Regulation of the human brain natriuretic peptide gene by GATA-4

Quan He*, Mariela Mendez*, and Margot C. LaPointe

Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Michigan 48202


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -myosin heavy chain (alpha -MHC) (22).

Studies have suggested that the GATA-4 element plays a role in alpha 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 alpha - and beta -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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 at -85 was accomplished by a two-step PCR protocol by use of the following oligonucleotides: mutant sense strand, 5'-GGCTGGTAAATCAGAGACTA-3' (-89/-70); mutant antisense strand, 5'-ATTTACCAGCCACATTCCGG-3' (-79/-98); wild-type -283/-264 sense strand (5'-CCAACCTAGGACCCCGGAGA-3'); and +83/+100 antisense strand (5'-GGGACTGCGGAGGCTGCT-3'). The final PCR product was digested with AvrII and BamHI to isolate the mutated fragment, which was then subcloned into 1818hBNPLuc cut with the same enzymes to generate 1818(mGATA)hBNPLuc. Mutation of base pairs was verified by sequencing. The same strategy was used to mutate the AP-1-like site at -111, resulting in a change from 5'-TGATCTCA-3' to 5'-TTTTCTCA-3'.

The -111 to -40 region of the hBNP promoter was generated by PCR and subcloned in the HindIII and BamHI sites of the luciferase expression vector described previously (17). A minimal thymidine kinase (TK) promoter (-49 to + 52) was generated by PCR and subcloned downstream from the hBNP fragment in the BamHI and BglII sites. This construction is referred to as 111/40TKLuc.

Nuclear 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 [gamma -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) alpha -MHC GATA (22): 5'-TGGGGACATGATAAGGA-3'.

For supershift assay, 1 µl of the GATA-4 antibody (Santa Cruz Biotechnology) was added to the nuclear extract for 1 h at room temperature before addition of the radiolabeled hBNP GATA oligonucleotide.

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). [gamma -32P]ATP was obtained from Du Pont-NEN (Boston, MA). Routine supplies and chemicals were obtained from Fisher (Chicago, IL) and Sigma.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 right-arrow G) essentially reduced luciferase activity to background levels (greater than 97% reduction).


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Fig. 1.   Potential regulatory elements in the proximal human brain natriuretic peptide (hBNP) promoter. Two functional nuclear protein-biding sites were designated as -97 M-CAT and -124 M-CAT. The other potential regulatory elements are GATA at -85 and activator protein-1 (AP-1)-like element at -111, when the proximal hBNP promoter sequence is compared with consensus sequences of regulatory motifs.



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Fig. 2.   Effect of mutation of the -85 GATA-4 site on hBNP promoter coupled to luciferase reporter gene (1818hBNPLuc) activity (1818). y-Axis, luciferase activity expressed as a percentage of 1818hBNPLuc; x-axis, hBNP constructs tested. Activity of 1818hBNPLuc construct was set to 100%. Each bar represents mean ± SE of 5 experiments. Normalized luciferase activity of 1818hBNPLuc was 8,632 ± 1,016; that of the GATA element in the promoter [1818(mGATA)hBNPLuc] (mGATA) was 293 ± 35. **P < 0.01 vs. 1818.

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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Fig. 3.   Effect of overexpressed GATA-4 on hBNP promoter activity in neonatal ventricular myocytes (NVM). y-Axis, luciferase activity [degree of increase vs. control (CONT), with CONT value arbitrarily set to 1]; x-axis, different expression vectors tested. A: effect of GATA-4 on 1818hBNPluc. CONT is 1818hBNPLuc in the absence of GATA-4. Each bar is mean ± SE of 6 experiments. *P < 0.05 for GATA-4 vs. CONT. B: effect of GATA-4 on 70hBNPLuc. CONT is -70hBNPLuc deletion construct in the absence of GATA-4. Each bar is mean ± SE of 4 experiments. C: effect of GATA-4 on 1818(mGATA)hBNPLuc. CONT is 1818(mGATA)hBNPLuc in the absence of GATA-4. Each bar is mean ± SE of 5 experiments.

We confirmed the presence of GATA-4 in NVM by Western blot. As can be seen in Fig. 4A, GATA-4 was detected in three different extracts. In contrast, GATA-5 and -6 were not detected (data not shown). In addition, GATA-4 was present in nuclear extracts. Nuclear extracts were immunoprecipitated with either GATA-5 or GATA-4 antibodies, and the resulting protein was subjected to GATA-4 Western blot (Fig. 4B). No GATA-4 was present in nuclear extract immunoprecipitated in the absence of primary antibody (lane 1) or in the presence of GATA-5 antibody (lane 2). GATA-4 was detected in extracts immunoprecipitated with GATA-4 antibody (lane 3).


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Fig. 4.   Western blot of GATA-4 protein in NVM. A: 3 cell lysates analyzed from NVM extracts. B: Western blot of GATA-4 in nuclear extracts. Nuclear extracts were either immunoprecipitated with GATA-4 (lane 3) or GATA-5 (lane 2) antibodies and then subjected to Western blot for GATA-4. Sample in lane 1 is the control, which was treated identically to the other samples except for the absence of a primary antibody.

Because the GATA-4 protein is present in NVM and its high endogenous level might interfere with the transactivation experiments, we verified our results in a different cell line. We first tested MEF cells for the presence of GATA-4 protein by Western blot but were unable to detect it (data not shown). We then co-transfected MEF3T3 cells with the GATA-4 expression vector and 1818hBNPLuc and found that GATA-4 transactivated the hBNP promoter 22-fold, whereas mutation of the GATA site reduced the effect of GATA-4 on hBNP promoter activity by ~50% (Fig. 5A). Finally, GATA-4 was able to transactivate the proximal -111 to -40 region of the hBNP promoter (111/40TKLuc) but had no effect on TKLuc (Fig. 5B).


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Fig. 5.   Effect of overexpressed GATA-4 on hBNP promoter activity in MEF3T3 cells; y- and x-axes expressed as in Fig. 3. A: values are means ± SE of 4 separate experiments. Normalized luciferase activity for 1818hBNPLuc was 2,185 ± 158; normalized activity for 1818(mGATA)hBNPLuc was 1,311 ± 168. **p < 0.01 for mGATA vs. 1818. B: values are means ± SE of n = 3 [thymidine kinase Luc (TKLuc)] and n = 6 (111/40TKLuc) separate experiments. *P < 0.05 vs. CONT and TKLuc. CONT, activity in absence of GATA-4 normalized to 1; 111/40TKLuc, -111/-40 region of hBNP coupled to TK promoter; TKLuc, TK promoter alone.

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 alpha -MHC GATA (lane 4) oligonucleotides but not by an unrelated oligonucleotide, Sp1 (lane 5). In addition, a 32P-labeled alpha -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 alpha -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 alpha -MHC GATA-protein complex.


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Fig. 6.   Gel shift analysis of the -85 GATA element. Lanes 1-5 show gel shift when hBNP GATA element is radiolabeled and used as a probe. Lane 1 is the negative control; lane 2 shows protein binding to labeled oligonucleotide; lanes 3 and 4 show effect of adding a 100-fold excess of cold hBNP GATA and alpha -myosin heavy-chain (MHC) GATA; lane 5 shows effect of 100-fold excess of a noncompetitor Sp1 DNA. Lanes 6-10 show gel shift pattern when alpha -MHC GATA element is radiolabeled and used as a probe. Lane 6 is the negative control; lane 7 shows protein binding to labeled alpha -MHC GATA element; lanes 8 and 9 show effect of adding a 100-fold excess of cold alpha -MHC GATA and hBNP GATA; lane 10 shows effect of 100-fold excess of a noncompetitor Sp1 DNA. NC, noncompetitor oligonucleotide (Promega). Each experiment was repeated at least twice with different preparations of nuclear extract.

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 alpha - and beta -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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Fig. 7.   Effect of isoproterenol (ISO) and cAMP on the proximal hBNP promoter GATA element. y-Axis, luciferase activity (degree of increase in presence and absence of a stimulus); x-axis, hBNPLuc construct tested. A: effect of 100 µM ISO. Values are means ± SE of 6 experiments. *P < 0.05 vs. 1818hBNPLuc. B: effect of 1 mM dibutyryl cAMP. Values are means ± SE of 5 experiments. **P < 0.01 vs. 1818hBNPLuc. CONT, unstimulated NVM (assigned a value of 1).

We next tested whether the alpha -adrenergic agonist PE also acts through the GATA element. Whereas 50 µM PE robustly activated the 1818hBNP promoter (14.9 ± 2.4-fold; n = 8), mutation of the GATA element had no effect (11.2 ± 1.8-fold; n = 5).

EMSA studies were done using nuclear extracts from NVM treated with ISO for 15 min and ISO and cAMP for 1 h. ISO increased GATA binding to the hBNP GATA element at 15 min (Fig. 8A, lanes 1-2), and at 1 h both ISO and cAMP increased binding (lanes 5-7). This binding activity was competed for by excess unlabeled hBNP GATA (lane 3), but not by mGATA (lane 4). Addition of a GATA-4 antibody to the nuclear extract reduced binding (Fig. 8B, lanes 1-2). Neither ISO nor cAMP increased Oct-1 binding to nuclear extracts (Fig. 8C).


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Fig. 8.   Electrophoretic mobility shift assay (EMSA), showing effect of ISO and cAMP on GATA-binding activity. A: nuclear extracts were prepared after treatment with ISO for 15 min and ISO and cAMP for 1 h. Lane 1, control nuclear extract + 32P-hBNP GATA oligonucleotide; lane 2, nuclear extract treated with ISO for 15 min; lane 3, control + 100-fold excess unlabeled hBNP GATA oligonucleotide; lane 4, control with 100-fold excess unlabeled hBNP mGATA oligonucleotide; lane 5, control nuclear extract + 32P-hBNP-GATA oligonucleotide; lane 6, extract treated with ISO for 1 h; lane 7, extract treated with cAMP for 1 h. NS, nonspecific binding. B: supershift (SS) assay. Lane 1, control nuclear extract; lane 2, extract + GATA-4 antibody. C: binding of Oct-1. Lane 1, control nuclear extract + 32P-Oct-1 oligonucleotide; lane 2, nuclear extract treated with ISO for 1 h; lane 3, nuclear extract treated with cAMP for 1 h; lane 4, control in the presence of 100-fold excess unlabeled Oct-1 oligonucleotide.

To determine whether ISO was increasing GATA-4 protein levels, Western blots were done. ISO treatment for either 1 or 24 h had no effect on GATA-4 protein levels (Fig. 9, A and B). Data from Western blots were analyzed by densitometry to confirm the absence of an effect on protein levels. Compared with controls (assigned a value of 1), the effect of ISO treatment for 1 h was 1.3 ± 0.2-fold (n = 5), and for 24 h it was 1.2 ± 0.2-fold (n = 6) (Fig. 9C).


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Fig. 9.   Effect of ISO on GATA-4 protein level. A: Western blot of NVM treated with ISO for 1 h. B: Western blot of NVM treated with ISO for 24 h. C, control. C: densitometry data of 5-6 separate experiments. y-Axis, degree of increase vs. control (CONT); x-axis, treatment.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, alpha -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 beta -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 3beta (GSK3beta ), 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 GSK3beta 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 beta -adrenergic activation of GATA factors, alpha -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.


    ACKNOWLEDGEMENTS

We thank Nicole Bart and Fangfai Wang for their excellent technical assistance.


    FOOTNOTES

* 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 283(1):E50-E57
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