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Am J Physiol Heart Circ Physiol 288: H2111-H2117, 2005. First published December 30, 2004; doi:10.1152/ajpheart.00838.2004
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PGE2-induced hypertrophy of cardiac myocytes involves EP4 receptor-dependent activation of p42/44 MAPK and EGFR transactivation

Mariela Mendez and Margot C. LaPointe

Hypertension and Vascular Research Division, Department of Medicine, Henry Ford Hospital, Detroit, Michigan

Submitted 18 August 2004 ; accepted in final form 22 December 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Upon induction of cyclooxygenase-2 (COX-2), neonatal ventricular myocytes (VMs) mainly synthesize prostaglandin E2 (PGE2). The biological effects of PGE2 are mediated through four different G protein-coupled receptor (GPCR) subtypes (EP1–4). We have previously shown that PGE2 stimulates cAMP production and induces hypertrophy of VMs. Because the EP4 receptor is coupled to adenylate cyclase and increases in cAMP, we hypothesized that PGE2 induces hypertrophic growth of cardiac myocytes through a signaling cascade that involves EP4-cAMP and activation of protein kinase A (PKA). To test this, we used primary cultures of VMs and measured [3H]leucine incorporation into total protein. An EP4 antagonist was able to partially block PGE2 induction of protein synthesis and prevent PGE2-dependent increases in cell surface area and activity of the atrial natriuretic factor promoter, which are two other indicators of hypertrophic growth. Surprisingly, a PKA inhibitor had no effect. In other cell types, G protein-coupled receptor activation has been shown to transactivate the epidermal growth factor receptor (EGFR) and result in p42/44 mitogen-activated protein kinase (MAPK) activation and cell growth. Immunoprecipitation of myocyte lysates demonstrated that the EGFR was rapidly phosphorylated by PGE2 in VMs, and the EP4 antagonist blocked this. In addition, the selective EGFR inhibitor AG-1478 completely blocked PGE2-induced protein synthesis. We also found that PGE2 rapidly phosphorylated p42/44 MAPK, which was inhibited by the EP4 antagonist and by AG-1478. Finally, the p42/44 MAPK inhibitor PD-98053 (25 µmol/l) blocked PGE2-induced protein synthesis. Altogether, we believe these are the first data to suggest that PGE2 induces protein synthesis in cardiac myocytes in part via activation of the EP4 receptor and subsequent activation of p42/44 MAPK. Activation of p42/44 MAPK is independent of the common cAMP-PKA pathway and involves EP4-dependent transactivation of EGFR.

prostaglandin E2; epidermal growth factor receptor; cyclooxygenase; G protein; ventricular myocyte; mitogen-activated protein kinase

CARDIAC HYPERTROPHY IS AN adaptive response of the heart to several cardiovascular diseases. Vasoactive molecules such as ANG II, endothelin-1, and phenylephrine are known mediators of cardiomyocyte hypertrophy. In vitro and in vivo studies have demonstrated that cardiac hypertrophy occurs along with induction of the hypertrophic program including reexpression of fetal genes [e.g., atrial natriuretic peptide (ANP), {beta}-myosin heavy chain, and skeletal muscle {alpha}-actin], activation of immediate early genes (e.g., c-fos, c-jun, and erg-1), and increased protein synthesis and morphological changes (e.g., cell size and sarcomeric structures; Refs. 31, 48, 54).

Cyclooxygenase-2 (COX-2) products have gained special interest as promoters of cell growth aside from their role in inflammation (3, 33, 51). In vivo studies (24) have shown that in the heart, COX-2 inhibition reduces hypertrophy and fibrosis in a mouse model of myocardial infarction. We have previously reported (33) that in an inflammatory setting, neonatal ventricular myocytes (VMs) preferentially produce prostaglandin E2 (PGE2) over other prostaglandins, and addition of PGE2 promotes their growth. In other cells, PGE2 has been implicated in tumor growth and mitogenesis (3, 23, 32, 51), and HEK-293 cells that overexpress both COX-2 and PGE2 synthase grow faster than normal cells (34).

The biological effects of PGE2 are mediated via four different G protein-coupled receptor (GPCR) subtypes (EP1–4). EP1 activation results in Ca2+ mobilization, and the EP3 receptor is coupled to a G{alpha}i protein that leads to decreased cAMP levels. Both EP2 and EP4 are coupled to G{alpha}s, and their activation results in increased cAMP levels (37). There is evidence that EP subtypes are involved in cell growth. The EP1 receptor subtype has been implicated in growth of keratinocytes (56), primary cultures of hepatocytes (22), and breast cancer cells (19). However, our previous findings (33) demonstrate that EP4 is more likely than EP1 to be involved in PGE2-induced protein synthesis in cardiac myocytes. In agreement with this, in vitro (46, 47, 51) and in vivo (35) reports have implicated the EP4 receptor as a mediator of cell growth.

Activation of G{alpha}s-coupled receptors has been described (63, 64) to mediate hypertrophic growth of cardiac myocytes through cAMP and downstream activation of mitogen-activated protein kinases (MAPKs). cAMP mediates growth-promoting signals by activation of protein kinase A (PKA) in other cell types (52, 61). However, activation of MAPKs occurs by two additional mechanisms that include 1) direct effects of cAMP (independent of PKA activation; Refs. 20, 28, 42); and 2) GPCR transactivation of epidermal growth factor receptors (EGFRs; Refs. 12, 41, 59). Thus in the present study, we examined the signaling cascade responsible for PGE2 induction of protein synthesis in cardiac myocytes. In contrast to what we hypothesized originally, we found that PGE2-induced protein synthesis in cardiac myocytes involves phosphorylation of p42/44 MAPK by a mechanism independent of cAMP and PKA activation that involves EP4-mediated EGFR transactivation.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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Cell culture. Primary cultures of neonatal VMs were derived from digestion of 1- to 2-day-old neonatal Sprague-Dawley rat hearts (Charles River; Kalamazoo, MI) as described previously (25). After 40 h in culture with DMEM plus 10% fetal bovine serum (GIBCO), the medium was changed to serum-free medium supplemented with glutamine, insulin, selenium, and transferrin for 24 h. Cells were incubated with the different treatments for 48 h for [3H]leucine-incorporation studies and for 5 min for p42/44 MAPK and EGFR phosphorylation. In the protocols where different antagonists were used, cells were pretreated for 1 h before the addition of PGE2. This protocol was approved by the Henry Ford Hospital Institutional Animal Care and Use Committee.

Protein isolation, EGFR immunoprecipitation, and Western blot. Protein was isolated from cardiac myocytes using lysis buffer as described previously (26) that was supplemented with protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, 1 mM sodium vanadate, 10 mM sodium fluoride, and 20 mM glycerophosphate). For immunoprecipitation experiments, 300 µg of total protein was resuspended in lysis buffer that contained protease inhibitors and was incubated overnight at 4°C with 10 µl of EGFR antibody (Santa Cruz) and 100 µl of protein A agarose beads. Immunoprecipitates were washed four times with lysis buffer and one time with 10 mM Tris·HCl (pH 7.6). Beads were resuspended in 30 µl of Western loading buffer and boiled for 5 min. Samples were rapidly centrifuged, and supernatants were electrophoresed in a 7.5% acrylamide gel and transferred onto a polyvinylidene difluoride membrane. Membranes were incubated with anti-phosphotyrosine antibody (1:2,000 dilution) in 5% milk solution prepared in Tris-buffered saline with 0.1% Tween (TBST) for 1.5 h and were then washed. The appropriate secondary antibody linked to horseradish peroxidase (1:1,000 dilution) was used for chemiluminescent detection with enhanced chemiluminescence Western blot reagents (Amersham Pharmacia Biotech). The signal was detected by exposure to Fuji RX film and quantified by laser densitometry.

Assay for p42/44 MAPK and immunoblotting. Before VMs were treated, the cells were washed every 15 min for 1 h with serum-free medium to decrease basal p42/44 MAPK phosphorylation levels. When the EP4 antagonist AG-1478 (AG) or H-89 was used, the washing medium was supplemented with the antagonist to result in 1 h of pretreatment. Cells were then treated in serum-free medium for 5 min at 37°C, and the reaction was stopped with ice-cold lysis buffer supplemented with the above-mentioned inhibitors. Protein from each sample (15–20 µg) was electrophoresed in 7.5% acrylamide gel and transferred to polyvinylidene difluoride membranes, which were developed with a selective primary antibody (1:2,000 dilution) against phosphorylated p42/44 MAPK (Cell Signaling). As a control for protein loading, the same membranes were stripped and reblotted with a nonphosphorylated p42/44 MAPK antibody.

Protein synthesis and cell surface-area quantification. Protein synthesis was determined by incorporation of [3H]leucine into trichloroacetic acid-insoluble material in 48 h as described previously (33). VMs were pretreated with the antagonists for 1 h, and then PGE2 was added for another 48 h. For each treatment in each experiment, counts per minute from triplicate filters were averaged.

For cell surface measurement, 30,000 VMs were plated in fibronectin-coated glass covers, serum deprived for 24 h, treated for 48 h as described for protein synthesis, and visualized by staining with an anti-actin antibody. In brief, after the treatments, the cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS for at least 15 min. Cells were then permeabilized in 0.1% Triton X-100 in PBS for 15 min and blocked for 1 h in 1% BSA-TBST (with 0.1% Tween). Cells were incubated with the primary antibody (diluted 1:100 in 1% BSA-TBST) for 60 min and then washed with PBS. The secondary antibody (donkey anti-goat Alexa Fluor 488) was diluted 1:250 in 1% BSA-TBST for 1 h. After two washes with PBS, samples were mounted in Fluoromount-G (Southern Biotechnology Associates). Immunofluorescence was detected using a fluorescence microscope (Nikon Eclipse E600) attached to a SPOT camera (Diagnostic Instruments). Cell areas of at least 40 cells, corresponding to three independent experiments, in randomly selected microscopic fields were analyzed by planimetry using NIH Image J software by an observer who was unaware of the treatment groups.

Transient transfection and luciferase assay. Transfection was performed by electroporation, and luciferase activity was assayed as described previously (15, 27). The human ANP promoter (–2593 to +18 relative to the start site of transcription) cloned upstream of the luciferase cDNA in the pMG-1 vector was described previously (10). Plasmid DNA (6 µg per 6 x 106 VMs) was transfected, and the cells were equally distributed on a 12-well plate (0.5 x 106 cells/well). After 40 h, the medium was changed to serum-free DMEM; 24 h later, the cells were treated with PGE2 and the EP4 antagonist. After a 24-h treatment period, VMs were lysed and assayed for luciferase activity using Promega reagents in an OptoComp 1 luminometer. Relative light units from triplicate wells were averaged. Relative light unit values in control (untreated) lysates were normalized, and the values of treated lysates were compared with controls to yield the fold increase.

Chemicals. PGE2 was obtained from Cayman, and L-[3,4,4-3H(N)]leucine was purchased from NEN/DuPont (Boston, MA). PD-98053 (PD), SB-203580 (SB), and AG were obtained from Calbiochem (San Diego, CA), forskolin and H-89 were from Biomol, and the EPAC activator 8-(4-chlorophenylthio)-2'-O-methyladenosine (8-CPT-2Me)-cAMP was from Tocris. GM-6001 (GM; Ilomastat) was from Chemicon International. Anti-phospho- and total p42/44 MAPK antibodies were purchased from Cell Signaling; EGFR (1005) and the actin (sc-1616) antibody were from Santa Cruz; and anti-phosphotyrosine antibody was from Upstate. For immunofluorescence, secondary antibody was obtained from Molecular Probes (Eugene, OR). The EP4 antagonist was kindly provided by Dr. Robert Young (Merck Frosst; Kirkland, Quebec, Canada). All other laboratory supplies and chemicals were obtained from Sigma, Fisher, and VWR Scientific.

Statistics. Data are expressed as means ± SE. Differences in mean values were analyzed by one-way ANOVA using the Student-Newman-Keuls method for pair-wise multiple comparisons. P < 0.05 was considered significant.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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PGE2 increases protein synthesis in myocytes via EP4 receptors. We have previously reported that PGE2 induces protein synthesis in VMs (33). We tested the ability of the EP4 antagonist L-161982 (10 µmol/l) to prevent induction of [3H]leucine incorporation by PGE2 (1 µmol/l) as a measure of protein synthesis. We found that treatment with the EP4 antagonist for 48 h blunted PGE2 induction of protein synthesis by 44% (control, 1; PGE2, 1.9 ± 0.1-fold; PGE2 with EP4 antagonist, 1.5 ± 0.2-fold; Fig. 1A).



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  		Fig. 1. Effects of the EP4 antagonist L-161982 on prostaglandin (PG)E2-induced myocyte growth. A: effects of the EP4 antagonist on PGE2-induced protein synthesis. Amount of [3H]leucine incorporation into total protein over 48 h is indicated [expressed as fold increase compared with control (CONT), which was arbitrarily set to 1]. Bars indicate means ± SE for seven different experiments. *P < 0.05 vs. control; #P < 0.05 vs. PGE2. B: effects of the EP4 antagonist on PGE2-induced transactivation of the human ANP promoter (–2593 hANPLuc). C: effects of the EP4 antagonist on PGE2-increased cell size in ventricular myocytes. Areas were quantified by computer-assisted planimetery of actin-stained cell slides; n = 41 (control), 37 (PGE2), and 29 [PGE2 with EP4 antagonist (EP4ant)]. For B and C, *P < 0.01 vs. control; #P < 0.01 vs. PGE2.

 
In addition to increased protein synthesis, cardiac hypertrophy is characterized by reexpression of fetal genes such as ANP and increased cell size (31, 58). To test the effect of PGE2 on ANP gene transcription, the human ANP promoter coupled to a luciferase reporter gene (–2593 hANPLuc) was transiently transfected into VMs, and cells were treated with PGE2 for 24 h. We found that PGE2 stimulated hANP promoter activity, and this could be blocked by selective inhibition of the EP4 receptor subtype (control, 1; PGE2, 1.9 ± 0.4-fold; PGE2 with EP4 antagonist, 0.6 ± 0.1-fold; Fig. 1B). In addition, 48 h of treatment with PGE2 increased cell size by 1.4-fold compared with the control, which was significantly decreased by pretreatment with the EP4 antagonist (Fig. 1C).

PGE2 induces protein synthesis independently of PKA and adenylate cyclase activation. Because the EP4 receptor is coupled to a Gs protein that leads to cAMP generation (7, 37, 43), we tested whether PKA mediates PGE2-induced protein synthesis. The PKA inhibitor H-89 (1 µmol/l) had no effect on PGE2 induction of protein synthesis (data not shown). In addition, neither activation of adenylate cyclase with forskolin (50 µmol/l) nor inhibition of adenylate cyclase with SQ-22536 (10 µmol/l) had any effect on PGE2-induced protein synthesis (data not shown).

PGE2-induced protein synthesis involves EP4-dependent activation of p42/44 MAPK but not p38 MAPK. We further investigated which other signaling molecules could mediate the induction of protein synthesis. We first tested whether the PGE2 signaling cascade involves p42/44 MAPK activation. Using Western blot analysis, we found that PGE2 caused rapid phosphorylation of p42/44 MAPK (control, 1; PGE2, 3.2 ± 0.5-fold; n = 10; P < 0.01), which was inhibited by 69% by the EP4 antagonist (n = 6; Fig. 2).



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  		Fig. 2. Effects of PGE2 and an EP4 antagonist on phospho- and total p42/44 MAPK. Phosphorylation of p42/44 MAPK (top) was as follows: control, 1; PGE2, 3.2 ± 0.5-fold; PGE2 with EP4 antagonist, 1.7 ± 0.4-fold; *P < 0.01 vs. control; #P < 0.05 vs. PGE2. Membranes used for phospho-p42/44 MAPK Western blotting were stripped and reprobed with an anti-total p42/44 MAPK. Representative blots are shown (bottom).

 
To study the involvement of p42/44 MAPK activation as a downstream mediator of PGE2-induced protein synthesis, we tested whether the selective p42/44 MAPK inhibitor PD (25 µM) could prevent PGE2-induced [3H]leucine incorporation. PD completely blocked the effect of PGE2 (Fig. 3); however, inhibition of p38 MAPK with SB (10 µM) had no effect (control, 1; PGE2, 1.6 ± 0.2-fold; PGE2 with PD, 1.6 ± 0.2-fold; n = 3).



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  		Fig. 3. Effects of p42/44 MAPK inhibition on PGE2-induced protein synthesis. Effect of the p42/44 MAPK inhibitor PD-98052 (PD). Incorporation of [3H]leucine into total protein over 48 h is expressed as fold increase compared with control, which was arbitrarily set to 1. *P < 0.01 vs. control; #P < 0.01 vs. PGE2; PGE2 with PD-98052 vs. control, nonsignificant; n = 3.

 
PGE2-induced p42/44 MAPK activation and protein synthesis require transactivation of EGFR. Because increasing evidence suggests that receptor tyrosine kinase transactivation links GPCR and MAPK activation, we questioned whether PGE2 could transactivate the EGFRs and mediate the effect of PGE2 on protein synthesis in cardiac myocytes. By immunoprecipitation of myocyte lysates, we found that stimulation with PGE2 for 5 min induced phosphorylation of EGFRs, and blockade of EP4 inhibited it (Fig. 4).



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  		Fig. 4. Effects of PGE2 on epidermal growth factor receptor (EGFR) activation. Total cell lysates were immunoprecipitated with an anti-EGFR antibody and blotted with a phospho-tyrosine antibody as described (see METHODS). A semiquantitative analysis of pooled data is shown (top). Although not shown in the graph, there were no changes in total EGFR (measured in density units). Control, 1; PGE2, 0.86 ± 0.3; PGE2 with EP4 antagonist, 0.94 ± 0.5. *P < 0.05 vs. control; #P < 0.05 vs. PGE2; n = 3. A representative blot showing PGE2-induced EGFR phosphorylation and blockade by the EP4 antagonist is shown (bottom). Band 1, control; band 2, PGE2 stimulation; and band 3, PGE2 with EP4 antagonist. Positive control was a lysate of EGF-stimulated A431 cells (Upstate).

 
To identify the EGFR as a mediator of PGE2-induced protein synthesis, we tested the effect of the EGFR inhibitor AG on PGE2-induced [3H]leucine incorporation. We observed that 5 µmol/l AG completely blocked PGE2 induction of protein synthesis (control, 1; PGE2, 1.5 ± 0.1-fold; PGE2 with AG, 1 ± 0.1-fold; n = 5; Fig. 5). Finally, we found that inhibition of EGFR activation with AG decreased PGE2-dependent activation of p42/44 MAPK by 64% (n = 3), which indicates that p42/44 MAPK activation is downstream from EGFR activation (Fig. 6). Consistent with our previous findings, inhibition of PKA with H-89 had no effect on PGE2-induced p42/44 MAPK activation (data not shown).



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  		Fig. 5. Effects of the EGFR receptor inhibitor AG-1478 (AG) on PGE2-induced protein synthesis. Incorporation of [3H]leucine into total protein over 48 h was measured and expressed as fold increase vs. control, which was arbitrarily set to 1. *P < 0.01 vs. control; #P < 0.01 vs. PGE2.

 


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  		Fig. 6. Effects of EGFRs on PGE2-induced p42/44 MAPK activation. Effect of the EGFR inhibitor AG-1478 is shown (top). *P < 0.01 vs. control; n = 3. Representative Western blot is shown (bottom). In each experiment, the membranes were stripped and reblotted with an antibody against total p42/44 MAPK. *P < 0.01 vs. control; #P < 0.01 vs. PGE2.

 
PGE2-dependent transactivation of EGFRs requires EGFR agonist ectoshedding. Transactivation of EGFRs has been described to occur by metalloproteinase-dependent ectoshedding of EGFR agonists (59). Inhibition of the metalloproteinases with GM (4 µM) prevented the PGE2-dependent growth response (control, 1; PGE2, 1.7 ± 0.1-fold; PGE2 with GM, 1.2 ± 0.1-fold; n = 9; Fig. 7).



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  		Fig. 7. Involvement of metalloproteinase-dependent ectoshedding of EGFR agonist in PGE2 stimulation of protein synthesis. Effects of the metalloproteinase inhibitor GM-6001 are shown. *P < 0.01 vs. control; #P < 0.01 vs. PGE2.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
When cardiac myocytes undergo hypertrophy, protein synthesis is elevated as are the activities of early response genes (e.g., c-myc, fos, etc.) and fetal markers (e.g., ANP, brain natriuretic peptide, myosin light chain, etc.; Refs. 31, 58). In our study, we found that PGE2 stimulation of myocyte hypertrophy involved increased protein synthesis, cell surface area, and ANP transcription, and those effects were mediated by EP4. EP4 was coupled to EGFR transactivation as well as activation of p42/44 MAPK. The fact that PGE2 has growth-promoting effects in vitro provides some explanation for in vivo studies using COX-2 inhibitors, where COX-2 inhibition after myocardial infarction improved function, decreased hypertrophy and fibrosis (24), and decreased fibroblast proliferation (49).

Prostaglandin receptors belong to the seven-transmembrane-domain receptor family coupled to heterotrimeric G proteins. So far, four different PGE2 receptor subtypes have been described, which couple to different intracellular signaling cascades (37). We previously reported (33) that a selective inhibitor of the EP1 and EP2 receptor subtypes does not affect PGE2 stimulation of cardiomyocyte growth. We have extended those studies using a specific EP4 antagonist that implicates EP4 as the EP receptor subtype that mediates myocyte growth (30); this is in agreement with studies showing that the EP4 receptor mediates growth and invasiveness of colon cancer cells (35, 46, 51).

Although PGE2 results in cAMP production by myocytes (33), neither adenylate cyclase nor PKA seems to be involved in mediating the effect of PGE2. The role of cAMP in cell growth remains controversial. Its effect as a modulator of cell growth strictly depends on cell type (52). In cardiac myocytes, activation of G{alpha}s-coupled {beta}-adrenergic receptors by isoproterenol promotes growth (64). This effect is partially mediated through cAMP-dependent PKA phosphorylation and subsequent activation of potential growth-promoting signals such as MAPK (52, 61), activation of L-type Ca2+ channels (13, 53), and mobilization of Ca2+ (45, 53). We have previously shown that intracellular cAMP increases after myocytes are treated with PGE2 and that exogenous addition of high concentrations of cell permeable cAMP increases protein synthesis in cardiac myocytes (33). However, our results with a PKA inhibitor, an adenylate cyclase inhibitor, and an adenylate cyclase activator demonstrate that neither cAMP nor PKA is likely to mediate the hypertrophic effect of PGE2 in cardiac myocytes. We have previously shown that blockade of the EP2 subtype or activation of the prostacyclin receptor, both of which are coupled to adenylate cyclase, had no effect on PGE2-induced protein synthesis (33). Therefore, activation of different membrane-bound enzyme pools may account for the differential effects of cAMP when produced by EP4 receptor activation vs. exogenous addition or activation of other GPCRs.

It has been shown (20, 28, 42) that cAMP activates p42/44 MAPK independently of PKA through a novel pathway that involves activation of the exchange proteins directly activated by cAMP (EPACs). This does not seem to be the case in cardiac myocytes, because blocking cAMP elevation with an adenylate cyclase inhibitor had no effect on PGE2-induced protein synthesis. In addition, challenging the cells with different concentrations of the EPAC activator 8-CPT-2Me-cAMP (10–60 µmol/l) did not increase [3H]leucine incorporation (M. Mendez and M. C. LaPointe, unpublished observations).

We show here that PGE2 was able to activate the EGFR, and that EGFR inhibition blocked PGE2-induced protein synthesis. Supporting our findings, PGEs was previously reported to transactivate EGFRs in gastric epithelial cells and colon cancer cells (40). Since then, other groups have demonstrated that EGFRs mediate the effects of PGE2 on cell migration (8) and cell growth (39, 50). Activation of EGFRs has been shown to be essential for normal heart function (16). In vivo studies have implicated EGFR in cardiac hypertrophy and remodeling (17, 18, 55). Recently, EGFRs were identified as mediators of hypertrophic agents by linking stretch-mediated myocyte growth and activation of genes that serve as markers of hypertrophy such as brain natriuretic peptide (1). Thus our studies would indicate that EGFRs are involved in multiple aspects of cardiac hypertrophy and are likely to mediate the effects of COX-2 products in failing heart.

EGFRs have been shown to be activated at the plasma membrane and to be continually active even in late endosomes (14, 60), endocytosis being indispensable for EGFR-dependent downstream activation of ERK (57). In cardiac myocytes, COX-2 and the membrane-localized PGE2 synthase-1 are localized in a perinuclear compartment (33). In addition, EP subtypes are reportedly present in the nuclear membrane (4, 5). Thus it is tempting to speculate that intracellular PGE2 production acting via nuclear EP receptors maintains EGFR in an active state and thereby amplifies the signal that started at the plasma membrane.

It is not clear how EP4 receptors transactivate EGFRs in VMs. Several mechanisms and intracellular mediators have been described. GPCR activation by different agonists may stimulate membrane-bound metalloproteinases with subsequent release of EGFR agonists from the plasma membrane (2, 41, 59). In addition, GPCR-dependent activation of the tyrosine kinases Src and Pyk can directly activate EGFRs (21, 29). Our data showed that the metalloproteinase inhibitor GM completely blocked PGE2-induced cell growth, which suggests that release of plasma membrane-preformed EGFR agonists is likely to mediate EGFR transactivation in our cells. At the concentration we used, the metalloproteinase inhibitor has been shown to prevent mechanical stress-induced release of heparin-binding (HB)-EGF into the culture media of neonatal cardiac myocytes (1). The EGFR agonist HB-EGF has been suggested to play an important role as a mediator of cardiac hypertrophy (2) and heart function (16). In addition, its gene expression is upregulated in a myocardial infarction model in rats (55). Although our data do not directly identify HB-EGF as the only mediator, it is likely to be part of PGE2-induced hypertrophic signaling.

We demonstrated here that inhibition of EP4 and EGFRs prevented PGE2-induced MAPK phosphorylation by 69 and 64%, respectively. In addition, a selective p42/44 MAPK inhibitor was able to completely block PGE2-induced protein synthesis, whereas a p38 inhibitor had no effect, thereby identifying ERK1/2 as a downstream mediator of PGE2 and EGFRs. Because both EP4 and EGFR inhibitors prevented p42/44 MAPK phosphorylation to the same extent, our data suggest that PGE2-induced p42/44 MAPK activation is downstream of EP4 and EGFRs. In agreement with our results, p42/44 MAPK has been implicated in GPCR-dependent regulation of cardiac hypertrophy (9). In contrast, p38 MAPK involvement in cardiac growth is more controversial. Although it has been reported to mediate the hypertrophic effects of a variety of other GPCR agonists (11, 38), recent work with genetically modified animal models suggests that p38 MAPK does not mediate cardiac hypertrophy in vivo (6, 62).

In our studies, the EP4 antagonist did not completely inhibit PGE2 induction of protein synthesis. However, this partial blockade does not seem to be due to incomplete antagonism of EP4 receptors. We measured PGE2-induced cAMP production in the absence and presence of EP1 and EP2 antagonists to determine the relative contribution of EP4 activation to total cAMP. We found that the EP4 subtype was responsible for 50% of the PGE2-induced cAMP levels, and this increase was completely prevented by pretreatment of VMs with the concentration of EP4 antagonist used in the present experiments (M. Mendez and M. C. LaPointe, unpublished observations). However, another explanation for the partial inhibitory effect could be chemical instability of the antagonist over a 48-h treatment period.

The partial effect of the EP4 antagonist on PGE2-induced p42/44 MAPK activation (~70% inhibition) might point to the participation of another EP subtype. To date, four splice variants of the EP3 subtype have been described (36, 44); however, their presence in cardiac myocytes is unknown. Therefore, the possible small contribution of EP3 in myocyte growth remains to be elucidated.

Together, our observations indicate that PGE2 acting through EP4 transactivates the EGFR, which mediates PGE2 induction of myocyte growth via the p42/44 MAPK pathway. Thus in conditions where cardiac hypertrophy is accompanied by inflammation, additional targets to control the disease process might include PGE2 synthesis and EP4.


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This work was supported by National Institutes of Health Grant P01 HL-28982.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. LaPointe, Hypertension and Vascular Research Division, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202-2689 (E-mail: mlapoin1{at}hfhs.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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