©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Dissociation of p44 and p42 Mitogen-activated Protein Kinase Activation from Receptor-induced Hypertrophy in Neonatal Rat Ventricular Myocytes (*)

(Received for publication, September 17, 1995)

Ginell R. Post (§) David Goldstein Donna J. Thuerauf (1) Christopher C. Glembotski (1) Joan Heller Brown (¶)

From the Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636 Molecular Biology Institute and Department of Biology, San Diego State University, San Diego, California 92182-0057

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In response to hormones and mechanical stretch, neonatal rat ventricular myocytes exhibit a hypertrophic response that is characterized by induction of cardiac-specific genes and increased myocardial cell size. Hypertrophic stimuli also activate mitogen-activated protein kinase (MAPK), an enzyme thought to play a central role in the regulation of cell growth and differentiation. To determine if MAPK activation is sufficient for acquisition of the molecular and morphological features of cardiac hypertrophy we compared four agonists that stimulate G protein-coupled receptors. Whereas phenylephrine and endothelin transactivate cardiac-specific promoter/luciferase reporter genes, increase atrial natriuretic factor (ANF) expression, and promote myofilament organization, neither carbachol nor ATP induces these responses. Interestingly, all four agonists activate both the p42 and the p44 isoforms of MAPK. Furthermore, the kinetics of MAPK activation are not different for the hypertrophic agonist phenylephrine and the nonhypertrophic agonist carbachol. Transient transfection of myocytes with dominant-interfering mutants of p42 and p44 MAPK failed to block phenylephrine-induced ANF expression, although Ras-induced gene expression was inhibited by expression of the mutant MAPK constructs. Moreover, PD 098059, an inhibitor of MAPK kinase, blocked phenylephrine-stimulated MAPK activity but not ANF reporter gene expression. Thus, MAPK activation is not sufficient for G protein receptor-mediated induction of cardiac cell growth and gene expression and is apparently not required for transcriptional activation of the ANF gene.


INTRODUCTION

The mitogen-activated protein kinases (MAPK) (^1)are a family of serine/threonine protein kinases thought to play a central role in cell proliferation and differentiation. There are several isoforms of MAPK(1) , the best characterized of which are the p42 MAPK (Erk2) and p44 MAPK (Erk1) isoforms. Growth factors, phorbol esters, and hormones regulate MAPK activity through a series of phosphorylation events(2) . Upon activation, MAPK translocates to the nucleus and can phosphorylate a variety of nuclear transcription factors, suggesting that MAPK plays an important role in transducing cytoplasmic signals to nuclear responses (3, 4, 5, 6) . For receptor tyrosine kinases, the signaling pathways leading to MAPK activation and the requirement for MAPK in various cellular responses have been well documented. In contrast, the mechanisms by which G protein-coupled receptors activate the MAPK pathway and the involvement of MAPK in G protein receptor signaling is far less clear. Additionally, while MAPK has been shown to be important in responses such as neuronal differentiation in PC12 pheochromocytoma cells and mitogenesis in fibroblasts(7, 8, 9) , the role of MAPK in terminally differentiated cells has not been well studied.

In nondividing ventricular myocytes, the molecular and phenotypic changes associated with cardiac hypertrophy can be induced by treatment with phenylephrine (PE) or endothelin (ET). These agonists interact with seven transmembrane-spanning receptors to activate phospholipase C and subsequently protein kinase C(10, 11, 12) . Other agonists that interact with receptor tyrosine kinases, such as bFGF, are also effective at inducing cardiac hypertrophy(13) . Therefore, both G protein-linked and tyrosine kinase receptors stimulate pathways that elicit changes associated with hypertrophy. In neonatal myocytes PE, ET, angiotensin II and bFGF have been shown to activate two closely related MAPK isoforms, p42 MAPK and p44 MAPK(14, 15, 16, 17) . It has been proposed that the signaling pathways utilized by G protein-coupled receptors and tyrosine kinase receptors converge at MAPK and that MAPK plays a central role in cardiac hypertrophy(15) . Recently, an interfering mutant of MAPK was shown to block PE-induced ANF promoter activation(16) , further implicating MAPK in the signaling pathway that leads to cardiac hypertrophy.

In this study we examined four agonists known to activate different G protein-coupled receptors and compared their abilities to induce various features of the hypertrophic phenotype and to activate MAPK in neonatal ventricular myocytes. We demonstrate that ET, PE, carbachol (CCh), and ATP all activate the p42 and p44 isoforms of MAPK, but that only PE and ET induce ANF and MLC-2 expression and organization of myofilaments. We further demonstrate that the kinetics of MAPK activation are not different for the hypertrophic agonist PE and the nonhypertrophic agonist CCh. Finally, we demonstrate that neither transient expression of dominant interfering mutants of MAPK nor pharmacological blockade of MAPK activation inhibits PE-induced ANF promoter activity. We conclude from our studies that MAPK activation may not be necessary and is clearly not a sufficient signal for induction of cardiac gene expression and the changes in morphology associated with in vitro hypertrophy of cardiomyocytes.


EXPERIMENTAL PROCEDURES

Cell Culture

Neonatal ventricular myocytes were prepared from hearts of 1-3-day-old Sprague-Dawley rats as described previously (18, 19) . Briefly, ventricles were trisected, pooled, and myocytes dissociated in collagenase and pancreatin solution, and myocardial cells were purified on a Percoll step gradient. Cells were plated at 3.5-4 times 10^4/cm^2 on gelatin-coated plates or 25-mm etched coverslips in Dulbecco's modified Eagle's medium: medium 199 (Life Technologies, Inc.) (4:1) containing 10% horse serum, 5% fetal calf serum, and antibiotics for 24 h.

Transient Transfection and Reporter Gene Assays

For calcium-phosphate transfections, myocytes were exposed to a cDNA-calcium-phosphate precipitate 24 h post-plating as described previously(19) . Either the full-length (3003 base pairs) or a 638-base pair fragment of the rat ANF promoter(20) , or a 2.7-kilobase pair fragment of the MLC-2 promoter (21) fused to firefly luciferase cDNA were used as reporter genes. Transfected cDNA consisted of the appropriate reporter gene (2.5-3 µg) and 1.5-2 µg of CMV/beta-galactosidase, which was used as a control for transfection efficiency except in experiments where beta-galactosidase activity was differentially induced by the experimental manipulations. Following transfection, cells were washed extensively and maintained in serum-free medium or in the presence of ET (10 nM), PE (100 µM plus 2 µM propranolol to block beta-adrenergic receptors), CCh (300 µM), or ATP (50 µM). Cells were incubated for 48 h and then harvested in a 0.5% Triton X-100 buffer, and luciferase activity of the lysate was measured in a Berthold luminometer(22) .

In some experiments an alternative protocol developed by Sprenkle et al.(23) was used to examine ANF expression following short term agonist treatment. Briefly, freshly isolated ventricular myocytes at a density of 1 times 10^6/ml of medium were transfected by electroporation with 10 µg of the ANF/luciferase reporter gene and 3 µg of cytomegalovirus/beta-galactosidase. After a 12-14-h post-electroporation recovery in serum-containing medium, myocytes were washed and medium replaced with serum-free medium. Twenty-four hours later, the medium was again replaced with serum-free medium and cells were treated ± PE for 6 h. Cell lysates were prepared and luciferase and beta-galactosidase activities determined.

Immunofluorescence Techniques

Indirect immunofluorescence analysis was performed using a modification of a previously described procedure(24) . Myocardial cells were plated on coverslips precoated with laminin (Sigma) and gelatin and maintained in serum-free medium for 24 h prior to addition of agonist. Forty-eight hours after incubation with agonist, the cells were rinsed with phosphate-buffered saline, fixed with 3% paraformaldehyde, permeabilized with 0.3% Triton X-100, incubated with 1% bovine serum albumin to block nonspecific sites, and then with MLC-2 antiserum(25) . Following several washes, coverslips were incubated with a fluorescein-conjugated goat anti-rabbit IgG (Cappel). ANF expression was detected using a mouse monoclonal antibody against ANF and a rhodamine-conjugated, goat anti-mouse antibody (Cappel). MLC-2 organization and ANF expression were photographed using a 63times Plan-apochromat objective (Zeiss).

MAPK Assays and Immunodetection

After plating, myocardial cells were washed and maintained in serum-free medium for 24 h prior to experimentation. Cells were then treated with agonist for the indicated times, washed with cold phosphate-buffered saline, and lysed in Tris buffered saline containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 500 µM Na(3)VO(4), and 1 mM sodium pyrophosphate. For immunocomplex kinase assays, cell lysates were incubated with agarose-conjugated Erk1 or Erk2 antibody (Santa Cruz Biotechnology) for 90 min at 4 °C. After washing, the agarose complex was resuspended in 30 µl of kinase buffer containing 30 mM HEPES, pH 7.4, 1 mM dithiothreitol, 10 mM MgCl(2), 20 µM ATP, 60 µg/ml myelin basic protein (MBP), and 4 µCi of [-P]ATP. After incubation at 30 °C for 12 min, reactions were terminated by the addition of Laemmli sample buffer(26) . Samples were boiled, phosphorylated MBP isolated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and phosphate incorporation quantitated by radioanalytic scanning (AMBIS). For investigations using the MAPK kinase inhibitor, myocytes were pretreated with the indicated concentrations of PD 098059 or Me(2)SO for 20 min prior to addition of PE.

MAPK activity was also examined using an in-gel kinase assay with modifications of the procedure described by Kameshita and Fujisawa (27) . Briefly, cells were treated with agonist, cell lysates prepared, and proteins resolved in 10% SDS-polyacrylamide gels containing 0.5 mg/ml MBP. After electrophoresis, the gels were washed with 20% 2-propanol in 50 mM HEPES, pH 7.6, then with 5 mM mercaptoethanol in HEPES buffer. Proteins were denatured by washing the gels with 6 M urea in Tris buffer and then renatured by washing in HEPES buffer containing 0.04% Tween 20 at 4 °C. After preincubation of the gel for 30 min at 4 °C in 20 mM HEPES, 2 mM dithiothreitol, 10 mM MgCl(2), pH 7.6, in situ phosphorylation of MBP was performed in the same buffer containing 6.7 µCi/ml [-P]ATP at 30 °C for 2 h. After extensive washing in 5% trichloroacetic acid, 10% sodium pyrophosphate, gels were dried, exposed to film, and phosphate incorporation quantitated by radioanalytic scanning of gels.


RESULTS

We compared the abilities of four G protein-coupled receptor agonists to induce promoter activities of two cardiac-specific genes in neonatal rat ventricular myocytes. Myocytes were transiently transfected with ANF or MLC-2 promoter/luciferase reporter genes containing regions of the promoter previously shown to be sufficient for PE-inducible expression(20) . Myocardial cells were then stimulated with either ET (10 nM), the adrenergic receptor agonist PE (100 µM plus 2 µM propranolol to block beta-adrenergic effects of PE), the stable acetylcholine analog CCh (300 µM) or ATP (50 µM) for 48 h. As shown in Fig. 1(upper panel), CCh and ATP did not increase ANF-luciferase activity, indicating that these agonists did not transactivate the ANF promoter. In contrast, ET and PE markedly increased ANF luciferase expression. Likewise, ET and PE were effective activators of MLC-2 reporter gene expression, while CCh and ATP were not (Fig. 1, lower panel).


Figure 1: Differential effects of endothelin, phenylephrine, carbachol, and ATP on ANF and MLC-2 gene expression. Neonatal rat ventricular myocytes were transfected with an ANF promoter/luciferase (upper panel) or an MLC-2 promoter/luciferase (lower panel) reporter gene and then incubated for 48 h with either no drug, 10 nM ET, 100 µM phenylephrine (with 2 µM propranolol to block beta-adrenergic receptors), 300 µM carbachol, or 50 µM ATP. Luciferase activity was normalized to beta-galactosidase activity for each sample and agonist-induced increases expressed as -fold stimulation relative to no drug treatment. Values are the mean ± S.E. of data from three experiments performed in triplicate.



The hypertrophic response in ventricular cardiac myocytes is also characterized by increases in myofilament organization and cell size. To assess these morphological changes, myocytes cultured on coverslips were incubated with ET, PE, CCh, or ATP for 48 h and subsequently fixed and immunostained for MLC-2 or ANF using specific antisera and secondary fluorescent antibodies as described previously(22) . Myocardial cells cultured in the presence of ET and PE showed organization of MLC-2 into contractile units, while myofilaments in CCh- and ATP-treated cells remained disorganized and the cells appeared smaller in size (Fig. 2, upper panel). In addition, ANF protein expression was induced by ET and PE, but not in response to CCh or ATP (Fig. 2, lower panel). The lack of effect of ATP extends the findings of Zheng et al.(28) , demonstrating that ATP does not induce [^14C]phenylalanine incorporation into protein or increase cell size in ventricular myocytes. Moreover, these data corroborate results obtained from transfection experiments and demonstrate that myofilament organization and ANF protein expression, like transcriptional activation of the MLC-2 and ANF reporter genes, are selectively induced by a subset of G protein-coupled receptor agonists.


Figure 2: Differential effects of endothelin, phenylephrine, carbachol, and ATP on ANF protein expression and myofilament organization. Myocytes plated on glass coverslips were incubated in serum-free medium alone (control) or medium containing 10 nM ET, 100 µM phenylephrine, 300 µM carbachol, or 50 µM ATP for 48 h. The cells were processed for immunofluorescent analysis using antibodies against MLC-2 (upper panels) or ANF (lower panels) as described under ``Experimental Procedures.'' Cells were photographed using a Zeiss Axiophot fluorescent microscope with a 63times oil immersion Apochromat objective.



Since it has been suggested that agents which induce cardiac hypertrophy signal through a MAPK pathway(12, 14, 15, 16, 17, 29, 30) , we reasoned that MAPK might be activated by ET and PE, but not CCh and ATP. To test this hypothesis, myocytes were treated with CCh, PE, ATP, or ET and MAPK was immunoprecipitated with a p44 MAPK antibody (Santa Cruz Biotechnology). As shown in Fig. 3, (upper panel), ET induced a 6-fold increase in MAPK activity, and all other agonists increased MAPK activity 3-4-fold. Thus, the activation of p44 MAPK did not correlate with the hypertrophic potential of these G protein-coupled receptor agonists. Western analysis of immunoprecipitated proteins showed that the p44 MAPK antibody used in these studies (Fig. 3) weakly immunoprecipitates the 42-kDa form of MAPK (data not shown). Since these experiments did not assess the total MAPK activity present in cardiac myocytes (which express both the p44 and p42 isoforms of MAPK (data not shown and (14) )), we also used a p42 MAPK antibody to examine the possibility that p42 MAPK might be differentially regulated by hypertrophic and nonhypertrophic agonists. As shown in Fig. 3, (lower panel), p42 MAPK was also activated by all of the agonists. The responses to PE (2.6-fold) and ET (3.4-fold) were, on average, greater than that observed for CCh (1.5-fold); however, nearly identical increases in p42 MAPK activity were induced by PE (2.6-fold) and ATP (2.1-fold), agonists with divergent effects on hypertrophic responses. Extracts from agonist-treated neonatal ventricular myocytes were also analyzed for MAPK activity using in-gel MBP phosphorylation assays. All four agonists were also found to induce p42 and p44 MBP kinase activity in these in-gel MBP kinase assays (data not shown). These results indicate that the initial activation of specific MAPK isoforms does not differentiate the ability of agonists to induce hypertrophic responses in ventricular myocytes.


Figure 3: Carbachol, phenylephrine, ATP, and endothelin stimulate p44 and p42 MAPK activity. Myocytes were either untreated or exposed to agonist for 5 min and cell lysates immunoprecipitated with an anti-p44 MAPK antibody (upper panel) or an anti-p42 antibody (lower panel). MAPK activity was determined in an in vitro kinase assay using MBP as substrate. After separation of [P]MBP by SDS-PAGE, radioactivity was quantitated by radioanalytic scanning of gels. Representative autoradiograms are shown. Agonist-induced increases in [P]MBP are expressed as -fold stimulation relative to no drug treatment. Values are the mean ± S.E. of data from four experiments performed in duplicate or triplicate.



Neuronal differentiation in PC12 cells and cellular proliferation in fibroblasts have been correlated with sustained MAPK activation(8, 31) . Since changes in gene expression and morphology occur over extended times, the kinetics of PE- and CCh-induced MAPK activation in neonatal rat ventricular myocytes were determined. Agonist-induced MAPK activity was found to be maximal 5 min after the addition of agonist and remained slightly elevated until returning to control levels at 18 h (Fig. 4). Importantly, the time course of CCh-induced MAPK activity was nearly superimposable on that of PE, suggesting that sustained MAPK activation does not account for the ability of PE to elicit changes in gene expression associated with cardiac hypertrophy.


Figure 4: Carbachol and phenylephrine show similar kinetics of MAPK activation. Myocytes were treated with 100 µM PE (bullet) or 300 µM CCh (circle) for the times shown. Cell lysates were prepared, SDS-PAGE sample buffer was added, and lysates were boiled prior to separation in 10% SDS-PAGE containing 0.5 mg/ml MBP. In situ phosphorylation of MBP was assayed as described under ``Experimental Procedures.'' Results are expressed as -fold activation relative to unstimulated cells. Values are the mean ± S.E. of data from two experiments performed in duplicate.



It has recently been reported that expression of an interfering mutant of p44 MAPK prevents PE-induced ANF promoter activity(16) . However, our results suggesting that MAPK activation is insufficient to induce cardiac hypertrophy led us to reexamine the requirement for MAPK in alpha(1)-adrenergic receptor (alpha(1)-AR)-induced gene expression. Myocytes were transiently transfected with the cDNA for either a dominant-interfering p42 (K52RErk2) or p44 (K71RErk1) MAPK protein along with the ANF-luciferase reporter gene and stimulated with PE for 48 h. As shown in Fig. 5, PE-induced ANF reporter gene expression was not blocked by expression of either of the dominant-interfering MAPKs. In the same experiments, we cotransfected cells with activated Ras ([Val]Ras), which transactivates the ANF promoter(32) . In contrast to their effect on PE, both mutant MAPK proteins significantly inhibited Ras-induced ANF reporter gene expression (Fig. 5), demonstrating that the mutant MAPK constructs were functional. Thus, alpha(1)-AR-induced ANF expression can occur in a MAPK-independent manner.


Figure 5: Interfering mutants of MAPK block Ras- but not PE-induced activation of the ANF promoter. Myocytes were transfected with 6 µg of the dominant-negative p44 MAPK (K71RErk1), dominant-negative p42 MAPK (K52RErk2), or backbone vector (pCEP4) and 2 µg of activated Ras ([Val]Ras) or its backbone vector (pDCR) along with the ANF reporter gene. Cells were extensively washed, then immediately stimulated with PE for 48 h (or maintained in serum-free medium for Ras-transfected cells). Luciferase activity was determined and normalized to protein since activated Ras significantly increases beta-galactosidase activity. The data represent the mean ± S.E. of three experiments performed in quadruplicate.



MAPK activation requires phosphorylation on both threonine and tyrosine residues by the dual specificity kinase, MAPK kinase or MEK (MAPK/Erk activating kinase)(33, 34) . To further assess the role of MAPK in alpha(1)-AR-mediated ANF induction, a cell-permeable MEK inhibitor, PD 098059 (2-(2`-amino-3`methoxyphenyl)-oxanaphalen-4-one) (35, 36) was used to pharmacologically block MAPK activation. Myocytes were pretreated with various concentrations of PD 098059 or Me(2)SO for 20 min prior to the addition of PE. Cell lysates were prepared 5 min later and MAPK activity determined using either immunocomplex kinase (Fig. 6A) or in-gel assays (Fig. 6B). PE-induced MAPK activation was inhibited by about 50% using 3 µM PD 098059 and was fully inhibited by 10 µM PD 098059 (Fig. 6).


Figure 6: The MAPK kinase inhibitor PD 098059 blocks PE-induced MAPK activation. Serum-deprived myocytes were treated with the indicated concentrations of PD 098059 for 20 min prior to the addition of 100 µM PE (plus 2 µM propranolol) for 5 min. Cells were lysed and MAPK activity was determined using immunocomplex kinase following immunoprecipitation with a p44 MAPK antibody (A) or in-gel kinase (B) assays as described under ``Experimental Procedures.'' Results shown in A are the means ± S.E. of data from 3-5 experiments performed in duplicate. Data are expressed as -fold stimulation relative to unstimulated (0.1% Me(2)SO) controls. PD 098059 induced a concentration dependent decrease in basal MAPK (not shown). In B, a representative in-gel MBP kinase assay demonstrates that PE-stimulated p42 and p44 MAPK activation is reduced to basal levels by 10 µM PD 098059.



Two transfection protocols that differ in the duration of agonist treatment were used to determine whether inhibition of MAPK prevented PE-induced ANF reporter gene expression. In the protocol used in the experiments described above and in our previous studies, cells were transfected with ANF/luciferase reporter constructs, washed, maintained in serum-free medium or treated with PE, and luciferase activity measured in cell lysates prepared 48 h later (Fig. 7, A and B). In the second protocol, cells were transfected by electroporation with ANF/luciferase reporter constructs, serum-deprived for 24 h and then treated with PE for 6 h (Fig. 7, C and D). The effects of PD 098059 on ANF promoter activity after 6 or 48 h of PE treatment were then assessed using either the full-length (3003 base pairs, Fig. 7, B and D) or 638-base pair (Fig. 7, A and C) ANF promoter/luciferase constructs. Regardless of the construct or the duration of PE treatment, PD 098059 failed to block PE-stimulated luciferase expression, consistent with the conclusion that PE can induce ANF expression independently of MAPK activation.


Figure 7: MAPK inhibition with PD 098059 does not prevent PE-induced ANF reporter gene expression. Myocytes were transiently transfected with either the 638-base pair or full-length (3003-base pair) ANF promoter/luciferase reporter gene. Cells preincubated with 10 µM PD 098059 or 0.1% Me(2)SO (Ctl) for 20 min were maintained in serum-free medium without (open bars) or with PE (filled bars) for 48 h (A and B) or 6 h (C and D). In A and B, myocytes were transfected using calcium-phosphate, then treated with MEK inhibitor (or Me(2)SO) ± PE. In C and D, myocytes were transfected by electroporation, serum-deprived for an additional 24 h, and then incubated with PD 098095 ± PE. Luciferase activity was measured in cell lysates and normalized to protein (A and B) or beta-galactosidase activity (C and D). The data in A, B, and D are the means ± S.E. of triplicate samples from three experiments. The data in D are from one experiment performed in triplicate.




DISCUSSION

As shown previously, PE (37) or ET (10) induce ANF and MLC-2 expression, myofilament organization, and increases in cardiac myocyte size. The purinergic receptor agonist ATP, reported not to increase protein synthesis or augment myocardial cell size(28) , also failed to induce ANF or MLC-2 reporter gene expression or increase myofilament organization. A stable ATP analog, ATPS, was also ineffective (not shown), suggesting that the lack of response to ATP is not due to hydrolysis of this nucleotide agonist. Carbachol also did not induce ANF or MLC-2 promoter activity (22) or lead to myofilament organization in cardiomyocytes. The failure of CCh to elicit a response through endogenous muscarinic cholinergic receptors (mAChRs) is not likely to result from depletion of this agonist from the medium since we have shown that CCh can induce cardiac-specific gene expression in myocytes transfected with cDNA for the M(1)mAChR(22) . Therefore, we reasoned that these agonists, which have divergent effects on cardiac hypertrophy, must signal through distinct pathways.

Hormones and mechanical stimuli shown to induce hypertrophic responses in neonatal ventricular myocytes (e.g. PE, ET, angiotensin II, bFGF, and stretch) have also been shown to activate MAPK(12, 14, 15, 16, 17, 29) . This observation, together with evidence demonstrating the role of MAPKs in growth regulation, suggests that MAPK could be a key mediator of cardiac hypertrophy. However, we found that MAPK activation does not correlate with the hypertrophic potential of an agonist since it is stimulated not only by PE and ET but also by CCh and ATP. Our finding that ET is a more effective activator of MAPK than PE is in agreement with an earlier study comparing these agonists on MAPK activation in ventricular myocytes(12) . However, PE is not significantly more effective than ATP as an activator of MAPK (Fig. 3). Therefore, although there are quantitative differences in the magnitude of MAPK activation by the four agonists, the extent of MAPK activation does not appear to correlate with the hypertrophic potential of these agonists.

In PC12 cells, epidermal growth factor and nerve growth factor both induce a rapid and transient activation of MAPK; however, only nerve growth factor induces neuronal differentiation and causes sustained (>6 h) MAPK activation(8, 38) . Likewise, a biphasic response with a transient, followed by a more prolonged activation phase, characterizes the response of CCL39 lung fibroblasts to the mitogenic agonist thrombin but not the nonmitogenic agonist CCh(31) . However, in cultured neonatal ventricular myocytes, MAPK activation in response to PE (Fig. 6) and ET (15) peaks at 5 min and the extent of MAPK activity at 1-18 h is the same for PE and CCh. Thus, sustained MAPK activation cannot explain the divergent effects of these agonists.

The observation that both hypertrophic and nonhypertrophic agonists activate MAPK suggests that additional signaling pathways are required to induce the phenotypic features of cardiac hypertrophy. Stimulation of alpha(1)-AR or endothelin receptors increases phosphoinositide hydrolysis in cardiac myocytes(10, 11, 39) . Our previous studies using antibodies (40) and pertussis toxin (^2)to block G protein function suggest that PE and ET regulate phosphoinositide (PI) hydrolysis through receptor interactions with G. Carbachol and ATP have also been shown to increase PI turnover in cardiac preparations(42) , but in the neonatal rat ventricular myocyte we find that CCh (22) and ATP (data not shown) induce only modest increases in PI hydrolysis (<2-fold) relative to PE and ET, which markedly (15-fold) increase inositol phosphate accumulation(11) . Thus, the hypertrophic potential of G protein-coupled receptor agonists appears to correlate with the ability of the agonist to activate phospholipase C through receptor interactions with G(q).

The preferential ability of G(q)-coupled receptors to induce cardiac-specific gene expression and morphological changes is consistent with other data implicating G(q) in hypertrophic responses(10, 24, 41) . For example, transient expression of a constitutively activated form of Galpha(q) induces ANF and MLC-2 reporter gene expression and blockade of endogenous G(q) function by microinjection of inhibitory Galpha(q) antibodies inhibits PE-induced ANF expression and cellular hypertrophy(40) . In addition, we have shown that cardiac specific gene expression can be stimulated through heterologously expressed wild type and chimeric M(1)mAChRs that effectively couple to G(q), but not through heterologously expressed mAChRs that couple to G(i)(22) . Overall, the data suggest that either activation of G(q) and the sequelae of phospholipase C activation (i.e. Ca release and protein kinase C activation) or activation of other signaling pathways unique to G(q)-coupled receptors are required for mediating cardiac hypertrophy.

The question of whether MAPK activation is a necessary, albeit an insufficient, signal may be more controversial. Our data in this regard differ from those published by Thorburn et al.(16) . Although we used similar methodologies for myocyte preparation and transfection, we failed to observe significant inhibition of ANF-reporter gene expression when myocytes were cotransfected with the dominant-interfering p44 MAPK construct that significantly inhibited cardiac gene expression in their studies. In addition, we did not observe inhibition of PE-induced ANF gene expression with cotransfection of the interfering mutant of the p42 MAPK isoform (Fig. 5) or when both p42 and p44 mutant MAPK constructs were coexpressed (data not shown). We demonstrated that transactivation of the ANF promoter by constitutively activated Ras was inhibited by expression of both p42 and p44 dominant interfering MAPK constructs, suggesting that the mutant MAPK proteins are expressed and can functionally block Ras-mediated transcriptional responses. These constructs also block PE-induced c-fos-luciferase reporter gene expression (data not shown), indicating that the dominant-interfering MAPK proteins are expressed at sufficient levels to block agonist-induced transcriptional responses. One difference between our studies and those published earlier is that we examined transactivation of the 638-base pair minimal ANF promoter (20) rather than full-length ANF (3003-base pair) promoter. When we examined the effect of interfering MAPK mutants on PE-induced activation of the full-length ANF promoter/luciferase gene, we found inconsistent inhibition (data not shown). Another difference is that Thorburn et al. normalized luciferase activity to coexpressed beta-galactosidase activity. When our data are expressed in the same way, we find at most a 10% inhibition of PE-induced ANF expression by these interfering MAPK mutants.

As a second, independent line of investigation to assess the role of MAPK in alpha(1)-AR-induced ANF expression, we used PD 098059, a newly described cell-permeable inhibitor of the MAPK kinase (MEK) (35, 36) . This compound has been shown to block MEK-mediated tyrosine phosphorylation and activation of MAPK, while not affecting other tyrosine kinases (e.g. Src) or serine/threonine kinases (e.g. protein kinase A, protein kinase C, or Raf)(36) . When cardiac myocytes were treated with PD 098059, a dose-dependent inhibition of PE-stimulated MAPK activation was observed. At 10 µM PD 098059, PE-induced MAPK activity was reduced to basal levels. There was no inhibition of PE-induced ANF reporter gene expression, mediated through either the 638- or 3003-base pair promoter, in response to short (6 h) or long term (48 h) agonist treatment. These findings clearly indicate that PE-induced ANF expression can occur independently of MAPK activation.

In summary, the present studies demonstrate that CCh and ATP, which are not hypertrophic agonists in this model system, activate p42 and p44 MAPK to an extent similar to that for the hypertrophic agonists PE and ET. Neither dominant-interfering forms of MAPK nor pharmacological inhibition of MAPK activation prevent the activation of ANF reporter gene expression by alpha(1)-AR stimulation. These results indicate that activation of p42 and p44 MAPK can be dissociated not only from the morphological changes associated with alpha(1)-AR-mediated hypertrophy(16) , but also from G protein-coupled receptor effects on cardiac gene expression.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Research Grants HL28143 (to J. H. B.), NS 25037 (to C. C. G.), and HL46345 (to J. H. B. and C. C. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a postdoctoral fellowship from the American Heart Association-California Affiliate.

To whom correspondence should be addressed: Dept. of Pharmacology, UCSD School of Medicine, 9500 Gilman Dr., La Jolla, CA 92093-0636. Tel.: 619-534-2595; Fax: 619-822-0041.

(^1)
The abbreviations used are: MAPK, mitogen-activated protein kinase; ET, endothelin; PE, phenylephrine; bFGF, basic fibroblast growth factor; CCh, carbachol; alpha(1)-AR, alpha(1)-adrenergic receptor; mAChR, muscarinic cholinergic receptor; MBP, myelin basic protein; PAGE, polyacrylamide gel electrophoresis; ATPS, adenosine 5`-O-(thiotriphosphate); ANF, atrial natriuretic factor; MEK, MAPK/Erk activating kinase.

(^2)
R. Hilal-Dandan, M. T. Ramirez, S. Villegas, A. Gonzalez, Y. Endo-Mochizuki, J. H. Brown, and L. L. Brunton, submitted for publication.


ACKNOWLEDGEMENTS

We thank Drs. Ken Chien, Melanie Cobb, and Kirk Knowlton for the reporter genes and dominant-negative MAPK expression plasmids. We also thank Drs. Kirk Knowlton and Steven Post for critical reading of the manuscript and M. Teresa Ramirez and Frances Putkey for their contributions to this study.


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