuclear receptors present in chromatin (23). These
receptors associate with cognate DNA recognition elements, termed
thyroid hormone response elements (TREs), as monomeric, homodimeric, or
heterodimeric (typically partnered with the related retinoid X receptor
(RXR)) complexes. These, in turn, interact with coregulatory nuclear
proteins that promote enhanced (coactivators) or reduced (corepressors)
transcriptional activity. The p160 family of coactivators
(e.g. glucocorticoid receptor-interacting protein
(GRIP1/TIF2/NCoA-2), the steroid receptor coactivator (SRC1/NCoA-1),
and the coactivator for the nuclear hormone receptor
(ACTR/pCIP/Rac3/AIB1/TRAM-1)) associate with the liganded TR and
promote assembly with additional regulatory proteins (e.g.
CBP (cAMP-responsive element-binding
protein-binding protein and p300) harboring
histone acetyltransferase activity that is capable of modifying
chromatin structure in a manner that leads to increased gene
transcription (24). The TR has also been shown to interact with a
second class of coactivators collectively termed the TRAP
(TR-associated protein)-DRIP (vitamin D receptor-interacting
protein) complex (25). This complex is capable of promoting
receptor-dependent transcription on naked DNA, presumably
by fostering contacts with the core transcriptional apparatus (26, 27).
There are two corepressor molecules (i.e. nuclear receptor
corepressor (NCoR) (28, 29) and SMRT (silencing mediator for retinoid and thyroid
hormone receptors) (29, 30)), each of which forms a suppressor complex
with the unliganded TR, as well as other proteins possessing histone
deacetylase activity, and represses transcription.
In this study, we have identified the BNP gene as a target of thyroid
hormone action in the myocardial cell. Interaction of the liganded TR
with the BNP gene promoter takes place largely through a single TRE
located ~1 kb upstream from the start site of transcription. This
interaction provides the substrate for assembly of various coactivator
and corepressor complexes that control transcription of this gene.
Materials--
[ Cell Culture--
Ventricular myocytes were prepared from
1-day-old neonatal rat hearts by alternate cycles of 0.05% trypsin
digestion and mechanical disruption as described previously (31). Cells
were cultured in Dulbecco's modified Eagle's medium containing 10%
enriched calf serum, 2 mM glutamine, 100 units/ml
penicillin, and 100 mg/ml streptomycin for 24 h. Cells were
changed to serum-free medium containing 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin prior to initiation of
each experiment.
BNP Radioimmunoassay--
The culture medium of each well was
collected after 24 h of treatment and centrifuged to remove
cellular debris; the supernatant was taken for assay. Radioimmunoassay
was performed according to the instructions provided by the
manufacturer (Phoenix Pharmaceuticals) using rabbit antiserum
specific for rat BNP-45 and 125I-labeled rat BNP-45.
Protein Synthesis--
Protein synthesis was assessed by
measuring [3H]leucine incorporation into cultured
ventricular myocytes. Cells were cultured in 24-well plates for 24 h and then changed to serum-free medium and subjected to treatment for
48 h. Cells were pulsed with [3H]leucine in
leucine-free medium for the final 4 h of the treatment period,
washed three times with phosphate-buffered saline (PBS), and extracted
with 10% trichloroacetic acid at 4 °C for 30 min. Cell residues
were rinsed in 95% ethanol, solubilized in 0.25 N NaOH for
2 h, and neutralized with 2.5 mM HCl plus 1 mM Tris-HCl (pH 7.5). Radioactivity was measured in a
liquid scintillation counter and normalized to cell number.
RNA Isolation and Northern Blot Analysis--
Total RNA was
isolated from cultured ventricular myocytes using RNAzol (LPS
Industries Inc., Moonachie, NJ). Fifteen µg of RNA was
size-fractionated on a 1.2% agarose gel containing 2% formaldehyde,
transferred to a nitrocellulose membrane, and hybridized simultaneously
with 32P-labeled 640-bp rat BNP cDNA and
32P-labeled 1.3-kilobase pair glyceraldehyde-3-phosphate
dehydrogenase cDNA. The membrane was washed and exposed to x-ray
film. Autoradiographic signals were scanned and quantified by NIH
Image. Normalized data are presented as the ratio of BNP to
glyceraldehyde-3-phosphate dehydrogenase mRNA levels in individual samples.
Plasmid Construction and Site-directed Mutagenesis--
The
construction of Transfection and Luciferase Assay--
Freshly prepared
ventricular myocytes were transiently transfected with the indicated
reporters (6 µg/1.5 × 107 cells) and expression
vectors (see figure legends for DNA concentrations) by electroporation
(Gene-Pulser, Bio-Rad) at 280 mV and 250 microfarads. DNA content in
individual cultures was normalized with pUC18. Cells were plated
(1.25 × 106/well) and cultured in Dulbecco's
modified Eagle's medium/H-21 containing 10% enriched calf serum for
24 h. Cells were then changed to serum-free medium overnight prior
to treatment. Cells were harvested and lysed in 200 µl of cell
culture lysis reagent (Promega). Cell lysates were processed for the
luciferase assay as described (32) using a commercially
available kit (Promega). Luciferase levels were normalized for
concentrations of soluble protein in the extracts. To ensure
reproducibility, experiments were repeated with three to five
independent cell preparations.
Immunostaining--
Ventricular myocytes were cultured in
four-chamber slides for 24 h and then changed to serum-free medium
and subjected to treatment for 48 h. Cells were washed with PBS
and incubated with 3.7% paraformaldehyde at room temperature for 20 min, followed by PBS containing 0.2% Triton for 2 min. Cells were
blocked with PBS containing 0.2% bovine serum albumin and 0.1 µg/ml
normal horse IgG and incubated with mouse anti-rat sarcomeric
Expression and Purification of GST Fusion Proteins from
Escherichia coli--
GST fusion expression vectors were
transformed into the BL21 strain of E. coli (Stratagene),
expanded in suspension culture, and induced with 1 mM
isopropyl- Electrophoretic Mobility Shift Assay
(EMSA)--
Oligonucleotides encoding wild-type and mutant TREs (both
sense strand) were as follows (TRE-like sequence is underlined, and
mutagenized bases are identified by lowercase letters): wild type,
5'-CGATCTCCTGACCTCGTGATCCGACCGCCTCG-3';
and mutant,
5'-CGATCTCCTtAtCaCGcGATCCGACCGCCTCG-3'. Oligonucleotides used for scanning mutagenesis were as follows: wild
type, 5'-CGATCTCCTTGACCTCGTGATCCGACCGCCTCG-3'; M1,
5'-CGATCTCCTTaAaaTCGTGATCCGACCGCCTCG-3'; M2,
5'-CGATCTCCTTGACCTCGTaATaaGACCGCCTCG-3'; M3,
5'-CGATCTCCTTGACCTCGTGATCCataaGCCTCG-3'; M4,
5'-CGATCTCCTTGACCTCGTGATCCGACCaaaTCG-3'; and M5,
5'-CGATaTaaTTGACCTCGTGATCCGACCGCCTCG-3'. hTR Statistics--
Data were analyzed using one-way analysis of
variance and the Newman-Keuls test to assess significance.
We began by examining the ability of T3 to increase
expression of the BNP gene in neonatal rat ventricular myocytes. As
shown in Fig. 1A, exposure to
T3 for 48 h resulted in a >6-fold increment in
release of BNP immunoreactivity from cultured myocytes. This was
accompanied by a 3-fold increase in steady-state BNP mRNA transcript levels (Fig. 1B). For reference, endothelin (ET),
a well documented activator of BNP gene expression (39, 40) and
hypertrophy (41) in this in vitro model, effected an almost 8-fold increase in BNP mRNA levels; however, the combination of ET
plus T3 resulted in an increase in transcript levels
(~20-fold) that was clearly greater than additive, implying a
synergistic interaction between these two agonists. T3
effected a modest increase (2-3-fold in this experiment) in BNP
promoter activity, whereas ET increased activity by ~10-fold (Fig.
1C). Once again, ET plus T3 synergistically
increased promoter activity by >60-fold.
1000 and
987
relative to the transcription start site. Both TR homodimers and
TR·retinoid X receptor heterodimers associated with this element in an electrophoretic mobility shift assay. Protein fragments harboring the LXXLL motifs of the
coactivators GRIP1 and SRC1 or TRAP220 interacted predominantly with
the TR·retinoid X receptor heterodimeric pair in a
ligand-dependent fashion. Both TR homodimers and
heterodimers in the unliganded state selectively associated with
glutathione S-transferase-nuclear receptor corepressor fragments harboring one of three receptor interaction domains containing the sequence (I/L)XX(I/V)I. These
interactions were dissociated following the addition of T3.
Collectively, these findings identify the BNP gene as a potential model
for the investigation of TR-dependent gene regulation in
the heart.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-adrenergic receptors (9),
-myosin heavy chain
(
-MHC) (2, 3, 10),
-myosin heavy chain (
-MHC) (2, 3, 10, 11), Na+/K+-ATPase (12), voltage-gated
K+ channels (13), phospholamban (14), type V and VI
adenylyl cyclases (15), Na+-Ca2+ exchanger (16,
17), and the thyroid hormone receptor (TR) itself (3).
T3-dependent effects appear to be unique from
those associated with so-called pathological hypertrophy
(e.g. that due to hemodynamic overload in vivo)
in that T3 treatment of the pathologically hypertrophied
myocardium produces a shift in the gene expression profile away from
that which is typically identified with pathological hypertrophy alone
(18) and toward a profile more closely resembling the physiological
hypertrophy associated with exercise training (3).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP,
[
-32P]ATP, and [3H]leucine were
purchased from PerkinElmer Life Sciences. Endothelin-1 and the BNP
radioimmunoassay kit were obtained from Phoenix Pharmaceuticals, Inc.
T3 was obtained from Sigma. Anti-human (h) TR
and
anti-hRXR
antibodies were purchased from Santa Cruz Biotechnology,
Inc. The TNT T7 Quick-Coupled Transcription/Translation
system was purchased from Promega. All oligonucleotides were
synthesized by Invitrogen. Other reagents were obtained through
standard commercial suppliers.
1595hBNP-luciferase has been described previously
(32). Expression vectors for human TR
(33), TR
(34), RXR
(35),
and GRIP1 (36) have also been described previously, as have expression
vectors encoding the GST-NCoR-His fusion proteins of the three NCoR
receptor interaction domains (RIDs) (37). The latter included RID1
(NCoR residues 2231-2321), RID2 (residues 2034-2114), and RID3
(residues 1888-2031). GST-GRIP1 (amino acids 618-770) was amplified
by standard PCR methodology from pSG5-GRIP1 using synthetic primers
incorporating EcoRI and SalI sites at the
5' and 3' termini of the coding sequence, respectively. Amplified DNA
was restricted with EcoRI and SalI and cloned
into the EcoRI and SalI sites of the bacterial
expression vector pGEX-5X (Amersham Biosciences). The GST-mutant GRIP1
(mGRIP1) expression vector was constructed in a similar fashion, except
that pSG5-mGRIP1 was used as a template. mGRIP1 contains double alanine
substitutions of two leucines (LXXLL
LXXAA)
in nuclear receptor boxes 2 (residues 685-701) and 3 (residues
740-756) (36). The latter are required for TR binding to GRIP1.
GST-TRAP220 has been reported previously by Yuan et al.
(38). GST-SRC1 was constructed by linking residues 632-752 of SRC1 to
six His residues at the carboxyl terminus of the fragment. An
NdeI site was then added to the amino terminus and a
SalI site to the carboxyl terminus of the chimeric fragment using conventional PCR methodology. The resultant fragment was cloned
between the NdeI and SalI sites of pGEX-5X.
Site-directed mutagenesis was carried out with the QuikChange kit
(Stratagene, La Jolla, CA) using conditions recommended by the
manufacturer. The sequence of the TRE mutagenic primer (sense strand)
was as follows (mutagenized bases are identified by lowercase letters): 5'-CGATCTCCTtAtCaCGcGATCCGACCGCCTCG-3'.
-actinin antibody (EA-53, Sigma) at 4 °C overnight. Cells were
washed three times with PBS and incubated with Texas Red-conjugated
horse anti-mouse secondary antibody (Vector Labs, Inc., Burlingame, CA)
at room temperature for 30 min. After three consecutive washes, cells were mounted with Vectashield mounting medium and viewed by
fluorescence microscopy. Linear dimensions and surface areas of
individual cells were calculated using semiautomatic computer-assisted
planimetry from two-dimensional images.
-D-thiogalactopyranoside. Cells were pelleted;
sonicated in 50 mM Tris-HCl (pH 7.5), 150 mM
NaCl, and 0.05% Tween 20; and centrifuged at 15,000 × g. The supernatant was decanted, added to 300 µl of
glutathione-Sepharose beads, and rotated at 4 °C for 1 h. The
mixture was transferred to a 10-ml Poly-Prep chromatography column
(Bio-Rad) and washed three times with PBS containing 0.1% Nonidet
P-40. Elution buffer containing 50 mM Tris-HCl (pH 8.0), 5 mM glutathione, and 1 mM dithiothreitol was
added to the column, and the elute was collected in 200-µl fractions.
Protein concentration was measured using Coomassie reagent. The peak
fractions were pooled and dialyzed against 50 mM Tris-HCl
(pH 8.0) at 4 °C overnight. GST fusion protein was aliquoted and
stored at
80 °C prior to use.
or hRXR
was
synthesized in vitro using the TNT T7
Quick-Coupled Transcription/Translation system according to the
manufacturer's instructions. Two µl of each in vitro
translated protein was incubated with purified
32P-end-labeled, double-stranded oligonucleotide in binding
reaction buffer containing 10 mM HEPES (pH 7.9), 50 mM KCl, 2 mM EDTA, 2.5 mM
dithiothreitol, 10% glycerol, 0.05% Nonidet P-40, and 0.5 µg of
poly(dI-dC) at room temperature for 30 min. For competition experiments, a 10-50-fold molar excess of unlabeled,
double-stranded oligonucleotide was added to the binding reaction. For
cofactor assembly experiments, varying amounts of GST-GRIP1, GST-SRC1, GST-TRAP220, or GST-NCoR RID1-3 fragments were added to the binding reaction in the absence or presence of 10 nM
T3. For immunoperturbation experiments, 1 µg of
polyclonal antibody directed against TR
or RXR
was included in
the reaction. All samples were resolved on 5% nondenaturing
polyacrylamide gels. Gels were dried and exposed to x-ray film to
generate autoradiographs.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of T3 and ET, alone or in
combination, on immunoreactive BNP secretion, BNP mRNA
levels, and BNP promoter activity in neonatal rat ventricular
myocytes. After 24 h of culture, cells were changed to
serum-free medium and treated with 10 nM T3,
100 nM ET, or both in combination for 48 h.
A, the medium was collected and subjected to
radioimmunoassay. B, 15 µg of total RNA was subjected to
blot hybridization analysis. Blots were hybridized simultaneously with
radiolabeled BNP and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) probes. Autoradiographs were quantified by NIH Image
and normalized for glyceraldehyde-3-phosphate dehydrogenase. Pooled data from four independent experiments are
presented as means ± S.D. C, cells were transfected
with 1 µg of 1595hBNP-luciferase, and luciferase activity was
measured 48 h later. Pooled data from three independent
experiments are presented as means ± S.D. Basal luciferase levels
at this concentration of transfected DNA were 427 ± 46 light
units/µg of protein. *, p < 0.01 versus
control; #, p < 0.05 versus control; **,
p < 0.01 versus ET.
Given the known ability of both T3 and ET to promote
distinct forms of hypertrophy of myocardial cells in vitro
(2, 41) and in vivo (4, 42), we examined the effects of
these agents on conventional markers of hypertrophy in the cultured
myocyte model. As shown in Fig.
2A, T3 and ET
stimulated protein synthesis, assessed as [3H]leucine
incorporation, by ~2.5- and 4-fold, respectively. The combination of
both agents was roughly additive, with a net induction of
~7-fold. Morphologically, both T3 and ET increased cell
size and sarcomeric organization in these cultured myocytes (Fig.
2B). The effect was somewhat more pronounced with ET
versus T3. Calculation of linear dimensions of
cells cultured under each of these conditions showed that ET increased
both the length and width of the individual myocytes, whereas
T3 preferentially affected the length (Fig. 2C).
Myocyte surface area was increased by both agents. In each instance,
the combination of both agents led to an increase in cell dimensions
and enhanced sarcomeric definition to a level that surpassed that with
either agent alone, supporting the protein synthesis data described
above.
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T3 effected a dose-dependent increment in BNP
promoter activity that peaked at 108 M, with
a maximal induction of 5-6-fold (Fig.
3A). Amplification of the
response was seen following cotransfection with either hTR
or hTR
(Fig. 3B). Both TR
and TR
are known
to be expressed endogenously in neonatal rat cardiac myocytes (3).
Maximal induction with TR
was ~7.5-fold in this series of studies,
whereas that with TR
was in the range of 4-fold.
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As noted above, the liganded TR is thought to exert most of its
transcriptional effects through association with its cognate TRE. This
leads to subsequent association with one of several coactivators that,
in turn, promote downstream events (histone acetylation, complex
assembly on the promoter, etc.), resulting in increased transcriptional
activity. As shown in Fig. 4, the addition of T3 to the cultures resulted in a 3-fold
increment in BNP-luciferase activity, which was increased to 5-fold
following cotransfection with the hTR expression vector and to
9-fold following cotransfection with both hTR
and the heterodimeric
partner RXR. Little or no suppression of basal activity by unliganded
TRs was observed, suggesting that the majority of the T3
effect that was observed corresponded to positive enhancement of
promoter activity rather than relief of basal repression. Of note,
cotransfection of the coactivator molecule GRIP1 together with TR
and RXR resulted in a significant increase in basal promoter activity
(this was dependent on the concentration of GRIP1 expression vector
added to the cells), which increased even further following the
addition of T3. The relative induction (i.e.
-fold induction) seen following GRIP1 cotransfection was lower than
that seen in cells transfected with TR
alone or TR
·RXR,
reflecting the elevation in basal promoter activity; however, the
absolute magnitude of the inductions was clearly greater in those
cultures cotransfected with GRIP1. The elevation in basal promoter
activity likely reflects the ability of the p160 coactivators to
amplify the activity of other non-ligand-dependent transcription factors (43) that may control the activity of this
promoter.
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Next, we attempted to identify the location of the TRE responsible for
trafficking the liganded TR signal on the BNP gene promoter. Careful
scrutiny of the known sequence for candidate sites revealed a potential
TRE at 1000 that showed a high degree of homology to the consensus
TRE (Fig. 5A) (44). Our
initial impression was that the TRE was arrayed as two direct repeats in reverse orientation. The 5'-repeat is a complete match to the consensus half-site (TGACCT; AGGTCA on the lower strand). There were
possible matches to a 3'-repeat (four of six positions) in both
direct repeat (DR) 2 (TGATCC; GGATCA on the lower strand) and
DR7 (CGACCG; CGGTCG on the lower strand) configurations.
Introduction of several nucleotide changes across this element (see
Fig. 5A for description of mutations) resulted in virtually
complete abrogation of the T3-dependent
induction of the BNP gene promoter (Fig. 5B) while exerting
no significant effect on basal promoter activity. Note that the bulk of
the nucleotide changes were introduced in the upstream consensus
half-site. Interestingly, this same mutation resulted in a partial
reduction (~40-45%) in ET-dependent activation of the
BNP promoter (Fig. 5C), suggesting a potential explanation for the synergistic interaction between ET and T3 noted
above.
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Using an EMSA, we explored the ability of the TR and RXR to associate
with this TRE in vitro. As shown in Fig.
6A, TR homodimers associated
with a labeled 32-bp oligonucleotide encoding the TRE from the BNP
promoter. This association was completely inhibited by wild-type
sequence (i.e. unlabeled, but otherwise identical double-stranded nucleotide), but not by a mutant sequence identical to
that produced for the functional studies carried out for Fig. 5. The
RXR did not bind appreciably to the TRE, but the TR·RXR heterodimer
bound well and migrated in a position that was readily separated from
the homodimer. Once again, unlabeled wild-type sequence (even when
reduced to 10-fold excess), but not mutant sequence, proved capable of
disrupting this interaction. Of note, when mutations were introduced
into the candidate half-sites of the DR2 element alluded to above (Fig.
6B), only those in the consensus half-site (M1)
significantly impaired binding of the heterodimer to DNA (Fig.
6C). Mutations in the putative non-consensus DR2 half-site
(M2), as well as two additional downstream sites in or near the
putative DR7 half-site (M3 and M4) and one upstream site (M5), had no
effect on heterodimer binding to the TRE. When the analysis was
repeated for TR homodimer binding, M1 again proved most critical for
the DNA-protein interaction. M2, M4, and M5 had no effect, whereas M3
(in sequences within the possible DR7 half-site) also showed a
significant reduction in homodimer binding (Fig.
6C).
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Because the BNP TRE did not adhere to the exact DR4 consensus, we
further examined the properties of the BNP TRE-bound TR homodimeric and
heterodimeric complexes in EMSAs. The TRE-bound TR·RXR heterodimers
also proved capable of interacting with members of the p160 class of
coactivator proteins in this EMSA. As shown in Fig.
7A, the heterodimer, as well
as the TR homodimer, associated with the TRE in the absence of ligand.
The addition of T3 effected a modest alteration in the
binding intensity and migration pattern (slightly faster migration
pattern) of the heterodimeric complex. The addition of a GST-linked
GRIP1 fragment (amino acids 618-770), which contains the
LXXLL motif required for association with the TR (36), led
to the appearance of a slower mobility band on the gel. The addition of
ligand enhanced this band considerably and resulted in a reduction in
the intensity of the signal identified with the isolated heterodimeric
complex. The mutant GRIP1 construct mGRIP1, with a mutation in the
LXXLL motif (36), was, as expected, unable to interact with
the heterodimeric complex in the presence or absence of ligand. A
receptor-binding fragment of SRC1 (amino acids 632-752), another
member of the p160 coactivator family, also showed evidence of modest
interaction with the heterodimeric complex in the absence of ligand;
and again, the addition of T3 resulted in amplification of
the putative receptor·coactivator complex. Finally, the heterodimer
also appeared to be capable of associating with a GST-linked fragment
of TRAP220 (amino acids 622-701), a subunit of the TRAP complex that
has previously been shown to interact with the TR·RXR heterodimer
(38), putatively providing a molecular link to the core transcriptional
machinery. As shown in Fig. 7A, TRAP220, like the p160
coactivators, associated with the heterodimer to a modest degree in the
absence of ligand; and again, this association increased dramatically
following the addition of T3. The addition of
T3 effected a significant reduction in TR homodimer
association with the BNP TRE (Fig. 7B); however, as with the
heterodimer described above, there was evidence of a slight interaction
of the unliganded homodimer with the GST-GRIP1, GST-SRC1, and
GST-TRAP220 fragments, and this interaction increased modestly, albeit
not to the levels seen with the heterodimer, following the addition of
T3.
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To provide assurance that the coactivator-shifted complexes
did, in fact, contain the TR·RXR heterodimer, we perturbed the shifted complexes with antibodies directed against hTR and hRXR
. As shown in Fig. 8, anti-TR
antibody
successfully supershifted the TR·RXR complex as well as the
GST-GRIP1- and GST-SRC1-shifted complexes. Antibody directed against
the RXR was similarly effective in supershifting these same three
complexes, implying that both the TR and RXR are present in the
p160-dependent complexes that assemble in the presence of
ligand.
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Next, we investigated whether binding of GRIP1 and TRAP220 is mutually
exclusive in the context of TR·RXR bound to DNA. Previous studies
have suggested that the p160 coactivators and TRAP220 bind in a
competitive fashion to receptor in solution (45). As shown in Fig.
9, the addition of increasing amounts of
GRIP1 protein reduced TRAP220 binding to the DNA-bound heterodimer and vice versa. This suggests that these two coactivators, although structurally unrelated and possessed of independent regulatory activity, operate through association with the same or a similar interface on the receptor. Of incidental note, in addition to promoting
assembly of the heterodimer into a slower migrating complex, TRAP220
also increased the levels of TR monomer bound to DNA, implying that
this interaction promotes a fundamental alteration in receptor
conformation.
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Finally, it is known that the unliganded TR displays a propensity for
association with the transcription-suppressing corepressors NCoR (27,
28) and SMRT (29, 30). In the case of NCoR, this is thought to occur
through association of the receptor(s) with one or more RID boxes
present in the corepressor protein (37, 46-48). Recent studies
indicate that the TR employs three RID boxes in the NCoR molecule to
promote this association, whereas the unliganded estrogen receptor and
RXR largely employ RID1 and RID3 to effect stable interaction with
NCoR (37). As shown in Fig. 10, the TR
homodimer proved capable of interacting strongly with each of the three
RID box-containing fragments (definition of the exact
sequence incorporated in these individual constructs is presented under
"Experimental Procedures"). In each case, the addition of
T3 predictably resulted in abrogation of the TR-NCoR interaction as well as dissociation of the TR homodimer from the BNP
TRE. Interestingly, the unliganded TR·RXR heterodimer also proved
capable of associating with the individual NCoR fragments. There was
preferential association of the heterodimer with RID2 and, to a lesser
extent, RID3, with little or no interaction with RID1. This suggests
that the TR has the capacity to preferentially interact with selected
domains in the NCoR molecule depending on whether it engages the TRE as
a homodimeric or heterodimeric complex.
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DISCUSSION |
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Several key findings emerge from this study. First, it provides the first identification of the BNP gene promoter as a target of thyroid hormone action in the heart. Second, it demonstrates that the effects of T3 and ET, a more conventional hypertrophic agonist in this in vitro model (41), interact functionally in promoting both the morphological changes associated with hypertrophy and activation of the BNP gene promoter. Third, it identifies the TRE in the BNP gene that provides the substrate for assembly of coregulatory complexes on this promoter. Finally, it identifies several features of complex assembly that appear to be unique to the DNA-associated TR.
The BNP response was temporally linked to the development of the hypertrophic phenotype, supporting the hypothesis that activation of BNP gene transcription serves as a marker of hypertrophy in this model (19). The function of BNP in the in vivo setting of thyroid hormone excess is unknown. BNP could optimize cardiac performance through reduction in filling pressures and afterload (49). Alternatively, it could act to control the progression and magnitude of the hypertrophic response through a local negative feedback loop (50, 51), thereby limiting cardiac fibrosis, chamber remodeling, and progression to cardiomyopathy. The cardiac hypertrophy associated with thyroid hormone excess is notably deficient in accompanying fibrosis (52).
The gene expression profile in pathological hypertrophy
(i.e. that associated with hemodynamic overload in
vivo or G protein-coupled receptor activation in vitro)
closely resembles that associated with hypothyroidism in the cardiac
myocyte, whereas physiological hypertrophy has been linked to changes
in gene expression that more closely resemble those associated with
thyroid hormone administration (3, 53). This dichotomy has led some
investigators to suggest that pathological hypertrophy may represent a
state of functional hypothyroidism within the cardiac myocyte. In fact,
Kinugawa et al. (3) recently documented reductions in
endogenous TR levels in both phenylephrine-induced hypertrophy in
cultured myocytes as well as pathological hypertrophy generated by
constriction of the rat ascending aorta in vivo. In a
separate study, Kinugawa et al. (54) demonstrated a
reduction in TR1 levels in failing human ventricular tissue with a
coincident increase in the levels of TR
2, a TR isoform that does not
bind ligand, yet is capable of functioning in a dominant-negative mode
to inhibit TR action. TR
1 levels were unchanged relative to
age-matched controls. These data remain controversial in that others
have published conflicting findings (55-57), but they raise the
intriguing possibility that relative TR deficiency may account for the
functional hypothyroid picture that dominates the gene expression
profile in pathological hypertrophy. Of note, Chang et al.
(18) showed that thyroid hormone (in this case, thyroxine)
administration to rats with left ventricular hypertrophy post-aortic
banding normalized myocardial function with improved contractility,
faster relaxation, and increased concentrations of
-MHC and SERCA
proteins relative to those without thyroxine treatment. These data
support the model discussed above and suggest that exogenous thyroid
hormone can suppress at least some of the phenotypic features
associated with pathological hypertrophy.
Although both ET and T3 appear to stimulate activation of hypertrophic markers in these neonatal myocytes, the phenotype of myocytes treated with these agents is unique; and by inference, the qualitative features of the hypertrophy are distinct. T3 promoted elongation of the myocytes with only a marginal increase in the overall surface area. On the other hand, ET appeared to increase both the length and width of the individual myocytes. The combination of both agents promoted an increase in total cell dimension and a dramatic increase in sarcomeric organization relative to either treatment alone. It is conceivable that these differences are related to the distinct phenotypic changes that accompany T3-induced versus pathological (e.g. overload-induced) hypertrophy in vivo.
Similar to the findings reported here, Kinugawa et al. (3)
demonstrated an additive effect of T3 and the hypertrophic
agonist phenylephrine in increasing protein synthesis in cardiac
myocyte cultures. However, unlike our results, they found that
T3 and phenylephrine acted in opposing fashion in
regulating thyroid hormone-responsive genes in these cells.
T3 increased -MHC and SERCA levels and suppressed
-MHC, whereas phenylephrine increased
-MHC and reduced
-MHC
and SERCA. The combination of both T3 and phenylephrine led
to an intermediate response. A similar dichotomy was noted upon
comparison of exercise (physiological)-induced versus aortic
constriction (pathological)-induced hypertrophy in vivo,
with the gene expression profile in the former more closely resembling
that of T3 treatment in vitro. In this study, ET
and T3 operated in synergistic fashion to drive expression
of the BNP gene and activation of the hBNP promoter, implying that
thyroid hormone and G protein-coupled receptors can operate in parallel rather than opposing fashion in governing gene expression. The mechanism underlying the synergy between ET and T3 remains
unclear. At least a portion of the ET prohypertrophic activity resides outside the TRE because deletions down to within 198 bp of the transcription start site, which effectively eliminate the TRE from the
promoter, retain the ability to respond to ET (data not shown). On the
other hand, the data presented in Fig. 5C indicate that the
TRE mutant displays a subnormal response to ET (~50% of the control
response), implying that ET may indirectly influence signaling through
the TR·TRE complex. The mechanism underlying this effect is unknown,
but could involve alterations in TR binding to DNA, recruitment of
coregulators to the TR·TRE complex, or assembly of the complex with
the core transcriptional apparatus.
Kinugawa et al. (3) also demonstrated TR
isoform-specific effects on individual promoters in their study, with
-MHC, SERCA, and TR
1 responding primarily to TR
1 and
-MHC,
as well as overall myocyte size, falling under the control of TR
1.
Based on the findings presented in Fig. 3B, the BNP gene
appears to fall in the former category, primarily under the control of
TR
1; however, this finding is difficult to substantiate without
concomitant measurement of exogenous (transfectant-expressed) TR
levels, and additional studies using isoform-specific agonists will be
required to provide definitive support for this conclusion.
The TR typically associates with direct repeats of the sequence AGGTCA
in a DR4 configuration (33), although numerous exceptions have been
described in the literature (58). The TRE that we have identified in
the BNP gene promoter does not conform readily to this typical
TR-binding configuration. The upstream consensus half-site (TGACCT;
AGGTCA on the lower strand) appears to be absolutely required for both
TR DNA binding and functional activity. Sequence requirements for
monomer binding to the second "half-site" are either very liberal
or nonexistent for heterodimer binding because mutations upstream and
downstream from the consensus half-site had no effect on binding to
DNA. Heterodimer-binding functional TREs consisting solely of
half-sites have been described in other systems (55-57), where it is
assumed that relatively nonspecific contacts between contiguous DNA and
one of the partners are sufficient to stabilize the complex. Sequence
requirements for homodimer binding to the BNP TRE may be slightly more
stringent because downstream mutations in the M3 region led to a
significant reduction in TR-binding activity (Fig. 6, B and
C). One candidate half-site (CGACCG; CGGTCG on the lower
strand) that is affected by this mutation is arrayed as a direct repeat
with the consensus half-site with a 7-nucleotide spacer. The rather
limited restrictions on TR binding to this site raise the possibility
that other nuclear receptors may employ this same regulatory element to
activate or suppress BNP gene transcription. In fact, our preliminary
studies suggest that peroxisome proliferator-activated receptor-
(but not vitamin D receptor) also associates with this element
(data not shown); however, mutation of this site does not interfere with peroxisome proliferator-activated
receptor-
-dependent regulation of the BNP promoter (data
not shown), suggesting that the signal transduction system underlying
the latter is not as straightforward as that for the TR. Moreover, the
unusual nature of the BNP TRE may suggest why unliganded TRs failed to
suppress basal BNP promoter activity; the extent of suppression of
basal promoter activity varies according to the exact topology of the
TR DNA-binding site (58).
Despite its unusual configuration, both homodimeric (TR·TR) and heterodimeric (TR·RXR) complexes bound avidly to the TRE in the unliganded state. Moreover, the addition of ligand preferentially dissociates the homodimer (but not the heterodimer) from DNA, as reported previously for other TREs (59). Both the BNP TRE-bound homodimer and heterodimer associated with the p160 coactivator (GRIP1 and SRC1) and TRAP220 binding motifs; and in all instances, this association was stimulated by the addition of ligand. However, the interaction was considerably stronger (both in the presence and absence of the ligand) with the heterodimer. Of note, the presence of the TRAP220 binding motif appeared to promote the binding of the TR monomer to DNA in either the presence or absence of ligand, a property not shared by the p160 coactivators, suggesting that TRAP220 promotes a fundamental and unique alteration in receptor conformation. Despite this and the apparent assembly of a TR·TRAP220 complex in the presence of ligand, the affinity of the isolated homodimer for DNA was reduced significantly in the presence of ligand.
It has been shown that the p160 coactivators and TRAP220 bind sequentially to the liganded TR complex to trigger increased transcriptional activity (25, 26, 60, 61). Binding of the liganded DNA-bound heterodimer to GRIP1 versus TRAP220 is competitive in nature, implying that the LXXLL motifs in both fragments associate with a common receptor interface, a finding that supports those of Treuter et al. (45), who found a similar competitive interaction between TRAP220 and TIF2 (transcriptional intermediary factor-2), the human homolog of GRIP1. Based on the studies presented in Fig. 9, it appears that the affinity of the heterodimer for these two coactivators fragments is similar. If differential affinities of these coactivators for the TR·RXR complex do, in fact, exist in the intact cell, our findings may reflect a requirement for additional regulatory factors, not present in these assays, that might serve to amplify or reduce the affinity of the individual coactivators for the TR. Alternatively, the affinity of these fragments for the TR may not mirror that of their respective holoproteins.
The BNP TRE-bound homodimeric and heterodimeric complexes also
interacted with corepressors. The ability of each of three RID boxes to
associate with the unliganded but DNA-associated homodimeric TR was
approximately equivalent, supporting the previous studies carried out
using radiolabeled TR and GST-RID in solution (37). This stands in
contrast to the results obtained for hRXR (with helix 12 deleted),
which bound RID1 well and showed very little binding to RID3 and
almost no binding to RID2, and for human estrogen receptor-
(with helix 12 deleted), which showed binding to RID1 and RID3, but not
to RID2 (37). Binding to the unliganded DNA-bound TR·RXR heterodimer
was distinct from each of the patterns in that RID2 established the
strongest contact with the receptors, whereas RID3 displayed a lower
level of binding, and RID1 showed effectively no binding at all. There
was also an apparent reduction in the level of RID binding to the TR
heterodimeric versus homodimeric complex, although this may
reflect, in part, a reduced level of heterodimer versus
homodimer association with the TRE itself. Collectively, these data
suggest that association of unliganded receptors (estrogen
receptor versus RXR versus homodimeric TR
versus heterodimeric TR) with the transcriptional
corepressors leads them to adopt conformations that permit formation of
unique receptor-corepressor contacts. Similar heterogeneity in
receptor-corepressor binding has been noted by Cohen et al.
(47), who found that the retinoic acid receptor binds preferentially to
SMRT, whereas the TR prefers NCoR (47). They attributed these selected
interactions to sequence differences in the region in and around RID2
(termed N2 in their study) versus the equivalent region (S2)
in SMRT as well as the presence of a TR-specific binding domain termed
N3 (RID3 in this study) in NCoR that is absent in SMRT (48). Zamir et al. (62) have shown that the orphan nuclear receptor
RevErb associates with NCoR (but not SMRT), also presumably based on differential binding affinities for the available binding motifs in
these two corepressor proteins. Collectively, these studies support the
notion that different affinities for individual receptor-RID interactions may dictate the selectivity of receptor-corepressor assembly on the promoters of target genes and the subsequent regulation of transcriptional activity. As a corollary of this, heterogeneity in
RID box binding raises the possibility that peptidomimetic agents could
be developed that selectively target specific receptor-corepressor interactions.
In summary, the BNP gene has been identified as a transcriptional
target for the liganded TR in the cardiac myocyte. The TRE responsible
for this activity in the BNP promoter is positioned approximately 1 kb
upstream from the transcription start site and displays the capacity to
interact with the TR and to assemble both coactivator and corepressor
complexes in vitro. This system may prove to be an excellent
model for investigation of thyroid hormone-sensitive gene expression in
the heart.
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ACKNOWLEDGEMENT |
---|
We are grateful to Dr. Feng Wang for assistance with a number of the transfection analyses.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL35753 and American Heart Association Grant 9950062N.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.
¶ To whom correspondence should be addressed: Diabetes Center, 1109 HSW, University of California, 3rd and Parnassus Ave., San Francisco, CA 94143-0540. Tel.: 415-476-2729; Fax: 415-564-5813; E-mail: gardner@itsa.ucsf.edu.
Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M207593200
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ABBREVIATIONS |
---|
The abbreviations used are: T3, triiodothyronine; SERCA, sarcoplasmic endoplasmic reticulum Ca2+-ATPase; MHC, myosin heavy chain; TR, thyroid hormone receptor; BNP, brain natriuretic peptide; TRE, thyroid hormone response element; RXR, retinoid X receptor; NCoR, nuclear receptor corepressor; h, human; PBS, phosphate-buffered saline; GST, glutathione S-transferase; RID, receptor interaction domain; EMSA, electrophoretic mobility shift assay; ET, endothelin; DR, direct repeat.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Klein, I.,
and Ojamaa, K.
(2001)
N. Engl. J. Med.
344,
501-509 |
2. |
Deng, X. F.,
Rokosh, D. G.,
and Simpson, P. C.
(2000)
Circ. Res.
87,
781-788 |
3. |
Kinugawa, K.,
Yonekura, K.,
Ribeiro, R. C.,
Eto, Y.,
Aoyagi, T.,
Baxter, J. D.,
Camacho, S. A.,
Bristow, M. R.,
Long, C. S.,
and Simpson, P. C.
(2001)
Circ. Res.
89,
591-598 |
4. | Basset, A., Blanc, J., Messas, E., Hagege, A., and Elghozi, J. L. (2001) J. Cardiovasc. Pharmacol. 37, 163-172[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Kobori, H.,
Ichihara, A.,
Miyashita, Y.,
Hayashi, M.,
and Saruta, T.
(1999)
J. Endocrinol.
160,
43-47 |
6. | Arai, M., Otsu, K., MacLennan, D. H., Alpert, N. R., and Periasamy, M. (1991) Circ. Res. 69, 266-276[Abstract] |
7. |
Hartong, R.,
Wang, N.,
Kuiokawa, R.,
Lazar, M.,
Glass, C. K.,
Apriletti, J. W.,
and Dillmann, W. H.
(1994)
J. Biol. Chem.
269,
13021-13029 |
8. | Gardner, D. G., Gertz, B. J., and Hane, S. (1987) Mol. Endocrinol. 1, 260-265[Abstract] |
9. |
Hoit, B. D.,
Khoury, S. F.,
Shao, Y.,
Gabel, M.,
Liggett, S. B.,
and Walsh, R. A.
(1997)
Circulation
96,
592-598 |
10. | Izumo, S., Lompre, A. M., Matsuoka, R., Koren, G., Schwartz, K., Nadal-Ginard, B., and Mahdavi, V. (1987) J. Clin. Invest. 79, 970-977[Medline] [Order article via Infotrieve] |
11. | Ojamaa, K., Klemperer, J. D., MacGilvray, S. S., Klein, I., and Samarel, A. (1996) Endocrinology 137, 802-808[Abstract] |
12. | Gick, G. G., Melikian, J., and Ismail-Beigi, F. (1990) J. Membr. Biol. 115, 273-282[Medline] [Order article via Infotrieve] |
13. |
Ojamaa, K.,
Sabet, A.,
Kenessey, A.,
Shenoy, R.,
and Klein, I.
(1999)
Endocrinology
140,
3170-3176 |
14. | Kiss, E., Jakab, G., Kranias, E. G., and Edes, I. (1994) Circ. Res. 75, 245-251[Abstract] |
15. | Ojamaa, K., Klein, I., Sabet, A., and Steinberg, S. F. (2000) Metabolism 49, 275-279[Medline] [Order article via Infotrieve] |
16. | Boerth, S. R., and Artman, M. (1996) Cardiovasc. Res. 31, E145-E152[CrossRef][Medline] [Order article via Infotrieve] |
17. | Hojo, Y., Ikeda, U., Tsuruya, Y., Ebata, H., Murata, M., Okada, K., Saito, T., and Shimada, K. (1997) J. Cardiovasc. Pharmacol. 29, 75-80[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Chang, K. C.,
Figueredo, V. M.,
Schreur, J. H.,
Kariya, K.,
Weiner, M. W.,
Simpson, P. C.,
and Camacho, S. A.
(1997)
J. Clin. Invest.
100,
1742-1749 |
19. | Nakagawa, O., Ogawa, Y., Itoh, H., Suga, S., Komatsu, Y., Kishimoto, I., Nishino, K., Yoshimasa, T., and Nakao, K. (1995) J. Clin. Invest. 96, 1280-1287[Medline] [Order article via Infotrieve] |
20. | Mukoyama, M., Nakao, K., Saito, Y., Ogawa, Y., Hosoda, K., Suga, S., Shirakami, G., Jougasaki, M., and Imura, H. (1990) N. Engl. J. Med. 323, 757-758[Medline] [Order article via Infotrieve] |
21. | Hystad, M. E., Geiran, O. R., Attramadal, H., Spurkland, A., Vege, A., Simonsen, S., and Hall, C. (2001) Acta Physiol. Scand. 171, 395-403[CrossRef][Medline] [Order article via Infotrieve] |
22. | Kohno, M., Horio, T., Yasunari, K., Yokokawa, K., Ikeda, M., Kurihara, N., Nishizawa, Y., Morii, H., and Takeda, T. (1993) Metabolism 42, 1059-1064[Medline] [Order article via Infotrieve] |
23. | Zhang, J., and Lazar, M. A. (2000) Annu. Rev. Physiol. 62, 439-466[CrossRef][Medline] [Order article via Infotrieve] |
24. | Jenkins, B. D., Pullen, C. B., and Darimont, B. D. (2001) Trends Endocrinol. Metab. 12, 122-126[CrossRef][Medline] [Order article via Infotrieve] |
25. | Ito, M., and Roeder, R. G. (2001) Trends Endocrinol. Metab. 12, 127-134[CrossRef][Medline] [Order article via Infotrieve] |
26. | Ito, M., Yuan, C. X., Okano, H. J., Darnell, R. B., and Roeder, R. G. (2000) Mol. Cell 5, 683-693[Medline] [Order article via Infotrieve] |
27. | Horlein, A. J., Naar, A. M., Heinzel, T., Torchia, J., Gloss, B., Kurokawa, R., Ryan, A., Kamei, Y., Soderstrom, M., Glass, C. K., and Rosenfeld, M. G. (1995) Nature 377, 397-404[CrossRef][Medline] [Order article via Infotrieve] |
28. | Chen, J. D., and Evans, R. M. (1995) Nature 377, 454-457[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Ordentlich, P.,
Downes, M.,
Xie, W.,
Genin, A.,
Spinner, N. B.,
and Evans, R. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2639-2644 |
30. |
Park, E. J.,
Schroen, D. J.,
Yang, M.,
Li, H.,
Li, L.,
and Chen, J. D.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3519-3524 |
31. |
Wu, J.,
LaPointe, M. C.,
West, B. L.,
and Gardner, D. G.
(1989)
J. Biol. Chem.
264,
6472-6479 |
32. |
LaPointe, M. C.,
Wu, G.,
Garami, M.,
Yang, X. P.,
and Gardner, D. G.
(1996)
Hypertension
27,
715-722 |
33. | Umesono, K., and Evans, R. M. (1989) Cell 57, 1139-1146[Medline] [Order article via Infotrieve] |
34. |
Feng, W.,
Ribeiro, R. C.,
Wagner, R. L.,
Nguyen, H.,
Apriletti, J. W.,
Fletterick, R. J.,
Baxter, J. D.,
Kushner, P. J.,
and West, B. L.
(1998)
Science
280,
1747-1749 |
35. | Kliewer, D. A., Umesono, K., Heyman, R. A., Mangelsdorf, D. J., Dyck, J. A., and Evans, R. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1448-1452[Abstract] |
36. |
Ding, X. F.,
Anderson, C. M.,
Ma, H.,
Hong, H.,
Uht, R. M.,
Kushner, P. J.,
and Stallcup, M. R.
(1998)
Mol. Endocrinol.
12,
302-313 |
37. |
Marimuthu, A.,
Feng, W.,
Tagami, T.,
Nguyen, H.,
Jameson, J. L.,
Fletterick, R. J.,
Baxter, J. D.,
and West, B. L.
(2002)
Mol. Endocrinol.
16,
271-286 |
38. |
Yuan, C. X.,
Ito, M.,
Fondell, J. D.,
Fu, Z. Y.,
and Roeder, R. G.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7939-7944 |
39. |
Liang, F.,
and Gardner, D. G.
(1998)
J. Biol. Chem.
273,
14612-14619 |
40. |
Liang, F.,
Lu, S.,
and Gardner, D. G.
(2000)
Hypertension
35,
188-192 |
41. | Ito, H., Hirata, Y., Adachi, S., Tanaka, M., Tsujino, M., Koike, A., Nogami, A., Marumo, F., and Hiroe, M. (1991) J. Clin. Invest. 92, 398-403 |
42. | Ito, H., Hiroe, M., Hirata, Y., Fujisaki, H., Adachi, S., Akimoto, H., Ohta, Y., and Marumo, F. (1994) Circulation 89, 2198-2203[Abstract] |
43. |
Rogatsky, I.,
Zarember, K. A.,
and Yamamoto, K. R.
(2001)
EMBO J.
20,
6071-6083 |
44. | Umesono, K., Murakami, K. K., Thompson, C. C., and Evans, R. M. (1991) Cell 65, 1255-1266[Medline] [Order article via Infotrieve] |
45. |
Treuter, E.,
Johansson, L.,
Thomsen, J. S.,
Warnmark, A.,
Leers, J.,
Pelto-Huikko, M.,
Sjoberg, M.,
Wright, A. P.,
Spyrou, G.,
and Gustafsson, J. A.
(1999)
J. Biol. Chem.
274,
6667-6677 |
46. |
Webb, P.,
Anderson, C. M.,
Valentine, C.,
Nguyen, P.,
Marimuthu, A.,
West, B. L.,
Baxter, J. D.,
and Kushner, P. J.
(2000)
Mol. Endocrinol.
14,
1976-1985 |
47. |
Cohen, R. N.,
Putney, A.,
Wondisford, F. E.,
and Hollenberg, A. N.
(2000)
Mol. Endocrinol.
14,
900-914 |
48. |
Cohen, R. N.,
Brzostek, S.,
Kim, B.,
Chorev, M.,
Wondisford, F. E.,
and Hollenberg, A. N.
(2001)
Mol. Endocrinol.
15,
1049-1061 |
49. | Yoshimura, M., Yasue, H., Morita, E., Sakaino, N., Jougasaki, M., Kurose, M., Mukoyama, M., Saito, Y., Nakao, K., and Imura, H. (1991) Circulation 84, 1581-1588[Abstract] |
50. |
Kishimoto, I.,
Rossi, K.,
and Garbers, D. L.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
2703-2706 |
51. |
Tamura, N.,
Ogawa, Y.,
Chusho, H.,
Nakamura, K.,
Nakao, K.,
Suda, M.,
Kasahara, M.,
Hashimoto, R.,
Katsuura, G.,
Mukoyama, M.,
Itoh, H.,
Saito, Y.,
Tanaka, I.,
Otani, H.,
and Katsuki, M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4239-4244 |
52. | Yao, J., and Eghbali, M. (1992) Circ. Res. 71, 831-839[Abstract] |
53. |
Rupp, H.,
and Wahl, R.
(1990)
J. Appl. Physiol.
68,
973-978 |
54. |
Kinugawa, K.,
Minobe, W. A.,
Wood, W. M.,
Ridgway, E. C.,
Baxter, J. D.,
Ribeiro, R. C.,
Tawadrous, M. F.,
Lowes, B. A.,
Long, C. S.,
and Bristow, M. R.
(2001)
Circulation
103,
1089-1094 |
55. | Kurokawa, R., Yu, V. C., Naar, A., Kyakumoto, S., Han, Z., Silverman, S., Rosenfeld, M. G., and Glass, C. K. (1993) Genes Dev. 7, 1423-1435[Abstract] |
56. |
Yen, P. M.,
Ikeda, M.,
Wilcox, E. C.,
Brubaker, J. H.,
Spanjaard, R. A.,
Sugawara, A.,
and Chin, W. W.
(1994)
J. Biol. Chem.
269,
12704-12709 |
57. | Ikeda, M., Rhee, M., and Chin, W. W. (1994) Endocrinology 135, 1628-1638[Abstract] |
58. |
Yoh, S. M.,
and Privalsky, M. L.
(2001)
J. Biol. Chem.
276,
16857-16867 |
59. |
Yen, P. M.,
Darling, D. S.,
Carter, R. L.,
Forgione, M.,
Umeda, P. K.,
and Chin, W. W.
(1992)
J. Biol. Chem.
267,
3565-3568 |
60. |
Sharma, D.,
and Fondell, J. D.
(2000)
Mol. Endocrinol.
14,
2001-2009 |
61. |
Sharma, D.,
and Fondell, J. D.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
7934-7939 |
62. | Zamir, I., Harding, H. P., Atkins, G. B., Horlein, A., Glass, C. K., Rosenfeld, M. G., and Lazar, M. A. (1996) Mol. Cell. Biol. 16, 5458-5465[Abstract] |