Prostaglandin E2 Inhibits LPS‐Induced Monocyte Chemoattractant Protein 5 in Adult Cardiac Fibroblasts Through its EP4 Receptor via Reduced Akt and NF‐κB Signaling
Authors: Bryson Timothy Dean , Ross Jacob E , Peterson Edward , Harding Pamela 
Published: June 2021

Prostaglandin E2 (PGE2) signals through 4 G‐protein coupled receptor sub‐types (EP1–EP4) to elicit a variety of pathophysiological effects. We previously reported that mice lacking the EP4 receptor in cardiomyocytes (EP4 KO) develop heart failure with a phenotype of dilated cardiomyopathy. These mice also have increased levels of monocyte chemoattractant protein‐5 (MCP‐5), in their left ventricles. We recently published that overexpression of the EP4 receptor improves cardiac function in a model of myocardial infarction; associated with anti‐inflammatory effects. Moreover, conditioned media from EP4 KO cardiomyocyte cultures increased macrophage migration in vitro, which was attenuated with a MCP‐5 neutralizing antibody. In a variety of cell types, PGE2 antagonizes lipopolysaccharide (LPS)‐induced secretion of chemokines/cytokines. We therefore hypothesized that PGE2 inhibits LPS‐induced MCP‐5 secretion in adult mouse cardiac fibroblasts via its EP4 receptor. Our hypothesis was tested using 16–20‐week old mouse adult ventricular fibroblasts treated with LPS (10 μg/ml). Media and cells were harvested after 1, 2, 4, and 24 hrs of LPS 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) and this stimulation persisted for at least 24 hrs (p < 0.001). Pre‐treatment with PGE2 or an EP4 agonist significantly reduced LPS‐stimulated MCP‐5 by 24 hours (p< 0.01). Both the LPS‐stimulation of MCP‐5 and its inhibition by PGE2‐EP4 takes 4 hours to be detectable, suggesting that PGE2 via EP4 signaling may regulate MCP‐5 transcription. To test this, we examined MCP‐5 mRNA levels after 1, 2, 4, and 24 hours post LPS stimulation. After 1 hour of LPS stimulation, MCP‐5 mRNA significantly increased compared to vehicle (4.49 ± 0.71 vs. 1.00, respectively; 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 < 0.005). PGE2 treatment also attenuated MCP‐5 mRNA 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). PGE2 and the EP4 agonist only reduced LPS‐stimulated MCP‐5 mRNA at the 1 hr time point, suggesting that EP4 receptor activation leads to downstream signaling that quickly downregulates MCP‐5. Western blot showed PGE2‐EP4 reduced LPS stimulated phosphorylation of Akt and IκBα, indicating reduced NF‐κB activation. Furthermore, blocking NF‐κB with Cardamonin (10 μM) reduced LPS‐stimulated MCP‐5 after 24 hours of treatment (1.71 ± 0.41 vs. 9.11 ± 3.09 in LPS treated cells; p<0.005). Our data also show that the inhibitory effects of PGE2 or the EP4 agonist are independent of cAMP, JNK, p‐44/42, or p38 pathways. Therapeutically, our study may help shed light on the mechanism(s) regulating chemokine production in cardiac fibroblasts.Support or Funding InformationPH: NIH grant [5P01HL028982] (sub‐project 2). TB: NIH T32 Detroit Cardiovascular Training Grant (5T32HL12082205)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.