siRNA knockdown of GPR18 receptors in BV-2 microglia attenuates N-arachidonoyl glycine-induced cell migration

Journal of Molecular Signaling

Heather B Bradshaw¹  Jeremy Page¹  James Wager-Miller¹  Douglas McHugh¹ 

[1] Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN, 47405, USA

关键词: Microglia;    Endocannabinoid;    Cellular migration;    Abnormal cannabidiol receptor;    NAGly;    GPR18;   

Others  :  802918
DOI  :  10.1186/1750-2187-7-10

 received in 2012-03-15, accepted in 2012-07-11,  发布年份 2012

【 摘 要 】

Background

Neurons are known to employ the endogenous cannabinoid system to communicate with other cells of the CNS. Endocannabioid signaling recruits microglia toward neurons by engaging cannabinoid CB₂ and abnormal cannabidiol (Abn-CBD) receptors. The Abn-CBD receptor is a prominent atypical cannabinoid receptor that had been discriminated by means of various pharmacological and genetic tools but remained to be identified at the molecular level. We recently introduced N-arachidonoyl glycine (NAGly) signaling via GPR18 receptors as an important novel signaling mechanism in microglial-neuronal communication. NAGly is an endogenous, enzymatically oxygenated metabolite of the endocannabinoid N-arachidonoyl ethanolamide (AEA). Our recent studies support strongly two hypotheses; first that NAGly initiates directed microglial migration in the CNS through activation of GPR18, and second that GPR18 is the Abn-CBD receptor. Here we present siRNA knockdown data in further support of these hypotheses.

Findings

A GPR18-targetting siRNA pSUPER G418 GFP cDNA plasmid was created and transfected into BV-2 microglia. Successfully transfected GFP⁺ GPR18 siRNA BV-2 microglia displayed reduced GPR18 mRNA levels and immunocytochemical staining. Cell migration induced by 1 μM concentrations of NAGly, O-1602 and Abn-CBD were significantly attenuated in GFP⁺ cells.

Conclusions

Our data provide definitive evidence that these compounds, characteristic of Abn-CBD receptor pharmacology, are acting via GPR18 in BV-2 microglia. A fuller understanding of the hitherto unidentified cannabinoid receptors such as GPR18; their molecular interactions with endogenous ligands; and how phytocannabinoids influence their signaling is vital if we are to comprehensively assess the function of the endogenous cannabinoid signaling system in human health and disease.

【 授权许可】

   
2012 McHugh et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Schlicker E, Kathmann M: Modulation of transmitter release via presynaptic cannabinoid receptors. Trends Pharmacol Sci 2001, 22:565-572.
• [2]Pertwee RG, Ross RA: Cannabinoid receptors and their ligands. Prostaglandins Leukot Essent Fatty Acids 2002, 66:101-121.
• [3]Walter L, Franklin A, Witting A, Wade C, Xie Y, Kunos G, Mackie K, Stella N: Nonpsychotropic cannabinoid receptors regulate microglial cell migration. J Neurosci 2003, 23:1398-1405.
• [4]Franklin A, Stella N: Arachidonylcyclopropylamide increases microglial cell migration through cannabinoid CB2 and abnormal-cannabidiol-sensitive receptors. Eur J Pharmacol 2003, 474:195-198.
• [5]Begg M, Pacher P, Bátkai S, Osei-Hyiaman D, Offertáler L, Mo FM, Liu J, Kunos G: Evidence for novel cannabinoid receptors. Pharmacol Ther 2005, 106:133-145.
• [6]Mackie K, Stella N: Cannabinoid receptors and endocannabinoids: evidence for new players. AAPS J 2006, 8:E298-E306.
• [7]Járai Z, Wagner JA, Varga K, Lake KD, Compton DR, Martin BR, Zimmer AM, Bonner TI, Buckley NE, Mezey E, et al.: Cannabinoid-induced mesenteric vasodilation through an endothelial site distinct from CB1 or CB2 receptors. Proc Natl Acad Sci USA 1999, 96:14136-14141.
• [8]Offertáler L, Mo F-M, Batkai S, Liu J, Begg M, Razdan RK, Martin BR, Bukoski RD, Kunos G: Selective ligands and cellular effectors of a G protein-coupled endothelial cannabinoid receptor. Mol Pharmacol 2003, 63:699-705.
• [9]Mo FM: Offertáler, Kunos G: Atypical cannabinoid stimulates endothelial cell migration via a Gi/Go-coupled receptor distinct from CB1, CB2 or EDG-1. Eur J Pharmacol 2004, 489:21-27.
• [10]Vaccani A, Massi P, Colombo A, Rubino T, Palolaro D: Cannabidiol inhibits human glioma cell migration through a cannabinoid receptor-independent mechanism. BJP 2005, 144:1032-1036.
• [11]McHugh D, Hu SS-J, Rimmerman N, Juknat A, Vogel Z, Walker JM, Bradshaw HB: N-Arachidonoyl glycine potently drives directed BV-2 microglial migration through GPR18, the putative abnormal cannabidiol receptor. BMC Neurosci 2010, 11:44. BioMed Central Full Text
• [12]McHugh D, Page J, Dunn E, Bradshaw HB: Δ9-Tetrahydrocannabinol and N-arachidonoyl glycine are full agonists at GPR18 receptors and induce migration in human endometrial HEC-1B cells. BJP 2012, 165:2414-2424.
• [13]Bradshaw H, Rimmerman N, Hu S, Benton V, Stuart J, Masuda K, Cravatt B, O’Dell D, Walker JM: The endocannabinoid anandamide is a precursor for the signaling lipid N-arachidonoyl glycine by two distinct pathways. BMC Biochem 2009, 10:14. BioMed Central Full Text
• [14]Burstein SH, Rossetti RG, Yagen B, Zurier RB: Oxidative metabolism of anandamide. Prostaglandins Other Lipid Mediat 2000, 61:29-41.
• [15]Huang SM, Bisogno T, Petros TJ, Chang SY, Zavitsanos PA, Zipkin RE, Sivakumar R, Coop A, Maeda DY, De Petrocellis L, et al.: Identification of a new class of molecules, the arachidonyl amino acids, and characterization of one member that inhibits pain. J Biol Chem 2001, 276:42639-42644.
• [16]Sheskin T, Hanus L, Slager J, Vogel Z, Mechoulam R: Structural Requirements for Binding of Anandamide-Type Compounds to the Brain Cannabinoid Receptor. J Med Chem 1997, 40:659-667.
• [17]Kohno M, Hasegawa H, Inoue A, Muraoka M, Miyazaki T, Oka K, Yasukawa M: Identification of N-arachidonylglycine as the endogenous ligand for orphan G-protein-coupled receptor GPR18. Biochem Biophyl Res Comm 2006, 347:827-832.
• [18]Oh DY, Yoon JM, Moon MJ, Hwang J-I, Choe H, Lee JY, Kim JI, Kim S, Rhim H, O’Dell DK, et al.: Identification of Farnesyl Pyrophosphate and N-Arachidonylglycine as Endogenous Ligands for GPR92. J Biol Chem 2008, 283:21054-21064.
• [19]Wagner JA, Varga K, Jarai Z, Kunos G: Mesenteric vasodilation mediated by endothelial anandamide receptors. Hypertension 1999, 33:429-434.
• [20]Carp H: Mitochondrial N-formylmethionyl proteins as chemoattractants for neutrophils. J Exp Med 1982, 155:264-275.
• [21]Chadwick VS, Mellor DM, Myers DB, Selden AC, Keshavarzian A, Broom MF, Hobson CH: Production of peptides inducing Chemotaxis and lysosomal enzyme release in human neutrophils by intestinal bacteria in vitro and in vivo. Scand J Gastroenterol 1988, 23:121-128.
• [22]McHugh D, Tanner C, Mechoulam R, Pertwee RG, Ross RA: Inhibition of human neutrophil chemotaxis by endogenous cannabinoids and phytocannabinoids: evidence for a site distinct from CB1 and CB2. Mol Pharmacol 2008, 73:441-450.