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Am J Physiol Cell Physiol 292: C1248-C1255, 2007. First published November 15, 2006; doi:10.1152/ajpcell.00624.2005
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CELLULAR METABOLISM

Involvement of oxidants and AP-1 in angiotensin II-activated NFAT3 transcription factor

Victoria C. Tu, Haipeng Sun, G. Tim Bowden, and Qin M. Chen

Department of Pharmacology, Arizona Cancer Center, College of Medicine, University of Arizona, Tucson, Arizona

Submitted 13 December 2005 ; accepted in final form 4 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cardiomyocyte hypertrophy is associated with multiple pathophysiological cardiovascular conditions. Recent studies have substantiated the finding that oxidants may contribute to the development of cardiomyocyte hypertrophy. Activation of the nuclear factor of activated T cells-3 (NFAT3) transcription factor has been shown to result from endocrine inducers of cardiomyocyte hypertrophy such as angiotensin II (ANG II) and serves as an important molecular regulator of cardiomyocyte hypertrophy. In this study, we found that antioxidant enzyme catalase and antioxidants N-acetyl-L-cysteine, {alpha}-phenyl-N-tert-butylnitrone, and lipoic acid prevent ANG II from activating NFAT3 promoter-luciferase. H2O2 induces a time- and dose-dependent activation of NFAT3 transcription factor. A dominant negative form of NFAT3 transcription factor inhibited H2O2 from activating NFAT3 promoter. An inhibitor of ERKs, but not phosphoinositide 3-kinase or p38 MAPKs, blocked NFAT3 activation by H2O2. The NFAT3 binding site in the promoters of most genes contains a weak activator protein-1 (AP-1) binding site adjacent to the core consensus NFAT binding sequence. ERK inhibitor PD98059 was found previously to inhibit AP-1 activation by H2O2. Inactivation of AP-1 transcription factor by cotransfection of a dominant negative c-Jun, TAM67, prevented H2O2 or ANG II from activating NFAT3 promoter. NFAT3 promoter containing the core NFAT cis-element without AP-1 binding site failed to show activation by H2O2 treatment. Our data suggest that hypertrophy inducers ANG II and H2O2 may activate NFAT3 in cardiomyocyte through an AP-1 transcription factor-dependent mechanism.

activator protein-1; nuclear factor of activated T cells-3; cardiomyocytes; hypertrophy; antioxidants

CARDIOVASCULAR DISEASE IS the most deadly disease worldwide. In the United States, heart attack occurs in about one million Americans and claims lives among one-third of them each year. Hypertension comprises a major risk factor for heart attack. Inhibition of the renin-angiotensin system is a necessary therapy for many hypertensive patients. Angiotensin II (ANG II), an end product of the renin-angiotensin system, is an eight-amino acid peptide that can bind to its receptor, AT1 or AT2, on the surface of cells. These G protein-coupled receptors initiate a cascade of signaling events upon ligand binding. In the myocardium, ANG II has been shown to cause cardiomyocyte hypertrophy.

Cardiomyocyte hypertrophy is associated with an altered profile of gene expression. Activation of several transcription factors, including the nuclear factor of activated T cells-3 (NFAT3), contributes in part to the changes in gene expression associated with hypertrophy (28, 29). NFAT3 belongs to the NFAT family of transcription factors, discovered as important regulators of immune responses in mammals (8, 25, 38). All members of the NFAT family contain an NH2-terminal transactivation domain, a conservative NFAT homologous domain, and a DNA binding domain that has a partial Rel homology. Several members of the NFAT family contain an additional transactivation domain in the COOH-terminal region. Four members of NFATs have been identified: NFAT1 (NFATp/NFATc2), NFAT2 (NFATc/NFATc1), NFAT3, and NFAT4. NFAT1 and NFAT2 are expressed predominantly in lymphoid tissues. While NFAT4 is mainly expressed in the thymus, the expression of NFAT3 occurs primarily in nonlymphoid tissues.

Nuclear translocation constitutes a necessary step in the mechanism of NFAT activation. NFATs usually reside in the cytosol and are constitutively phosphorylated by glycogen synthase kinase-3 (GSK3) at the conserved SPXX repeat motifs located in the regulatory domain (8, 15, 25, 33, 38). This domain contains a calcineurin docking site with a conserved sequence of PxIxIT. Calcineurin, a Ca2+- and calmodulin-dependent serine/threonine phosphatase (also called protein phosphatase 2B or PP2B), can dephosphorylate SPXX motifs and serine-rich regions located in the regulatory domain (8, 25, 38). As a result of dephosphorylation, NFAT proteins refold and expose a nuclear localization sequence. The calcineurin/NFAT3 pathway can be activated by classical hypertrophy inducers, including ANG II, endothelin-1 (ET-1), and catecholamines (27, 49). Inhibitors of calcineurin have been reported to prevent cardiac hypertrophy in several experimental models (21, 27, 29, 31, 32, 41). It is commonly believed that these hypertrophy inducers activate phospholipase C (PLC) on binding to their G protein-coupled receptors, resulting in release of phosphoinositol from membrane phospholipids. Phosphoinositol in turn triggers an increase in cytoplasmic Ca2+ concentration. As a result, calcineurin and therefore NFAT3 are activated on exposure of cells to ANG II and other hypertrophy inducers.

Recent evidence suggests that binding of ANG II to its receptor in the plasma membrane of cells is coupled to activation of a membrane-associated NADPH oxidase (12, 13). This enzyme produces superoxide from oxygen and NADPH. In vascular smooth muscle cells, oxidants appear to mediate ANG II-induced signaling events and hypertrophy (12). Oxidant production contributes to ANG II-induced gene expression, including activation of NFATs in cardiac fibroblasts (11). In cardiomyocytes, evidence suggests that oxidants may mediate hypertrophy induced by ANG II (39). Oxidants have been shown to induce hypertrophy of cardiomyocytes in vitro (4, 40). Whether oxidants mediate the signaling pathways and NFAT3 activation induced by ANG II in cardiomyocytes has not been demonstrated.

We found that the majority of cardiomyocytes in culture can survive a pulse treatment with low or mild doses of H2O2 but become enlarged over a course of 4–7 days (4). H2O2 activates the phosphoinositide 3-kinase (PI3K) pathway and MAPK pathways (45, 46). Because PI3K is a kinase regulating multiple signaling pathways including PLC, it is likely that H2O2 may increase intracellular Ca2+ concentration. In fact, intracellular Ca2+ overload has been demonstrated with H2O2 treatment in cardiomyocytes (20). Transient activation of calcineurin has been observed in cardiomyocytes following treatment with H2O2 (7), suggesting that oxidants may activate NFATs. H2O2 has been shown to activate three branches of MAPKs, i.e., JNKs, p38, and ERKs, among which ERKs appear to regulate the activation of activator protein-1 (AP-1) transcription factor (46). AP-1 transcription factor has been shown to be essential for NFAT activation in the immune system (8, 24, 25, 38). With these lines of evidence, we test the involvement of oxidants and AP-1 transcription factor in NFAT3 activation induced by ANG II in cardiomyocytes.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and reagents. Chemicals were purchased from Sigma (St. Louis, MO) unless otherwise indicated. Stabilized H2O2 (Sigma, H-1009) was used, and the concentration of the stock was verified by absorbency at 240 nm (OD240 = 1.0 for 0.023 M H2O2). PD98059, SB202190, and LY249002 were obtained from Calbiochem (La Jolla, CA).

Cell culture and H2O2 or ANG II treatment. Cardiomyocytes were prepared from 1- to 2-day-old neonatal Sprague-Dawley rats (Harland, Indianapolis, IN) as previously described (7, 45). The cardiomyoctes were seeded at a density of 5 x 104 cells/cm2 and plated in DMEM with 1 mM pyruvate, 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 units/ml streptomycin. Cells were pretreated with inhibitors for 1 h and then treated with H2O2 for 10 min. After H2O2 treatment, oxidized medium was changed to fresh 0.5% FBS-DMEM with or without inhibitor added back. When ANG II was used, cells were pretreated with antioxidants for 30 min before ANG II addition. After 12 h, the medium was changed, and ANG II and antioxidants were added back for 24-h harvesting.

Transfection and NFAT luciferase assay. Cardiomyocytes were seeded at 0.5 x 106 per well in six-well plates (~960-mm2 culture area per well). At 24 h after plating, cells were incubated 5 h with 0.8 µg of the NFAT luciferase reporter construct pNFAT-luc (Stratagene, La Jolla, CA) and 0.4 µg of the NFAT3 expression vector RSV-NFAT3 per well using 3 µl of FuGene-6 liposomes (Roche, Mannheim, Germany). The transfection efficiency is generally low (3–10%) in cardiomyocytes and can vary because of the nature of primary culture. The low transfection efficiency and different batches of primary cultures of cardiomyocytes contribute to the variation in the data of promoter reporter gene assay. To correct for transfection efficiency, we cotransfected cardiomyocytes with the pRL-TK plasmid (0.04 µg/well), which encodes a Renilla luciferase gene under the control of a thymidine kinase (TK) promoter. After transfection, the cells were placed in 10% FBS-DMEM for 24 h and subsequently in 0.5% FBS-DMEM for 18–24 h before H2O2 treatment, or in 0% FBS-DMEM for 18–24 h before ANG II treatment. When cells were cotransfected with catalase, Tam67, or a dominant negative form of NFAT (dnNFAT), the amount of pNFAT-luc, RSV-NFAT3, or pRL-TK DNA was reduced to 50%. The activity of Firefly vs. Renilla luciferase was measured using a dual luciferase assay kit (Promega, Madison, WI) with a luminometer (Turner Designs, Sunnyvale, CA). Results were expressed as the ratio of the relative light unit (RLU) from the readings of Firefly vs. Renilla luciferase.

EMSA. Cardiomyocytes were harvested, and nuclear extracts were prepared as described previously (46). Briefly, cardiomyocytes were lysed on ice with lysis buffer (10 mM KCl, 1.5 mM MgCl2, 0.15% NP-40, 10 mM HEPES, pH 7.9, 0.5 mM DTT, 0.5 mM PMSF). After microscopic examination for the completion of cell lysis, the nuclei were collected by centrifugation at 2,000 g and resuspended in an ice-cold buffer [1.5 mM MgCl2, 0.2 mM EDTA, 26% glycerol (vol/vol), 5 mM HEPES, pH 7.9, 0.5 mM DTT, 0.5 mM PMSF]. NaCl was added to a final concentration of 0.3 M. After 30-min incubation on ice, samples were centrifuged at 20,000 g for 10 min for collection of nuclear extracts. DNA binding reactions were carried for 30 min at 4°C in a reaction volume of 20 µl containing 8 µg of nuclear protein, 4% glycerol, 5 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 10 mM Tris·HCl, pH 7.5, 0.05 mg/ml poly(dI-dC), and 30,000 counts/min (cpm) 32P-labeled oligonucleotide probes (GGAGGAAAAACTGTTTCATACAGAAGGCGT). When supershift assay was performed, nuclear extracts were preincubated with antibodies against NFAT3 from Dr. Nancy Rice (22) for 1 h in the reaction buffer at 4°C before the radiolabeled oligonucleotide probe was added to the reaction mixture. For competition assay, 50x unlabeled NFAT oligonucleotide probe was added to the binding mixture. The binding complexes were separated on nondenaturing 5% polyacrylamide gels. The gels were vacuum dried for autoradiography.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Antioxidants inhibit ANG II from activating NFAT3 transcription factor. NFAT3 is the major form of NFAT family member expressed in cardiomyocytes (38). ANG II has been shown to activate NFAT3 transcription factor in cardiomyocytes (29). To determine a role of reactive oxygen species (ROS) in mediating ANG II-induced NFAT3 activation, we exposed cells to three cell-permeable antioxidants: N-acetyl-L-cysteine (NAC), {alpha}-phenyl-N-tert-butylnitrone (PBN), and lipoic acid. NAC is a precursor of reduced glutathion, and PBN is a free radical spin trap, while lipoic acid, a dithiol compound serving as a mitochondrial bioenergic enzyme cofactor, is known to exhibit antioxidant properties. Lipoic acid has been shown to reduce myocardial injury and cardiac hypertrophy in a transgenic hypertensive animal model (26). Unlike commonly known antioxidant vitamins, such as vitamin E and ascorbic acid, which become prooxidants because of oxidation in the air or tissue culture medium, the three compounds used in this study retain their antioxidant potential in tissue culture medium. Cardiomyocytes transfected with an NFAT promoter-luciferase construct were pretreated with NAC (10 mM), PBN (5 mM), or lipoid acid (200 µM) for 30 min before addition of ANG II (1 µM). Measurements of luciferase activity at 24 h after ANG II treatment indicate that NAC, PBN, and lipoic acid were capable of preventing ANG II from activating the NFAT3 promoter (Fig. 1A). A similar response was observed with ANG II at a 100 nM concentration.


Figure 1
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  		Fig. 1. Antioxidants inhibit ANG II from activating nuclear factor of activated T cells-3 (NFAT3) transcription factor in cardiomyocytes. Rat neonatal cardiomyocytes were transfected with the NFAT luciferase reporter construct (pNFAT-luc) and serum starved as described in MATERIALS AND METHODS. The cells were pretreated with N-acetyl-L-cysteine (NAC; 10 mM), {alpha}-phenyl-N-tert-butylnitrone (PBN; 5 mM), or lipoid acid (LPA; 200 µM) for 30 min before addition of 1 µM ANG II. After 12 h, cells were placed in fresh medium with newly added ANG II and corresponding antioxidants. After a total of 24 h of incubation, the cells were harvested for luciferase activity assay (A). For catalase cotransfection experiment, cells were treated with ANG II for a total of 24 h with one medium change at 12 h (B). The data are presented as means ± SD of the ratios of NFAT luciferase over Renilla luciferase, in relative light units (RLU), from 3 independent experiments (n = 3). Ctr, control.

 
ANG II has been shown to produce superoxide by activation of a plasma membrane-associated NADPH oxidase (13). Cardiomyocytes express cytosolic superoxide dismutase, which can convert superoxide to H2O2 (48). Expression of catalase allows the removal of H2O2. We performed a cotransfection experiment in which an expression vector of catalase was introduced into cardiomyocytes at the time of transfection of the NFAT promoter-luciferase construct. The results show that cotransfection with catalase vector prevented ANG II from activating the NFAT promoter (Fig. 1B).

We have used a luciferase reporter gene construct containing four repeats of a NFAT binding site derived from the interleukin-2 (IL-2) promoter for studying NFAT activation by ANG II (18). Because of the low expression level of NFAT3 in cardiomyocytes and low transfection efficiency of primary cultured cardiomyocytes, the NFAT3 expression vector RSV-NFAT3 was cotransfected with the NFAT promoter-luciferase construct to augment the promoter activity. Without NFAT3 expression, the basal and induced levels of NFAT3 promoter-luciferase were low (Table 1), suggesting that induction of NFAT3 promoter activity by ANG II was indeed associated with the activation of NFAT3 transcription factor.


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  		Table 1. Verification of the promoter in pNFAT-luc as the target of NFAT3 transcription factor

 
The data with NAC, PBN, lipoic acid, and catalase suggest that ROS may activate the NFAT3 transcription factor in cardiomyocytes. To test this, cardiomyocytes were treated with 100 µM H2O2 for 10 min and harvested at various time points after for measurement of NFAT3 activation using a promoter-reporter gene assay. Measurements of luciferase activities indicated an activation of NFAT promoter within 2 h after H2O2 treatment (Fig. 2A). Luciferase activity reached a peak of 2.5-fold at 4–6 h and started to decline 8 h after H2O2 treatment (Fig. 2A). At 24 h after treatment, the activity returned toward to the basal level (Fig. 2A). For dose response studies, five groups of cardiomyocytes were treated with H2O2 at various doses for 10 min and harvested 6 h after treatment. A dose-dependent increase of luciferase activity was observed with 50–150 µM H2O2 (Fig. 2B). The optimal dose for activation of NFAT promoter was 100 µM. At 200 µM, H2O2 was less efficient in activating NFAT promoter compared with at 100–150 µM (Fig. 2B). These results imply that H2O2 at a defined dose range is able to activate NFAT transcription factor in cardiomyocytes within a specific time frame.


Figure 2
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  		Fig. 2. H2O2 induces time (A)- and dose (B)-dependent activation of NFAT3 transcription factor. At 48 h after transfection with pNFAT-luc, cells were treated with H2O2 at 100 µM (A) or at various doses (B) for 10 min before being placed in fresh medium. Cells were collected at indicated time points (A) or at 6 h (B) for dual luciferase assay. Data represent means ± SD of the ratios of NFAT luciferase over Renilla luciferase, in RLU, from 3 independent experiments (n = 3).

 
EMSA was used to verify the activation of NFAT3 transcription factor. The binding of NFAT3 transcription factor to its cis-element of DNA was detected at 2 and 3 h after H2O2 treatment (Fig. 3). To test the specificity of DNA binding by NFAT3 transcription factor, we used an antibody against NFAT3 for a supershift assay. Preincubation of nuclear extract with the antibody eliminated the DNA binding, indicating the specificity of H2O2-induced NFAT3 binding to the cis-element of DNA (Fig. 3). The band representing the binding of NFAT3 to the cis-element of DNA can also be eliminated by excessive unlabeled oligonucleotide probe in gel shift assay, further indicating the specificity (Fig. 3). The bands showing lower molecular weight and not cleared by the NFAT3 antibody or unlabeled probe likely result from nonspecific protein-DNA interaction.


Figure 3
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  		Fig. 3. H2O2 induces NFAT3 binding to DNA cis-element. Cardiomyocytes were treated with 100 µM H2O2 for 10 min and placed in fresh medium until indicated time point. Nuclear extracts were prepared for EMSA with or without anti-NFAT3 antibodies or excess unlabeled probe, as described in MATERIALS AND METHODS. Arrow indicates specific binding of NFAT3 protein to DNA. Image represents 1 of 3 independent experiments.

 
To further verify that the NFAT3 transcription factor is activated by H2O2, we used a dominant negative form of NFAT3 (dnNFAT3). H2O2 treatment caused a consistent induction of NFAT3 luciferase between independent experiments, although the activity of luciferase may vary, in part, because of low transfection efficiency of cardiomyocytes (Fig. 4). The dnNFAT3 expression vector contains a gene encoding a truncated form of NFAT3 with remaining amino acids 1–130. Chow et al. (6) indicated that this dnNFAT blocks nuclear translocation of NFAT proteins by interfering with calcineurin. Inhibitors of calcineurin have been shown to prevent ANG II from activating NFAT3 (29). When pNFAT-luc was cotransfected with the dnNFAT3 expression vector at various amounts, we found an increasing inhibitory efficiency from 0.2 to 0.8 µg of dnNFAT3 plasmid DNA (Fig. 4). The data suggest that H2O2 indeed activates the NFAT3 transcription factor.


Figure 4
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  		Fig. 4. Dominant negative NFAT3 (dnNFAT3) inhibits H2O2-induced NFAT luciferase. Cardiomyocytes were transfected with pNFAT-luc with or without cotransfection of dnNFAT3. At 48 h after transfection, cells were treated with 100 µM H2O2 for 10 min and then placed in fresh medium for 6 h before harvesting for dual luciferase assay. Data represent means ± SD of the ratios of NFAT luciferase over Renilla luciferase, in RLU, from 3 independent experiments (n = 3).

 
AP-1 in H2O2-induced NFAT3 activation. Previous studies from our laboratory (45, 46) demonstrated that PI3K, p38 MAPK, and ERKs were activated by H2O2 and contributed to the development of hypertrophy. The pharmacological inhibitors of these signaling molecules allow us to explore whether PI3K, p38, or ERKs participate in NFAT3 regulation. NFAT promoter-luciferase construct-transfected cells were pretreated with the PI3K inhibitor LY249002 (20 µM), the p38 MAPK inhibitor SB210190 (10 µM), or the MEK1/ERK inhibitor PD98059 (50 µM) before H2O2 treatment. These drugs at indicated doses have been shown previously to inhibit their corresponding targets in cardiomyocytes (45, 46). The results from NFAT promoter-luciferase activity assay indicate that neither LY249002 nor SB210190 had an inhibitory effect (Fig. 5, A and B). In contrast, PD98059 prevented H2O2 from activating NFAT3 promoter (Fig. 5C).


Figure 5
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  		Fig. 5. Effect of pharmacological inhibitors on H2O2-induced NFAT3 activation. At 48 h after transfection of pNFAT-luc, cells were pretreated with 20 µM LY249002 (LY; A), 10 µM SB202190 (SB; B), or PD98059 at indicated doses (C) for 1 h before a 10-min treatment of H2O2 at 100 µM. Samples were collected at 6 h after H2O2 treatment for dual luciferase assay. Data represent means ± SD of the ratios of NFAT luciferase over Renilla luciferase, in RLU, from 3 independent experiments (n = 3).

 
PD98059 has been shown previously to inhibit H2O2 from activating AP-1 transcription factor (46). The NFAT binding site in the IL-2 promoter (GGAGGAAAAACTGTTTCATACAGAAGGCGT) contains a weak AP-1 binding site (TGTTTCA) adjacent to the core NFAT consensus sequence (GGAAA). The cooperation between NFAT and AP-1 has been shown to be an important mechanism of NFAT-dependent gene expressions in immune cells (24). Since ERKs regulate the activation of AP-1 transcription factor and were found to inhibit NFAT promoter activation by H2O2, we tested whether AP-1 transcription factors mediate NFAT activation induced by H2O2.

We used a dominant negative mutant of c-Jun, TAM67. TAM67 lacks the transactivation domain of c-Jun but is able to dimerize with c-Jun or c-Fos family members to inhibit AP-1 activity (2, 43). This construct has been shown to inhibit AP-1 in various cellular responses (9, 10). We cotransfected cardiomyocytes with a TAM67 expression vector and the NFAT promoter-luciferase construct. The data show that H2O2-induced NFAT luciferase activity was blocked by TAM67 (Fig. 6). This inhibitory effect was TAM67 dose dependent, and a complete inhibition was observed with 0.8 µg of TAM67 plasmid (Fig. 6A).


Figure 6
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  		Fig. 6. Requirement of activator protein-1 (AP-1) transcription factor in H2O2-induced NFAT3 activation. TAM67 or control vector at indicated amounts of DNA was cotransfected with pNFAT-luc (A). Two different luciferase reporter gene constructs, pNFAT-luc and M-luc, were transfected into cells within the same experiment for comparison of luciferase activity (B). At 48 h after transfection, cells were treated with 100 µM H2O2 for 10 min and then placed in fresh medium. Samples were collected at 6 h after H2O2 treatment for dual luciferase assay. Data represent means ± SD of the ratios of NFAT luciferase over Renilla luciferase, in RLU, from 3 independent experiments (n = 3).

 
To further verify that the increased NFAT promoter-luciferase activity observed with H2O2 treatment is AP-1 dependent, we used a NFAT promoter-luciferase construct that does not contain an AP-1 binding site adjacent to the NFAT3 core consensus sequence. This construct (M-Luc) has a NFAT-driven minimal promoter composed of three repeats of consensus NFAT binding site (TGGAAAAATAT) upstream of a thymidine kinase minimal promoter (29). After transfection of this AP-1-lacking NFAT promoter-luciferase construct, cardiomyocytes were treated with H2O2 for luciferase assay. The results show that H2O2 failed to activate the AP-1-lacking NFAT3 promoter (Fig. 6B).

To test that AP-1 also mediates ANG II-induced NFAT3 activation in cardiomyocytes, we performed TAM67 cotransfection experiments for measurements of NFAT promoter activation through luciferase reporter constructs. Although the activity of luciferase varies among independent experiments, the data suggest that TAM67 cotransfection can inhibit NFAT3 promoter activation by ANG II treatment (Fig. 7).


Figure 7
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  		Fig. 7. Dominant negative c-Jun inhibits ANG II from activating NFAT3. TAM67 or control vector (0.4 µg of plasmid DNA) was cotransfected with pNFAT-luc into cardiomyocytes. At 48 h after transfection, cells were treated with 1 µM ANG II for a total of 24 h with one medium change and ANG II being added back at 12 h. Cells were harvested for dual luciferase assay. Data represent means ± SD of the ratios of NFAT luciferase over Renilla luciferase, in RLU, from 3 independent experiments (n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study found that compounds exhibiting antioxidant properties, i.e., NAC, PBN, and lipoic acid, prevent ANG II from activating the transcription factor NFAT3 in cardiomyocytes. Consistent with the hypothesis that oxidants mediate NFAT3 activation, we found that H2O2 induces activation of NFAT3 transcription factor in a narrow dose range (50–150 µM) within 1–8 h. A chemical inhibitor of ERKs, PD98059, inhibited H2O2 from activating NFAT3. In contrast, the PI3K inhibitor LY249002 and the p38 MAPK inhibitor SB202190 failed to show an inhibitory effect on H2O2-induced NFAT3 activation. A dominant negative form of c-Jun, TAM67, abolished NFAT3 activation. Because PD98059 inhibits the activation of AP-1 transcription factors by H2O2 (46), our data suggest a role of AP-1 downstream of ERKs in the H2O2-induced NFAT3 activation in cardiomyocytes.

Cooperation of NFATs with other families of transcription factors is an important character of NFATs in regulating gene expression. There is evidence that AP-1 transcription factor is activated during the process of cardiac hypertrophy (30). In immune cells, NFAT1 and NFAT2 regulate the expression of cytokines including IL-2, IL-3, IL-4, IL-5, interferon-{gamma} (IFN-{gamma}), and tumor necrosis factor-{alpha} (TNF-{alpha}) and cell surface receptors such as CD40L and FasL (25, 34). Activation of the transcription of many of these genes results from the partnership of NFATs and AP-1 transcription factors (8, 24, 25, 38). For example, the expression of IL-2, IL-3, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), and FasL genes has been reported to require the interaction of NFATs with AP-1 (23). The AP-1 protein capable of mediating the activation of NFATs is usually the heterodimer of c-Fos:c-Jun (5, 24). For other genes, such as TNF-{alpha}, the AP-1 binding site is occupied by the c-Jun:ATF2 heterodimer in conjunction with NFATs (23). In either case, the core NFAT consensus sequence (GGAAA) is in a close proximity with a weak AP-1 binding site (TGTTTCA). The cooperation of NFATs and AP-1 to DNA binding greatly enhances the stability of the ternary complex compared with the DNA binding affinity of either NFATs or AP-1 alone (38). In fact, neither NFAT nor AP-1 alone binds to its consensus sequence in the composite promoter with high affinity (3, 19, 36). The binding of AP-1 or NFATs to their corresponding cis-element in the composite promoter is characterized by a relatively high dissociation rate. Deletion or mutation of the AP-1 cis-element results in a loss of NFAT transcription factor binding to the NFAT cis-element (24). A mutant NFAT protein unable to interact with c-Fos:c-Jun heterodimers failed to activate the NFAT promoter (23, 36). Interestingly, NFATs do not appear to interact with AP-1 proteins well in the absence of DNA (3, 19, 36). Because of this caveat, conventional immunochemical methods cannot be used to demonstrate the interaction between NFATs and AP-1. Regardless, although the synergistic interaction between NFATs and AP-1 transcription factors is a well-established phenomenon in immune cells, whether this interaction is present in cardiomyocytes has not been addressed previously. In cardiomyocytes, because hypertrophy inducers activate AP-1 as well as NFAT3, it becomes important to evaluate the cooperation between NFAT-3 and AP-1 in regulating gene expression associated with cardiomyocyte hypertrophy.

The cooperative physical interaction between NFATs and AP-1 has been found to regulate most but not all genes containing an NFAT binding consensus sequence in the promoter region (23, 24). In cardiomyocytes, NFAT3 has been shown to interact with the GATA transcription factor to regulate the expression of B-type natriuretic peptide (BPN) (29), a gene that elevates its expression in the early stage of cardiac hypertrophy. A potential binding site has been found in the BPN promoter (29). In addition, NFAT binding sites can appear to be NF-{kappa}B like, such as in the TNF-{alpha} promoter-{kappa}3 site (GGAGAACCC=NFAT/NF-{kappa}B). This NF-{kappa}B-like site also appears in the promoter region of the HIV-1 LTR gene (GGGACTTTCC), which is situated next to an Sp1 consensus sequence. The difference in the partners or DNA binding habit causes NFATs to regulate the expression of different sets of genes.

Recent evidence suggests that MAPKs participate in the regulation of NFATs and cardiac hypertrophy. Harris et al. (16) found that the Raf-1/ERKs pathway is essential for cardiomyocyte survival and cardiac hypertrophy in response to pressure overload. Tsatsanis et al. (44) reported that PD98059 prevented the Tpl-2 kinase from inducing NFAT activation in T-lymphocytes. Ichida and Finkel (18) demonstrated that activating the Ras/MEK1/ERK pathway results in NFAT activation in cardiomyocytes. MEKK3, an upstream kinase regulating MAPKs including ERKs, has been found to mediate ANG II-induced NFAT activation in T-lymphocytes (1). These reports are consistent with our finding in H2O2-induced NFAT3 activation. In our case, we have linked ERKs to activation of AP-1 transcription factor (46), which appears to serve as an important mechanism regulating NFAT3 activation. Additional evidence in the literature supporting this link includes the finding that MEKK3 regulates AP-1 as well as NFAT activation (1, 47). The interaction of NFAT3 with AP-1 probably occurs in the nuclei following nuclear translocation of NFAT3. Transfection experiments using green fluorescent protein-conjugated NFAT3 indicate that NFAT3 normally distributes in the nuclei as well as in the cytoplasm of cardiomyocytes (18). Therefore, an activated AP-1 transcription factor could recruit existing NFAT3 in the nuclei onto the NFAT/AP-1 composite site in DNA to cause transcriptional activation.

Proinflammatory interleukins, many of which are regulated by NFATs in cooperation with AP-1 transcription factors in lymphocytes, are indicated to play a role in myocardial remodeling after cardiac infarction, where oxidants are often overproduced (37). This suggests the possibility that oxidant-induced NFAT activation may serve as a mechanism of regulating interleukin expression in the myocardium following ischemia or ischemic reperfusion. Evidence suggests that interleukins, such as IL-6 and IL-1, contribute to myocardium remodeling, cardiac hypertrophy, and ultimately heart failure (17, 35, 42). The plasma level of these interleukins has been found elevated in hypertensive and heart failure patients (14, 37). If oxidants indeed induce or mediate the expression of these interleukins through an NFAT3-dependent mechanism in cardiomyocytes, there is additional evidence supporting that oxidants contribute to cardiac hypertrophy. It appears that the mechanism of oxidant-induced cardiomyocyte hypertrophy involves multiple pathways, some of which may intervene and overlap with those from other well-established hypertrophy inducers.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Burroughs Wellcome Foundation, the American Heart Association, the American Federation for Aging Research, the Arizona Disease Control Research Commission, and National Institutes of Health Grants R01-ES-010826 and RO1-HL-076530-01.


   ACKNOWLEDGMENTS
 
We thank Dr. Jeffery Molkentin (Cincinati Children's Hospital) for the RSV-NFAT3 expression vector and NFAT promoter-luciferase construct (M-Luc), Dr. Roger Davis (University of Massachusetts Medical School) for the dnNFAT3 construct, Dr. Margaret Briehl (University of Arizona) for the catalase expression vector, and Dr. Nancy R. Rice (National Cancer Institute at Frederick) for the NFAT3 polyclonal antibody.

Current address for V. C. Tu: Dept. of Toxicology, Avon Products, Inc., 1 Avon Pl., Suffern, NY 10901.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Q. M. Chen, Dept. of Pharmacology, Arizona Cancer Center, College of Medicine, Univ. of Arizona, 1501 N. Campbell Ave., Tucson, AZ 85724 (e-mail: qchen{at}email.arizona.edu)

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.


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