Published online before print April 29, 2004, doi: 10.1161/01.RES.0000129701.14494.52
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© 2004 American Heart Association, Inc.
Molecular Medicine |
Endothelin-1–Dependent Nuclear Factor of Activated T Lymphocyte Signaling Associates With Transcriptional Coactivator p300 in the Activation of the B Cell Leukemia-2 Promoter in Cardiac Myocytes
From the Department of Cardiovascular Medicine (T Kawamura, T.M., M.A., E.I.-K., H.W., N.S., T Kita), Graduate School of Medicine, Kyoto University, Kyoto, Japan; Division of Translational Research (K.O., K.H.), Kyoto Medical Center, National Hospital Organization, Kyoto, Japan.
Correspondence to Koji Hasegawa, MD, PhD, Division of Translational Research, Kyoto Medical Center, National Hospital Organization, 1-1 Mukaihata-cho Fukakusa, Fushimi-ku, Kyoto 612-8555, Japan. E-mail koj{at}kuhp.kyoto-u.ac.jp
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Abstract |
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Endothelin-1 (ET-1) is a potent survival factor that protects cardiac myocytes from apoptosis. ET-1 induces cardiac gene transcription and protein expression of antiapoptotic B cell leukemia-2 (bcl-2) in a calcineurin-dependent manner. A cellular target of adenovirus early region 1A (E1A) oncoprotein, p300 also activates bcl-2 transcription in cardiac myocytes and is required for their survival. p300 acts as a calcineurin-regulated nuclear factors of activated T lymphocytes (NFATc), downstream targets of calcineurin. In addition, the bcl-2 promoter contains multiple NFAT consensus sequences. These findings prompted us to investigate the role of NFATc in ET-1–dependent and p300-dependent bcl-2 transcription in cardiac myocytes. In primary cardiac myocytes prepared from neonatal rats, mutation of 2 NFAT sites within the bcl-2 promoter completely abolished the ET-1– and p300-induced increases in the activity of this promoter. We show here that p300 markedly potentiates the binding of NFATc1 to the bcl-2 NFAT element by interacting with NFATc1 in an E1A-dependent manner. On the other hand, stimulation of cardiac myocytes with ET-1 causes nuclear translocation of NFATc1, which interacts with p300 and increases DNA binding. Expression of E1A did not change the cardiac nuclear localization of NFATc1 but blocked its interaction with p300, DNA binding, and bcl-2 promoter activation. These findings suggest that ET-1–dependent NFATc signaling associates with p300 in the transactivation of bcl-2 gene in cardiac myocytes.
Key Words: transcription • calcineurin • NFAT • endothelin-1 • cardiomyocytes
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Introduction |
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Endothelin-1 (ET-1) is a 21-residue peptide originally isolated from supernatants of vascular endothelial cells.1 ET-1 acts not only as a vasoconstrictive and growth-promoting peptide but also as a survival factor against apoptosis in smooth muscle cells,2 fibroblasts,3 and cardiac myocytes.4,5 This antiapoptotic effect is mainly mediated through a type A-receptor dependent pathway that involves Gq protein.4 Subcellular signaling pathways for the ET type A receptor include phosphatidylinositol 3-kinase- and mitogen-activated protein kinase-mediated pathways.4,6 In addition to these pathways, common among different cell types, ET-1 stimulation results in an increase in intracellular calcium levels.7 Calcium-activated phosphatase, calcineurin, dephosphorylates the serine residues of the calcineurin-regulated nuclear factors of activated T lymphocytes (NFATc) and unmasks its nuclear localization sequences. These modifications induce the translocation of cytoplasmic NFATc to the nuclei.5,8 The calcineurin/NFATc pathway not only mediates changes in gene expression during cardiac hypertrophy9 but also is required for ET-1– and phenylephrine-mediated protection against myocardial cell apoptosis.5,10,11 ET-1 stimulation induces expression and gene transcription of antiapoptotic protein B cell leukemia-2 (bcl-2) in cardiac myocytes. This induction is attenuated by an inhibitor of calcineurin, cyclosporin A, suggesting a role of calcineurin-dependent signaling.5 However, the precise mechanisms by which ET-1-dependent signaling transactivates the bcl-2 gene in cardiac myocytes are unknown.
p300 is a transcriptional coactivator that governs gene expression patterns by being recruited to target genes through association with specific transcription factors.12–14 Several lines of evidence, including ours, suggest that p300 plays an important role in the physiological and pathological growth of cardiac myocytes.15–19 In addition, p300 appears to regulate apoptosis in various cell types. In contrast to a proapoptotic role of p300 in fibroblasts,20 analysis of adenovirus early region 1A (E1A) mutants suggests that endogenous p300 is required for the survival of differentiated cardiac myocytes.15,21 Recently, we reported that forced expression of p300 in cardiac myocytes protected these cells from doxorubicin-induced apoptosis and increased the expression of bcl-2 protein and the activity of its promoter. This raises the question of which transcription factor(s) become activated by interacting with p300 during the transactivation of the bcl-2 gene. p300 protein has been reported to act as a coactivator of NFATc, a downstream target of calcineurin.22,23 In addition, the bcl-2 promoter contains multiple NFAT consensus sequences. These findings prompted us to investigate whether NFATc plays a role in the p300-mediated transactivation of the bcl-2 gene in cardiac myocytes. And if so, given the fact that ET-1 also transactivates the bcl-2 gene in cardiac myocytes in a cyclosporin A-dependent manner, it would be possible that ET-1-mediated calcineurin/NFATc signaling is somehow related to a p300-dependent pathway during the transactivation of the bcl-2 gene. The present study was performed to test these hypotheses.
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Materials and Methods |
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Plasmid Constructs
Expression vectors plasmid cytomegalovirus ß-galactosidase (pCMVß-gal; (Santa Cruz Biotechnology) and pCMVwtp30012 (a gift from Dr Richard Eckner, University of Zurich, Switzerland) contain the cytomegalovirus promoter/enhancer fused to ß-gal cDNA and a full-length human p300 cDNA, respectively. The plasmid construct pbcl-2luciferase (luc; Dr Linda M. Boxer, Stanford University School of Medicine, California) is a firefly luciferase-reporter plasmid driven by a DNA fragment containing sequences –1796- to –999-bp relative to the translation start site of the human bcl-2 gene.24 The reporter plasmid p wild-type bcl-2-luciferase (pwtbcl-2-luc) and plasmid p mutant bcl-2-luciferase (pmutbcl-2-luc) with comparable length were generated by amplifying nucleotides –1796- to –1126-bp of the human bcl-2 promoter by the polymerase chain reaction and subcloning into a BamHI-BglII–cleaved pXP2.25 In pmutbcl-2-luc, 2 NFAT sites at nucleotides –1145- to –1126-bp were mutated. p Rous sarcoma virus (RSV) chloramphenicol acetyl transferase (CAT) contains a bacterial CAT gene driven by the RSV long-terminal repeat sequence.18 p calcineurin–regulated transcription factor nuclear factor of activated thymocytes (pNFATc1; a gift from Dr Ken-ichi Arai, University of Tokyo, Japan) is a murine NFATc1 expression plasmid.26 pwtE1A is an expression vector for wt 12S E1A. pdel2-36E1A was derived from pwtE1A, which bears an amino-terminal deletion of E1A 12S between amino acid residues 2 and 36.13
Cell Culture, Transfection, and Luciferase/CAT Assays
Primary ventricular cardiac myocytes from neonatal rats were prepared as described previously.18 These cells were cotransfected with the indicated amounts of DNA using Lipofectamine Plus (Life Technologies) and subjected to assays for luciferase and CAT activities as described previously.18
COS7 cells were maintained in DMEM with 10% FBS. The cells were washed twice with serum-free medium and then transfected with the indicated amounts of DNA using lipofectamine (Life Technologies) as described previously.18
Western Blotting and Immunoprecipitation
Protein extracts were prepared from primary neonatal rat cardiac myocytes as described previously.18 Immunoprecipitation and Western blotting for p300, E1A, and ß-actin were performed as described previously.18 Briefly, aliquots of the lysates containing 100 µg of protein were immunoprecipitated by incubating with an anti-NFATc1 monoclonal antibody (Santa Cruz Biotechnology), anti-p300 polyclonal antibody (Santa Cruz Biotechnology), or normal mouse IgG in low-stringency buffer for 16 hours at 4°C, and then incubated with protein G beads (Amersham Biosciences) for 2 hours at 4°C. The precipitate was washed 4x in the same buffer and subjected to Western blotting by using a monoclonal antibody against p300 (Upstate Biotechnology) or anti-NFATc1 monoclonal antibody (Santa Cruz Biotechnology).
Electrophoretic Mobility Shift Assays
Double-stranded oligonucleotides were designed to contain NFAT motifs from the bcl-2 promoter as follows; wild-type bcl-2 NFAT (Wt-NFAT): 5'-CCTTTTTAGGAAAAGAGGGAAAAAATAAAACCC-3', and mutant bcl-2 NFAT (Mut-NFAT) 5'-CCTTTTTAGCTCCCGAGGCTCCCAATAAAACCC-3'. Electrophoretic mobility shift assays (EMSAs) were performed as described previously.18
Immunocytochemistry
The cardiac myocytes were grown on flask-style chambers with glass slides (Nalgen Nunc) and costained for NFATc1 and cardiac ß-myosin heavy chain using the indirect immunofluorescence method as described previously5 with a minor modification. Briefly, after fixation, the cells were incubated with goat anti-NFATc1 polyclonal antibody (Santa Cruz Biotechnology) at a dilution of 1:50, followed by incubation with anti-goat fluorescence-conjugated secondary antibody (ICN Biomedicals) at a dilution of 1:200. Subsequently, these slides were stained with mouse anticardiac ß-myosin heavy chain monoclonal antibody (Novocastra Laboratories) at a dilution of 1:50 and the anti-mouse rhodamine-conjugated secondary antibody (Chemicon) at a dilution of 1:50.
Statistical Analysis
Data are presented as means±SE. Statistical comparisons were performed using unpaired 2-tailed Student t tests or ANOVA with Scheffe test where appropriate, with a probability value of <0.05 taken to indicate significance.
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Results |
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p300 Is Involved in ET-1–Responsive Transcription of the bcl-2 Gene in Cardiac Myocytes
To determine whether p300 is involved in ET-1–responsive bcl-2 transcription, primary neonatal cardiac myocytes were cotransfected with pbcl-2luc and 1 of pCMVß-gal, pwtE1A, or pd2–36E1A. Then these cells were incubated with ET-1 (0.1 µmol/L) or saline as a control for 48 hours. As shown in Figure 1A, ET-1 increased the relative luciferase activity of pbcl-2luc by 2.5-fold in ß-gal–expressing cardiac myocytes. Expression of E1A, which disrupts p300 function, not only reduced the level of bcl-2 promoter activity at the basal state but also completely blocked the ET-1–responsive bcl-2 transcription. In contrast, expression of mutant E1A (d2–36E1A), which lacks a p300 binding domain, did not inhibit the bcl-2 promoter activity at the basal or ET-1–stimulated state compared with ß-gal expression. These findings suggest that p300 is involved in ET-1–responsive transcription of the bcl-2 gene in cardiac myocytes.
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NFAT Sites Play Critical Roles in Both p300- and ET-1–Dependent bcl-2 Transcription in Cardiac Myocytes
A human bcl-2 promoter contains 2 NFAT consensus motifs at nucleotides –1145- to –1126-bp relative to the translation start site. To investigate the role of these sites in p300- and ET-1–responsive bcl-2 transcription in cardiac myocytes, primary cardiac myocytes from neonatal rats were transfected with a luciferase reporter driven by nucleotides –1796- to –1126-bp of the bcl-2 promoter (pwtbcl-2-luc; Figure 1B). ET-1 increased the relative luciferase activity of pwtbcl-2-luc in ß-gal–expressing cells (Figure 1B) to a similar extent, as observed in the preceding experiment by pbcl-2luc (Figure 1A). Cotransfection of pCMVwtp300 further increased the pwtbcl-2-luc activity. Because pwtbcl-2-luc and pbcl-2luc respond to ET-1 and p300 to a similar extent, we defined the specific role of NFAT sites in the context of pwtbcl-2-luc. Stimulation with ET-1 in addition to pCMVwtp300 cotransfection further potentiated the increase in the pwtbcl-2-luc activity. However, mutation of the 2 NFAT sites within the bcl-2 promoter completely abolished the ET-1– and p300-induced increase in the promoter activity (pmutbcl-2-luc; Figure 1C). Thus, these NFAT sites are required for p300- and ET-1-responsive bcl-2 transcription in cardiac myocytes.
p300 Interacts With NFATc1
To determine whether p300 interacts with NFATc1, and if so, whether expression of E1A, which disrupts p300 function, perturbs this interaction, we performed immunoprecipitations followed by Western blotting. COS7 cells were transfected with either pCMVß-gal or pwtE1A in addition to an expression plasmid encoding p300 (pCMVwtp300) and 1 encoding NFATc1 (pNFATc1). As shown in Figure 2A, the expression levels of p300 and NFATc1 before immunoprecipitation were similar between ß-gal– and E1A-expressing cells. Extracts from these cells were subjected to immunoprecipitation with anti-NFATc1 antibody, followed by Western blotting using anti-p300 antibody. As shown in the top of Figure 2B, an interaction between p300 and NFATc1 was observed (lane 1). Expression of E1A disrupted this interaction (lane 2). The anti-p300 antibody was stripped, and then the membrane was reprobed with the anti-NFATc1 antibody. NFATc1 was similarly immunoprecipitated with anti-NFATc1 antibody in these 2 groups (Figure 2B, bottom). As shown in Figure 2C, we confirmed these observations by reciprocal experiments: immunoprecipitation using anti-p300 antibody followed by Western blotting with anti-NFATc1 antibody (bottom) and with anti-p300 antibody (top). No protein was immunoprecipitated with control IgG (data not shown). Thus, NFATc1 interacts with p300 in an E1A-dependent manner.
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p300 Increases DNA-Binding Activity of NFATc1
To determine whether p300 modulates the DNA-binding activity of NFATc1, EMSAs were performed. COS7 cells were transfected with pNFATc1 in the presence or absence of pwtE1A and pCMVwtp300, as indicated. Total amounts of transfected DNA in each group were kept constant by cotransfecting pCMVß-gal. Extracts from these cells were subjected to EMSAs using a radiolabeled double-stranded oligonucleotide containing the bcl-2 NFAT sites as a probe. As shown in Figure 3A and 3B, the intensity of the retarded band was markedly increased in extracts from p300-expressing cells (lane 2) compared with those from ß-gal–expressing cells (lane 1). Coexpression of E1A in addition to p300 blocked the p300-mediated increase in intensity (lane 3). As shown in lanes 4 and 5 of Figure 3A, the retarded band represented sequence-specific binding, as evidenced by the fact that it was competed out by an excess of unlabeled wild-type bcl-2 NFAT oligonucleotide (lane 5) but not by the same amount of unlabeled oligonucleotide containing the bcl-2 NFAT sites with mutation (lane 4). To further confirm that the retarded band represents an interaction of the probe with NFATc1, we performed supershift experiments. The retarded band was supershifted by an anti-NFATc1 antibody (lane 7) but not by normal mouse IgG (lane 6). As shown in Figure 3C, the expression levels of NFATc1 were similar among the 3 extracts. These findings demonstrate that p300 increases the DNA-binding activity of NFATc1 in association with p300/NFATc1 interaction.
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p300 and NFATc1 Synergistically Activate the bcl-2 Promoter
The p300/NFATc1 association and p300-mediated increase in NFATc1/DNA binding suggest that p300 might be directly involved in NFATc1-dependent transactivation of the bcl-2 gene. To test this hypothesis, we cotransfected into COS7 cells pbcl-2-luc together with pNFATc1 alone or in combination with pCMVwtp300. Forty-eight hours later, we measured the bcl-2 reporter activity. As shown in Figure 4A, the transfection of either pNFATc1 (lane 3) or pCMVwtp300 (lane 2) alone increased the bcl-2 promoter activity only modestly. However, the cotransfection of both pCMVwtp300 and pNFATc1 induced a marked stimulation of the bcl-2 promoter activity (lane 4). As shown in Figure 4B, p300 is similarly expressed in extracts from pCMVwtp300- and pCMVß-gal–transfected cells (lane 2) and in those from pCMVwtp300- and pNFATc1-transfected cells (lane 4). NFATc1 levels were similar between lanes 3 (pNFATc1- and pCMVß-gal–transfected cells) and 4 (pNFATc1- and pCMVwtp300-transfected cells). These findings demonstrate that p300 and NFATc1 synergistically activate the bcl-2 promoter.
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ET-1 Induces the Association of NFATc1 With p300 in Cardiac Nuclei
Next, we examined whether stimulation of cardiac myocytes with ET-1 induced an association between p300 and NFATc1. Primary cardiac myocytes from neonatal rats were transfected with pwtE1A or pCMVß-gal as a control and stimulated with ET-1 (0.1 µmol/L) or saline as a control for 48 hours. As shown in Figure 5, immunofluorescence microscopy demonstrated that ET-1 stimulation markedly changed the localization of NFATc1 from the cytoplasm to the nucleus, in accord with our previous report. Although ET-1–mediated translocation was prevented by cyclosporin A, an inhibitor of calcineurin, E1A expression did not change the location of NFATc1 in cardiac nuclei. To confirm that the translocation occurred in cardiac myocytes, these cells were also stained for cardiac ß-myosin heavy chain. As shown in the second line from the top of Figure 5, myofibrils of these cells were clearly stained for cardiac ß-myosin heavy chain. We confirmed the specificity of double staining in the right 2-row panels. Accordingly, extracts from these cells were subjected to immunoprecipitation with anti-NFATc1 antibody, followed by Western blotting using the anti-p300 antibody. As shown in Figure 6A, transfecting different doses of an E1A expression vector resulted in dose-dependent E1A expression in cardiac myocytes. As shown in the top of Figure 6B, the interaction between p300 and NFATc1 was markedly increased in extracts from ET-1–stimulated cardiac myocytes (lane 2) compared with those from saline-treated cells (lane 1). Transfection of an E1A expression vector dose-dependently blocked the ET-1–mediated increase in the p300/NFATc1 interaction (lanes 3 and 4). The levels of NFATc1 proteins immunoprecipitated with anti-NFATc1 antibody were similar among these 4 groups (Figure 6B, bottom). Similar results were obtained by performing reciprocal experiments (Figure 6C; ie, immunoprecipitation with anti-p300 antibody, followed by Western blotting with anti-NFATc1 antibody [bottom] and with anti-p300 antibody [top]). No protein was immunoprecipitated with control IgG (data not shown). These findings demonstrate that ET-1 induces the association of NFATc1 with p300 in cardiac nuclei.
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ET-1 Induces NFATc1/DNA Binding in Cardiac Myocytes
To determine whether stimulation of cardiac myocytes with ET-1 modulates the DNA-binding activity of NFATc1 in association with its interaction with p300, we performed EMSAs. The same extracts used to examine the interaction between NFATc1 and p300 in Figure 6 were subjected to EMSAs using the bcl-2 NFAT site as a probe. As shown in Figure 7A and 7B, the intensity of C1 and C2 were markedly increased in extracts from ET-1–stimulated cells (lane 2) compared with those from saline-stimulated cells (lane 1). Expression of E1A, which perturbed the p300/NFATc1 association, dose-dependently blocked the ET-1–mediated increase in the intensity (lane 3 and 4). C3 exhibited similar tendency with C1 and C2. As shown in Figure 7A, these retarded bands (C1, C2, and C3) represented sequence-specific binding (lanes 5 and 6), and were immunoreactive with an anti-NFATc1 antibody (lane 8) but not with normal mouse IgG (lane 7). Furthermore, administration of an anti-p300 antibody eliminated C1, a complex migrating most slowly, but not C2 or C3 (lane 9). These findings suggest that C1 comprises NFATc1 and p300.
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Discussion |
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The present study investigated the role of NFATc in p300- and ET-1–induced transcription of the gene encoding an antiapoptotic molecule, bcl-2, in cardiac myocytes. We showed here that mutation of NFAT sites within the bcl-2 promoter completely abolished both p300- and ET-1–responsive bcl-2 transcription in cardiac myocytes, suggesting a critical role of NFATc in these processes. To date, 5 NFAT isoforms have been identified and designated NFATc1 (also known as NFAT2 or NFATc), NFATc2 (NFAT1 or NFATp), NFATc3 (NFAT4 or NFATx), NFATc4 (NFAT3), and NFAT5. Among these, only NFAT5 appears to be constitutively nuclear and not to be regulated by calcineurin.27 NFATc1–NFATc4 bind the consensus DNA sequence through a Rel homology domain, and all 4 of these factors are expressed in cardiac myocytes.11 The present study demonstrated that NFATc1 binds to the NFAT site within the bcl-2 promoter in a sequence-specific manner. We also demonstrated that p300 acts as a coactivator of NFATc1 in the transcription of the bcl-2 gene. However, our results do not rule out possible roles of NFATc2–NFATc4 in bcl-2 transcription. It was reported that the NH2-terminal region of NFATc2 directly interacts with the cAMP response element binding protein-binding protein (CBP) 4 region of CBP/p300, which includes the E1A-binding site.22 Therefore, further studies are needed to examine the roles of each member of NFATc1–NFATc4 in p300- and ET–1-responsive bcl-2 transcription in cardiac myocytes.
ET-1 is a potent survival factor against apoptosis in various cell types.2–5 This antiapoptotic effect is mediated mainly through Gq protein-coupled ET-1 type A receptor.4 Activation of Gq increases intracellular calcium levels and subsequently activates the calcineurin pathway.5,9,11 The present study demonstrated that stimulation of cardiac myocytes with ET-1 induces the translocation of cytoplasmic NFATc1 into the nucleus. This translocation may contribute to the ET–1-induced association of NFATc1 with p300 in cardiac nuclei. In addition, ET-1 is functionally coupled to activation of protein kinase C and mitogen-activated protein kinases.6 p300 has been reported to be posttranslationally modified (phosphorylated) by activation of protein kinase C or mitogen-activated protein kinases.28 These signaling proteins may be involved in promoting the association of p300 with NFATc1 in cardiac nuclei. Our data also demonstrate that association of p300 with NFATc1 increases its DNA binding. p300 not only provides a bridge between NFATc1 and the basal transcriptional machinery but also possesses histone acetyltransferase activity.14,29 By this activity, p300 is also able to acetylate a number of transcription factors and enhance their DNA-binding activity.14 However, further studies are needed to elucidate the precise mechanisms by which p300 regulates NFATc1/DNA binding.
Recently, it has been reported that NFAT transcription factors account for the protective effects of calcineurin activation in cardiac myocytes.10,11 Given the potent ability of bcl-2 to block apoptosis,30 p300/NFATc-dependent bcl-2 transcription may provide a mechanism by which NFAT activation causes protective effects in cardiac myocytes. However, there are diverse mechanisms by which apoptosis may be regulated in various cell types. In fact, calcineurin and p300 are proapoptotic in some situations.20,31 Therefore, the precise roles of p300/NFATc in bcl-2 transcription in the context of other cell types should be clarified by further investigations. Interestingly, both p300 and NFATc associate with a cardiac zinc finger transcription factor, GATA-4. These associations (p300/GATA-4 and NFATc/GATA-4) are involved in regulating myocardial cell growth.9,17–19 Cardiac overexpression of p300 or an activated form of NFATc results in heart failure.9,17 Therefore, maintenance of adequate activation of p300- and NFATc1-dependent pathways would be required for treatment of heart failure. To establish an appropriate heart failure therapy by modulating p300 and calcineurin, further studies on the precise regulation of these pathways in different types of heart failure are needed.
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Acknowledgments |
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This work was supported in part by Advanced and Innovational Research Program in Life Science and grants to T.K. and K.H. from the Ministry of Education, Science, and Culture of Japan. We thank S. Nagata for excellent technical assistance.
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Footnotes |
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Original received October 27, 2003; revision received April 14, 2004; accepted April 15, 2004.
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