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Am J Physiol Heart Circ Physiol 290: H1740-H1746, 2006. First published January 20, 2006; doi:10.1152/ajpheart.00904.2005
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Regulation of Cardiovascular Functions by Eicosanoids and Other Lipid Mediators

PGE2 stimulates human brain natriuretic peptide expression via EP4 and p42/44 MAPK

Jian-Yong Qian, Alicia Leung, Pamela Harding, and Margot C. LaPointe

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

Submitted 23 August 2005 ; accepted in final form 13 January 2006


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin E2 (PGE2) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its EP4 receptor. We hypothesized that PGE2, acting through EP4, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE2 increased hBNP promoter activity 3.5-fold. An EP4 antagonist reduced the stimulatory effect of PGE2 but not an EP1 antagonist. Because EP4 signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE2 stimulation of the hBNP promoter. H-89 at 5 µM decreased PGE2 stimulation of BNP promoter activity by 100%. Because p42/44 MAPK mediates the effect of PGE2 on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. PGE2 stimulation of the hBNP promoter was inhibited by a MEK1/2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of PGE2. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE2 stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE2 stimulates the BNP promoter mainly via EP4, PKA, Rap, and p42/44 MAPK.

EP receptor; cardiac myocytes; hypertrophy; signaling pathways

BRAIN NATRIURETIC PEPTIDE (BNP), one of three members of the natriuretic peptide family, is a cardiac hormone composed of 32 amino acids (in the human heart) and has diuretic, natriuretic, vasodilator, and antifibrotic properties (22). BNP is synthesized and secreted constitutively by the adult heart (primarily the ventricle) (2, 35). It is also regarded as a marker gene of hypertrophy. Circulating levels of BNP are elevated as a result of myocardial infarction (MI), hypertrophy, or heart failure (1, 35). Plasma BNP concentrations also serve as a biochemical marker of left ventricular dysfunction and a neuroendocrine marker of heart failure (24, 40). Patients with heart failure infused with BNP have reduced preload and afterload, increased stroke volume, and enhanced natriuresis and diuresis (3); thus it would appear that this cardiac hormone has beneficial and compensatory effects that modulate the progression of cardiac dysfunction. For example, BNP has antifibrotic actions in the heart (16, 31, 44). It may also have antigrowth actions in the vasculature, operating either directly or indirectly through stimulation of C-type natriuretic peptide synthesis (33, 37, 38).

PGE2 is a proinflammatory prostanoid that acts as an autocrine/paracrine mediator to signal changes within the immediate environment where it is released. It is synthesized from arachidonic acid by cyclooxygenase (COX-1 and COX-2) and PGE2 synthase (25). In cardiac myocytes, induction of COX-2 by the proinflammatory cytokine interleukin-1beta was accompanied by induction of the PGE2 synthase mPGES-1, resulting in the release of micromolar amounts of PGE2 (11). PGE2 has growth-promoting effects in many cell types, most notably colorectal carcinoma cell proliferation and migration (36). We previously showed that exogenous PGE2 increased protein synthesis in myocytes, a marker of hypertrophic growth (26). Moreover, in a mouse model of myocardial infarction, inhibition of COX-2 decreased myocyte cross-sectional area, an index of hypertrophy (19). In addition to regulation of protein synthesis, PGE2 also stimulated synthesis and secretion of atrial natriuretic peptide (ANP) (10) and ANP promoter activity in myocytes (27); however, no signaling mechanisms were identified in those studies. Thus, in the present study, we focused on the intracellular mechanisms involved in PGE2 regulation of BNP gene expression.

PGE2 exerts its biological actions through four specific heteromeric G protein-coupled receptors (GPCRs), EP1, EP2, EP3, and EP4, which differ in structure, ligand-binding properties, activation of signal transduction pathways, and tissue distribution (32). The mRNAs for EP2, EP3, and EP4 have been detected in the heart of several species, including humans, and EP4 is the most abundantly expressed EP subtype (47). EP1 is linked to a Gq{alpha} protein and its activation results in Ca2+ mobilization; the EP3 receptor is coupled to a Gi{alpha} protein, leading to decreased cAMP levels. Activation of EP4 and EP2 leads to accumulation of intracellular cAMP through interaction with a cholera-sensitive Gs{alpha} protein. EP4 has also been linked to activation of p42/44 MAPK (4), and in cardiac myocytes it is involved in regulation of protein synthesis (27). Because the role of EP4 signaling pathways in the regulation of gene expression in cardiac myocytes has not been investigated to our knowledge, this was the focus of the present study.


   METHODS
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 METHODS
RESULTS
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Cell culture. Preparation of neonatal ventricular myocytes (NVM) from rat pups was approved by the Henry Ford Hospital Institutional Animal Care and Use Committee. Primary cultures of NVM were derived from digestion of hearts from 1- to 2-day-old neonatal Sprague-Dawley rats (Charles River Laboratories, Kalamazoo, MI) as described previously (20). NVM were placed in 12-well plates (0.5 x 106 cells/well) or 10-cm plates (6 x 106 cells) and cultured in DMEM (GIBCO) containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mmol/l glutamine, 0.1 mmol/l bromodeoxyuridine, and 10% FBS (HyClone) for 40 h. They were maintained in serum-free DMEM (SF-DMEM plus penicillin, streptomycin, glutamine, insulin, transferrin, and selenium) for 24 h before adding test compounds.

Transient transfection and luciferase activity assay. Transfection and luciferase activity assay were described previously (21). Briefly, freshly isolated NVM were transiently transfected by electroporation in PBS-glucose at 0.28 V and 250 µF with a Bio-Rad gene pulser. One microgram of 1818hBNPLuc [a plasmid encoding the hBNP promoter (from position –1818 to +100) driving expression of a luciferase reporter gene] (21), {Delta}73Luc (the same DNA construct as 1818hBNPLuc but without the hBNP promoter), or CRE-luciferase (2 cAMP and PKA-responsive elements cloned upstream from a minimal thymidine kinase-driven luciferase reporter; Clontech) was transfected per 106 cells. To test the effect of the dominant-negative mutants of Raf (dnRaf), Ras (dnRas), and Rap (dnRap) on hBNP promoter activity, 1818hBNPLuc (0.33 µg/106 cells) was cotransfected with dnRaf, dnRas, dnRap (3.3 µg/106 cells), or a control vector. Forty hours after transfection and incubation in 10% FBS-DMEM, the cells were starved in SF-DMEM for 24 h and then treated with PGE2 or other agents for 24 h. Effects of pharmacological inhibitors were tested by treating cells for 1 h before addition of PGE2. Finally, NVM were harvested, lysed, and assayed for luciferase activity (relative light units or RLU) with the Promega luciferase assay system and an OptoComp 1 luminometer (MGM) according to the manufacturer's protocol. Duplicate aliquots of cell lysates from triplicate wells were averaged. Sample protein concentrations were also assayed using Coomassie protein reagent (Pierce).

DNA constructs. The chimeric hBNP-luciferase reporter gene construct (1818hBNPLuc) has been described previously (21). dnRaf (Raf 301) and dnRas (Ras N17) were obtained from Dr. Michael Karin (Univ. of California at San Diego). dnRap (Rap N17) was obtained from Dr. M. Olah (Univ. of Cincinnati Medical School). The generation and efficacy of these constructs have been described previously (5, 9, 17). Western blot analysis of NVM transfected with dnRas and dnRap showed that protein levels were increased 12 ± 4.4-fold and 8.8 ± 1.9-fold, respectively.

Chemicals. PGE2, butaprost (EP2 agonist), sulprostone (EP1/EP3 agonist), and SC-19220 (EP1 antagonist) were from Cayman. L-161982 (EP4 antagonist) was provided by Merck-Frosst (gift of Dr. Robert Young). U-0126 (MEK1/2 inhibitor), SB-203580 (p38 MAPK inhibitor), and SP-600125 (JNK inhibitor) were purchased from Calbiochem, and H-89 (PKA inhibitor) was from Biomol. Effective concentrations of these chemicals were either determined in our laboratory or obtained from the literature (11, 27, 46). Other standard laboratory supplies and reagents were obtained from Fisher, VWR, Promega, and Sigma.

Analysis of BNP mRNA by real-time PCR. We analyzed BNP and beta-actin mRNA by real-time RT-PCR, which produced cDNAs of 241 bp (BNP) and 342 bp (beta-actin). PCR conditions and the sequences of primers and probes were reported previously (19). In brief, total RNA of NVM was extracted in Tri-Reagent (MRC) according to the manufacturer's instructions. After treating the samples with DNase I and reverse transcription, we performed real-time PCR using a QuantiTect Probe PCR kit (Qiagen) and a Roche LightCycler (version 2.0). Data analysis was performed with LightCycler software (version 4.0). Gene-specific standard curves were generated using a linearized plasmid containing the cDNA of interest and serially diluting it to generate concentrations ranging over five orders of magnitude. The LightCycler software calculates the amount of cDNA in a given sample (copies/µl) compared with the standard curve. BNP cDNA was normalized to beta-actin cDNA.

Statistical analysis. Luciferase activity from each sample was normalized to its protein levels (RLU/mg protein). Control values were arbitrarily set to 1.0 and compared with other treatment groups to determine the fold increase. Data are expressed as means ± SE and were analyzed either by Student's t-test or by 1-way ANOVA, with multiple pairwise comparisons made by the Student-Newman-Keuls method. P < 0.05 was considered significant.


   RESULTS
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 METHODS
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DISCUSSION
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PGE2 activates the hBNP promoter via the EP4 receptor. To determine whether PGE2 stimulates the hBNP promoter, we measured luciferase activity in lysates of transiently transfected NVM. As indicated in Fig. 1A, PGE2 increased hBNP promoter activity in a dose-dependent manner at 1.0 and 5.0 µM. However, 1 µM PGE2 had no effect on the promoterless construct (control = 1-fold; PGE2 = 1.2 ± 0.1-fold; n = 4; P = NS).


Figure 1
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  		Fig. 1. PGE2 activates the human brain natriuretic peptide (hBNP) promoter via EP4. The y-axis is relative luciferase activity [fold increase compared with control (CONT), arbitrarily set to 1], and the x-axis is treatment. A: dose response for PGE2. Each bar represents the mean ± SE of 6–10 separate experiments. B: effect of an EP4 antagonist on PGE2. The cells were pretreated with 10 µM L-161982 (an EP4 antagonist) and then cotreated with 1 µM PGE2 for 24 h. Each bar represents the mean ± SE of 12 separate experiments. P values are indicated. P = not significant (NS) for L-161982 and PGE2 vs. either control or L-161982 alone. C: effect of an EP4 antagonist on sulprostone. The cells were pretreated with 10 µM L-161982 and then cotreated with 0.1 µM sulprostone for 24 h. Each bar represents the mean ± SE of 4–7 separate experiments. P values are indicated.

 
We next investigated the involvement of EP4 in stimulation of the hBNP promoter using L-161982, an EP4 antagonist. L-161982 (10 µM) reduced the stimulatory effect of PGE2 to a level not statistically different from control (Fig. 1B), suggesting that EP4 plays a major role in the effect of PGE2. To test the specificity of the EP4 antagonist, we examined its effect on stimulation of the hBNP promoter by sulprostone (a metabolism-resistant EP3 agonist), using a concentration that had a stimulatory effect similar to 1 µM PGE2. Treatment with 0.1 µM sulprostone stimulated hBNP promoter activity 2.1-fold, which was unaffected by the EP4 antagonist (Fig. 1C).

PGE2 increases BNP mRNA. To confirm that BNP promoter activity represented an increase in BNP transcription, we used real-time RT-PCR to investigate the effects of PGE2 on BNP mRNA. Treating NVM with 1 µM of PGE2 for 4 h increased BNP mRNA 2.2-fold (Fig. 2). Agarose gel electrophoresis of the PCR products revealed cDNA fragments of the correct size (BNP, 241 bp; beta-actin, 342 bp; data not shown).


Figure 2
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  		Fig. 2. PGE2 increases BNP mRNA in neonatal ventricular myocytes (NVM). NVM were treated with PGE2 for 4 h. BNP mRNA was evaluated by real-time RT-PCR and normalized to beta-actin. The y-axis is BNP/beta-actin ratio [fold increase compared with control, arbitrarily set to 1]. Each bar represents the mean ± SE of 5 separate experiments. P value is indicated.

 
EP1 and EP2 are not involved in hBNP promoter activation. Because PGE2 can bind to all four EP receptors (EP1–4) (32), we investigated the effects of other EP agonists and antagonists. As shown in Fig. 3A, the EP1 antagonist SC-19220 (10 µM) had no effect on PGE2-stimulated luciferase activity. To test EP2 involvement, we used the EP2 agonist butaprost (0.01 to 10 µM) but found that it had no effect on hBNP promoter activity (Fig. 3B). As a control, we tested the efficacy of 1 µM butaprost by measuring cAMP generation (26). After stimulation with butaprost for 30 min, there was a fourfold increase in intracellular cAMP (from 0.44 ± 0.16 to 1.92 ± 0.1 pmol/5 x 105 cells; n = 3; P < 0.001). Together these data suggest that neither EP1 nor EP2 is involved in PGE2 stimulation of the hBNP promoter.


Figure 3
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  		Fig. 3. EP1 and EP2 are not involved in PGE2 stimulation of the hBNP promoter. The y-axis is relative luciferase activity (fold increase compared with control, arbitrarily set to 1), and the x-axis is treatment. A: effect of an EP1 antagonist (SC-19220, 10 µM). Each bar represents the mean ± SE of 7 separate experiments. P values are indicated. B: effect of an EP2 agonist (butaprost, 0.01–10.0 µM). Each bar represents the mean ± SE of 5–8 separate experiments. P = NS between groups.

 
PGE2 stimulation of the hBNP promoter involves PKA. EP4 can couple to cAMP-PKA pathways through Gs{alpha} protein (39), and PGE2 increases cAMP in cardiac myocytes (26). In addition, cAMP-dependent activation of PKA may result in subsequent growth-promoting signaling pathways (48). Hence, we examined whether the traditional cAMP-PKA pathway mediates PGE2 stimulation of the hBNP promoter. We first tested the efficacy of H-89 using CRE-luciferase (CRE-Luc), which contains a binding site for CRE binding protein (CREB), a cAMP- and PKA-activated transcription factor. Forskolin (10 µM), an activator of adenylate cyclase, stimulated CRE-Luc twofold, while 5 µM H-89 inhibited the effect by 100% (Fig. 4A). The promoterless construct was not responsive to forskolin (data not shown). Additionally, we found that 1 µM H-89 did not affect PGE2 stimulation of the hBNP promoter (Fig. 4B) but 5 µM reduced the effect by 100%.


Figure 4
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  		Fig. 4. Effect of protein kinase A (PKA) inhibition on hBNPLuc and CRE-luciferase. The y-axis is luciferase activity expressed as fold increase vs. control (arbitrarily set to 1; represents luciferase activity in unstimulated cells), and the x-axis is treatment. A: NVM were transfected with CRE-luciferase and treated with PGE2 and the PKA inhibitor H-89 (1 and 5 µM); n = 12 for forskolin and n = 4 for all other treatments. P values are indicated. B: NVM were transfected with 1818hBNPLuc and treated with PGE2 and the PKA inhibitor H-89 (1 and 5 µM); n = 8 for PGE2 and n = 4 for all other treatments. P values are indicated.

 
p42/44 MAPK mediates PGE2 stimulation of the hBNP promoter. We have previously shown that PGE2 activates p42/44 MAPK and stimulates protein synthesis in cardiac myocytes (27) and that p38 MAPK is involved in regulation of the hBNP promoter by many different stimuli (18). In addition, a study of EP4-overexpressing cells has indicated that EP4 couples to growth-promoting pathways (8). Thus we tested the roles of the three MAPK signaling cascades in PGE2 regulation of BNP promoter activity. We first studied whether PGE2 activates p42/44 MAPK and confirmed our previous study (27) showing that stimulation of NVM with PGE2 for 5 min enhanced p42/44 MAPK phosphorylation (data not shown). When we administered the MEK1/2 inhibitor U-0126 (10 µM), PGE2 stimulation of the hBNP promoter was significantly inhibited (Fig. 5A). To verify this finding, we investigated the effect of Raf, an upstream activator of MEK1/2. As shown in Fig. 5B, dnRaf also inhibited the effect of PGE2, confirming that p42/44 MAPK mediates hBNP promoter activation. In contrast, neither the p38 MAPK inhibitor SB-203580 (10 µM) (Fig. 6A) nor SP-600125 (10 µM), a JNK inhibitor, was able to block PGE2 stimulation of the hBNP promoter. Paradoxically, JNK inhibition enhanced the effect of PGE2 (Fig. 6B).


Figure 5
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  		Fig. 5. p42/44 MAPK mediates PGE2 stimulation of the hBNP promoter. The y-axis is luciferase activity expressed as fold increase vs. control (arbitrarily set to 1, represents luciferase activity in unstimulated cells), and the x-axis is treatment. A: effect of a MEK1/2 antagonist (U-0126, 10 µM). Each bar represents the mean ± SE of 5 separate experiments. B: effect of dominant negative Raf (dnRaf). 1818hBNPLuc was cotransfected with dnRaf or a control expression vector and then treated with PGE2 (1 µM). Each bar represents the mean ± SE of 4 separate experiments. P values are indicated.

 

Figure 6
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  		Fig. 6. Effects of p38 MAPK and JNK inhibitors on PGE2 stimulation of the hBNP promoter. The y-axis is luciferase activity expressed as fold increase vs. control (arbitrarily set to 1, represents luciferase activity in unstimulated cells), and the x-axis is treatment. A: effect of a p38 MAPK inhibitor. The cells were treated with a p38 MAPK inhibitor (SB-203580, 10 µM). Each bar represents the mean ± SE of 4 separate experiments. P values are indicated. B: effect of a JNK inhibitor. The cells were treated with a JNK inhibitor (SB-600125, 10 µM). Each bar represents the mean ± SE of 5 separate experiments. P values are indicated.

 
The small GTPase Rap is involved in the effect of PGE2. Either Rap or Ras can activate Raf, thereby stimulating p42/44 MAPK (12, 17). We tested the involvement of these small GTPases in PGE2 stimulation of the BNP promoter. dnRap inhibited PGE2 stimulation of the hBNP promoter by 42% (n = 4, P < 0.05) (Fig. 7A), but dnRas had no effect (Fig. 7B). Overexpression of dnRap and dnRas was confirmed by Western blot, as reported in METHODS.


Figure 7
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  		Fig. 7. Effect of the small GTPases Rap and Ras. The y-axis is luciferase activity expressed as fold increase vs. control (arbitrarily set to 1, represents luciferase activity in unstimulated cells), and the x-axis is treatment. A: effect of dominant negative Rap (dnRap). 1818hBNPLuc was cotransfected with dnRap or a control expression vector and then treated with PGE2 (1 µM). Each bar represents the mean ± SE of 4 separate experiments. P values are indicated. B: effect of dominant negative Ras (dnRas). 1818hBNPLuc was cotransfected with dnRas or a control expression vector and then treated with PGE2 (1 µM). Each bar represents the mean ± SE of 3 separate experiments. P values are indicated.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
BNP is an antifibrotic hormone and a marker gene of cardiac hypertrophy (15). It is also a good predictor of left ventricular dysfunction and is used to treat acute heart failure (1, 3, 24, 35, 40). Hence, investigation of the signaling pathways that regulate BNP expression may improve our understanding of the underlying mechanisms of cardiac hypertrophy and heart failure. Many prohypertrophic and proinflammatory molecules regulate BNP gene expression (18). PGE2 is a well-known inflammatory mediator and growth factor, acting through EP1–4 receptors. We have shown that PGE2 is the main prostaglandin released by interleukin-1beta-stimulated cardiac myocytes and increases protein synthesis and cell size by EP4 and p42/44 MAPK (26). Therefore, it is reasonable to expect that PGE2 regulates BNP expression via similar pathways. Indeed, in the present study we found that PGE2 stimulates the hBNP promoter through activation of EP4. Because PGE2 stimulated the BNP promoter and mRNA to a similar extent (~2- to 3-fold), we believe this stimulation occurs at the transcriptional level.

EP receptors are GPCRs, a seven-transmembrane domain receptor family coupled to heteromeric G proteins. So far, four EP receptors have been described, which couple to different intracellular signaling cascades (32). EP1 is linked to Gq{alpha} and elevated intracellular calcium levels, and EP3 couples to Gi{alpha} and decreased cAMP, whereas EP2 and EP4 are coupled to Gs{alpha} and result in increased cAMP (32). Although EP2 and EP4 are both linked to Gs{alpha}, there are significant differences in their signaling capabilities. For example, in cells overexpressing EPs, EP2 couples to PKA-dependent signaling cascades (6, 39). In contrast, EP4 binding to PGE2 produces less cAMP (7) and results in rapid internalization linked to downstream activation of p42/44 MAPK (4, 8), similar to the beta2-adrenergic receptors.

Our pharmacological approach showed that EP1 and EP2 do not mediate PGE2 stimulation of the hBNP promoter. This supports our previous finding that a selective inhibitor of the EP1 and EP2 receptor subtypes does not affect PGE2 stimulation of protein synthesis in NVM (26). EP4 plays a major role in PGE2 regulation of BNP expression, because the EP4 antagonist inhibited PGE2 stimulation of the hBNP promoter. In agreement with these data, we have found that PGE2 treatment of cardiac myocytes increases protein synthesis and cell size mainly through EP4 (27). Moreover, EP4 is involved in growth of other cell types. In models of colon cancer, either knockout or inhibition of EP4 decreased cancer growth (30). Because the EP4 antagonist did not completely inhibit PGE2 stimulation of the hBNP promoter, it is possible that another EP subtype plays a minor role. The EP3 agonist sulprostone stimulated hBNP promoter activity, but at 0.1 µM it did not activate EP4. Thus EP3 may play a minor role in BNP gene regulation. We are in the process of developing an EP4 small interfering RNA to confirm our findings.

EP4 has been shown to couple to the cAMP-PKA cascade. cAMP can modulate cell growth, depending on the cell type (43). Although PGE2 resulted in cAMP production by myocytes (26), neither PGE2 stimulation of protein synthesis (27) nor isoproterenol regulation of the hBNP promoter (14) was affected by 1 µM H-89. In accord with our previous data, 1 µM H-89 had no effect on PGE2 stimulation of the hBNP promoter in the present study. In contrast, 5 µM H-89 reduced the effect of PGE2 by 100%. An examination of the hBNP 5'-flanking sequence for potential CRE sites (TGACGTCA) found no consensus elements, although there was a partial site (CTTCGTCA) located at –839 (21). This might suggest an indirect effect of PKA on the BNP promoter or nonspecific effects of H-89 at high concentrations. Nonetheless, using EP2- and EP4-overexpressing HEK cells, investigators have demonstrated differences between the two receptors in cAMP production, PKA dependence, and signaling to downstream effectors (e.g., Tcf/Lef and CREB) (6, 7). Thus it appears that EP4 signaling may be distinct in different cell types.

MAPK cascades are among the most thoroughly studied signal transduction systems and play critical roles in cell differentiation, growth, and apoptosis as well as regulation of various transcription factors and gene expression (42). In mammalian systems, the family is composed of three distinct MAPK modules. These include the p42/44 MAPK (ERK1/2) cascade, which preferentially regulates cell growth and differentiation, as well as the JNK and p38 MAPK cascades, which function mainly in stress responses like inflammation and apoptosis (41). We demonstrated here that PGE2 stimulated p42/44 MAPK phosphorylation. Inhibition of p42/44 MAPK using both a p42/44 MAPK inhibitor and dnRaf blocked PGE2 stimulation of hBNP promoter activity. In accordance with our results, p42/44 MAPK has been implicated in GPCR-dependent regulation of cardiac hypertrophy both in vivo and in vitro (27).

In contrast to p42/44 MAPK, p38 MAPK involvement in cardiac hypertrophy is uncertain. p38 MAPK mediated the hypertrophic effects of a variety of other GPCR agonists (34), but in genetically modified animal models p38 MAPK did not mediate cardiac hypertrophy in vivo (49). Our study showed that p38 MAPK is not involved in PGE2 stimulation of the hBNP promoter. We also investigated the role played by JNK. A JNK inhibitor, SP-600125, did not inhibit PGE2 stimulation of the hBNP promoter but rather enhanced it, suggesting that there may be cross-talk between p42/44 MAPK and JNK, as MAPKs have been reported to oppose each other in regulating ANP expression (34). Thus we hypothesized that activation of JNK would in turn inactivate p42/44 MAPK and that JNK inhibition would reverse this process, allowing for enhanced activation of p42/44 MAPK. However, in preliminary studies we found no effect of the JNK inhibitor on PGE2 activation of p42/44 MAPK (Qian J-Y and LaPointe MC, unpublished observations). Thus this observation with the JNK inhibitor requires further study.

Presently, we have not completely clarified how EP4 links to p42/44 MAPK activation. In cells overexpressing EP4, activation of p42/44 MAPK is mediated by beta-ARK, beta-arrestin, Src, and small GTPases (23). The small GTPases Ras and Rap1 have different functions. Ras acts through Raf-1 to activate MEK and p42/44 MAPK, while Rap1 acts via B-Raf. The second messenger cAMP can activate Rap1 either via a cAMP-dependent exchange factor (Epac) or a cAMP-PKA pathway (12, 17). Recent studies have shown that Epac participates in cardiac hypertrophy in vitro (28). In the present study, Rap was involved in PGE2 stimulation of the hBNP promoter but Ras was not. Thus our data suggest that PGE2 activates EP4, which couples to Rap and B-Raf, resulting in downstream activation of p42/44 MAPK. The question now is whether EP4 signaling involves Epac and/or PKA activation of Rap.

It is also unclear how p42/44 MAPK activates BNP gene expression. Transcription factors such as Elk-1 and GATA-4 have been reported to mediate hypertrophic gene transcription in cardiac myocytes (29). In addition, p42/44 MAPK has been shown to activate GATA-4 (45), and GATA-4 activates the hBNP promoter (13). Because prohypertrophic growth factors and proinflammatory cytokines enhance BNP gene expression by targeting the proximal region of the hBNP promoter, it is likely that GATA-4 is a primary target of PGE2 action.

In summary, our data indicate that PGE2 regulates the BNP promoter via EP4 with downstream activation of the p42/44 MAPK cascade in cardiac myocytes. To our knowledge, these are the first data to demonstrate that PGE2 enhances BNP gene expression. Our findings support a link between PGE2, BNP, and cardiac hypertrophy, which may enable us to better understand the signaling mechanisms underlying the hypertrophic effects of PGE2 in myocytes.


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This work was supported by National Heart, Lung, and Blood Institute Grant P01-HL-28982.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. LaPointe, Hypertension and Vascular Research Division, Henry Ford Hospital, 2799 W. 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.


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