Prostaglandin E₂ and an EP4 Receptor Agonist Inhibit LPS-Induced Monocyte Chemotactic Protein 5 Production and Secretion in Mouse Cardiac Fibroblasts via Akt and NF-κB Signaling.

Abstract

BACKGROUND:

Prostaglandin E2 (PGE₂) signals through 4 separate G-protein coupled receptor sub-types to elicit a variety of physiologic and pathophysiological effects. We have previously reported that mice lacking the EP4 receptor in the cardiomyocytes develop heart failure with a phenotype of dilated cardiomyopathy. Also, these mice have increased levels of chemokines, like MCP-5, in their left ventricles. We have recently reported that overexpression of the EP4 receptor could improve cardiac function in the myocardial infarction model. Furthermore, we showed that overexpression of EP4 had an anti-inflammatory effect in the whole left ventricle. It has also been shown that PGE₂ can antagonize lipopolysaccharide-induced secretion of chemokines/cytokines in various cell types. We therefore hypothesized that PGE₂ inhibits lipopolysaccharide (LPS)-induced MCP-5 secretion in adult mouse cardiac fibroblasts via its EP4 receptor.

Methods and Results:

Our hypothesis was tested using isolated mouse adult ventricular fibroblasts (AVF) treated with LPS. Pre-treatment of the cells with PGE₂ and the EP4 agonist CAY10598 resulted in reductions of the pro-inflammatory response induced by LPS. Specifically, we observed reductions in MCP-5 secretion. Western blot analysis showed reductions in phosphorylated Akt and IκBα indicating reduced NF-κB activation. The anti-inflammatory effects of PGE₂ and EP4 agonist signaling appeared to be independent of cAMP, p-44/42, or p38 pathways.

Conclusion:

Exogenous treatment of PGE₂ and the EP4 receptor agonist blocked the pro-inflammatory actions of LPS. Mechanistically, this was mediated via reduced Akt phosphorylation and inhibition of NF-κB.

1. INTRODUCTION

Chemokines are a group of small (8-14 kDa) chemoattractant molecules that regulate the trafficking of various leukocytes, mainly neutrophils, monocytes, lymphocytes, and eosinophils to various tissues [1]. Chemokines bind to about 20 different receptors, all of which belong to the seven transmembrane G-protein class of receptors [2]. One particular chemokine, monocyte chemoattractant protein-5 (MCP-5), is the mouse analogue to human MCP-1, and is a potent chemoattractant for monocytes and macrophages [3]. Several studies have implicated the significance of MCP-1 in diseases characterized by infiltrating monocytes, especially myocardial ischemia [4–6]. Far less is known about the murine MCP-5, however.

Prostaglandin E₂ (PGE₂) signals through 4 distinct receptor subtypes (EP1, EP2, EP3, and EP4) to elicit a variety of effects. EP2 and EP4 both increase cAMP levels in the cell via adenylate cyclase activation, whereas EP3 inhibits cAMP production. EP1 increases Ca²⁺ levels in the cell [7, 8]. The differences in signaling of these receptors in cardiac tissue have still not been studied in depth. Previously, our lab developed a mouse line in which the EP4 receptor is deleted specifically in the cardiomyocytes. These mice develop a phenotype of dilated cardiomyopathy with reduced ejection fraction with age, as well as pockets of interstitial infiltrate within the left ventricle. Gene array data showed that these mice have increased chemokine/cytokines in their left ventricles, including CCL12 (MCP-5) [9]. Very recently, we reported that overexpression of the EP4 receptor improves cardiac function in a myocardial infarction (MI) model. Moreover, we showed that overexpression of the EP4 receptor was associated with an anti-inflammatory effect shown by reduced levels of MCP-1 with increased IL-10 in the left ventricle [10]. Precisely how PGE₂ exerts its anti-inflammatory effects in the heart has not been elucidated. PGE₂ signaling through its EP4 receptor, however, has been shown to have anti-inflammatory effects in organ systems and cell types other than the heart [11–16]. Specifically, PGE₂-EP4 signaling has been shown to inhibit bacterial lipopolysaccharide (LPS) signaling [16], but this result has not been reported for isolated adult mouse cardiac fibroblasts.

Damage-associated molecular patterns (DAMPs), such as LPS, activate pattern recognition receptors (PRRs) of the toll-like receptor (TLR) family, particularly TLR-2 and 4 [17]. Activation of TLRs characteristically leads to the transcriptional activation of genes which encode pro-inflammatory cytokines and other mediators of inflammation by transcription factors such as nuclear factor κB (NF-κB). Both TLR-2 and TLR-4 have been shown to be expressed in cardiac myocytes and fibroblasts [18]. Cardiac fibroblasts play a particularly important role in cardiac injury due to their ability to maintain the integrity of the extracellular network. Furthermore, cardiac fibroblasts play an important role in secreting and responding to cytokines/chemokines in response to injury [19].

In this current study, we take an in vitro approach to examine whether treatment with exogenous PGE₂ in cardiac fibroblasts affects chemokine production. We hypothesized that PGE₂ inhibits LPS-induced MCP-5 secretion in adult mouse cardiac fibroblasts via its EP4 receptor.

2. METHODS

2.1. Animal Use

16-20 wks. old male C57BI/6 mice used for cardiac cell isolation were from Jackson labs. The isolation of adult ventricular fibroblasts (AVF) was previously described by us [9]. All studies involving the use of animals were approved by the institutional review committee at Henry Ford Hospital, in accordance with federal guidelines.

2.2. Chemicals

EP1 antagonist (SC-51089), EP2 agonist (Butaprost), EP2 antagonist (TG4-155), EP4 agonist (CAY10598), EP4 antagonists (GW627368X, L-161, 982), and prostaglandin E₂ were from Cayman Chemical (Ann Arbor, MI). EP3 antagonist (L798, 106) and cardamonin were obtained from Tocris (Minneapolis, MN). IKK inhibitor III was from Millipore Sigma (Burlington, MA). Tri-reagent was obtained from Molecular Research Center (Cincinnati, OH). All other drugs were obtained from Sigma Aldrich.

2.3. Treatment of Isolated Cardiac Fibroblasts

For all AVF experiments, the cells were passaged to p3 followed by serum starvation for at least 16 hours. Time Course Experiment; cells were pre-treated with vehicle (ethanol), PGE₂ (1 μM), or the EP4 agonist (CAY10598; 1 μM) for 1 hour. After 1 hour, cells were treated with lipopolysaccharide (LPS; 10 μg/ml) for 1,2, 4, and 24 hours. Media was collected and snap-frozen in liquid nitrogen for ELISA analysis. Cells were scraped in 1 ml of Tri-reagent and snap frozen for future RNA isolation and real time RT-PCR. EP receptor antagonist experiment; In another set of experiments, the cells were first pre-treated with the EP1 antagonist SC-51089 (1 μM), EP2 antagonist TG4-155 (0.5 μM), EP3 antagonist L798, 106 (1 μM), or an EP4 antagonist GW627368X or L-161, 982 (1 μM) prior to treatment as described above for 24 hrs. In separate experiments at shorter time points (0, 5, 15, 30 mins), protein lysate was collected for Western blot analysis by scraping the cells on ice with lysis buffer containing protease and phosphatase inhibitors (Roche). Dibutyryl cAMP (db cAMP); cells were pre-treated with vehicle (DMSO), Dibutyryl cAMP (100 μM), or the EP4 agonist (1 μM) for 1 hour. After 1 hour, cells were treated with LPS (10 μg/ml) for 24 hours. Media was collected and snap-frozen for ELISA analysis. PI3K Inhibitor (Wortmannin and LY290042); cells were pre-treated with vehicle (DMSO and ethanol), PGE₂ (1 μM), or Wortmannin (1 μM) or LY290042 (10 μM) for 1 hour. After 1 hour, cells were treated with LPS (10 μg/ml) for 24 hours. Media was collected and snap-frozen for ELISA analysis. NF-κB inhibition (cardamonin and IKK inhibitor III); cells were pre-treated with vehicle (ethanol), cardamonin (10 μM), or IKK inhibitor III (10 μM), for 1 hour. After 1 hour, LPS (10 μg/ml) was added to the appropriate wells for 24 hours. Media was collected and snap-frozen for ELISA analysis and protein lysates were collected for western blot analysis. JNK inhibitor (SP600125); cells were pre-treated with vehicle (ethanol), or SP600125 (10 μM) for 1 hour. After 1 hour, the cells were treated with vehicle (ethanol and DMSO), PGE₂ (1 μM), or the EP4 agonist (1 μM) for 1 hour. After 1 hour, cells were treated with LPS (10 μg/ml) for 24 hours. Media was collected and snap-frozen in liquid nitrogen for ELISA analysis. Protein lysates were collected for western blot analysis.

2.4. MCP-5 Secretion Analyses

MCP-5 ELISA analysis of media samples was performed according to the manufacturer’s instructions (Ray BioTech; Catalog No. ELM-MCP5). For all ELISA experiments, data was collected as pg/ml, corrected for the amount of protein in each sample and then presented as fold of vehicle. This kit is specific for MCP-5 and shows no cross reactivity with a number of different cytokines/chemokines, including MCP-1. The limit of detection for this kit is 0.5 pg/ml.

2.5. Polymerase Chain Reaction

Measurement of MCP-5 mRNA expression was performed by real-time RT-PCR using a SYBR green method as follows. 1 μg of DNase I-treated RNA sample was reverse transcribed using random primers and Omniscript reverse transcriptase kit (Giagen; Valencia, CA). 2 μL of the reverse transcription reaction was amplified in a Roche version 2.0 lightcycler PCR machine (Roche; Indianapolis, IN) using SYBR green dye and specific primers against MCP-5 (sense; 5’-CAAGAGRATCACCAGCAGCAGG-3’. Antisense; 5’-TGCTTGAGGTGGTTGTGGAA-3’) and the house keeping gene GAPDH (sense; 5’-CAAGGTCATCCCAGAGCTG-3’. Antisense; 5’-TGTCATCATACTTGGCAGGTT-3’). Primers were purchased from TIB MolBiol (Adelphia, NJ). It has been reported that stimulation with LPS in other cell types can affect GAPDH expression [20]. In our hands, the average GAPDH crossing points did not change with LPS stimulation, thus GAPDH was used as a control (17.00 ± 0.22 for vehicle treated mice vs. 17.37 ± 0.38 for LPS treated mice. p=0.796). To determine the mRNA expression of EP1, EP2, EP3, and EP4 in the fibroblasts, 2 μg of RNA was reverse transcribed in the same manner described above. 2 μl of the reverse transcription reaction was amplified in a Bio-Rad PTC-100 thermal cycler using Taq polymerase. PCR conditions were: 94°C for 30 sec, 58–62°C for 30 sec, 72°C for 90 sec and a final extension at 72°C for 5 min. EP1 Sense; 5’-ATCATGGTGGTKTCGTGCATCT-3’. Antisense; 5’-CATGGGTCCAGGATCTGGTT-3’. EP2 Sense; 5’-TATGCTCCYTGCCTTTCAC-3’. Antisense: 5’-CTCAGTGAAGTCCGACAACAG-3’. EP3 (Qiagen, NM_011196.2). EP4 Sense; 5’-GTGCAGAGATCCAGATGGTCA-3’. EP4 Antisense; 5’-ATCYGGGTTTCTGCTGATGTC-3’.

2.6. MCP-5 mRNA Stability

Measurement of MCP-5 mRNA stability was determined using real time RT-PCR (see methods 2.5). AVF were pre-treated with PGE₂, EP4 agonist, or vehicle for 1 hour. After a 1-hour pre-treatment, cells were treated with LPS (10 μg/ml) for 2 hours. Previous experiments in our lab have determined that 2 hours is adequate to stimulate MCP-5 mRNA. After LPS treatment, AVF were treated with 5, 6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) for 0 and 30 minutes to block transcription. The cells were harvested in Tri-reagent and stored at −80°C for further PCR analysis.

2.7. Western Blot Analysis

Phosphorylated IκBα (p-IκBα; 1:1000), phosphorylated Akt (p-Akt; 1:1000), phosphorylated JNK (p-JNK; 1:1000), phosphorylated p44/42 (p-p44/42; 1:1000) and phosphorylated p38 (p-p38; 1:1000) were measured by western blot using a method previously described by us [21]. Phosphorylated IκBα, p-Akt, p-JNK, p-p44/42, and p-p38 were corrected to total IκBα, Akt, JNK, p44/42 and p38 respectively. All antibodies were from Cell Signaling (Danvers, MA). Western blot was quantified using NIH Image J software (Bethesda, MD). Phosphorylated protein was corrected to total and then corrected to vehicle. LPS stimulation at 15 minutes was set to 100% and the effect of PGE₂ and the EP4 agonist are presented as the percentage of LPS stimulation.

2.8. Statistical Analysis

All statistics were performed by a statistician in the Department of Public Health Sciences of Henry Ford Hospital using the statistical package SAS Version 9.4. For all tests, a two-sample Wilcoxon test with the Fligner-Policello correction for unequal variances was used. We also used a Hochberg’s method for multiple testing. For the western blot analysis, a paired T-test was used. A p-value < 0.05 was considered as evidence of a statistically significant difference for experimental data with the p values being two-sided.

3. RESULTS

3.1. Expression of the EP Receptors in Adult Mouse Cardiac Fibroblasts.

Figure 1 shows the expression profile of the EP receptors in cultured adult mouse cardiac fibroblasts. All four of the EP receptors are expressed and this experiment was repeated on two separate occasions using different preparations with the same result.

Figure 1:

Representative 2 % Agarose gel of PCR products. Product sizes (in bp) are as follows: EP1; 142, EP2; 186, EP3; 134, EP4; 139.

3.2. PGE₂ and EP4 Receptor Agonist Inhibit MCP-5 Secretion after LPS-Stimulation

To first determine the effect of PGE₂ and the EP4 agonist on LPS-stimulation of MCP-5 secretion, we performed a time course experiment, analyzing the concentration of MCP-5 in media at 1,2, 4, and 24 hours after treatment with LPS (10 μg/ml). Cells were pre-treated with PGE₂ or the EP4 agonist for 1 hr prior to stimulation. On average, the baseline MCP-5 concentration in the media was 61.23 ± 19.51 pg/ml and this rose to 1470.51 ± 163.21 pg /ml after 24 hrs of LPS treatment. All ELISA results are presented as fold of vehicle control. After 1 or 2 hours of LPS stimulation, there was no detectable increase in MCP-5 in the media compared with vehicle treated cells (0.95 ± 0.14 vs. 1.00 ± 0.0 in vehicle treated cells; p=0.423) and (1.38 ± 0.23 vs. 1.00 ± 0.0 in vehicle treated cells; p=0.240), respectively. After 4 hours of LPS treatment however, there was a significant increase in MCP-5 in the media (2.27 ± 0.42 fold vs. 1.00 ± 0.0 in vehicle treated cells; p=0.012). PGE₂ significantly reduced LPS-stimulated MCP-5 (1.23 ± 0.18 vs. 2.27 ± 0.42 in LPS treated cells; p=0.005). There was also a tendency for a decrease in LPS-stimulated MCP-5 when cells were pre-treated with the EP4 agonist, although this data did not reach statistical significance (1.77 ± 0.24 vs. 2.27 ± 0.42 in LPS treated cells; p=0.36). Figure 2A shows that by 24 hours, LPS stimulation of MCP-5 remained significantly elevated (3.38 ± 0.36 vs. 1.00 ± 0.0 in vehicle treated cells; p=0.005). At this time point, both PGE₂ and the EP4 agonist significantly reduced MCP-5 production after LPS stimulation (1.18 ± 0.12 in PGE₂ pre-treated cells vs. 3.38 ± 0.36 in LPS treated cells; p=0.009 and 1.53 ± 0.20 in EP4 agonist pre-treated cells vs. 3.38 ± 0.36 in LPS treated cells; p=0.009). Since PGE₂ and the EP4 agonist both reduce MCP-5 production after LPS stimulation, it suggests that the effect of PGE₂ is mediated via the EP4 receptor. To test this, we pre-treated the cells with an EP4 antagonist (GW627368X) prior to treatment with PGE₂ or the EP4 agonist and LPS for 24 hours (Figure 2B). Surprisingly, GW627368X did not reverse the inhibitory effect of PGE₂, suggesting that the effects are mediated via other mechanisms rather than activation of the EP4 receptor. However, the inhibitory effect of the EP4 agonist was significantly attenuated with GW627368X (Figure 2C), suggesting that the EP4 agonist is specific and its inhibitory effects are indeed through the EP4 receptor (3.80 ± 0.22 in LPS treated vs. 1.36 ± 0.15 in LPS + CAY10598 treated; p<0.005 and 2.97 ± 0.16 in LPS + CAY10598 + GW627368X treated vs. 1.36 ± 0.15 in LPS + CAY10598 treated; p=0.000009). We confirmed this data using another potent and specific EP4 antagonist, L-161, 982, and again found there was no effect on the ability of PGE₂ to reduce LPS-stimulated MCP-5. However, this alternative compound also fully reversed the effects of the EP4 agonist, confirming its specificity. Moreover, we pretreated AVF with antagonists to either EP1, EP2, or EP3 receptors. Antagonism of these receptors also had no effect on the ability of PGE₂ to reduce LPS-stimulated MCP-5 secretion suggesting they are not responsible for the inhibition of LPS-stimulated MCP-5 by PGE₂ (EP1: 0.89 ± 0.08 in LPS + PGE₂ vs. 0.73 ± 0.06 in LPS + SC-51089 + PGE₂; EP2: 0.89 ± 0.08 in LPS + PGE₂ vs. 1.04 ± 0.08 in LPS + TG4-155 + PGE₂; EP3: 0.89 ± 0.08 in LPS + PGE₂ vs. 0.55 ± 0.06 in LPS + L798, 106 + PGE₂). Altogether, these data suggest that the inhibitory effect of PGE₂ is EP receptor independent. None of the EP receptor antagonists alone affected basal MCP-5 secretion.

Figure 2.

A: Mouse MCP-5 ELISA analysis data. AVF were pre-treated for 1 hour with PGE₂ (1 μM) or the EP4 agonist (1 μM) followed by treatment with LPS (10 μg/ml) for 24 hours. All results were obtained as pg/ml and corrected for the amount of protein in each well. Data is presented as fold of vehicle treatment (set to 1.0) ± SEM. ** p<0.01 vs. vehicle, ++ p< 0.01 vs. LPS. N=5

B: AVF were pre-treated for 1 hour with the EP4 antagonist GW627368X (GW; 1 μM). Cells were then treated with or without PGE₂ (1 μM) for 1 hour, followed by treatment with LPS (10 μg/ml) for 24 hours. All results were obtained as pg/ml and corrected for the amount of protein in each well. Data is presented as fold of vehicle treatment (set to 1.0) ± SEM. *p<0.05 vs. vehicle, + p<0.05 vs. LPS. N=6-9

C: AVF were pre-treated for 1 hour with the EP4 antagonist GW627368X (1 μM). Cells were then treated with the EP4 agonist (1 μM) for 1 hour, followed by treatment with LPS (10 μg/ml) for 24 hours. All results were obtained as pg/ml and corrected for the amount of protein in each well. Data is presented as fold of vehicle treatment (set to 1.0) ± SEM. ***p<0.005 vs. vehicle, +++ p<0.005 vs. LPS, $$$ p<0.005 vs. LPS + EP4 agonist. N=6.

D: Real time RT-PCR analysis for MCP-5 mRNA expression. AVF were pre-treated for 1 hour with PGE₂ (1 μM) or the EP4 agonist (1 μM) followed by treatment with LPS (10 μg/ml) for 1 hour. All results were obtained as fold induction and corrected to the housekeeping gene GAPDH. Data is presented as fold of vehicle treatment, means ± SEM. ** p<0.01 vs. vehicle. +++p<0.005 vs. LPS. N=5.

E: Real time RT-PCR analysis for MCP-5 mRNA stability. AVF were pre-treated with PGE₂ (1 μM) for 1 hour, followed by treatment with LPS (10 μg/ml) for 2 hours. After 2 hours, transcription was blocked using DRB (60 μM) and the decay of MCP-5 mRNA was assessed after 30 minutes. Data is presented as fold of LPS-stimulated MCP-5. N=6.

3.3. PGE₂ and EP4 Agonist Reduce MCP-5 mRNA after LPS-Stimulation

Both LPS-stimulation of MCP-5 secretion and its inhibition by PGE₂ and the EP4 agonist appears to take at least 4 hours to be detectable, suggesting that PGE₂ and the EP4 agonist may regulate MCP-5 transcription in addition to its secretion. To test this, we performed a time course experiment examining MCP-5 mRNA levels after 1, 2, 4, and 24 hours post LPS stimulation. We also examined the effects of PGE₂ and its EP4 receptor agonist at these time points. Figure 2D shows that after 1 hr of LPS stimulation, MCP-5 mRNA increased over 4-fold compared to vehicle (4.49 ± 0.71 vs. 1.00 in vehicle treated cells; p=0.005). Treatment with the EP4 agonist significantly blunted the response to LPS (2.09 ± 0.82 vs. 4.49 ± 0.71 in LPS-treated cells; p=0.004). Similarly, at the same time point, PGE₂ treatment attenuated MCP-5 levels after LPS stimulation, although this failed to reach statistical significance (2.68 ± 1.04 vs. 4.49 ± 0.71 in LPS-treated cells; p=0.186). LPS stimulation resulted in a significant elevation in MCP-5 mRNA levels throughout all time points; however, there was no significant effect of either PGE₂ or the EP4 agonist at any other time points after 1 hr. These data suggest that PGE₂ treatment and EP4 receptor activation leads to downstream signaling that quickly downregulates MCP-5 mRNA. Since previous reports have suggested that LPS can directly affect the stability of pro-inflammatory chemokine/cytokine mRNA transcripts [22–24], we performed an experiment using the transcriptional inhibitor DRB (60 μM). Figure 2E shows that after 30 minutes of DRB treatment, there was no effect of PGE₂ on LPS-stimulated MCP-5 mRNA (1.10 ± 0.18 vs. 1.02 ± 0.23 in LPS-stimulated cells; ns). These data suggest that the inhibition of LPS-stimulated MCP-5 is not due to an effect on MCP-5 mRNA stability.

3.4. Inhibition of NF-κB abolishes LPS-induced secretion of MCP-5

It is well known that NF-κB is an important transcription factor in regulating cytokine production [25–27]. Typically, the IκB complex sequesters NF-κB in the cytoplasm. Upon phosphorylation by IκB Kinase (IKK), IκBα is targeted for degradation allowing NF-κB p65 subunit to translocate into the nucleus to regulate transcription [25]. Therefore, we used p-IκBα as a surrogate of NF-κB activation. To determine if NF-κB is involved in regulating transcription of MCP-5, we pre-treated AVF with the NF-κB inhibitor, cardamonin. Figure 3A shows that, as expected, 24 hours of LPS stimulation significantly increased MCP-5 production compared with vehicle treatment (9.11 ± 3.09 vs. 1.00 ± 0.0 in vehicle treated cells; p=0.003). Pre-treatment with cardamonin greatly reduced the MCP-5 produced after LPS treatment (1.71 ± 0.41 vs. 9.11 ± 3.09 in LPS treated cells; p=0.001). Furthermore, we confirmed the specificity of this inhibitor by examining phosphorylation of IκBα. The western blot in figure 3B clearly shows that after 24 hours of LPS + cardamonin treatment, p-IκBα is reduced compared to LPS alone. To confirm our findings, we repeated these experiments using an IKK inhibitor (IKKi). Similar to cardamonin, pre-treatment with the IKK inhibitor significantly reduced LPS-stimulation of MCP-5, (3.49 ± 0.34 in LPS vs. 1.02 ± 0.16 in LPS + IKKi; p=0.00000001). Together, these data suggest that NF-κB plays a major role in regulating the production of MCP-5 in LPS-stimulated cardiac fibroblasts.

Figure 3.

A: Mouse MCP-5 ELISA analysis. AVF were pre-treated with the NF-κB inhibitor, cardamonin (10 μM) for 1 hour, followed by treatment with LPS (10 μg/ml) for 24 hours. Data was obtained as pg/ml of MCP-5, corrected to the amount of protein in each well and presented as fold of vehicle ± SEM. *** p<0.005 vs. vehicle. +++ p< 0.005 vs. LPS. N=6.

B: Representative western blot for phosphorylated IκBα and total IκBα used as a loading control.

3.5. PGE₂ and The EP4 Agonist Reduce LPS Stimulation of MCP-5 by Inhibiting NF-κB Signaling

NF-κB appears to be a major factor in regulating MCP-5 production after LPS challenge. Furthermore, the attenuation of MCP-5 production by PGE₂ and the EP4 receptor agonist only occur under LPS stimulation and not under basal conditions. Therefore, we sought to determine at which point in the inflammatory signaling cascade PGE₂ and/or EP4 signaling is eliciting its effects. The western blot analysis in figure 4 shows that after 15 minutes of LPS treatment, p-IκBα was reduced with PGE₂ and the EP4 agonist. Pretreatment of AVF with PGE₂ resulted in a 45% decrease in LPS-stimulated p-IκBα (55.2 ± 12.0 % vs. 100 ± 0.0 % in LPS treated cells; p<0.005). Likewise, when the cells were pretreated with the EP4 agonist, there was a 32% decrease in LPS-stimulated p-IκBα (68.5 ± 10.6 % vs. 100 ± 0.0 % in LPS treated cells; p<0.05). By 30 minutes p-IκBα remains greatly elevated with LPS treatment, however there was no significant reduction when cells were pretreated with PGE₂ or the EP4 agonist, suggesting that their inhibition on NF-κB signaling may be a rapid signaling event, climaxing at 15 minutes (415.91 % ± 157.5 vs. 100 % ± 0.0 at 15 minutes; p=0.07).

Figure 4:

Top panel. Representative western blot analysis using antibodies against phosphorylated IκBα. AVF were pre-treated with PGE₂ (1 μM) and the EP4 agonist (1 μM) for 1 hour, followed by treatment with LPS (10 μg/ml) for 15 minutes. Data obtained was corrected to total IκBα. Bottom panel. LPS stimulation at 15 mins was set to 100% and data was calculated as a percentage of the LPS stimulation. *p<0.05, ** p<0.01 vs. LPS. N=6.

3.6. Inhibition of MCP-5 via PGE₂ and the EP4 Agonist is independent of the JNK, p38, and p44/42 signaling pathways

Since LPS treatment can stimulate c-Jun N-terminal kinase (JNK) signaling, p44/42 and p38 MAPK pathways in other cell types leading to cytokine production [28], we examined whether these pathways played a role in AVF. Figure 5A shows that LPS treatment stimulates p-JNK. We observed an inhibitory effect of LPS-stimulated phosphorylation of JNK with PGE₂ and EP4 agonist treatment, although this data did not reach statistical significance (27.3 % reduction; 72.71 ± 17.9 % in PGE₂ treated vs. 100 ± 0.0 % in LPS treated cells and a 20 % reduction; 80.11 ± 22.7 % in EP4 agonist treated vs. 100 ± 0.0 % in LPS treated cells, respectively). The stimulation of p-JNK persisted for at least 30 minutes (141.2 ±57.8 in LPS treated cells vs. 100 ± 0.0 % in LPS treated cells at 15 minutes). Similarly, PGE₂ and the EP4 agonist each had a 30 % reduction on the LPS-stimulation of p-JNK with values of 70.2 ± 11.2 % and 71.32 ± 12.3 %, respectively. To examine whether the reduction in p-JNK with PGE₂ and the EP4 agonist after LPS stimulation affects MCP-5 production/secretion, we treated AVF with the JNK inhibitor, SP600125. Treatment with PGE₂ and the EP4 agonist reduced LPS-stimulated MCP-5 production as expected (1.4 ± 0.21 in PGE₂ treated vs. 4.7 ± 0.15 in LPS treated cells; p<0.005 and 2.2 ± 0.21 in EP4 agonist treated vs. 4.7 ± 0.15 in LPS; p<0.005). However, as shown in figure 5B, when cells were pretreated with the JNK inhibitor (SP600125), there was an additional inhibitory effect, greater than the effect of PGE₂ or the EP4 agonist alone (0.6 ± 0.03 in LPS + PGE₂ + SP600125 vs. 1.4 ± 0.21 in LPS + PGE₂; p<0.005 and 0.9 ± 0.04 in LPS + EP4 + SP600125 vs. 2.2 ± 0.21 in LPS + EP4; p<0.005). Furthermore, figure 5C shows that after LPS stimulation (3.6 ± 0.47 vs. 1.0 ± 0.03 in vehicle; p< 0.005), there was a slight reduction in MCP-5 production with the JNK inhibitor alone, however this data did not reach statistical significance (2.7 ± 0.32 vs. 3.6 ± 0.47 in LPS treated cells; p=0.12). Together these data suggest that JNK signaling may play a small role in LPS signaling. However, both PGE₂ and EP4 signaling appear to be acting through a pathway separate of JNK signaling to reduce MCP-5 production/secretion. Additionally, phosphorylation of p44/42 and p38 also increased after LPS treatment but neither PGE₂ nor the EP4 agonist had any effect (figures 5D and 5E, respectively).

Figure 5.

A: top panel. Representative western blot for phosphorylated JNK. AVF were pre-treated with PGE₂ (1 μM) and the EP4 agonist (1 μM) for 1 hour, followed by treatment with LPS (10 μg/ml) for 15 and 30 minutes. Bottom panel. LPS stimulation at 15 mins was set to 100% and data was calculated as a percentage of the LPS stimulation at 15 mins. N=4.

(B/C): Mouse MCP-5 ELISA analysis. AVF were pre-treated with the JNK inhibitor, SP600125 (10 μM), PGE₂ (1 μM), and/or the EP4 agonist (1 μM) for 1 hour, followed by treatment with LPS (10 μg/ml) for 24 hours. Data was obtained as pg/ml of MCP-5, corrected to the amount of protein in each sample and presented as fold of vehicle ± SEM. *** p<0.005 vs. vehicle. +++ p< 0.005 vs. LPS, $$$ p< 0.005 vs. LPS + PGE₂, &&& p< 0.005 vs. LPS + EP4 ag. B, N=4-6/group. C, N=12-14/group.

D: top panel. Representative western blot analysis using antibodies against phosphorylated p44/42. AVF were pre-treated with PGE₂ (1 μM) and the EP4 agonist (1 μM) for 1 hour, followed by treatment with LPS (10 μg/ml) for 15 and 30 minutes. Data obtained was corrected to total p44/42. bottom panel. LPS stimulation at 15 mins was set to 100% and data was calculated as a percentage of the LPS stimulation. N=4.

E: top panel. Representative western blot analysis using antibodies against phosphorylated p38. AVF were pre-treated with PGE₂ (1 μM) and the EP4 agonist (1 μM) for 1 hour, followed by treatment with LPS (10 μg/ml) for 15 and 30 minutes. Data obtained was corrected to total p38. Bottom panel. LPS stimulation at 15 mins was set to 100% and data was calculated as a percentage of the LPS stimulation. N=4.

3.7. Inhibition of MCP-5 via PGE₂ and the EP4 Agonist is Independent of cAMP

One of the major downstream signaling pathways of the EP4 receptor is activation of adenylate cyclase and increased cAMP production. We sought to determine if PGE₂ or the EP4 agonist contributes to the reduction of LPS-induced MCP-5 production via this pathway. Using ELISA, we found that LPS stimulation for 24 hours increased MCP-5 secretion (figure 6A). Importantly, when cells were pre-treated with dibutyryl cAMP (db cAMP), the cell permeable analogue of cAMP, there was no reduction of LPS-stimulated MCP-5 secretion (1.95 ± 0.17 in LPS + db cAMP vs. 2.29 ± 0.20 in LPS treated cells; p=0.190). Interestingly, db cAMP alone increased MCP-5 secretion (2.55 ± 0.35 vs. 1.00 ± 0.0 in vehicle treated cells; p=0.001). This data suggests that the inhibitory effect of the EP4 agonist is not occurring through cAMP production. Activation of the EP2 receptor is also linked to cAMP [29]. However, Figure 6B shows that when we treated AVF with the EP2 agonist Butaprost (500 nM), we observed no change in LPS-stimulated MCP-5 (2.89 ± 0.43 in LPS treated cells vs. 2.04 ± 0.18 in LPS + Butaprost treated cells; ns). This data suggests that the inhibitory effect of PGE₂ is at least independent of EP2 signaling.

Figure 6.

A: Mouse MCP-5 ELISA analysis data. AVF were pre-treated for 1 hour with the EP4 agonist (1 μM), or dibutyryl cAMP (db CAMP; 100 μM) followed by treatment with LPS (10 μg/ml) for 24 hours. All results were obtained as pg/ml and corrected for the amount of protein in each well. Data is presented as fold of vehicle treatment ± SEM. *** p<0.005 vs. vehicle. +++ p< 0.005 vs. LPS. N=10.

B: Mouse MCP-5 ELISA analysis data. AVF were pre-treated for 1 hour with the EP2 agonist Butaprost (500 nM) followed by treatment with LPS (10 μg/ml) for 24 hours. All results were obtained as pg/ml and corrected for the amount of protein in each well. Data is presented as fold of vehicle treatment ± SEM. *** p<0.005 vs. vehicle. N=6-8.

3.8. Inhibition of MCP-5 via PGE₂ and the EP4 Agonist is Partly Dependent on the PI3K Pathway

PGE₂ via EP4 signaling can also activate the PI3K-Akt pathway [30]. We therefore investigated the effects of the PI3K inhibitor Wortmannin on LPS-stimulated MCP-5 production. As expected, LPS increased MCP-5 secretion after 24 hours (figure 7A; 4.04 ± 0.98 in LPS treated cells vs. 1.00 ± 0.0 in vehicle treated cells; p=0.037), but pretreatment with Wortmannin did not affect the ability of either PGE₂ or the EP4 agonist to reduce the LPS-stimulation of MCP-5 (0.96 ± 0.22 in LPS + PGE₂ + Wortmannin vs. 4.04 ± 0.98 in LPS treated cells; p=0.050 and 1.74 ± 0.51 in LPS + EP4+ Wortmannin vs. 4.04 ± 0.98 in LPS treated cells; p=0.019). In a separate experiment (figure 7B), we show that under basal conditions Wortmannin had significant effect on MCP-5 secretion itself (0.60 ± 0.17 in Wortmannin vs. 1.00 ± 0.0 in vehicle treated cells; p=0.043). Furthermore, the treatment with Wortmannin significantly reduced MCP-5 secretion after LPS stimulation suggesting that the PI3K-Akt pathway itself plays a role in the inflammatory response to LPS (1.45 ± 0.19 in LPS + Wortmannin vs. 2.44 ± 0.44 in LPS treated cells; p=0.012). To confirm our findings, we repeated this experiment using a separate PI3K inhibitor, LY290042 (10 μM). Figure 7C shows pre-treatment with LY290042 did not affect the ability of PGE₂ or the EP4 agonist to reduce LPS-stimulated MCP-5 (0.53 ± 0.09 in PGE₂ treated vs. 2.33 ± 0.24 in LPS treated cells; p<0.005 and 0.59 ± 0.13 in EP4 agonist treated vs. 2.33 ± 0.24 in LPS treated cells; p<0.005). However, LY290042 pre-treatment significantly reduced LPS-stimulated MCP-5 on its own (0.68 ± 0.13 vs. 2.33 ± 0.24 in LPS treated cells; p<0.005). To determine if PGE₂ and/or the EP4 agonist is acting through the Akt pathway, we performed Western blot analysis of p-Akt. Figure 7D shows that treatment with PGE₂ and the EP4 agonist had a significant reduction on LPS stimulation of p-Akt after 15 mins (36.80 ± 4.5 % in PGE₂ treated vs. 100 ± 0.0 % in LPS treated cells; p<0.005 and 47.40 ± 6.5 in EP4 agonist treated vs. 100 ± 0.0 % in LPS treated cells; p< 0.005). These data suggest that inhibition of the Akt pathway by PGE₂ and an EP4 agonist plays a major role in the reduction of LPS-stimulated MCP-5.

Figure 7.

(A/B): Mouse MCP-5 ELISA analysis data. AVF were pre-treated for 1 hour with PGE₂ (1 μM), the EP4 agonist (1 μM), or the PI3K inhibitor, Wortmannin (1 μM) followed by treatment with LPS (10 μg/ml) for 24 hours. All results were obtained as pg/ml and corrected for the amount of protein in each well. Data is presented as fold of vehicle treatment ± SEM. *p<0.05, *** p<0.005 vs. vehicle. + p<0.05, ++ p<0.01, +++ p< 0.005 vs. LPS. N=3 in panel A, N=6 in panel B.

C: Mouse MCP-5 ELISA analysis data. AVF were pre-treated for 1 hour with PGE₂ (1 μM), the EP4 agonist (1 μM), or the PI3K inhibitor, LY290042 (10 μM) followed by treatment with LPS (10 μg/ml) for 24 hours. All results were obtained as pg/ml and corrected for the amount of protein in each well. Data is presented as fold of vehicle treatment ± SEM. *** p<0.005 vs. vehicle, +++ p< 0.005 vs. LPS. N=3.

D: Top panel. Representative western blot analysis using antibodies against phosphorylated Akt. AVF were pre-treated with PGE₂ (1 μM) and the EP4 agonist (1 μM) for 1 hour, followed by treatment with LPS (10 μg/ml) for 15 and 30 minutes. Data obtained was corrected to total Akt. Bottom panel. LPS stimulation at 15 mins was set to 100% and data was calculated as a percentage of the LPS stimulation. ***p<0.005 vs. vehicle. N=3.

4. Discussion

The results of this study show, for the first time, that MCP-5 secretion induced by treatment with LPS, can be inhibited by PGE₂ and an EP4 receptor agonist in isolated adult mouse cardiac fibroblasts. We have recently published that overexpression of the EP4 receptor in the cardiomyocytes was protective in a model of myocardial infarction [10]. In addition to the direct protective effects on cardiac function, we observed reduced inflammation in the whole heart. We speculated that overexpression of EP4 in the cardiomyocytes may have paracrine effects mediated by the cardiac fibroblasts. Our lab has data that shows the cardiac fibroblasts produce a much larger quantity of chemokines than the cardiac myocytes (unpublished data). Therefore, this study may provide some understanding of the anti-inflammatory effects we observed in the previous publication. In the present study, we hypothesized that PGE₂ inhibits LPS-induced MCP-5 secretion in adult mouse cardiac fibroblasts via its EP4 receptor.

The results from our current study show significant reductions in LPS-stimulated MCP-5 secretion after treatment with either PGE₂ or an EP4 agonist. Contrary to our hypothesis however, treatment with the EP4 antagonist, GW627368X did not reverse the effects of PGE₂, suggesting another mechanism may be involved. We obtained the same result using a second selective EP4 antagonist, L-161, 982. This unexpected data cannot be due to sub-maximal blockade of EP4 as both compounds completely reversed the effect of the EP4 agonist. Furthermore, EP receptor antagonists against EP1, EP2, and EP3 also had no effect on the ability of PGE₂ to inhibit LPS-stimulated MCP-5, suggesting PGE₂ is acting via an EP-receptor independent mechanism. To our knowledge, very few papers describe actions of PGE₂ that are independent of its EP receptors. However, Través et al. previously reported that PGE₂ reduces Ca²⁺ mobilization by an EP receptor-independent mechanism in human monocytes and macrophages [31]. Although the regulation of MCP-5 production and/or secretion is not well established, Eglite et al reported that depletion of Ca²+ in a basophil cell line reduced transcription and secretion of MCP-1, the human analogue of murine MCP-5 [32]. Thus, it is possible that PGE₂ is reducing [Ca²⁺]i and subsequently reducing MCP-5 secretion, although this is purely speculative and neither calcium dynamics nor its effects on MCP-5 regulation was investigated in our current study.

A limitation of our study is that we did not investigate the role of endogenous PGE₂. One would anticipate that treatment with LPS would rapidly induce COX-2 with a subsequent rise in PGE₂ as shown by Tsai et al in dermal fibroblasts [33]. In our study, LPS stimulation of MCP-5 secretion was not apparent until the 4 hr time point. It is tempting to speculate, therefore, that rapid induction of COX-2 and subsequent PGE₂ synthesis attenuates the secretion of MCP-5 at earlier time points. However, we have no evidence to support this idea. Moreover, we expect that the concentration of PGE₂ (1 μM) used in this study would greatly surpass the endogenous level of PGE₂. Furthermore, we cannot rule out the possibility that endogenously produced vs. exogenous PGE₂ have different signaling mechanisms and/or act at different locations. Future studies are necessary to elucidate exactly how PGE₂ is acting, upstream of p-Akt and NF-κB. Since both PGE₂ and the EP4 agonist similarly affect activation of Akt and NF-κB, it appears that their signaling converges at some point. This remains to be elucidated. Nonetheless, our study is important in showing that treatment with PGE₂ and/or an EP4 receptor agonist reduce pro-inflammatory signaling and production of the pro-inflammatory chemokine, MCP-5, which could have significant therapeutic benefits.

PGE₂ has been shown to have anti-inflammatory effects in cell types other than in the heart [11–15, 34] and it was shown in mouse macrophages to be exclusively via EP4 signaling [35]. Ngoc et al. examined PGE₂-EP4 signaling in a rat model of experimental autoimmune myocarditis (EAM) using an EP4 receptor agonist (EP4RAG). In vivo the authors determined that treatment with the EP4RAG significantly reduced infiltration of CD4⁺, but not CD8⁺ T cells in the left ventricle. Interestingly, the authors did not find a reduction in cytokine mRNA expression in the left ventricles of EAM rats treated with the EP4RAG. They show however that in vitro, spleen cells treated with myosin to induce EAM exhibit higher levels of MCP-1 in the supernatant, which is reduced with EP4RAG treatment [12]. Our study also supports an anti-inflammatory role for the EP4 receptor, in cardiac fibroblasts, a novel finding. With regard to the role of NF-κB, Miyatake et al. very recently reported that the C₂C₁₂ myotube cell line increased MCP-1 in a NF-κB dependent manner [36]. We also show that the transcription factor NF-κB plays an important role in regulating MCP-5, at least in response to LPS in cardiac fibroblasts. We have also been able to demonstrate in the current study that NF-κB activation is reduced with exogenous PGE₂ and EP4 agonist treatment by using two separate and specific inhibitors of NF-κB activation (Cardamonin, and IKK inhibitor VII). Importantly, the inhibitory effect of both PGE₂ and the EP4 receptor appears to be through reduction of Akt signaling and inhibition of NF-κB translocation, determined by reduced phosphorylation of IκBα.

The p38 and p44/42 MAPKs have been shown previously to become activated by a wide variety of inflammatory signals [37]. Furthermore, it has been demonstrated that LPS induces the phosphorylation of p38 in monocytes and macrophages [38, 39]. However, we find no evidence to support a role for either p38 or p44/42 phosphorylation in the inhibitory effects of PGE₂ and the EP4 agonist on LPS-induced MCP-5 secretion.

Evidence in the literature suggests that some chemokines and cytokines are rapidly released upon stimulation from intracellular secretory compartments. Oynebraten and colleagues showed that several chemokines, including MCP-1, were secreted in this manner in human endothelial cells [40]. Additionally, Catalfamo et al. reported that the chemokine RANTES is stored in intracellular vesicles and secreted rapidly in human CD8⁺ T-cells [41]. To our knowledge, there has been no report of this mechanism in cardiac fibroblasts. Furthermore, our data in this study would disagree with their findings. LPS stimulation only resulted in a significant increase in MCP-5 after 4 hours of treatment. Moreover, we observed changes in MCP-5 mRNA after 1 hr. of LPS stimulation, suggesting transcription is necessary. Evidence also suggests several cytokines are regulated at the post transcriptional level [42, 43]. However, we have shown in this study that the stability of the MCP-5 mRNA is not altered with exogenous PGE₂ or EP4 agonist treatment combined with LPS treatment. Together, these data would suggest that it is not a rapid release of MCP-5 via secretory vesicles. Instead, the data suggests that transcription of MCP-5 needs to take place.

cAMP is a well-known second messenger in the EP2 and EP4 signaling cascade [29]. Largo et al. showed that in human synovial fibroblasts, PGE₂ reduces MCP-1 production after IL-1β stimulation via its EP4 receptor [15]. Importantly, they show that this was due to production of cAMP, since the inhibitory effect on MCP-1 was reversed using a cAMP inhibitor. This is in contrast to our current data, which shows that treatment with dibutyryl cAMP alone actually stimulated the production of MCP-5 significantly. Also, pre-treatment of the AVF with Butaprost, the EP2 agonist, had no effect on LPS-stimulated MCP-5. Furthermore, the combination of LPS and dibutyryl cAMP had no inhibitory effect on MCP-5 production. Another study conducted by Takayama et al. utilized human macrophages and they showed that PGE₂ inhibited the production of chemokines via the EP4 receptor after LPS stimulation [14]. Furthermore, in agreement with our data, they showed that this was independent of cAMP-PKA signaling, although the exact mechanism was unclear. Therefore, our current study may shed light on the mechanism by showing the involvement of reduced NF-κB activation. Minami and colleagues [44] showed that in LPS stimulated RAW264.7 cells, forced expression of an EP4 receptor-associated protein mitigated the pro-inflammatory response. Specifically, they showed that the EP4 receptor-associated protein resulted in reduced NF-κB activation by directly interacting with the p105 subunit. This study differs from ours in that we show treatment with exogenous PGE₂ or EP4 agonist, reduces phosphorylated Akt and IκBα.

Studies in tissues other than the heart have also implicated the PI3K-Akt signaling pathway in playing an important role in regulating LPS-induced inflammation [45, 46]. Our data agrees with the literature in that treatment with the PI3K inhibitors, Wortmannin or LY290042, reduced LPS-stimulated MCP-5. Furthermore, inhibition of the PI3K pathway did not affect the ability of PGE₂ or the EP4 agonist to reduce LPS-stimulated MCP-5, nor was there an additional inhibitory effect, suggesting they are signaling through the same pathway. Western blot analysis of p-Akt revealed that PGE₂ and the EP4 agonist had a significant reduction of LPS-stimulated p-Akt after 15 mins. In contrast, treatment with the JNK inhibitor, SP600125, in combination with both PGE₂ and the EP4 agonist resulted in a further reduction in LPS-stimulated MCP-5. These data suggest that the JNK signaling pathway plays some role in regulating MCP-5 production. However, the inhibitory effect of PGE₂ and the EP4 agonist is occurring via a different pathway, presumably through Akt signaling.

Clinically, the use of an EP4 receptor agonist in the treatment of heart failure has merit as being a potential therapy. The use of an EP4 agonist has been tested clinically in healthy dogs [47, 48] and in humans [49] and was determined safe to use. However, the use of an EP4 agonist in humans under heart failure conditions has not yet been examined. We speculate that the use of an EP4 agonist in heart failure conditions may prove beneficial due to its anti-inflammatory properties and ability to reduce cytokines/chemokine production, potentially reducing chronic cardiac remodeling via effects on fibroblasts. The process of cardiac remodeling post MI is of critical importance to the survival of patients clinically [50]. Cytokines and chemokines are produced rapidly following myocardial ischemia [51, 52] and not only have direct effects on cardiac contractility [53], but also have major effects chronically on extracellular matrix remodeling [54], integrin expression [55], and angiogenesis [56]. Since cardiac fibroblasts are the most abundant non-cardiomyocyte population in the heart, they contribute substantially to the activation of the inflammation cascade following MI [57]. Evidence from clinical studies suggests that patients with chronically elevated serum inflammatory biomarkers, like MCP-1, have increased mortality even without new coronary events [58]. Therefore, this study is physiologically and clinically relevant by elucidating the mechanism of LPS-stimulated MCP-5 production in the cardiac fibroblasts via reduced Akt phosphorylation and NF-κB in response to PGE₂ and/or an EP4 agonist treatment.

Figure 8:

Scheme depicting the proposed mechanism of PGE₂ and EP4 inhibition of LPS-induced MCP-5. Also shown are the various agonists and antagonists used in the study. Filled lines indicated activation, dashed lines indicate inhibition, dashed/dotted lines indicate inhibition via an unknown pathway.

Highlights.

• PGE₂ and an EP4 agonist reduce MCP-5 production in cardiac fibroblasts.

• PGE₂ and EP4 agonist reduce LPS induced NF-κB activation and PI3K-Akt.

• Anti-inflammatory effect of PGE₂ and EP4 agonist is independent of p44/42 and p38.