prostaglandin-d2 and glyceryl-2-arachidonate

Proinflammatory macrophages are key mediators in several pathologies; thus, controlling their activation is necessary. The endocannabinoid system is implicated in various inflammatory processes. Here we show that in macrophages, the newly characterized enzyme α/β-hydrolase domain 6 (ABHD6) controls 2-arachidonoylglycerol (2-AG) levels and thus its pharmacological effects. Furthermore, we characterize a unique pathway mediating the effects of 2-AG through its oxygenation by cyclooxygenase-2 to give rise to the anti-inflammatory prostaglandin D2-glycerol ester (PGD2-G). Pharmacological blockade of cyclooxygenase-2 or of prostaglandin D synthase prevented the effects of increasing 2-AG levels by ABHD6 inhibition in vitro, as well as the 2-AG-induced increase in PGD2-G levels. Together, our data demonstrate the physiological relevance of the interaction between the endocannabinoid and prostanoid systems. Moreover, we show that ABHD6 inhibition in vivo allows for fine-tuning of 2-AG levels in mice, therefore reducing lipopolysaccharide-induced inflammation, without the characteristic central side effects of strong increases in 2-AG levels obtained following monoacylglycerol lipase inhibition. In addition, administration of PGD2-G reduces lipopolysaccharide-induced inflammation in mice, thus confirming the biological relevance of this 2-AG metabolite. This points to ABHD6 as an interesting therapeutic target that should be relevant in treating inflammation-related conditions, and proposes PGD2-G as a bioactive lipid with potential anti-inflammatory properties in vivo.

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acids; Biphenyl Compounds; Carbamates; Cell Line; Cells, Cultured; Cyclooxygenase 2; Endocannabinoids; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Esters; Female; Gene Expression; Glycerides; Glycerol; Inflammation; Interleukin-1beta; Intramolecular Oxidoreductases; Lipocalins; Lipopolysaccharides; Macrophage Activation; Macrophages; Male; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Prostaglandin D2; Reverse Transcriptase Polymerase Chain Reaction

2-Arachidonyl glycerol (2-AG) is an endogenous arachidonic acid derivative released on demand from membrane precursors. 2-AG-mediated suppression of interleukin (IL)-2 depends on cyclooxygenase 2 (COX-2) metabolism and peroxisome proliferator-activated receptor γ (PPARγ) activation. 15-Deoxy-Δ¹²,¹⁴-prostaglandin J₂-glycerol ester (15d-PGJ₂-G), a putative COX-2 metabolite of 2-AG, acts as a PPARγ ligand and produces IL-2 suppression in activated Jurkat T cells, in part, by decreasing nuclear factor of activated T cells (NFAT) transcriptional activity. The objective of the present studies was to investigate the mechanism by which 15d-PGJ₂-G modulates NFAT activity to suppress IL-2. 15d-PGJ₂-G treatment decreased phorbol 12-myristate 13-acetate (PMA)/calcium ionophore (I₀)-induced NFAT DNA binding to the human IL-2 promoter and nuclear NFAT2 accumulation. It is noteworthy that 15d-PGJ₂-G treatment increased active nuclear HDM2 (human homolog of the oncoprotein and E3 ubiquitin ligase murine double minute 2) expression, whereas there was no change in the expression of glycogen synthase kinase 3β, both of which regulate NFAT. 15d-PGJ₂-G and other PPARγ agonists, such as rosiglitazone and ciglitazone, decreased PMA/I₀-mediated elevation in intracellular calcium concentration ([Ca²⁺](i)) in activated Jurkat cells. We were surprised to find that the PPARγ antagonists 2-chloro-5-nitro-N-4-pyridinylbenzamide (T0070907) and 2-chloro-5-nitrobenzanilide (GW9662) also decreased the PMA/I₀-mediated elevation in [Ca²⁺](i) in activated T cells. In addition, the presence of T0070907 plus 15d-PGJ₂-G produced an additive decrease in PMA/I₀-mediated elevation of [Ca²⁺](i), suggesting that the 15d-PGJ₂-G effects on calcium might be either PPARγ-independent or -dependent on occupation of the PPARγ ligand binding domain. Collectively, our findings suggest that 15d-PGJ₂-G increases active nuclear HDM2, which could lead to a decrease in NFAT2 and IL-2 suppression.

Topics: Animals; Arachidonic Acids; Calcium; Cell Nucleus; DNA-Binding Proteins; Endocannabinoids; Female; Glycerides; Glycerol; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Interleukin-2; Jurkat Cells; Lymphocyte Activation; Mice; NFATC Transcription Factors; PPAR gamma; Promoter Regions, Genetic; Prostaglandin D2; Proto-Oncogene Proteins c-mdm2; T-Lymphocytes

Endocannabinoids have both anti-inflammatory and neuroprotective properties against harmful stimuli. We previously demonstrated that the endocannabinoid 2-arachidonoylglycerol (2-AG) protects hippocampal neurons by limiting the inflammatory response via a CB(1) receptor-dependent MAPK/NF-κB signalling pathway. The purpose of the present study was to determine whether PPARγ, an important nuclear receptor, mediates 2-AG-induced inhibition of NF-κB phosphorylation and COX-2 expression, and COX-2-enhanced miniature spontaneous excitatory postsynaptic currents (mEPSCs).. By using a whole-cell patch clamp electrophysiological recording technique and immunoblot analysis, we determined mEPSCs, expression of COX-2 and PPARγ, and phosphorylation of NF-kB in mouse hippocampal neurons in culture.. Exogenous and endogenous 2-AG-produced suppressions of NF-κB-p65 phosphorylation, COX-2 expression and excitatory synaptic transmission in response to pro-inflammatory interleukin-1β (IL-1β) and LPS were inhibited by GW9662, a selective PPARγ antagonist, in hippocampal neurons in culture. PPARγ agonists 15-deoxy-Δ(12,14) -prostaglandin J(2) (15d-PGJ(2)) and rosiglitazone mimicked the effects of 2-AG on NF-κB-p65 phosphorylation, COX-2 expression and mEPSCs, and these effects were eliminated by antagonism of PPARγ. Moreover, exogenous application of 2-AG or elevation of endogenous 2-AG by inhibiting its hydrolysis with URB602 or JZL184, selective inhibitors of monoacylglycerol lipase (MAGL), prevented the IL-1β- and LPS-induced reduction of PPARγ expression. The 2-AG restoration of the reduced PPARγ expression was blocked or attenuated by pharmacological or genetic inhibition of the CB(1) receptor.. Our results suggest that CB(1) receptor-dependent PPARγ expression is an important and novel signalling pathway in endocannabinoid 2-AG-produced resolution of neuroinflammation in response to pro-inflammatory insults.

Topics: Anilides; Animals; Arachidonic Acids; Benzodioxoles; Biphenyl Compounds; Cannabinoid Receptor Modulators; Cells, Cultured; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Endocannabinoids; Excitatory Postsynaptic Potentials; Glycerides; Hippocampus; Inflammation; Interleukin-1beta; Lipopolysaccharides; Mice; Monoacylglycerol Lipases; Neurons; NF-kappa B; Phosphorylation; Piperidines; PPAR gamma; Prostaglandin D2; Receptor, Cannabinoid, CB1; Rosiglitazone; Signal Transduction; Synaptic Transmission; Thiazolidinediones

2-Arachidonyl glycerol (2-AG) is an endogenous arachidonic acid derivative that binds cannabinoid receptors CB1 and CB2 and is hence termed an endocannabinoid. 2-AG also modulates a variety of immunological responses, including expression of the autocrine/paracrine T cell growth factor interleukin (IL)-2. The objective of the present studies was to determine the mechanism responsible for IL-2 suppression by 2-AG. Because of the labile properties of 2-AG, 2-AG ether, a nonhydrolyzable analog of 2-AG, was also used. Both 2-AG and 2-AG ether suppressed IL-2 expression independently of CB1 and CB2, as demonstrated in leukocytes derived from CB1/CB2-null mice. Moreover, we demonstrated that both 2-AG and 2-AG ether treatment activated peroxisome proliferator-activated receptor gamma (PPARgamma), as evidenced by forced differentiation of 3T3-L1 cells into adipocytes, induction of aP2 mRNA levels, and activation of a PPARgamma-specific luciferase reporter in transiently transfected 3T3-L1 cells. Consequently, the putative role of PPARgamma in IL-2 suppression by 2-AG and 2-AG ether was examined in Jurkat T cells. Concordant with PPARgamma involvement, the PPARgamma-specific antagonist 2-chloro-5-nitro-N-(4-pyridyl)-benzamide (T0070907) blocked 2-AG- and 2-AG ether-mediated IL-2 suppression. Likewise, 2-AG suppressed the transcriptional activity of two transcription factors crucial for IL-2 expression, nuclear factor of activated T cells and nuclear factor kappaB, in the absence but not in the presence of T0070907. 2-AG treatment also induced PPARgamma binding to a PPAR response element in activated Jurkat T cells. Together, the aforementioned studies identify PPARgamma as a novel intracellular target of 2-AG, which mediates the suppression of IL-2 by 2-AG in a manner that is independent of CB1 and/or CB2.

Topics: 3T3-L1 Cells; Adipogenesis; Animals; Arachidonic Acids; Benzamides; Cells, Cultured; Endocannabinoids; Fatty Acid-Binding Proteins; Female; Gene Expression; Glycerides; Humans; Interferon-gamma; Interleukin-2; Interleukin-4; Jurkat Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; NFATC Transcription Factors; Peroxisome Proliferator-Activated Receptors; PPAR gamma; Prostaglandin D2; Protein Binding; Pyridines; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Response Elements; T-Lymphocytes; Thiazolidinediones

Cyclooxygenases (COX) play an important role in lipid signaling by oxygenating arachidonic acid to endoperoxide precursors of prostaglandins and thromboxane. Two cyclooxygenases exist which differ in tissue distribution and regulation but otherwise carry out identical chemical functions. The neutral arachidonate derivative, 2-arachidonylglycerol (2-AG), is one of two described endocannabinoids and appears to be a ligand for both the central (CB1) and peripheral (CB2) cannabinoid receptors. Here we report that 2-AG is a substrate for COX-2 and that it is metabolized as effectively as arachidonic acid. COX-2-mediated 2-AG oxygenation provides the novel lipid, prostaglandin H(2) glycerol ester (PGH(2)-G), in vitro and in cultured macrophages. PGH(2)-G produced by macrophages is a substrate for cellular PGD synthase, affording PGD(2)-G. Pharmacological studies reveal that macrophage production of PGD(2)-G from endogenous sources of 2-AG is calcium-dependent and mediated by diacylglycerol lipase and COX-2. These results identify a distinct function for COX-2 in endocannabinoid metabolism and in the generation of a new family of prostaglandins derived from diacylglycerol and 2-AG.

Topics: Animals; Arachidonic Acids; Binding Sites; Cannabinoid Receptor Modulators; Cell Line; Cyclooxygenase 1; Cyclooxygenase 2; Endocannabinoids; Glycerides; Humans; Isoenzymes; Male; Membrane Proteins; Mice; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Sheep