EP2 Receptor Signaling Pathways Regulate Classical Activation of Microglia

Yi Quan,Jianxiong Jiang,Ray Dingledine

doi:10.1074/jbc.m113.455816

Abstract

Activation of EP2 receptors by prostaglandin E2 (PGE2) promotes brain inflammation in neurodegenerative diseases, but the pathways responsible are unclear. EP2 receptors couple to Gαs and increase cAMP, which associates with protein kinase A (PKA) and cAMP-regulated guanine nucleotide exchange factors (Epacs). Here, we studied EP2 function and its signaling pathways in rat microglia in their resting state or undergoing classical activation in vitro following treatment with low concentrations of lipopolysaccharide and interferon-γ. Real time PCR showed that PGE2 had no effect on expression of CXCL10, TGF-β1, and IL-11 and exacerbated the rapid up-regulation of mRNAs encoding cyclooxygenase-2, inducible NOS, IL-6, and IL-1β but blunted the production of mRNAs encoding TNF-α, IL-10, CCL3, and CCL4. These effects were mimicked fully by the EP2 agonist butaprost but only weakly by the EP1/EP3 agonist 17-phenyl trinor PGE2 or the EP4 agonist CAY10598 and not at all by the EP3/EP1 agonist sulprostone and confirmed by protein measurements of cyclooxygenase-2, IL-6, IL-10, and TNF-α. In resting microglia, butaprost induced cAMP formation and altered the mRNA expression of inflammatory mediators, but protein expression was unchanged. The PKA inhibitor H89 had little or no effect on inflammatory mediators modulated by EP2, whereas the Epac activator 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate acetoxymethyl ester mimicked all butaprost effects. These results indicate that EP2 activation plays a complex immune regulatory role during classical activation of microglia and that Epac pathways are prominent in this role.

Highlights

Microglial activation contributes strongly to brain inflammation
EP2 Activation Modulates Expression of Inflammatory Mediators in Rat Microglia—Resting state microglia were stimulated with 100 nM or 1 ␮M prostaglandin E2 (PGE2), 200 nM or 2 ␮M butaprost, or 10 ng/ml each LPS and IFN-␥ for 2 h, and the levels of
Ablation of COX-2 in forebrain neurons dampens brain inflammation after status epilepticus in part by reducing the induction of CCL3, CCL4, CXCL10, IL-11, and TNF-␣ [6]

Summary

Background

Results: The modulation of microglial cyclooxygenase-2, iNOS, and cytokine production by EP2 (PTGER2) activation is not blocked by a protein kinase A antagonist but is mimicked by an Epac agonist. The PKA inhibitor H89 had little or no effect on inflammatory mediators modulated by EP2, whereas the Epac activator 8-(4-chlorophenylthio)-2؅-O-methyladenosine 3؅,5؅-cyclic monophosphate acetoxymethyl ester mimicked all butaprost effects These results indicate that EP2 activation plays a complex immune regulatory role during classical activation of microglia and that Epac pathways are prominent in this role. A selective and brain-permeant EP2 antagonist has recently been shown to blunt inflammation in a pilocarpine epilepsy model [26] Based on these results, we asked whether modulation of innate immunity by EP2 activation in microglia proceeds through PKA or Epac. Our findings establish an immunomodulatory role for EP2 that proceeds largely through Epac pathways

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION