title: WWL70 attenuates PGE2production derived from 2-arachidonoylglycerol in microglia by ABHD6-independent mechanism.
abstract: α/β-Hydrolase domain 6 (ABHD6) is one of the major enzymes for endocannabinoid 2-arachidonoylglycerol (2-AG) hydrolysis in microglia cells. Our recent studies have shown that a selective ABHD6 inhibitor WWL70 has anti-inflammatory and neuroprotective effects in animal models of traumatic brain injury and multiple sclerosis. However, the role of ABHD6 in the neuroinflammatory response and the mechanisms by which WWL70 suppresses inflammation has not yet been elucidated in reactive microglia.
  
  
  
  The hydrolytic activity and the levels of 2-AG in BV2 cells were measured by radioactivity assay and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The expression of cyclooxygenase-2 (COX-2) and prostaglandin E2(PGE2) synthases in microglia treated with lipopolysaccharide (LPS) with/without WWL70 was determined by western blot and quantitative RT-PCR. The conversion of 2-AG to PGE2or PGE2-glyceryl ester (PGE2-G) was assessed by enzyme-linked immunoassay (EIA) or LC-MS/MS. The involvement of ABHD6 in PGE2production was assessed using pharmacological inhibitors and small interfering RNA (siRNA). The effect of WWL70 on PGE2biosynthesis activity in the microsome fraction from BV2 cells and experimental autoimmune encephalopathy (EAE) mouse brain was also examined.
  
  
  
  We found that WWL70 suppressed PGE2production in LPS-activated microglia via cannabinoid receptor-independent mechanisms, although intracellular levels of 2-AG were elevated by WWL70 treatment. This reduction was not attributable to WWL70 inhibition of ABHD6, given the fact that downregulation of ABHD6 by siRNA or use of KT182, an alternative ABHD6 inhibitor failed to suppress PGE2production. WWL70 attenuated the expression of COX-2 and PGES-1/2 leading to the downregulation of the biosynthetic pathways of PGE2and PGE2-G. Moreover, PGE2production from arachidonic acid was reduced in the microsome fraction, indicating that WWL70 also targets PGE2biosynthetic enzymes, which are likely to contribute to the therapeutic mechanisms of WWL70 in the EAE mouse model.
  
  
  
  WWL70 is an anti-inflammatory therapeutic agent capable of inhibiting PGE2and PGE2-G production, primarily due to its reduction of COX-2 and microsomal PGES-1/2 expression and their PGE2biosynthesis activity in microglia cells, as well as in the EAE mouse brain.
authors.affiliation: Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA.
authors.forename: Mikiei
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Biomedical Instrumentation Center, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA.
authors.forename: Sean
authors.initials: S
authors.lastname: Moran
authors.affiliation: Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Wen
authors.affiliation: Department of Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA.|Neuroscience Program, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA.
authors.forename: Kwame
authors.initials: K
authors.lastname: Affram
authors.affiliation: Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA.
authors.forename: Tinghua
authors.initials: T
authors.lastname: Chen
authors.affiliation: Department of Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA.|Neuroscience Program, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA.
authors.forename: Aviva J
authors.initials: AJ
authors.lastname: Symes
authors.affiliation: Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA. yumin.zhang@usuhs.edu.|Neuroscience Program, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA. yumin.zhang@usuhs.edu.
authors.forename: Yumin
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D001713:Biphenyl Compounds / Q000494:pharmacology*; D063386:Cannabinoid Receptor Agonists / Q000494:pharmacology; D063387:Cannabinoid Receptor Antagonists / Q000494:pharmacology; D002219:Carbamates / Q000494:pharmacology*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015232:Dinoprostone / Q000235:genetics / Q000378:metabolism*; D063388:Endocannabinoids / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D005989:Glycerides / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D051379:Mice; D017628:Microglia / Q000187:drug effects* / Q000378:metabolism; D008994:Monoacylglycerol Lipases / Q000378:metabolism*; D010880:Piperidines / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D000077285:Rimonabant
chemical_list: D001095:Arachidonic Acids; D001713:Biphenyl Compounds; D063386:Cannabinoid Receptor Agonists; D063387:Cannabinoid Receptor Antagonists; D002219:Carbamates; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D005989:Glycerides; D008070:Lipopolysaccharides; C584421:N-methyl-N-((3-(4-pyridinyl)phenyl)methyl)-4'-(aminocarbonyl)(1,1'-biphenyl)-4-yl ester, carbamic acid; D010880:Piperidines; D011720:Pyrazoles; D034741:RNA, Small Interfering; C094503:glyceryl 2-arachidonate; D051546:Cyclooxygenase 2; C527261:ABHD6 protein, mouse; D008994:Monoacylglycerol Lipases; D015232:Dinoprostone; D000077285:Rimonabant
keywords: 2-AG; ABHD6; Arachidonic acid; BV2 cells; COX-2; Endocannabinoid; Microglia; PGE synthase; PGE2; PGE2-glyceryl ester