Prostaglandin E receptor type 4-associated protein interacts directly with NF-kappaB1 and attenuates macrophage activation.
ABSTRACT Macrophage activation participates pivotally in the pathophysiology of chronic inflammatory diseases, including atherosclerosis. Through the receptor EP4, prostaglandin E(2) (PGE(2)) exerts an anti-inflammatory action in macrophages, suppressing stimulus-induced expression of certain proinflammatory genes, including chemokines. We recently identified a novel EP4 receptor-associated protein (EPRAP), whose function in PGE(2)-mediated anti-inflammation remains undefined. Here we demonstrate that PGE(2) pretreatment selectively inhibits lipopolysaccharide (LPS)-induced nuclear factor kappaB1 (NF-kappaB1) p105 phosphorylation and degradation in mouse bone marrow-derived macrophages through EP4-dependent mechanisms. Similarly, directed EPRAP expression in RAW264.7 cells suppresses LPS-induced p105 phosphorylation and degradation, and subsequent activation of mitogen-activated protein kinase kinase 1/2. Forced expression of EPRAP also inhibits NF-kappaB activation induced by various proinflammatory stimuli in a concentration-dependent manner. In co-transfected cells, EPRAP, which contains multiple ankyrin repeat motifs, directly interacts with NF-kappaB1 p105/p50 and forms a complex with EP4. In EP4-overexpressing cells, PGE(2) enhances the protective action of EPRAP against stimulus-induced p105 phosphorylation, whereas EPRAP silencing in RAW264.7 cells impairs the inhibitory effect of PGE(2)-EP4 signaling on LPS-induced p105 phosphorylation. Additionally, EPRAP knockdown as well as deficiency of NF-kappaB1 in macrophages attenuates the inhibitory effect of PGE(2) on LPS-induced MIP-1beta production. Thus, PGE(2)-EP4 signaling augments NF-kappaB1 p105 protein stability through EPRAP after proinflammatory stimulation, limiting macrophage activation.
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ABSTRACT: Objectives : The purpose of this study is to investigate the effect of Water Extract from Artemisiae Argi Folium (WAAF) on mouse macrophage Raw 264.7 cells stimulated by lipopolysaccharide (LPS). Methods : Cell viabilities were measured by MTT assay. And the intracellular productions of hydrogen peroxide (H2O2) were measured by dihydrorhodamine 123 assay. TNF- and IL-6 production from Raw 264.7 were measured by ELISA method. Results : The results of the experiment are as follows. 1. WAAF significantly increased the cell viability compared to the control group (treated with LPS only) at the concentrations of 10, 50, 100, 200, 400 ug/mL. 2. WAAF significantly increased the intracellular production of H2O2 compared to the control group at the concentrations of 50, 100, 200 ug/mL. 3. WAAF significantly decreased the production of TNF- compared to the control group at the concentrations of 100, 200 ug/mL. 4. WAAF significantly decreased the production of IL-6 compared to the control group at the concentrations of 50, 100, 200 ug/mL. Conclusions : WAAF could be supposed to have the immune-modulating activity related with the macrophage's immunoactivity.01/2009; 24(1).
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ABSTRACT: Extracellular nucleotides act as inflammatory mediators through activation of multiple purinoceptors. Under inflammatory conditions, the purinergic signalling is affected by various inflammatory mediators. We previously showed that prostaglandin (PG) E2 suppressed the elevation of intracellular Ca2+ concentration ([Ca2+]i) stimulated by P2X4, P2Y2, and P2Y6 receptors in J774 murine macrophages. In this study, we examined the mechanism of PGE2 inhibitory effects on P2Y6 receptor-mediated function in J774 cells. The P2Y6 receptor agonist UDP induced a sustained elevation of [Ca2+]i by stimulating the phospholipase C (PLC) signalling pathway. PGE2 inhibited [Ca2+]i elevation and phosphatidylinositol (PI) hydrolysis in a concentration-dependent manner. J774 cells highly expressed the E-type prostanoid 2 (EP2) receptor subtype, a Gs-coupled receptor. PGE2 and a selective EP2 receptor agonist caused cyclic AMP (cAMP) accumulation in J774 cells. The inhibitory effects of PGE2 on P2Y6 receptor-mediated responses were mimicked by the selective EP2 receptor agonist. Although EP2 receptor is linked to adenylyl cyclase activation, PGE2-induced inhibition of Ca2+ response and PI hydrolysis could not be mimicked by a lipophilic cAMP derivative, dibutyryl cAMP, or an adenylyl cyclase activator, forskolin. The inhibition of UDP-induced PLC activation by PGE2 was not affected by down-regulation of protein kinase C by phorbol-12-myristate-13-acetate treatment. PGE2 inhibited PLC activation induced by aluminium fluoride, but not by the Ca2+-ionophore, ionomycin. Finally, the inhibition of UDP-induced PLC activation by PGE2 was impaired by Gs knockdown using siRNA. These results suggest that EP2 receptor activation in macrophages negatively controls the Gq/11-PLC signalling through a Gs-mediated, but cAMP-independent signalling mechanism.
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ABSTRACT: Inflammation of adipose tissue induces metabolic derangements associated with obesity. Thus, determining ways to control or inhibit inflammation in adipose tissue has clinical interest. The present study tested the hypothesis that in mouse adipose tissue, endogenous prostaglandin E2 (PGE2) negatively regulates inflammation via activation of EP4 receptors. PGE2 (5-500nM) attenuated lipopolysaccharide (LPS)-induced mRNA and protein expression of chemokines, including interferon-γ-inducible protein 10 and macrophage-inflammatory protein-1α in mouse adipose tissue. A selective EP4 antagonist (L161,982) reversed, and two structurally different selective EP4 agonists [CAY10580 and CAY10598] mimicked these actions of PGE2. Adipose tissue derived from EP4-deficient mice did not display this response. These findings establish the involvement of EP4 receptors in this anti-inflammatory response. Experiments performed on adipose tissue from high-fat-fed mice demonstrated EP4-dependent attenuation of chemokine production during diet-induced obesity. The anti-inflammatory actions of EP4 became more important on high-fat diet, in that EP4 activation suppressed a greater variety of chemokines. Furthermore, adipose tissue and systemic inflammation was enhanced in high-fat fed EP4-deficient mice compared to wild-type littermates, and in high-fat fed untreated C57BL/6 mice compared to mice treated with EP4 agonist. These findings provide in vivo evidence that PGE2-EP4 signaling limits inflammation. In conclusion, PGE2, via activation of EP4 receptors, functions as an endogenous anti-inflammatory mediator in mouse adipose tissue, and targeting EP4 may mitigate adipose tissue inflammation. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.The Journal of Lipid Research 12/2014; DOI:10.1194/jlr.M054817 · 4.73 Impact Factor