Tyrosine Kinase and c-Jun NH₂-Terminal Kinase Mediate Hypertrophic Responses to Prostaglandin F₂ in Cultured Neonatal Rat Ventricular Myocytes

From the Department of Pharmacology (J.W.A., V.P.S., J.H.B.), University of California, San Diego, La Jolla, Calif, and the Department of Physiological Science (S.A.H.), University of California, Los Angeles.

Correspondence to Joan Heller Brown, PhD, Department of Pharmacology, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0636. E-mail jhbrown{at}ucsd.edu

	Abstract

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Abstract—Myocardial infarction results in focal areas of ischemia, hypoxia, necrosis, and decreased contractile function. To compensate for loss of contractile function, remaining viable myocytes undergo hypertrophic growth. Prostaglandin F₂ (PGF₂), which is released from cells of the myocardium during periods of stress such as hypoxia or ischemia/reperfusion, has recently been shown to stimulate hypertrophic growth in neonatal rat ventricular myocytes. In the present study, we determine which growth-related intracellular pathways are required for PGF₂ to induce morphological and genetic features characteristic of the hypertrophic phenotype. In cardiomyocytes, PGF₂ increases the hydrolysis of inositol phosphates and induces the translocation of protein kinase C to the myocyte membrane, consistent with PGF₂ receptor coupling to G_q. PGF₂ also activates the extracellular signal–regulated kinase (ERK) and p38 mitogen-activated protein kinase pathways. Surprisingly, studies using pharmacological inhibitors and transfection of dominant-interfering proteins demonstrate that PGF₂-induced myocyte hypertrophy occurs independent of either PKC, p38, or ERK pathways. Additional studies demonstrate that PGF₂ stimulates protein tyrosine phosphorylation and activates c-Jun NH₂-terminal kinase and suggest that these pathways mediate hypertrophic growth in response to PGF₂.

Key Words: prostaglandin • cardiac hypertrophy • extracellular signal-regulated kinase • c-Jun NH₂-terminal kinase • tyrosine kinase

	Introduction

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In the heart, regional episodes of ischemia due to coronary artery occlusion or myocardial infarction create areas of hypoxia, osmotic imbalance, and edema. Prostaglandins, including prostaglandin F₂ (PGF₂), are released from cells of the myocardium subsequent to experimental infarction. For example, regional myocardial ischemia induced by coronary artery occlusion was shown to cause rapid PGF₂ release into the coronary sinus of dogs.¹ Similarly, in adult rats subjected to experimental myocardial infarction induced by coronary ligation, significant cardiac hypertrophy developed and was accompanied by elevated levels of PGF₂ in the myocardium.² Furthermore, cultured rat ventricular myocytes subjected to short periods of hypoxia demonstrated a 6-fold increase in PGF₂ production compared with normoxic control myocytes.³

It is likely that the compensatory hypertrophy seen in animal models of myocardial ischemia results at least in part from the release of growth factors from damaged or stressed cells of the myocardium. We recently reported that PGF₂ is a potent stimulus for hypertrophic growth of ventricular myocytes in vitro.⁴ We suggested that PGF₂, released from cells of the myocardium during periods of hypoxia or osmotic stress, works through an autocrine/paracrine mechanism to activate specific cell surface receptors and cause hypertrophy in surrounding myocardial cells. However, the intracellular signaling pathways that transduce hypertrophic signals from the cardiac PGF₂ receptor have not been identified.

Defining the molecular mechanisms by which external stimuli are translated into intracellular events responsible for the hypertrophic phenotype is an active area of cardiovascular research. Agonists such as phenylephrine (PE), endothelin-1 (ET-1), and angiotensin II (Ang II) are known to interact with G_q-coupled receptors and cause changes in morphology and gene expression in cardiac myocytes representative of the hypertrophic phenotype.^{5 6 7} The receptors activated by these agonists have 7 transmembrane–spanning segments and interact with G_q to regulate phospholipase C, catalyze phosphoinositide hydrolysis, and activate protein kinase C (PKC). Reports from several laboratories have clearly demonstrated that a variety of hypertrophic responses can be induced by activation of G_q or PKC in cardiac myocytes.^{8 9 10 11}

Stimulation of G protein–linked receptors can also lead to activation of members of the mitogen-activated protein kinase (MAPK) family, thought to play a critical role in cell proliferation and differentiation. In the heart, hypertrophic interventions, such as mechanical stretch, and agonists, including Ang II, ET-1, and PE, stimulate the activities of MAPK family members, including extracellular signal–regulated kinase (ERK) and c-Jun NH₂-terminal kinase (JNK).^{7 12 13 14 15} Evidence for the role of ERK in G_q-linked hypertrophic responses comes from experiments using antisense oligonucleotides, pharmacological inhibitors, or dominant-interfering mutants of ERKs or their activators. For example, atrial natriuretic factor (ANF) expression and increased cell size in PE-treated neonatal rat ventricular myocytes were blocked by antisense oligodeoxynucleotides against ERK.¹⁶ However, studies using dominant-interfering ERK mutants transfected into ventricular myocytes demonstrated inhibition of PE-stimulated ANF expression, without any effect on myofibrillar organization.¹⁷ Similarly, activated Raf-1 kinase was shown to stimulate ANF expression via ERK, without effect on myofibrillar organization.¹⁸ In addition, various agonists increase ERK but do not induce hypertrophic responses in cardiac myocytes.¹⁹ Finally, cardiac-specific G_q overexpression induced hypertrophy in transgenic mice that is independent of increases in ERK activity.²⁰ Thus, there is growing evidence that activation of the ERK pathway cannot be responsible for the full spectrum of growth responses triggered by G_q-coupled receptors in cardiac myocytes.

Recently, several laboratories, including ours, have demonstrated that JNK, a member of the stress-activated protein kinase (SAPK) family, is activated by G protein–linked receptor agonists, is increased in myocytes by activated Ras, and is increased in association with Ras-induced hypertrophy in vivo.^{13 14 15} Importantly, we have recently demonstrated that expression of a mutant activated form of MKK7, a specific upstream activator of JNK, caused hypertrophy in cultured cardiac myocytes without increased ERK or p38 MAPK activity.²¹ Furthermore, studies with dominant-negative proteins have suggested that the JNK pathway mediates PE-induced changes in cardiac gene expression, consistent with a potential role for this pathway in ₁-adrenergic receptor–stimulated cardiac hypertrophy.^{13 22} Another member of the SAPK family, p38 MAPK, has recently been shown to cause hypertrophic growth in cardiac myocytes overexpressing MKK6, a kinase upstream from p38.^{23 24} In addition, pharmacological inhibition of p38 activation blocked PE-stimulated myocyte enlargement and sarcomere organization.²⁴ Thus, recent evidence points to a potential role for the SAPKs in mediating hypertrophic responses to G_q-coupled receptor activation in cardiac myocytes.

Despite the lack of intrinsic TK activity, G protein–coupled receptors, including those for PE, ET-1, and isoproterenol, have been shown to stimulate the phosphorylation of tyrosine residues on several target proteins.⁷ In addition, Ang II causes rapid tyrosine phosphorylation of the adaptor protein Shc and activation of the Src family of TKs in cardiac myocytes.²⁵ A requirement of TKs in G protein–mediated cardiac myocyte hypertrophy is suggested by experiments using pharmacological inhibitors. For example, hypertrophic responses to PE, -thrombin, or phosphatidic acid were blocked by inhibitors of TKs, including tyrphostin and genistein.^{26 27 28}

As a result of the conserved nature of transmembrane regions of the prostanoid receptor family, PGF₂ receptor cDNA clones were recently isolated from mouse and bovine cDNA libraries.^{29 30} Hydrophobicity plots based on the sequences of these clones are characteristic of receptors containing 7 transmembrane–spanning domains. In addition, when the PGF₂ receptor cDNA was transfected into COS cells, its activation led to increased inositol trisphosphate formation. A definitive connection was established between PGF₂ and G_q in Chinese hamster ovary (CHO) cells stably transfected with PGF₂ receptors (CHO-PGF₂).³¹ In these cells, PGF₂ elevated intracellular Ca²⁺ levels and stimulated phosphoinositide metabolism. Furthermore, microinjected antibodies to the subunit of G_q blocked the PGF₂-induced Ca²⁺ flux across CHO-PGF₂ cells. Thus, the PGF₂ receptor can initiate intracellular signals through activation of phospholipase C and generation of its downstream signaling molecules.

We examined the question of whether hypertrophic responses to PGF₂ in ventricular myocytes are mediated by the same signaling pathways described above. We show here that although PKC, p38 MAPK, and ERK are activated by PGF₂, they are not required for hypertrophy. We also demonstrate that this receptor causes activation of TKs and JNK and suggest that these pathways are needed to elicit both genetic and morphological responses characteristic of hypertrophy in cardiac myocytes.

	Materials and Methods

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Cell Culture
Neonatal rat ventricular myocytes were isolated and cultured as described previously.⁹ Briefly, hearts were obtained from 1- to 2-day-old Sprague-Dawley rat pups and digested with collagenase, and myocytes were purified by passage through a Percoll gradient. Cells were plated onto tissue culture dishes precoated with 1% gelatin and maintained overnight in 4:1 DMEM/medium 199 supplemented with 10% horse serum, 5% fetal calf serum, and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin).

Plasmid Constructs
The following reporter gene constructs were used in transfection experiments: a 638-bp fragment of the rat ANF promoter,³² a 2700-bp fragment of the myosin light chain-2 (MLC-2) promoter,³³ and a 249-bp fragment of the c-Jun promoter,³⁴ all fused to firefly luciferase cDNA. The expression plasmid encoding dominant-interfering MAPK kinase kinase 1 (MEKK1 [K432M]), a kinase inactive competitive inhibitory mutant originally characterized by Dr Gary Johnson (Fanger et al³⁵ ), was provided by Dr Michael Karin (University of California, San Diego). Dominant-interfering Raf-1 (K375R), mutated at the ATP binding site, was originally characterized by Dr U. Rapp (Bruder et al³⁶ ) and was provided by Dr James Feramisco (University of California, San Diego).

Transient Transfection
Myocytes in 6-well plates were transfected in serum-containing medium using a modified calcium phosphate transfection technique as described previously.⁹ A total of 6.6 µg of DNA was used per well, which consisted of the appropriate luciferase reporter (1.8 µg) and empty vector DNA or the plasmid-containing dominant-interfering mutant cDNA. After transfection, cells were washed extensively and then incubated for 48 hours in serum-free medium or in the presence of indicated agonists. To determine reporter gene activity, cells were lysed in a 1% Triton X-100 buffer, and luciferase activity and protein concentration were determined for each sample as described.⁹

Immunofluorescence Analysis
Indirect immunofluorescence was performed as previously described,³⁷ with minor modifications. Briefly, cells were cultured on laminin-coated (3.5 µg/cm², Sigma Chemical Co) chamber slides (Nunc) overnight in the presence of serum, washed, and incubated for a further 24 hours in serum-free media before treatment with agonists for 48 hours. Cells were fixed with 3.7% formaldehyde, permeabilized with 0.3% Triton X-100, blocked with 1% bovine serum albumin, and incubated sequentially with a rabbit polyclonal antibody against ANF (Peninsula Laboratories) and a fluorescein-conjugated goat anti-rabbit IgG (Cappel). Myocyte sarcomeres (F-actin) were labeled with rhodamine-conjugated phalloidin (Molecular Probes). Cellular ANF expression and myofilament organization were visualized on a Zeiss Axiovert 135 fluorescence microscope and photographed using a x63 Plan-apochromat objective (Zeiss).

Phosphoinositide Hydrolysis
Analysis of inositol phosphates in cultured ventricular myocytes was performed as previously described with minor modification.³⁸ Myocytes cultured overnight in the presence of serum were washed and incubated in serum-free medium containing 2 µCi/mL [³H]myo- inositol for 18 to 24 hours. Cells were washed to remove excess [³H]myoinositol and incubated in 20 mmol/L HEPES-buffered medium containing 10 mmol/L LiCl with or without agonists. At indicated time points, cells were washed with cold PBS and then lysed in ice-cold MeOH:0.1 mol/L HCl (1:1) for 30 minutes at 4°C. Cell lysates were transferred to columns containing Dowex 100–200 mesh (formate form) resin beads and washed with distilled H₂O; then total inositol phosphates were eluted with 8 mL of 1 mol/L ammonium formate and 100 mmol/L formic acid and quantified by liquid scintillation counting.

PKC Translocation
To determine relative levels of PKC in membrane and cytosolic fractions, myocytes were incubated in serum-free media for 24 hours before treatment with agonists. After 1 minute of agonist stimulation, cells were washed with cold PBS and separated into membrane and cytosolic fractions by methods previously described.³⁹ Fifty micrograms of cytosolic and 100 µg of membrane protein were analyzed per treatment group by 1-dimensional electrophoresis on 10% SDS-polyacrylamide gels according to the method of Laemmli.⁴⁰ Protein was transferred from gels to Immobilon membranes at 900 mA for 2 hours according to the method of Towbin et al.⁴¹ Immunoblotting with anti-PKC antibody (1:500 dilution, Transduction Laboratories) was performed as previously described.³⁹

Accumulation of cAMP
Myocytes were allowed to attach to 35-mm culture dishes for 18 hours in plating medium; after which, they were washed and incubated in serum-free media for 24 to 48 hours. Myocytes were then washed and incubated in 20 mmol/L HEPES containing 8 mg/mL DMEM and 2 mg/mL medium 199 and 100 µmol/L 3-isobutyl-1-methylxanthine for 15 minutes before adding agonists. After 2 minutes of stimulation with agonists, myocytes were washed with cold PBS and lysed with cold 10% trichloroacetic acid. Lysates were transferred to microfuge tubes and spiked with 1200 cpm [³H]cAMP. Ion exchange columns were prepared with Dowex AG 50–4X, 200–400 mesh, resin beads, and spiked lysates applied to columns, and the columns were washed with 2.5 mL distilled H₂O. cAMP was eluted from the washed columns with 4 mL distilled H₂O and dried down in a Speedvac concentrator (Savant Instruments, Inc). Recovery of the cAMP was 90% to 95%, as determined in control columns using [³H]cAMP. cAMP was measured by a competitive binding protein assay using aliquots of the dried sample resuspended in sodium acetate buffer.⁴²

ERK, p38 MAPK, and JNK Activity Assays
Kinase activity was measured as described previously.^{19 23} Briefly, myocardial cells were washed and maintained in serum-free medium for 24 hours before agonist treatment. Cells were treated with agonists for 5 minutes (ERK assay) or 20 minutes (p38 and JNK assays), washed with cold PBS, and lysed in Tris-buffered saline containing 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, 500 µmol/L Na₃VO₄, and 1 mmol/L sodium pyrophosphate. For immunocomplex kinase assays, cell lysates were incubated with ERK-1, p38, or JNK-1 antibodies (Santa Cruz Biotechnology) conjugated to protein A Sepharose (Pharmacia) and assayed for 15 minutes for ERK and 20 minutes for p38 and JNK at 30°C in kinase buffer containing [-³²P]ATP and substrates, myelin basic protein (Sigma) for ERK and p38, or GST–c-Jun (a gift from Dr M. Karin, University of California, San Diego) for JNK. Phosphorylated myelin basic protein or GST–c-Jun was analyzed by 15% SDS-PAGE, and ³²P incorporation was quantified by radioanalytic scanning (AMBIS).

Tyrosine Phosphorylation of Shc, FAK, and Src
After 18 hours in plating media, myocytes were washed and incubated in serum-free media for 18 to 24 hours before they were treated with agonists for the indicated times. Cells were washed with ice-cold PBS and then lysed in complete lysis buffer (50 mmol/L HEPES [pH 7.0], 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 1.5 mmol/L MgCl₂, 1 mmol/L EGTA, 10 mmol/L NaF, 10 mmol/L NaP₂O₇, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, 0.1 mmol/L leupeptin, and 0.1% SDS). Equal amounts of lysate (determined from protein concentration measured by Bradford assay) were immunoprecipitated with either polyclonal anti-phosphotyrosine (Bartholomew Sefton, Salk Institute, La Jolla, Calif), monoclonal Src (UBI), or monoclonal PY20 antibodies (Transduction Labs). After immunoprecipitation, samples were boiled in Laemmli buffer, and proteins were separated on 10% (focal adhesion kinase [FAK]) or 12% (Shc and Src) SDS-polyacrylamide gels, transferred to PVDF (Immobilon) nylon membranes, and immunoblotted with either monoclonal Shc (Transduction Labs), polyclonal FAK (Tony Hunter, Salk Institute), or polyclonal anti-phosphotyrosine (Src immunoprecipitation). Membranes were washed and then incubated with the appropriate secondary antibodies, followed by enhanced chemiluminescent detection of antibody binding performed using the Supersignal Western blotting system (Pierce).

[³H]Phenylalanine Incorporation
To estimate the relative rates of protein synthesis between groups of cells, [³H]phenylalanine incorporation into cellular protein was determined as previously described.⁴ Briefly, after the attachment period, myocytes were incubated in serum-free medium for 4 to 16 hours before the addition of chemical inhibitors. After incubation with various inhibitors for the indicated times, myocytes were stimulated with agonists for 24 hours. During the last 4 hours of stimulation, myocytes were switched into medium containing 0.36 mmol/L L-phenylalanine (Sigma) and 5 µCi/mL L-[2,3,4,5,6-³H]phenylalanine (Amersham Corp). Cells were rinsed 3 times with PBS and incubated in ice-cold 10% trichloroacetic acid for 30 minutes. Cell precipitates were washed 3 times with 10% trichloroacetic acid and solubilized in 1% SDS at 37°C for 1 hour. SDS-soluble protein was transferred to scintillation vials and counted on a liquid scintillation counter (Beckman LS 1801).

RNA Isolation and Northern Blot Analysis
RNA was isolated by the acid guanidinium thiocyanate–phenol–chloroform extraction method of Chomczynski and Sacchi.⁴³ Agarose gel electrophoresis, Northern blot hybridization, and densitometry were performed as described previously.⁴⁴ RNA was stained after transfer with methylene blue and photographed to demonstrate RNA quality and loading and transfer equality between lanes (see Figure 6C, 18S rRNA stain). c-fos–specific mRNA was identified by hybridization to a 1.0-kb HindIII/NcoI v-fos cDNA provided by Dr Michael Karin, University of California, San Diego. Membranes were washed to a final stringency of 0.1x SSPE and 0.1% SDS at 55°C. Film exposure was varied to obtain autoradiograms as close to the linear range of the film as possible.

View larger version (49K): [in this window] [in a new window]   Figure 6. Inhibition of PKC does not block PGF2-stimulated hypertrophic responses. A, After an initial plating period, myocytes were incubated in the presence or absence of 5 µmol/L GF109203X for 30 minutes before adding PMA (100 nmol/L) or PGF2 (100 nmol/L) to culture media. After 48 hours of treatment, myocytes were stained with phalloidin as described in Materials and Methods. Bar=50 µm. B, Myocytes incubated with [3H]phenylalanine (3H-Phe) for 20 hours were incubated in the presence or absence of GF109203X for 30 minutes before stimulation with agonists for 4 hours. PMA- and PGF2-stimulated myocytes were harvested, and the 3H-Phe incorporated into cellular protein was measured as described in Materials and Methods and compared with unstimulated myocytes in the presence or absence of inhibitor. Data represent mean±SE from 3 separate experiments in which measurements were made in triplicate. *P<0.05 vs absence of GF109203X. Methods identical to those described in panel A were used to evaluate the effect of PKC inhibition on ANF protein expression. After 48 hours of stimulation, myocytes were stained for ANF, and 300 to 400 myocytes were scored for ANF immunoreactivity per experimental group. The percentage of ANF-positive myocytes was not altered in unstimulated cells by the addition of inhibitor and was typically in the range of 2% to 4%. Data represent mean±SE from 3 separate experiments. C, Myocytes were incubated in the presence or absence of 5 µmol/L GF109203X (GF) for 30 minutes before stimulation with agonists. Total RNA was isolated, and Northern blots were performed as described in Materials and Methods. Before hybridization with c-fos cDNA probe, membranes were stained with methylene blue, and the band corresponding to 18S rRNA was photographed to assess RNA quality and loading and transfer efficiency.

	Results

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PGF₂ Stimulates ANF Protein Expression in Myocytes
PGF₂ treatment of cultured neonatal rat ventricular myocytes has previously been shown to stimulate development of a hypertrophic phenotype characterized by increased protein synthesis, myofibrillar gene expression, and myofilament organization.⁴ To extend this research, we examined ANF protein expression in response to PGF₂ treatment by immunostaining myocytes with polyclonal antibodies to ANF. Cells treated with PGF₂ show increased perinuclear staining indicative of increased ANF protein expression (Figure 1). This is consistent with a previous study by Gardner and Schultz,⁴⁵ who demonstrated that rat atrial and ventricular myocytes produced immunoreactive ANF on PGF₂ stimulation in vitro, and recent evidence from Lai et al,² who demonstrated increased ANF mRNA levels and ANF release into the media of cultured ventricular myocytes after PGF₂ treatment. Double staining with phalloidin also demonstrated increased myocyte size and organization of F-actin into myofilaments after PGF₂ treatment, similar to the organization of MLC-2 into myofilaments previously reported.⁴

View larger version (110K): [in this window] [in a new window]   Figure 1. PGF2 stimulates myofibrillar organization and ANF expression. Neonatal rat ventricular myocytes were stimulated with 100 nmol/L PGF2 for 48 hours. The myocytes were then fixed, permeabilized, and incubated with anti-ANF antibody and stained with phalloidin as described in Materials and Methods. Panels A and B show unstimulated myocytes (same field), and panels C and D show PGF2-stimulated myocytes (same field). Panels A and C show F-actin staining with phalloidin, and panels B and D show ANF staining. Bar=50 µm.

PGF₂ Stimulates Phosphoinositide Hydrolysis but Does Not Stimulate or Inhibit cAMP Accumulation in Myocytes
PGF₂ receptor stimulation causes phosphoinositide hydrolysis in a variety of cell types.^{46 47 48} Accordingly, we determined whether PGF₂ also stimulates the production of inositol phosphates in neonatal rat ventricular myocytes. In cells in which phosphoinositides were labeled with [³H]myoinositol, PGF₂ treatment resulted in a nearly 30-fold increase in total inositol phosphate accumulation, a response significantly greater than that to the ₁-adrenergic receptor agonist PE (Figure 2). Thus, like receptors for other hypertrophic agonists, (eg, Ang II, ET-1, and PE), the PGF₂ receptor appears to couple to G_q to regulate phospholipase C in cardiac myocytes.

View larger version (20K): [in this window] [in a new window]   Figure 2. PGF2 stimulates phosphoinositide hydrolysis. Myocytes were incubated in serum-free media containing 2 µCi/mL [3H]myoinositol for 24 hours, then washed, and switched to HEPES-buffered media containing 10 mmol/L LiCl and 100 nmol/L PGF2 or 100 µmol/L PE (plus 2 µmol/L propranolol to block ß-adrenergic receptors) for 30 minutes. Cell lysates were run over ion exchange columns, and total inositol phosphates were eluted and measured as described in Materials and Methods. Data represent average fold stimulation (±SE) from 9 determinations per experimental group. *P<0.05 vs control.

In noncardiac cells, it has been reported that the PGF₂ receptor is coupled to G_i as well as G_q, as evidenced by a decrease in cAMP with PGF₂ treatment.^{46 49} However, in our hands, PGF₂ alone did not affect cAMP levels, nor were the increased cAMP levels seen with isoproterenol treatment decreased by concurrent treatment with PGF₂ (data not shown). Thus, unlike the hypertrophic agonists ET-1 and Ang II, which inhibit adenylate cyclase in the heart via the pertussis toxin–sensitive G_i protein,^{50 51} the cardiac PGF₂ receptor does not appear to couple to G_i. To further rule out the possibility that the hypertrophic response to PGF₂ is mediated by signaling through members of the pertussis toxin–sensitive G_i/G_o family of heterotrimeric G proteins, myocytes were treated with pertussis toxin (at a concentration previously shown to block muscarinic receptor inhibition of adenylyl cyclase) before PGF₂ stimulation, and ANF-luciferase reporter gene expression was measured. We find that elevated luciferase activity in response to PGF₂ stimulation was unaffected by pretreatment of myocytes with pertussis toxin (data not shown). Thus, ANF expression in response to PGF₂ stimulation is not mediated by receptor coupling to a G_i/G_o family protein.

Translocation of PKC With PGF₂ Treatment
Previous studies from our laboratory have demonstrated that the isoform of PKC is translocated to the membrane by PE.³⁹ This translocation event is thought to be indicative of PKC activation. Accordingly, we determined whether PGF₂ stimulated the redistribution of PKC. Western blot analysis demonstrates that 1 minute of PGF₂ treatment results in increased immunoreactivity of PKC in the membrane fraction and a corresponding decrease in PKC in the cytosolic fraction of lysates prepared from myocyte cultures (Figure 3). Similarly, direct activation of PKC with phorbol 12-myristate 13-acetate (PMA) resulted in increased membrane-associated PKC and decreased cytosolic levels of PKC. These results suggest that PKC is activated by PGF₂ in cardiac myocytes.

View larger version (41K): [in this window] [in a new window]   Figure 3. PGF2 stimulates translocation of PKC. Myocytes were stimulated with 100 nmol/L PGF2 or 100 nmol/L PMA for 1 minute. Cell lysates were separated into cytosolic and membrane fractions as described in Materials and Methods. Membrane and cytosolic proteins were separated by SDS-PAGE, and PKC levels were detected by immunoblotting. Western blots are representative of 3 separate experiments.

Treatment of Myocytes With PGF₂ Causes Activation of JNK, ERK, and p38 MAPK
MAPK activation has been shown to be important in the regulation of myocyte gene expression. Accordingly, we examined activation of 2 members of the MAPK family, ERK-1 and JNK-1. We previously examined time courses of ERK and JNK activation by PE and demonstrated that ERK activation was maximal at 5 minutes¹⁹ and that JNK activation was maximal by 20 minutes of stimulation.¹³ These times were therefore used to assess ERK and JNK activation by PGF₂ and demonstrated a 4-fold increase in ERK-1 activity and a 2.6-fold increase in JNK-1 activity. p38 MAPK activity was also slightly increased (1.5-fold) after 20 minutes of PGF₂ stimulation (not shown). Thus, PGF₂ causes reproducible, albeit modest, activation of the ERK, p38, and JNK pathways (Figure 4).

View larger version (59K): [in this window] [in a new window]   Figure 4. PGF2 stimulates activation of JNK and ERK. Myocytes were mock-stimulated or stimulated with 100 nmol/L PGF2 for 20 minutes (JNK) or 5 minutes (ERK). Endogenous JNK and ERK proteins were immunoprecipitated and assayed for activity by measuring incorporation of [-32P]ATP into the GST–c-Jun or myelin basic protein (MBP) substrates as described in Materials and Methods. Data on chart represent the average fold stimulation relative to unstimulated controls (±SE) from 3 experiments similar to that shown in the inset (where C indicates control, and PG indicates PGF2). *P<0.05 vs control.

Treatment of Myocytes With PGF₂ Stimulates Tyrosine Phosphorylation of Shc, FAK, and Src
It was recently demonstrated that cellular TK signaling pathways were activated by the G_q-coupled Ang II receptor in cardiac myocytes.⁷ PGF₂ stimulation leads to phosphorylation of tyrosine residues on specific cellular proteins, includ-ing p125^FAK in NIH-3T3 cells,⁵² but it is not known whether this pathway is also activated by PGF₂ in cardiac myocytes. To address this question, we measured phosphorylation of tyrosine residues on p125^FAK, Shc, and Src. We observed a 2-fold increase in tyrosine phosphorylation of Shc and Src and a 3.4-fold increase in phosphorylation of p125^FAK within 1 minute of PGF₂ addition (Figure 5). Elevated levels of tyrosine phosphorylation in response to PGF₂ were sustained for >10 minutes. These findings indicate that PGF₂ leads to activation of protein TKs in neonatal ventricular myocytes.

View larger version (39K): [in this window] [in a new window]   Figure 5. PGF2 stimulates increased TK activity. Myocytes were stimulated with 100 nmol/L PGF2 for indicated time periods, cells were lysed, and protein concentration was analyzed by Bradford assay. Equal amounts of lysate protein were immunoprecipitated with anti-phosphotyrosine antibody as described in Materials and Methods. Immunoprecipitates were separated by SDS-PAGE, and the tyrosine-phosphorylated forms of Shc, Src, and FAK were detected by immunoblotting and chemiluminescence detection.

Inhibition of PKC With GF109203X
To evaluate the involvement of these intracellular pathways in PGF₂-stimulated myocyte hypertrophy, we blocked individual kinases with pharmacological inhibitors or expression plasmids for dominant-interfering mutants. The role of each kinase pathway was evaluated on the basis of the inhibition of changes in myocyte morphology, gene expression, and protein synthesis in response to PGF₂.

GF109203X (bisindolylmaleimide I), a synthetic derivative of staurosporine with improved selectivity for PKC over several serine/threonine kinases and TKs, acts as a competitive inhibitor of PKC with respect to ATP.⁵³ To evaluate its ability to block responses to PKC activation in myocytes, we demonstrated that it inhibits protein synthesis and myofibrillar organization in response to PMA treatment. The immediate-early gene c-fos, whose expression has been associated with a variety of models of cardiomyocyte hypertrophy including stimulation by PMA, was strongly induced by both PMA and PGF₂. However, inhibition of PKC with GF109203X, which completely blocked PMA-stimulated c-fos induction, did not inhibit the PGF₂-stimulated c-fos response (Figure 6C). The apparent potentiation of PGF₂-stimulated c-fos induction by GF109203X is reproducible, but its relevance is not currently understood. Similarly, induction of the hypertrophic phenotype in response to PGF₂ was not significantly blocked by PKC inhibition, since increased myofibrillar organization, ANF expression, and [³H]PE incorporation were still observed (Figure 6A and 6B). Similar results were obtained when myocytes were pretreated with a different inhibitor of PKC, chelerythrine, which acts on the catalytic domain of the kinase (data not shown). Thus, it appears that although PGF₂ stimulates PKC translocation, it is not required for PGF₂-stimulated cardiomyocyte hypertrophy.

Pharmacological Inhibition of ERK and p38 Activation
PD098059 is an inhibitor of MAPK kinase (MEK), the enzyme that activates ERK-1 and ERK-2.⁵⁴ We previously demonstrated inhibition of ERK activation by PE in myocytes pretreated with 10 µmol/L PD098059.¹⁹ This concentration also completely blocked PGF₂-stimulated ERK activation in myocytes, whereas JNK activation was unaffected (data not shown). Inhibition of ERK activation with PD098059 did not, however, inhibit PGF₂-stimulated increases in myofibrillar organization as assessed by phalloidin staining (Figure 7A), nor did it inhibit protein synthesis as estimated by the incorporation of [³H]phenylalanine into cellular protein (Figure 7B). Furthermore, pretreatment of myocytes with PD098059 did not block the expression of endogenous ANF expression in response to PGF₂ (Figure 7B).

View larger version (64K): [in this window] [in a new window]   Figure 7. Inhibition of MEK does not block PGF2-stimulated hypertrophic responses. Myocytes were incubated in the presence or absence of 10 µmol/L PD098059 for 30 minutes before adding PGF2 (100 nmol/L) to culture media. Myofilament organization (A) and [3H]phenylalanine (3H-Phe) incorporation and ANF protein expression (B) were measured as described in Materials and Methods and in Figure 6. Data represent mean±SE from 3 separate experiments in which measurements were made in triplicate. *P<0.05 vs absence of PD098059. In panel A, bar=50 µm.

A specific inhibitor of p38 MAPK, SB203580 (Calbiochem), was tested for its ability to block myocyte ANF expression and myofilament organization in response to PGF₂ stimulation. At concentrations previously demonstrated to inhibit hypertrophy in cardiac myocytes stimulated by MKK6 or PE,^{23 24} we saw no affect on PGF₂-stimulated ANF immunoreactivity or myofilament organization (not shown). Thus, neither ERK nor p38 MAPK activation appear to be required for PGF₂-stimulated hypertrophic responses in cultured cardiac myocytes.

Inhibition of the JNK Pathway With Dominant-Interfering MEKK1 Expression Plasmid
To evaluate the role of JNK in PGF₂-stimulated ANF expression, a MEKK1 expression plasmid, mutated in its catalytic site, was cotransfected with ANF-luciferase reporter constructs. This dominant-interfering mutant likely inhibits JNK activation by binding to the upstream small G proteins (eg, Rac or Ras) required for activation of endogenous MEKK1.³⁵ The dominant-interfering MEKK1 mutant was recently shown to block PE-induced JNK activation in myocytes, as assessed by inhibition of a Jun-Gal reporter gene.¹³ Figure 8 shows that expression of dominant-negative MEKK1 markedly reduced PGF₂-induced ANF expression. Consistent with these results, dominant-interfering MEKK1 expression also inhibited PGF₂-stimulated MLC-2–luciferase and c-Jun–luciferase activity (data not shown). The dominant-interfering MEKK1 can also bind to Ras and thereby also inhibit activation of ERK.³⁵ In order to establish that inhibition of ERK activation by dominant-interfering MEKK1 is not responsible for the inhibitory effect on genetic markers of hypertrophic growth, we performed parallel experiments using dominant-interfering Raf-1, which binds Ras and inhibits the MEK/ERK pathway. Dominant-interfering Raf-1 expression did not affect PGF₂-induced ANF-luciferase as shown in Figure 8, nor did it inhibit MLC-2-luciferase or c-Jun–luciferase reporter activity (not shown). These data suggest that inhibition of the JNK pathway, but not the ERK pathway, blocks the induction of several genetic markers of hypertrophic growth by PGF₂.

View larger version (12K): [in this window] [in a new window]   Figure 8. Dominant-interfering MEKK inhibits PGF2-stimulated ANF-luciferase activity. Transient cotransfection of myocytes with plasmids expressing dominant-interfering mutant kinases and luciferase reporter plasmids were performed as described in Materials and Methods. After the transfection period, myocytes were washed thoroughly and then stimulated with 100 nmol/L PGF2 for 48 hours before harvesting cells and measuring luciferase activity in cell lysates. All treatment groups were normalized to unstimulated cells transfected with plasmids containing vector sequences and reporter gene alone. dnMEKK and dnRaf indicate dominant-negative MEKK1 and Raf, respectively. Data represent mean±SE from 3 separate experiments performed in triplicate. *P<0.05 vs control.

Inhibition of Protein TKs With Herbimycin
Pretreatment of myocytes with 1.5 µmol/L herbimycin, which binds irreversibly to thiol groups on TKs, inhibited PGF₂-induced myofibrillar organization, protein synthesis, and expression of endogenous ANF (Figure 9). Tyrphostin A25, another protein TK inhibitor that acts on the substrate binding site of the kinase, also blocked PGF₂-stimulated hypertrophic responses (data not shown). These results suggest that protein tyrosine phosphorylation is required for hypertrophic responses to PGF₂ in cardiac myocytes.

View larger version (55K): [in this window] [in a new window]   Figure 9. Inhibition of protein TKs blocks PGF2-stimulated hypertrophic responses. Myocytes were incubated in the presence or absence of 1.5 µmol/L herbimycin for 16 hours before adding 100 nmol/L PGF2 to the culture medium. Myofilament organization (A) and [3H]phenylalanine (3H-Phe) incorporation and ANF expression (B) were measured as described in Materials and Methods and in Figure 6. Data represent mean±SE from 3 separate experiments in which measurements were made in triplicate. *P<0.05 vs absence of herbimycin. In panel A, bar=50 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Stimulation of neonatal rat ventricular myocytes with PGF₂ activates a host of hypertrophic changes, including increased protein synthesis, myofibrillar organization, and expression of ANF and MLC-2 genes. The induction of these responses is characteristically seen on stimulation of myocytes with receptor agonists that stimulate phospholipase C.^{37 55 56} We find that activation of the PGF₂ receptor also stimulates phosphoinositide hydrolysis in cardiac myocytes. Phosphoinositide hydrolysis presumably occurs via G_q-mediated activation of phospholipase C and results in the production of signaling molecules, including inositol 1,4,5-trisphosphate and PKC. Experiments using pharmacological inhibitors and constitutively active mutants of PKC have established that this enzyme is important for growth regulation in cardiac myocytes.^{9 10} Reports from our laboratory and others have shown that the hypertrophic agonists PE and ET-1 cause a rapid transient increase in membrane-associated PKC in neonatal rat cardiac myocytes.^{39 57} Similarly, we have found that PGF₂ stimulation causes a rapid increase in membrane-associated PKC. Surprisingly, inhibitors of PKC (GF109203X and chelerythrine) that were effective at blocking myocyte hypertrophy induced by PMA had little effect on PGF₂-induced responses. These results suggest that PGF₂ activates PKC but does not require this protein kinase to mediate hypertrophy. Interestingly, we observed that inhibition of PKC with GF109203X blocked PE-stimulated myofibrillar organization and ANF expression (data not shown), indicating that hypertrophic responses to 2 G_q-coupled receptors may be mediated by distinct intracellular mechanisms.

PGF₂ also activates ERK in cultured myocytes. The 4-fold increase in ERK activation with PGF₂ stimulation is comparable in magnitude to the degree of stimulation seen in response to PE and ET-1.¹³ However, although PD098059 inhibited PGF₂-stimulated ERK activation (data not shown), it did not prevent any of the PGF₂-induced hypertrophic responses. In addition, cotransfection experiments with expression plasmids for dominant-interfering Raf-1 suggest that the Raf/MEK/ERK pathway does not mediate PGF₂-stimulated expression of genes associated with hypertrophic growth (eg, ANF, MLC-2, and c-Jun). Thus, activation of the Raf/MEK/ERK pathway by PGF₂ does not account for the increases in hypertrophic gene expression, protein synthesis, or myofibrillar organization induced by PGF₂.

Our experiments demonstrate that PGF₂ also activates JNK. The 2.6-fold stimulation is comparable to the magnitude of JNK activation reported in response to ₁-adrenergic receptor activation.¹³ Expression of dominant-interfering MEKK1 inhibits PGF₂-stimulated MLC-2–luciferase, ANF-luciferase, and c-Jun–luciferase reporter gene expression. These findings are consistent with recent work from our laboratory that implicates the JNK pathway as a mediator of Ras and PE-induced myocyte hypertrophy.¹³ They are also supported by studies from other laboratories demonstrating increases in cell size and ANF expression in myocytes transfected with activated MEKK⁵⁸ and inhibition of PE-stimulated ANF-luciferase activity in myocytes after blockade of the JNK pathway with dominant-negative MEKK1.²² Thus, it is apparent from our results that sequestration of upstream activators of MEKK1 (eg, Ras and Rac) by dominant-negative MEKK1 abrogates the expression of hypertrophic marker genes. In addition to JNK, p38 and ERK pathways could be inhibited by dominant-negative MEKK1; however, pharmacological inhibition of these kinase pathways with PD098059 or SB203580, respectively, did not prevent either genetic or morphological hypertrophic responses to PGF₂. Thus, although we cannot formally rule out the possibility that activation of other as-yet-undefined downstream targets of Rac or Ras could also be inhibited by dominant-negative MEKK1, we attribute the PGF₂-induced expression of hypertrophic marker genes to the JNK pathway.

PGF₂ was reported to stimulate mitogenesis and tyrosine phosphorylation of several cellular proteins, including FAK (p125^FAK) in NIH-3T3 cells.⁵² We have demonstrated that PGF₂ also stimulates tyrosine phosphorylation of Shc, Src, and FAK in neonatal rat cardiac myocytes. Thus, this putative G_q-coupled receptor appears to activate cytoplasmic TKs. The mechanism by which G protein–coupled receptors stimulate the activity of cytoplasmic TKs is currently unknown. It was recently suggested that activation of p21^ras by Ang II in neonatal rat cardiac myocytes involves tyrosine phosphorylation of Shc and an Src-related protein.²⁵ Our finding that the TK inhibitors herbimycin and tyrphostin block PGF₂-stimulated changes in gene expression and myocyte morphology further attests to the importance of TK function in cardiac growth regulation. However, further studies are needed to define the roles of the adaptor protein, Shc, and the TKs, Src and FAK, in G_q-coupled growth responses in cardiac myocytes.

Although the mechanism by which MAPKs, including JNK, are activated by G_q-coupled receptors is currently unknown, preliminary experiments in our laboratory have demonstrated that inhibition of TK activation with herbimycin prevents JNK activation by PGF₂ (J.W. Adams and J.H. Brown, unpublished data, 1998). These data suggest that TKs are upstream from JNK in this pathway and are consistent with the findings of Kawakami et al,⁵⁹ who demonstrated that Bruton's TK regulates JNK activity in mast cells, and results from Zohn et al,⁶⁰ who showed that inhibition of TK with genistein blocked Ang II–stimulated JNK activation in liver cells.

Hypoxia/reoxygenation activates multiple intracellular responses in cardiac myocytes, including activation of the Src family of nonreceptor TKs, activation of JNK, immediate-early gene induction, and increased protein synthesis.^{61 62 63 64} These pathways, or a subset thereof, are likely to mediate the reactive form of hypertrophy that is manifest in the myocardium after myocardial infarction. Given that PGF₂, now established as a distinct hypertrophic agent in the heart, is elevated in the myocardium of animals subjected to ischemia/reperfusion and that we show this ligand to activate pathways similar to those activated by ischemia/reperfusion, PGF₂ emerges as a likely mediator of the compensatory hypertrophy seen in animal models of myocardial ischemia. The etiology of compensatory cardiac hypertrophy is undoubtedly multifactorial, a consequence of complex interactions between a variety of cell types producing an assortment of chemical signals that may be involved during different stages of its development. A definitive role of PGF₂ in compensatory hypertrophy cannot be established until PGF₂ receptor antagonists are developed or the effect of PGF₂ receptor knockouts on cardiac function are analyzed. Nevertheless, our work establishes that PGF₂ can elicit a host of the features characteristic of hypertrophy in neonatal rat cardiac myocytes and that these are mediated intracellularly by TK and JNK pathways.

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-28143 and HL-46345 (Dr Heller Brown) and National Institutes of Health postdoctoral training grant 5 T32 HL-07444-15 (Dr Adams). We thank Anh Le for culturing the neonatal rat ventricular myocytes and David Goldstein and for technical help.

Received March 30, 1998; accepted May 18, 1998.

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