Prostaglandin E2 Inhibits Monocyte Chemotactic Protein 5 Production and Secretion in Mouse Cardiac Fibroblasts via EP4 Receptor
Authors: Bryson Timothy D , Szandzik David , Harding Pamela 
Published: November 2023

Monocyte Chemotactic Protein 5 (MCP‐5) is a chemokine that recruits monocytes and macrophages to sites of inflammation. Prostaglandin E2 (PGE2) is a local hormone that signals through four distinct G protein‐coupled receptors (EP1, EP2, EP3 and EP4) and was recently shown to have both pro and anti‐ inflammatory actions depending on which receptor sub type is stimulated. Although all four EP receptors are expressed in the heart, it is unclear what role they play in physiological and pathological conditions. Previously, we have shown that cardiomyocyte‐specific EP4 knockout (KO) mice have a phenotype of dilated cardiomyopathy coupled with elevated MCP‐5 mRNA in the left ventricle, suggesting that MCP‐5 may be regulated by PGE2 via its EP4 receptor and play a role in cardiac remodeling. Therefore, we hypothesized that PGE2 via EP4 receptor is anti‐inflammatory; inhibiting MCP‐5 in cardiac fibroblasts, which are a key cell type in the cardiac remodeling process. Primary cultures of cardiac fibroblasts from male C57 Bl/6 mice (16–21 weeks) were treated with vehicle, PGE2 (1 μM) or the EP4 receptor agonist (ONO‐AE1‐329; 1μM), in the presence or absence of lipopolysaccharide (LPS; 10 μg/μL), a known stimulator of MCP‐5 release. Media and cells were harvested after 1 hr, 2 hrs, 4 hrs, and 24 hrs of treatment. MCP‐5 secretion was determined by ELISA. LPS treatment for 4 hrs stimulated MCP‐5 secretion 2.3‐fold compared to vehicle (p<0.05, n=7) and this stimulation persisted for at least 24 hrs (p < 0.001). PGE2 treatment significantly attenuated this increase at both 4 hr and 24 hrs and reduced MCP‐5 to baseline levels (p<0.05). Similarly, the EP4 receptor agonist reduced LPS‐induced MCP‐5 secretion at both 4 hrs and 24 hrs. To determine if the decrease in production/secretion of MCP‐5 was due to PGE2 affecting gene expression, MCP‐5 mRNA was measured using real time RT‐PCR. MCP‐5 mRNA levels were markedly increased after LPS stimulation as early as 1 hour after treatment (3.8 fold that of vehicle, n=4) and remained elevated for 24 hrs. PGE2 treatment reduced LPS stimulation of MCP‐5 mRNA by 48% at 2 hours (n=5) and continued to diminish MCP‐5 mRNA throughout 24 hrs. Similarly, treatment with EP4 agonist attenuated the LPS‐induced increase in MCP‐5 mRNA at 2 hrs by 17% and continued to reduce MCP‐5 mRNA throughout 24 hrs. To elucidate how PGE2 and the EP4 agonist inhibit LPS‐induced MCP‐5 secretion or production, we treated cells with dibutyryl cAMP (100 μM) since the EP4 receptor is coupled to increases in cAMP. LPS‐stimulated MCP‐5 secretion was not altered by co‐treatment with dibutyryl cAMP. In conclusion, these data suggest that PGE2 acts through its EP4 receptor to affect MCP‐5 production, but this appears to be independent of cAMP. This inhibitory effect of PGE2 and the EP4 agonist may reduce cell migration to sites of inflammation during various cardiac pathologies and could potentially exert a cardioprotective effect.

Support or Funding Information

These studies were funded by NIH grant 5P01HL028982 (sub‐project 2) to P.H.