Distinct Protein Kinase A Anchoring Proteins Direct Prostaglandin E2 Modulation of Toll-like Receptor Signaling in Alveolar Macrophages

Abstract

Toll-like receptors (TLRs) direct a proinflammatory program in macrophages. One mediator whose generation is induced by TLR ligation is prostaglandin E 2 (PGE 2 ), which is well known to increase intracellular cAMP upon G protein-coupled receptor ligation. How PGE 2 /cAMP shapes the nascent TLR response and the mechanisms by which it acts remain poorly understood. Here we explored PGE 2 /cAMP regulation of NO production in primary rat alveolar macrophages stimulated with the TLR4 ligand LPS. Endogenous PGE 2 synthesis accounted for nearly half of the increment in NO production in response to LPS. The enhancing effect of PGE 2 on LPS-stimulated NO was mediated via cAMP, generated mainly upon ligation of the E prostanoid 2 receptor and acting via protein kinase A (PKA) rather than via the exchange protein activated by cAMP. Isoenzyme-selective cAMP agonists and peptide disruptors of protein kinase A anchoring proteins (AKAPs) implicated PKA regulatory subunit type I (RI) interacting with an AKAP in this process. Gene knockdown of potential RI-interacting AKAPs expressed in alveolar macrophages revealed that AKAP10 was required for PGE 2 potentiation of LPS-induced NO synthesis. AKAP10 also mediated PGE 2 potentiation of the expression of cytokines IL-10 and IL-6, whereas PGE 2 suppression of TNF-␣ was mediated by AKAP8-anchored PKA-RII. Our data illustrate the pleiotropic manner in which G protein-coupled receptor-derived cAMP signaling can influence TLR responses in primary macrophages and suggest that AKAP10 may coordinate increases in gene expression.Tissue macrophages play a pivotal role in innate immune responses. Via recognition receptors such as Toll-like receptors (TLRs), 3 these sentinel cells sense pathogens and other danger signals and then initiate a coordinated inflammatory response (1). The best-studied example is the recognition by TLR4 of LPS of Gram-negative bacterial cell walls. TLR-mediated inflammation is orchestrated by the activation of transcription factors including NFB (2). NFB-dependent genes that are critical to antimicrobial defense include proinflammatory cytokines and iNOS (3). NO is an important signaling molecule involved in regulating a wide range of biological activities in the neural, vascular, and immune systems. NO and its metabolites mediate a number of host defense functions of activated macrophages, including antimicrobial and tumoricidal activities, but are also implicated in the pathogenesis of tissue damage associated with acute and chronic inflammation (4).Ligation of TLRs also enhances the expression of COX-2, the enzyme responsible for the inducible generation of prostanoids including prostaglandin E 2 (PGE 2 ) (5, 6). PGE 2 is produced in abundance at sites of inflammation and infection and itself modulates many aspects of macrophage function (7). The immunomodulatory effects of PGE 2 largely result from its ability to increase intracellular cAMP through the stimulatory G protein (G s )-coupled E prostanoid (EP) receptors EP2 and EP4 (8). Increases in intracellular cA...

Prostaglandin E2Reduces Toll-Like Receptor 4 Expression in Alveolar Macrophages by Inhibition of Translation

“…We have previously shown that PGE 2 , also via a EP2-cAMP-PKA axis, can modulate cytokine production initiated by TLR4 ligation when added to AMs at the same time as LPS (16). This effect involves PKA regulation of TLR4 signaling pathway components that culminate in the activation of NF-kB.…”

“…The capacities of LTB 4 and PGE 2 to differentially influence macrophage functions have been linked to their abilities to ligate cognate G protein-coupled receptors (GPCRs) with resulting decreases and increases, respectively, in intracellular levels of the second messenger cAMP (13)(14)(15). We have previously shown that, through such increases in cAMP, PGE 2 can profoundly and rapidly suppress TLR4 pathway output by inhibiting downstream signaling events (16). However, whether PGE 2 and cAMP pathways can also influence the expression of TLR4 is not known.…”

SOCS1 is a negative regulator of metabolic reprogramming during sepsis

“…Western blotting was performed as previously described (83). Membranes were incubated with the following primary antibodies: STAT3 (1:1,000; #9139, clone 124H6), phospho-STAT3 (Tyr705; 1:250; #9145, clone D3A7), STAT1 (#9172), phospho-STAT1 (Tyr701; 1:500; #9167, clone 58D6), HIF-1α (1:1,000; #14179, clone D2U3T), and HK1 (1:1,000; #2024, clone C35C4) (all from Cell Signaling Technology) or β-actin (1:20,000; # A5441, clone UC15, Sigma-Aldrich), and then washed and incubated with appropriate fluorophore-conjugated secondary antibodies (1:2,000, anti-rabbit IgG, HRPlinked antibody, #7074, from Cell Signaling Technology).…”

PGE2 Induces Macrophage IL-10 Production and a Regulatory-like Phenotype via a Protein Kinase A–SIK–CRTC3 Pathway

“…Several studies have shown that agents that elevate cAMP can enhance IL-10 transcription (19)(20)(21)(22)(23)(24). One example of this is the PG PGE 2 .…”