Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Michigan 48202
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ABSTRACT |
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Brain natriuretic peptide (BNP) is a
cardiac hormone constitutively expressed in the adult heart. We
previously showed that the human BNP (hBNP) proximal promoter region
from 127 to
40 confers myocyte-specific expression. The proximal
hBNP promoter contains several putative cis elements. Here
we tested whether the proximal GATA element plays a role in basal and
inducible regulation of the hBNP promoter. The hBNP promoter was
coupled to a luciferase reporter gene (1818hBNPLuc) and transferred
into neonatal ventricular myocytes (NVM), and luciferase activity was measured as an index of hBNP promoter activity. Mutation of the putative GATA element at
85 of the hBNP promoter
[1818(mGATA)hBNPLuc] reduced activity by 97%. To study
transactivation of the hBNP promoter, we co-transfected 1818hBNPLuc
with the GATA-4 expression vector. GATA-4 activated 1818hBNPLuc, and
this effect was eliminated by mutation of the proximal GATA element.
Electrophoretic mobility shift assay showed that an oligonucleotide
containing the hBNP GATA motif bound to cardiomyocyte nuclear protein,
which was competed for by a consensus GATA oligonucleotide but not a
mutated hBNP GATA element. The
-adrenergic agonist isoproterenol and
its second messenger cAMP stimulated hBNP promoter activity and binding
of nuclear protein to the proximal GATA element. Thus the GATA element in the proximal hBNP promoter is involved in both basal and inducible transcriptional regulation in cardiac myocytes.
cardiomyocyte; gene regulation; adrenergic agonists; cyclic adenosine monophosphate
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INTRODUCTION |
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BRAIN NATRIURETIC PEPTIDE (BNP) is expressed in the adult heart and is primarily a ventricular hormone with actions similar to atrial natriuretic peptide (ANP), including natriuretic, diuretic, and vasodilator properties. The BNP gene is induced during myocardial infarction, cardiac hypertrophy, and heart failure, and circulating plasma BNP is a reliable biochemical marker of left ventricular dysfunction in these pathophysiological states (35, 39).
The proximal promoter region of the hBNP gene, located from 127 to
40 relative to the transcription start site, is important for
tissue-specific expression, because this region confers
myocyte-specific expression on a heterologous thymidine kinase promoter
(17). We have previously shown that this region of the
promoter contains M-CAT-like elements (at
97 and
124) that are
involved in both basal and inducible regulation of the gene
(11). The GATA and activator protein (AP)-1-like motifs at
85 (GATAAA) and
111 (TGATCTCA) are two other putative elements.
GATA-4, a member of the zinc finger GATA transcription factor family,
is involved in cardiac development (7, 8, 23). There are
six members in the GATA family. GATA-1, -2, and -3 are expressed in the
hematopoietic system, whereas GATA-4, -5, and -6 are expressed in the
heart (18, 40). GATA-4 has been shown to regulate
transcription of a number of genes expressed in the heart, including
cardiac troponin C (13) and I (31), rat ANP (6) and BNP (6, 38), the m2 subtype of the
acetylcholine receptor (33), the cardiac
Na+/Ca2+ exchanger NCX1 (32), and
cardiac-specific -myosin heavy chain (
-MHC) (22).
Studies have suggested that the GATA-4 element plays a role in
1-adrenergic agonist-induced endothelin-1 gene
expression in cardiac myocytes in vitro (26), as well as
in cardiac hypertrophy induced by adrenergic agonists or pressure
overload in vivo (10, 12, 34). A number of neurohumoral
agents contribute to hypertrophy of cardiac myocytes, including
-
and
-adrenergic agonists (25). Presently, it is not
known whether the proximal GATA site in the hBNP promoter is a target
of these neurohumoral agents.
We used transient co-transfection of an expression vector encoding
GATA-4 with the hBNP promoter coupled to a luciferase reporter gene,
mutagenesis of the putative GATA motif, and electrophoretic mobility
gel shift analysis (EMSA) to determine the functional relevance of this
cis element to the hBNP promoter. Our results suggest that
the GATA motif at 85 is functionally relevant in the regulation of
basal and inducible transcription.
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MATERIALS AND METHODS |
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Cell culture. All protocols using live animals were approved by the Henry Ford Institutional Animal Care and Use Committee in accordance with federal guidelines. Ventricular myocyte-enriched cultures were generated from Sprague-Dawley rat pups (Charles River, Kalamazoo, MI) as described previously (17). Neonatal ventricular myocytes (NVM) were separated from myocardial fibroblasts by differential plating. Ventricular myocytes were plated in DMEM (GIBCO) containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 0.1 mM 5'-bromo-2'-deoxyuridine, and 10% fetal bovine serum (GIBCO-BRL) for 40 h. Cultures were then maintained under serum-free conditions with DMEM supplemented with 5 mg/l insulin and transferrin and 2.5 mg/l selenium. After 24 h in serum-free medium, medium was changed and cells were treated for 1-24 h with adrenergic agonists, after which they were removed from the wells and lysed for assay of luciferase activity and protein.
Transfection and luciferase assay. Transfection and luciferase activity were assayed as described previously (17). Briefly, freshly isolated NVM were transiently transfected in PBS-glucose by electroporation at 280 V and 250 µF with a Bio-Rad gene pulser. For the hBNP-luciferase constructs, 1 µg was transfected per 3 × 106 cells. For the co-transfection studies, 2 µg of either GATA-4 or its control expression vector (1) were transfected together with 1818hBNPLuc. After transfection, the cells were aliquoted into 3 wells of a 12-well plate. Forty hours later, the medium was changed to serum-free DMEM; cells were treated with appropriate agents and then harvested, lysed, and assayed for luciferase activity (Luciferase Assay System, Promega) using an OptoComp 1 luminometer (MGM). Duplicate aliquots of cell lysate from triplicate wells were assayed and averaged. Luciferase activity was normalized to protein levels, as described previously (17).
A mouse embryonic fibroblast cell line, MEF3T3, was passaged in DMEM supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and transferred to a 6-well plate at 5 × 105 cells/well. On the following day, cells were transfected with 1 µg of 1818hBNPLuc and 2 µg of GATA-4 expression vector per well using FuGene6 transfection reagent, according to the manufacturer's protocol. The cells were lysed 48 h later and assayed for luciferase activity. At least two separate preparations of each plasmid were used for each experimental group. Data were expressed as means ± SE and analyzed by t-test or one-way ANOVA, with multiple pairwise comparisons made by the Student-Newman-Keuls method. A value of P < 0.05 was considered significant.Plasmid constructions and mutagenesis. The expression vector encoding GATA-4 was obtained from Dr. David Wilson (Washington University, St. Louis, MO). Chimeric hBNP-luciferase reporter gene constructs and deletions thereof have been described (17, 42). Polymerase chain reaction (PCR) was used to generate mutations in the hBNP proximal promoter. Oligonucleotides included restriction sites at their 5' and 3' borders to facilitate subcloning. The HindIII site on the sense primer and BamHI site on the antisense primer are not included in the following sequences.
Mutation of the GATA site atNuclear extracts and EMSA.
To detect binding of nuclear protein to putative cognate binding sites,
we used EMSA. Crude nuclear extracts were prepared from cultured NVM
(15 × 106 cells per 15-cm tissue culture dish) as
described previously (11). Oligonucleotides were
synthesized and their complementary strands annealed before their use
as probe or competitor. A [-32P]ATP 5' end-labeled
double-stranded oligonucleotide (0.0175 pmol) was used as a probe. For
hBNP GATA binding, nuclear extract (5-7 µg) was added to binding
buffer containing (1×) 10 mM HEPES (pH 7.9), 1 mM MgCl2,
50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 2.5 mg/ml
BSA, 0.05 µg/ml poly(dI-dC), and 30-50,000 cpm radiolabeled
probe at room temperature for 20 min. The reaction volume was 20 µl.
The Sp1 and Oct-1 oligonucleotides were obtained from Promega, and
binding studies were done according to the manufacturer's protocol.
DNA-protein complexes were separated out on a 4% nondenaturing acrylamide gel in 0.5× TBE buffer. After electrophoresis, the gel was
dried and exposed to X-ray film for 1-2 days. GATA-containing oligonucleotides used in EMSA were as follows: 1) hBNP GATA
(17): 5'-ATGTGGCTGATAAATCAGAGA-3';
2) mutated hBNP GATA (mGATA): 5'-ATGTGGCTGGTAAATCAGAGA-3'; 3)
-MHC GATA
(22): 5'-TGGGGACATGATAAGGA-3'.
Protein extraction and Western blot. Protein was isolated from NVM and MEF3T3 cells with the buffers and protease inhibitors described previously (15, 16). For detection of GATA-4, -5, and -6, we used a 1:2,000 dilution of antibody purchased from Santa Cruz. The appropriate secondary antibody (1:2,000) linked to horseradish peroxidase was used for chemiluminescent detection of the proteins. For immunoprecipitation of GATA-4 from nuclear extracts, we used the lysis buffers and inhibitors described for isolation of protein. An aliquot of nuclear extract was first precleared with preimmune serum. Then primary antibody was added to the nuclear extract and incubated overnight at 4°C. To precipitate the antibody-antigen complex, protein G-agarose (Protein G-plus, Calbiochem) was added for 1 h at room temperature. The complex was spun down at 2,500 rpm in a microfuge, and the pellet was washed three times. The pellet was then resuspended in sample buffer and boiled for 5 min, and the Sepharose G agarose was spun out. The supernatant was loaded on a gel and subjected to Western blot analysis. The signal was detected through exposure to Fuji RX film and analyzed by scanning densitometry (Molecular Analyst software, Bio-Rad).
Supplies and chemicals.
The MEF3T3 cell line was purchased from Clontech (Palo Alto, CA).
FuGene6 transfection reagent was obtained from Roche (Indianapolis, IN). The gel shift and luciferase assay systems were obtained from
Promega (Madison, WI). Isoproterenol (ISO), dibutyryl cAMP, and
L-phenylephrine (PE) were purchased from Sigma (St. Louis, MO). [-32P]ATP was obtained from Du Pont-NEN (Boston,
MA). Routine supplies and chemicals were obtained from Fisher (Chicago,
IL) and Sigma.
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RESULTS |
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The GATA element at 85 is involved in basal
transcriptional regulation.
We have shown that the proximal region of the hBNP promoter (
127 to
40) is highly active in myocytes and essentially inactive in
fibroblasts (17). We have previously identified two
functional M-CAT-like elements (
97 and
124) in the proximal
promoter region (Fig. 1)
(11), adjacent to a putative GATA regulatory
element. To test the relevance of the GATA element directly, we mutated it from TGATAA to TGGTAA within the context of the 1818hBNP
promoter and transferred mutant 1818(mGATA)hBNPLuc and wild-type
1818hBNPLuc into NVM. As shown in Fig. 2,
this one base pair mutation in the GATA motif (A
G) essentially
reduced luciferase activity to background levels (greater than 97%
reduction).
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GATA-4 transactivates the hBNP promoter.
To test whether the hBNP promoter is activated by the GATA-4
transcription factor, we co-transfected a GATA-4 expression vector with
1818hBNPLuc. As shown in Fig.
3A, overexpressed GATA-4
transactivated the hBNP promoter 2.3-fold. When the hBNP promoter was
deleted to position 70, eliminating the proximal GATA element at
85, GATA-4 overexpression had no effect (Fig. 3B).
Finally, we co-transfected the GATA-4 expression vector with
1818(mGATA) hBNPLuc and found that GATA-4's transactivation was
reduced to control levels (Fig. 3C).
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EMSA studies.
The GATA consensus motif WGATAR is bound by GATA
transcription factors, which contain a highly conserved DNA binding
domain consisting of two zinc finger motifs (21). To test
whether the proximal GATA element binds to nuclear proteins, we used
EMSA. As shown in Fig. 6, the
32P-labeled oligonucleotide spanning the 85 GATA element
bound to cardiomyocyte nuclear protein (lane 2), and this
binding was competed for by both 100-fold excess unlabeled hBNP
(lane 3) and
-MHC GATA (lane 4)
oligonucleotides but not by an unrelated oligonucleotide, Sp1
(lane 5). In addition, a 32P-labeled
-MHC
GATA consensus oligonucleotide probe also bound to cardiac myocyte
nuclear protein (lane 7), and this binding was competed for
by both excess unlabeled hBNP GATA and
-MHC (lanes 8 and
9) but not Sp1 (lane 10). In several replicates
of the experiment, the hBNP GATA-protein complex was similar to the
-MHC GATA-protein complex.
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Involvement of the GATA element in inducible regulation of the hBNP
promoter.
GATA-4 is involved in hypertrophic cardiac gene expression in cultured
cardiac myocytes (25). We tested whether the proximal GATA
element is necessary for inducible regulation of the hBNP promoter by
- and
-adrenergic agonists. We first tested the effect of
isoproterenol (ISO, 100 µM) and its second messenger cAMP (dibutyryl
cAMP, 1 mM) on the proximal hBNP promoter (111/40TKLuc) and found that
ISO stimulated it 4.6 ± 0.2-fold and cAMP stimulated it 3.3 ± 0.1-fold (n = 3 each). Mutation of the GATA element
reduced ISO and cAMP activation of the hBNP promoter by 60% (Fig.
7A) and 75% (Fig.
7B), respectively. In contrast, mutation of the AP-1-like
element at position
111 had no effect on either ISO or cAMP
activation of the 1818hBNP promoter (Fig. 7, A and
B).
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DISCUSSION |
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Our previous study on regulation of hBNP gene expression indicated
that the 1818hBNP promoter was more active in myocytes than in
fibroblasts (17). Moreover, we found that a region of the
proximal BNP promoter located between 127 and
40, which consists of
cis elements arranged in tandem, conferred cardiac myocyte
specificity. In the present study, we explored components involved in
basal transcriptional regulation of the hBNP gene, finding that the
proximal GATA site contributes to both basal activity of the hBNP
promoter and inducible regulation by ISO and cAMP.
Our data indicate that GATA-4, a zinc finger transcription factor,
activates the full-length hBNP promoter, consistent with its role in
cardiac-specific gene expression, as described previously for cardiac
troponin C, cardiac troponin I, -MHC, and rat ANP and BNP (6,
13, 22, 31, 38). Our mutagenesis studies indicate that the site
at
85 is required for basal hBNP promoter activity and
transactivation of the hBNP promoter by GATA-4 in NVM. This is
different from the rat BNP promoter. GATA-4 transactivation of the rat
BNP promoter is not diminished when either of the two proximal GATA
elements is mutated, but it is reduced by 50% when both are mutated
(38). Thus there are species-specific differences in the
importance of GATA to BNP gene regulation.
Because the GATA-4 protein is present in cardiac myocytes, we expected
that exogenous GATA-4 would only weakly transactivate the hBNP
promoter. As expected, basal expression of the hBNP promoter was lower
in fibroblasts than in myocytes and exogenous GATA-4 was a potent
activator. In NVM, GATA-4 was unable to activate 1818(mGATA)hBNPLuc,
but in fibroblasts (MEF3T3 cells) it had a partial effect. Thus it is
possible that fibroblasts would be a better cell line for uncovering
effects of other GATA elements in the hBNP promoter. The distal hBNP
promoter contains two consensus GATA elements (at positions 1368 and
1288) and a cluster of GATTA motifs between
1788 and
1686, and it
is possible that one or more of these elements contributes to GATA-4 transactivation.
GATA proteins play a role in inducible gene expression (10, 12, 34, 41) and cardiac hypertrophy (20). Because the BNP gene is induced during hypertrophy and its actions oppose growth stimuli, we hypothesized that hypertrophic factors would target the GATA site in the proximal hBNP promoter. Unexpectedly, the proximal GATA element in the hBNP promoter was unresponsive to PE. In the rat BNP promoter, mutation of either GATA element in the proximal promoter had little effect on PE activation, yet mutation of both reduced the effect of PE by 60% (37). The proximal hBNP promoter has only one GATA site, which is surrounded by a different arrangement of cis elements than the rat BNP promoter. This may account for the fact that PE has no effect on the GATA site in the hBNP promoter.
Both ISO and cAMP stimulated hBNP promoter activity in part through the
GATA element and activated binding to the GATA element in the absence
of any effect on GATA-4 protein levels. We have previously shown that
ISO and cAMP, acting through the small GTPase Rac and the tyrosine
kinase Src, but not protein kinase A, target the M-CAT element at
position 97 in the proximal hBNP promoter (11). In that
study, we suggested that the effect of cAMP might be mediated through
direct or indirect activation of Rac and Src. cAMP has been shown to
directly interact with proteins that control GTPase activity of Ras
family members (14).
Additional mechanisms have also been described. Morisco et al.
(30) showed that -adrenergic regulation of the rat ANP
promoter involves calcium/calmodulin-dependent kinase II (CaMKII) and
phosphatidylinositol (PI) 3-kinase activation of Akt. Akt's effect on
the ANP promoter is mediated by glycogen synthase kinase 3
(GSK3
), acting on GATA-4 (29). Preliminary data from
our laboratory implicate CaMKII, but not members of the MAPK family, in
ISO regulation of the hBNP promoter, suggesting that GSK3
may also
be involved (M.C. LaPointe, unpublished data). In addition, ISO's
effect on endothelin-1 expression seems to be mediated by the
calcium-activated phosphatase calcineurin and GATA-4 (27).
In contrast to the mechanisms described for
-adrenergic activation
of GATA factors,
-adrenergic stimulation of the endothelin-1 and rat
ANP promoters involves p42/44 or p38 MAPK phosphorylation of GATA-4
(5, 26). Thus GATA is a target for a number of different
signaling pathways involved in control of myocyte gene expression.
Transactivation by GATA-4 may involve cooperative interactions with other transcription factors. Bhalla et al. (3) reported that rat BNP promoter is cooperatively activated by GATA-4 and YY1, a 65-kDa multifunctional DNA-binding factor. Lee et al. (19) found that the cardiac tissue-restricted homeobox protein Csx/NKx2.5 combines with GATA-4 and participates in activation of ANP gene expression. Herzig et al. (12) reported that functional interaction between AP-1 and a GATA-4 transcription factor mediates pressure overload-induced angiotensin II receptor type 1a (AT1a) gene activation. GATA-4 works together with MEF2 (28), NFAT3 (24), serum response factor (2), FOG2 (36), and other GATA family members (4) to regulate cardiac gene expression. Regarding the hBNP promoter, whether there are cooperative interactions between GATA-4 and M-CAT-binding proteins, Jun, Jun family members, or other GATA family members is not known, but such an interaction might be possible, because the binding sites for the factors are in close proximity. In addition, it is possible that one of GATA's many coactivators is a target of signaling pathways activated by hypertrophic stimuli. A recent study has shown that CBP/p300, a coactivator of GATA-4, is activated by p42/44 MAPK in cardiac myocytes treated with phenylephrine (9).
In summary, we have identified a GATA element in the proximal hBNP promoter that contributes to basal regulation of the gene. Also, the GATA element is in part a target for signaling pathways activated by ISO and cAMP. Thus, by understanding basal and inducible transcriptional regulation of the hBNP gene in vitro, we may gain insight into the molecular mechanisms involved in its upregulation during cardiac hypertrophy and other pathophysiological conditions, such as myocardial infarction and heart failure.
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ACKNOWLEDGEMENTS |
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We thank Nicole Bart and Fangfai Wang for their excellent technical assistance.
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FOOTNOTES |
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* Q. He and M. Mendez contributed equally to this work.
This work was supported by National Institutes of Health Grants HL-03188 and HL-28982.
Address for reprint requests and other correspondence: M. C. LaPointe, Hypertension and Vascular Research Division, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202-2689 (E-mail: mclapointe{at}aol.com).
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.
March 19, 2002;10.1152/ajpendo.00274.2001
Received 21 June 2001; accepted in final form 15 March 2002.
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