INTRODUCTION

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The zinc finger-containing transcription factor GATA4 is an important regulator of tissue-specific gene expression in multiple mesodermally and endodermally derived tissues. In cardiac myocytes, GATA4 regulates expression of diverse genes including -myosin heavy chain (-MHC), -myosin heavy chain (-MHC), cardiac troponin-C, atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), cardiac troponin-I, sodium/calcium exchanger, cardiac-restricted ankyrin repeat protein, A1 adenosine receptor, m2 muscarinic receptor, and the myosin light chain 1/3 (reviewed in reference 22). In response to agonist or stress stimulation, many of the above listed genes are upregulated in cardiomyocytes as part of the hypertrophic response, suggesting that a common regulatory factor such as GATA4 would be an ideal factor for coordinating uniform alterations in stress-induced gene expression.

Analysis of the -MHC promoter in aortic-banded rats (pressure overload) revealed a proximal GATA binding site that mediated hypertrophy-induced expression in vivo (15). GATA4 was also implicated in regulating pressure overload-induced expression of the angiotensin type-1A receptor promoter in the adult rat heart (17). In neonatal cardiomyocyte cultures, electrical pacing-induced hypertrophy was associated with increased GATA4 mRNA levels (41), although GATA4 protein levels are not likely affected by hypertrophic stimulation (16, 20, 24). Alternatively, GATA4 transcriptional activity and DNA binding activity have been shown to be upregulated in response to hypertrophic stimuli (16, 17, 20, 24). In one report, as little as 15 min of arginine-vasopressin infusion in the adult rat was associated with enhanced GATA4 DNA binding activity in the heart (16). In cultured cardiomyocytes, phenylephrine (PE)-induced upregulation of the endothelin-1 promoter was associated with increased phosphorylation of GATA4, which was sensitive to PD98059, suggesting a role for MEK1/2-ERK1/2 in regulating GATA4 (24). Collectively, these various studies have suggested the hypothesis that GATA4 is regulated in the heart by stress-responsive signaling pathways such as the mitogen-activated protein kinase (MAPK) cascade.

The MAPK cascade consists of a series of successively acting protein kinases that include three well-characterized branches, the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and the p38 MAPKs (reviewed in reference 13). Signaling through each of these MAPK branches is initiated by diverse stress and mitogenic stimuli localized to the cell membrane or within the cytoplasm. Activation of ERKs, JNKs, and p38 MAPKs facilitates the phosphorylation of multiple transcriptional regulators such as myocyte enhancer factor-2, activating transcription factor-2, Elk-1, p53, nuclear factor of activated T cells, Max, c-Jun, and c-Myc (13). MAPK-mediated phosphorylation of these and other transcriptional regulators profoundly influences adaptive and inducible gene expression in many cell types. Members of the MAPK signaling cascade are also important regulators of cardiomyocyte hypertrophy (reviewed in reference 34), yet the downstream transcriptional mechanisms that alter cardiac gene expression have not been well characterized.

Here we demonstrate that GATA4 contains a conserved MAPK phosphorylation site at serine 105 within the transcriptional activation domain. Serine 105 of GATA4 is phosphorylated in response to agonist stimulation through MEK1-ERK1/2, but only weakly through JNK1/2 or p38 MAPKs. Phosphorylation-specific antisera directed against serine 105 of GATA4 implicated ERK1/2 as the relevant kinase. Mutagenesis of serine 105 in GATA4 attenuated agonist-induced transcriptional upregulation, as did the MEK1 inhibitor U0126. Finally, purified ERK2 protein directly phosphorylated serine 105 of bacterially purified GATA4. That GATA4 is a critical downstream effector of MEK1-ERK1/2 signaling was demonstrated through the inhibition of MEK1-induced cardiomyocyte hypertrophy with a dominant-negative engrailed-GATA4-expressing adenovirus.

	MATERIALS AND METHODS

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Western blotting. Protein extracts were generated from cultured cardiomyocytes or whole hearts and subjected to polyacrylamide gel electrophoresis and Western blotting as described previously (10). Primary antibodies and secondary antibodies were incubated overnight at room temperature in 5% milk and for 1 h at room temperature in 5% milk, respectively. Quantitative chemiluminescent detection was performed with Vistra enhanced chemifluorescence (Amersham) and scanned utilizing a Storm 860 (Molecular Dynamics, Sunnyvale, Calif.). Commercial antibodies used included anti-phospho-p38, anti-p38, anti-phospho-ERK1/2, anti-ERK1/2 (Cell Signaling, Beverly, Mass.), anti-GAPDH (Research Diagnostics Inc., Flanders, N.J.), and anti-GATA4 (Santa Cruz, Santa Cruz, Calif.).

Anti-phospho-GATA4(105) antibody generation. Phospho-specific rabbit antiserum was prepared against a GATA4 peptide consisting of YTPPPV-phospho-serine-PRFSFP (amino acids 99 to 111) at Research Genetics (Huntsville, Ala.). The resulting antiserum was cross-absorbed with the nonphosphorylated peptide YTPPPVSPRFSFP followed by immunoaffinity purification.

GST-GATA4 fusion proteins. To generate fusion proteins between glutathione S-transferase (GST) and GATA4, DNA sequences encoding amino acids 80 to 250 or 241 to 378 of GATA4 were amplified by PCR and subcloned into the EcoRI-XhoI sites of pGEX-4T-1 (Amersham Pharmacia Biotech). The full-length GST-GATA4 fusion cDNA (or the S105A mutant) encodes amino acids 1 to 441 of GATA4, which was subcloned into pGEX-4T-1 as an EcoRI fragment. All fusion proteins were expressed in Escherichia coli BL21 cells, precipitated with glutathione-Sepharose beads, and eluted with reduced glutathione (10 mM in 50 mM Tris, pH 8.0). The purity and concentration of each fusion protein were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using bovine serum albumin standards.

In vitro phosphorylation assays. In vitro phosphorylation of GST-GATA4 fusions was carried out at 30°C for 20 min using 25 ng of activated ERK2, p38, or JNK1 (Upstate Biotechnology) in a buffer containing 20 mM MOPS (pH 7.2), 25 mM -glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mM dithiothreitol supplemented with 75 mM MgCl₂, 500 µM ATP, and 10 µCi of [-³²P]ATP. Three hundred nanograms of each GATA4 fusion protein or 10 µg of myelin basic protein (MBP) (positive control) or recombinant c-Jun was used to examine ERK2-, p38- or JNK1-induced phosphorylation. Reactions were separated by SDS-polyacrylamide gel electrophoresis. Parallel phosphorylation reactions were performed in the absence of [-³²P]ATP and subjected to Western blot analysis with phospho-specific GATA4 antiserum.

EMSAs. Electrophoretic mobility shift assays (EMSAs) were performed using a double-stranded oligonucleotide containing two GATA motifs from the -MHC promoter as previously described (20). Briefly, 300 ng of bacterially generated GST-GATA4 (full-length) or GST-GATA4 S105A (full-length) were incubated with 50,000 cpm of ³²P-labeled double-stranded oligonucleotide, 1 µg of poly(dI-dC)-(dI-dC), and EMSA buffer (12 mM HEPES [pH 7.9], 4 mM Tris [pH 7.9], 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 12% glycerol, and 2 µg of aprotinin, leupeptin, and pepstatin/ml) at room temperature for 20 min in a 20-µl volume. A nondenaturing 5% polyacrylamide gel with 0.5× Tris-borate-EDTA was used to resolve the bound protein complexes from the free probe.

Cell culturing and transfections. Primary neonatal rat cardiomyocytes derived from the ventricles of 1- to 2-day-old Sprague-Dawley rat neonates were prepared as described previously (10). Cardiomyocytes were plated on gelatinized dishes and cultured in serum-free M199 medium containing 100 U of penicillin-streptomycin/ml and 2 mmol of L-glutamine/liter. Both cardiomyocytes and Cos-7 cells were transfected with Fugene 6 (Roche Diagnostics Corporation, Indianapolis, Ind.). Cultures were harvested 48 h after transfection and luciferase assays were performed as described previously (23). PE and MAPK inhibitors were added 24 h prior to harvest.

Plasmids. Plasmids used to generate AdGATA4, AdG4-Engr, and AdEngr were described previously (20). The activated MKK6b (Ser 207,211 Glu) and dominant-negative MKK3b (K-A) were a gift from Roger Davis, University of Massachusetts, Worcester, while MKK7 was a gift from E. Nishida, Kyoto University. Dominant-negative MEK1 (Ser 221 Ala) was a gift from C. J. Marshall, Institute of Cancer Research, London, United Kingdom. Each cDNA was subcloned as a HindIII fragment into pACCMCpLpA for subsequent viral production. To generate GAL4-GATA4, PCR-amplified GATA4 (amino acids 33 to 227) was ligated into pM1-GAL4 (amino acids 1 to 147) as an EcoRI fragment. Generation of the site-specific mutation at amino acid 105 was performed as previously described (23). The BNP luciferase reporter (116 to +83) was previously described and was a gift from Chris Glembotski, San Diego State, San Diego, Calif. (37).

Replication-deficient adenovirus infection. The procedures for generating and plaque purifying replication-deficient adenovirus were performed as previously described (10). Cardiomyocytes were infected at a multiplicity of infection of 100 PFU per cell for a single infection or at 50 PFU per cell for coinfections. Infection occurred for 2 h at 37°C in a humidified, 6% CO₂ incubator, and then the cardiomyocytes were placed in serum-free M199 medium for an additional 24 h prior to treatments or harvesting. Under these conditions approximately 98% of cells showed expression of each of the different viral gene inserts. The constitutively active MEK1- and dominant-negative MKK4-expressing adenoviruses were previously described (5, 6).

Immunocytochemistry. Cultured cardiomyocytes were prepared for immunocytochemistry against sarcomeric -actinin and ANF as described previously (10). Quantitation of cardiomyocyte cell surface area was performed on -actinin-stained cardiomyocytes using NIH Image software on a Sun system workstation. At least 100 cardiomyocytes in 15 to 30 fields at ×400 magnification were examined in three independent experiments. For quantitation of ANF expression, cardiomyocytes showing intense perinuclear staining were counted in at least 25 fields (n = 3).

Leucine incorporation assay. Determination of protein synthesis rates in cultured cardiomyocytes by [³H]leucine incorporation was described previously (31). Briefly, cardiomyocytes were infected with adenovirus, incubated 24 h to allow adequate expression, preincubated with leucine-free RPMI medium for 1 h followed again by incubation with 2.5 µCi of [³H]leucine/ml for 24 h. Plates were washed with phosphate-buffered saline and then 10% trichloroacetic acid was added at 4°C and incubated for 60 min to precipitate protein. The precipitate was washed twice in 95% ethanol and resuspended in 0.5 N NaOH and was measured by scintillation counting.

Statistical analysis. Data are expressed as means ± standard errors of the means. Differences between groups were evaluated for statistical significance using Student's t test for unpaired data or one-way analysis of variance followed by Bonferroni's posttest. P values of <0.05 were considered statistically significant. All statistical analyses were performed by using Instat 3.0 software (GraphPad, San Diego, Calif.).

	RESULTS

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GATA4 is phosphorylated at serine 105. We previously demonstrated that GATA4 DNA binding activity was up-regulated in pressure-overloaded rat hearts, suggesting that GATA4 is activated by stress responses in the heart (17). One potential mechanism involved in mediating this increase in GATA4 DNA binding activity is through direct phosphorylation by stress-activated kinases, which are important regulators of cardiomyocyte hypertrophy. Indeed, Morimoto et al. demonstrated that GATA4 was phosphorylated in response to -adrenergic stimulation of cardiomyocytes (24). Consistent with this finding, we also observed in vivo phosphorylation of GATA4 in response to hypertrophic agonist stimulation in cardiomyocytes; however, the relevant site(s) and potential modifying kinases are not known.

View larger version (52K): [in this window] [in a new window]   FIG. 1.   GATA4 contains a conserved MAPK phosphorylation site at serine 105. (A) Diagram of GATA4 showing the position of the zinc finger domains (Zn), the nuclear localization sequence and basic domain (nls), and the transcriptional activation domain (TAD). A MAPK recognition site containing serine 105 is conserved between mouse, human, and chicken GATA4. (B) Phospho-105-GATA4-specific antiserum was generated and used to examine GATA4 phosphorylation in response to -adrenergic stimulation with PE in cultured cardiomyocytes. Phosphorylation of endogenous GATA4 was upregulated by PE in control Adgal-infected cultures, while a similar pattern of up-regulation was observed when GATA4 was overexpressed by AdGATA4 infection. (C) Endogenous GATA4 was also phosphorylated at serine 105 in the hearts of PE-injected mice (3 h), compared to saline-treated control mice (n = 3 in each group). GATA4 protein levels did not vary.

ERK1/2 mediates phosphorylation of serine 105. The broad range of agonists identified above that promote GATA4 phosphorylation suggested the involvement of MAPK signaling effectors since they mediate diverse stress stimuli in many cell types (13). Cultured cardiomyocytes were coinfected with AdGATA4 and specific MAPK kinase-encoding adenoviruses in an attempt to implicate one or more kinase effectors in the phosphorylation of serine 105. The data demonstrate that coinfection with a constitutively active MEK1 adenovirus, which directly activates ERK1/2, promoted robust phosphorylation of GATA4 compared to Adgal-coinfected cardiomyocyte cultures (Fig. 2A). However, infection with a constitutively active MKK6 (activates p38) or MKK7 (activates JNK1/2) adenovirus did not result in the same degree of activation (Fig. 2A). GATA4 protein levels did not significantly vary in any of the coinfected cultures (Fig. 2A). Similar results were observed for three independent experiments. These data suggest that the MEK1-ERK1/2 pathway promotes phosphorylation of serine 105 in GATA4.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   MEK1-ERK1/2 signaling promotes phosphorylation of serine 105 in GATA4. (A) Cardiomyocyte cultures were coinfected with AdGATA4 and either Adgal, AdMEK1, AdMKK7, or AdMKK6. Twenty-four hours later, cultures were harvested and Western blotted with phospho-specific GATA4 antibody and then reprobed with GATA4 antibody. (B) Cardiomyocyte cultures were coinfected with AdGATA4 and either Adgal, Ad-dnMEK1, Ad-dnMKK4, or Ad-dnMKK3 and stimulated with PE for 3 h. Western blotting demonstrated reduction in PE-induced serine 105 phosphorylation with dominant-negative MEK1 (asterisk) but not with other dominant-negative MAPK kinase factors. (C) Quantitation of three independent experiments demonstrates a significant inhibition of PE-induced serine 105 phosphorylation only with Ad-dnMEK1 infection. *, P < 0.05 compared to Adgal infection.

View larger version (29K): [in this window] [in a new window]   FIG. 3.   ERK2 directly phosphorylates GATA4 in vitro. (A) Schematic representation of full-length GATA4 and the two different fusion proteins that were generated and purified from bacteria. (B) Polyacrylamide gel of [32P]ATP-labeled proteins. Three hundred nanograms of GST-GATA4 fusion protein, GST alone, or MBP was incubated with 25 ng of activated ERK2 protein for the labeling reaction. Only MBP and the GST-G4 fusion protein containing GATA4 amino acids 80 to 250 were efficiently labeled. (C) Western blot with GATA4 phospho-specific antiserum and ERK2-mediated in vitro-labeled GST and GST-GATA4 fusion proteins (300 ng). The asterisk shows the specific increase in ERK2-mediated phosphorylation of GST-GATA4 80-250.

Phosphorylation of serine 105 enhances transcriptional activation and DNA binding. MAPK-mediated phosphorylation of transcription factors can influence either DNA binding activity or transcriptional activation. To isolate effects specific for transcriptional activation apart from DNA binding, the GATA4 activation domain (amino acids 33 to 227) containing either the wild-type sequence or an S105A mutation was fused to the GAL4 DNA binding domain (Fig. 4A, bottom). These constructs were cotransfected into cultured cardiomyocytes along with the GAL4-luciferase reporter (G5E1b-luciferase). The data demonstrate that the S105A mutant had twofold less activity in unstimulated cardiomyocytes than in the wild-type fusion construct (Fig. 4A). Furthermore, PE induced a greater than fourfold activation of wild-type GAL4-GATA4, but not of the GAL4-GATA4 S105A mutant (P < 0.05) (Fig. 4A). PE-induced transcriptional activation of wild-type GAL4-GATA4 was blocked with the MEK1-ERK1/2 inhibitor U0126, but not with the p38 inhibitor SB202190 (n = 3) (Fig. 4A). Both the wild-type and S105A mutant constructs were expressed at comparable levels as assessed by Western blotting of protein extracts from transfected Cos-7 cells (data not shown). Collectively, these data suggest that serine 105 phosphorylation directly enhances the intrinsic transcriptional activation properties of GATA4 through a MEK1-ERK1/2-sensitive mechanism.

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Serine 105 in GATA4 mediates PE transcriptional responsiveness. (A) A plasmid encoding the GAL4 DNA binding domain fused to the wild-type (Wt) or S105A mutant GATA4 transcriptional activation domain (amino acids 33 to 227) was cotransfected into cardiomyocytes with the GAL4-luciferase reporter, G5E1b-luciferase. Cultures were either PE stimulated, left unstimulated, or treated with U0126 or SB202190 (SB) for 24 h. *, P < 0.05 versus GAL4; #, P < 0.05 versus wild type). (B) The BNP minimal promoter luciferase reporter was cotransfected into cultured cardiomyocytes with either wild-type GATA4 (Wt), S105A mutant GATA4, or empty vector (pCDNAI) and PE stimulated for 24 h and/or treated with U0126. *, P < 0.05 versus pCDNAI; #, P < 0.05 versus wild-type GATA4; , P < 0.05 versus PE plus wild-type GATA4.

View larger version (38K): [in this window] [in a new window]   FIG. 5.   ERK2-mediated phosphorylation of GATA4 enhances its DNA binding activity. (A) EMSA demonstrates that ERK2 enhances the DNA binding activity of a bacterially purified GST-GATA4 (full-length) wild-type (Wt) fusion protein but not GST-S105A mutant GATA4 (full-length). (B) Western blot analysis with phospho-specific GATA4 (serine 105) antiserum using the same protein reactions used for panel A demonstrates efficient ERK2-mediated phosphorylation of full-length GATA4, but not the S105A mutant GATA4 or GST. (C) Western blot analysis with GATA4 antibody using the same extracts from panel A demonstrates equal protein levels.

JNK and p38 only weakly phosphorylate serine 105 in GATA4. To determine if other MAPK terminal effectors can phosphorylate serine 105 in GATA4, purified wild-type and S105A mutant proteins were incubated with purified ERK2, p38, and JNK1 proteins in the presence of [³²P]ATP. Similar to the results shown in Fig. 3 with truncated GATA4, ERK2 efficiently phosphorylated full-length wild-type GATA4 (Fig. 6). However, full-length S105A mutant GATA4 was not significantly phosphorylated by ERK2, further demonstrating that serine 105 is the only relevant site for ERK-mediated phosphorylation (Fig. 6). As a control, ERK2 and p38 were each capable of phosphorylating MBP, while JNK1 showed efficient phosphorylation of recombinant c-Jun protein (Fig. 6). Incubation of both wild-type and S105A mutant GATA4 with either p38 or JNK1 revealed detectable phosphorylation by each kinase, although the stoichiometry of phosphorylation was not assessed. Coomassie brilliant blue staining of each gel revealed similar levels of loaded protein (data not shown). These results suggest that both p38 and JNK1 are capable of phosphorylating GATA4 in vitro. However, phosphorylation mediated by p38 and JNK1 showed only a minor preference for serine 105, suggesting that other serine-proline motifs within GATA4 are likely targeted by these two kinases (see Discussion). Taken together, these results indicate that ERK2 specifically targets serine 105 for phosphorylation, while p38 and JNK1 show only a minor effect.

View larger version (45K): [in this window] [in a new window]   FIG. 6.   ERK2, but not p38 and JNK, mediates phosphorylation of serine 105. (A) In vitro kinase reaction with bacterially purified wild-type (Wt) full-length GATA4 or the S105A mutant GATA4 incubated with purified ERK2 in the presence of [32P]ATP and subjected to SDS-polyacrylamide gel electrophoresis. Both proteins were also incubated with purified p38 (B) and JNK1 (C) protein to assess phosphorylation. MBP and recombinant c-Jun were used as positive controls in the kinase assays. The arrow shows the migration of the full-length GST-GATA4 fusion protein, although degradation products of faster migration were also visible.

GATA4 is required for mediating MEK1-ERK1/2-induced hypertrophy. We previously demonstrated that expression of activated MEK1 in the hearts of transgenic mice or by adenoviral gene transfer in cultured cardiomyocytes induces cardiac hypertrophy (5). However, the mechanism whereby ERK1/2 activation might promote cardiomyocyte hypertrophy has not been characterized. To determine if GATA4 is a necessary component of MAPK-induced cardiac hypertrophy, a dominant-negative GATA4 adenovirus was generated. A fusion protein was constructed consisting of N-terminal Flag epitope, the engrailed repressor domain (amino acids 2 to 298), and the GATA4 zinc finger DNA binding domain (amino acids 211 to 329) (20). A similar strategy was previously used to generate a dominant-negative Nkx2.5 transcription factor which blocked heart development in vivo (12). Western blotting of protein extracts from AdG4-Engr-infected cardiomyocytes confirmed the integrity and stability of the fusion protein (Fig. 7A). The engrailed-GATA4 fusion functions by binding DNA and blocking GATA-dependent transcriptional responses in cardiomyocytes, while the engrailed domain alone has no effect (20).

View larger version (29K): [in this window] [in a new window]   FIG. 7.   GATA4-engrailed blocks MEK1-ERK1/2-induced cardiomyocyte hypertrophy. (A) Western blotting with Flag monoclonal antibody demonstrates a protein of the predicted size from AdG4-Engr-infected cardiomyocytes. (B) Cell surface areas were quantified from each of the indicated adenoviral infected cardiomyocyte cultures. *, P < 0.05 versus Adgal; #, P < 0.05 versus AdMEK1 plus AdEngr. (C) The percentage of cells expressing ANF protein was quantified from each of the indicated adenoviral infected cardiomyocyte cultures. *, P < 0.05 versus Adgal; #, P < 0.05 versus AdMEK1 plus AdEngr. (D) [3H]leucine incorporation was measured in Adgal-, AdMEK1-, and AdMEK1-plus-AdG4-Engr-infected cardiomyocytes. *, P < 0.05 versus Adgal. All data represent the averages of three independent experiments.

View larger version (41K): [in this window] [in a new window]   FIG. 8.   GATA4-engrailed blocks MEK1-ERK1/2-induced sarcomeric organization and hypertrophy. Representative images of immunostained cardiomyocyte cultures infected with each of the indicated adenoviral constructs are shown. The left panels show anti--actinin antibody (orange) reactivity to demonstrate cardiomyocyte sarcomeres and gross morphology. The right panels show the same cells double immunostained for ANF (green), which appears in a perinuclear pattern.

	DISCUSSION

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Cardiac myocytes respond to a wide array of neural-humoral stimuli through G-protein-coupled receptors and/or receptor tyrosine kinases. Membrane receptors in turn activate discrete intermediate signal transduction pathways, which ultimately modify intracellular physiology and mediate inducible gene expression within the nucleus. However, the mechanisms whereby intracellular signaling cascades mediate alterations in cardiac gene expression are poorly understood. Here it is demonstrated that the cardiac-expressed transcription factor GATA4 is phosphorylated by the MEK1-ERK1/2 signaling pathway as a mechanism involved in altering hypertrophic gene expression within the heart.

Role of serine 105 phosphorylation in GATA4. GATA4 is a member of a subfamily that includes the closely related GATA5 and GATA6 genes. All three genes are expressed within cardiomyocytes of the embryonic heart, yet as development progresses, GATA5 expression is lost from the heart (28). While it is not known if other cardiac-expressed GATA factors are also posttranslationally modified in response to hypertrophic stimulation, the consensus phosphorylation site identified within GATA4 (serine 105) is not present in GATA5 or GATA6, suggesting alternative mechanisms of regulation. Noncardiac-expressed GATA family members, namely GATA1 and GATA2, have also been shown to undergo site-specific phosphorylation (8, 38). In fact, ERK MAPK was shown to directly phosphorylate GATA2 in hematopoietic progenitor cells and COS cells, although effects on DNA binding or transcriptional activation were not noted (38).

Evidence implicating GATA4 as a hypertrophic regulator. The identification of an ERK MAPK phosphorylation site in GATA4 suggests a transcriptional mechanism whereby MEK1-ERK1/2 signaling might mediate hypertrophy. If such a hypothesis is correct, GATA4 alone should also induce cardiomyocyte hypertrophy. Indeed, it has recently been demonstrated that adenoviral-mediated overexpression of GATA4 in cultured cardiomyocytes induces hypertrophy characterized by enhanced sarcomeric organization, increased cell surface area, and increased protein accumulation (20). In addition, transgenic mice with 2.5-fold GATA4 overexpression in the heart demonstrated cardiac hypertrophy, collectively suggesting that GATA factors are sufficient regulators of cardiomyocyte hypertrophy in vitro and in vivo (20).

Evidence implicating MEK1-ERK1/2 signaling in cardiac hypertrophy. The role that MEK1-ERK1/2 signaling plays in regulating the cardiac hypertrophic response is an area of ongoing investigation. ERK1/2 has been shown to be activated in cultured neonatal rat cardiomyocytes by agonist stimulation and cell stretching, suggesting a functional role in hypertrophy (3, 35). In support of this hypothesis, Glennon et al. demonstrated that ERK signaling was necessary for PE-induced cardiomyocyte hypertrophy using antisense oligonucleotides (14). Similarly, using the MEK1 inhibitor PD98059, a number of studies have concluded a role for ERK signaling in mediating aspects of cardiomyocyte hypertrophy (1, 7, 18, 19, 42). More significantly, adenoviral gene transfer of dominant-negative MEK1 in cultured cardiomyocytes was shown to block PE-, endothelin-1-, isoproterenol-, and stretch-induced hypertrophy, providing convincing evidence without the use of pharmacologic inhibitors (39). In vivo, transgenic mice expressing activated MEK1 in the heart showed constitutive ERK1/2 activation and prominent cardiac hypertrophy, demonstrating that the MEK1-ERK1/2 signaling pathway can initiate the hypertrophic response in vivo (5). Similar results were also reported in cultured cardiomyocytes infected with an adenovirus expressing activated MEK1 (5, 39).