siRNA knockdown of GPR18 receptors in BV-2 microglia attenuates N-arachidonoyl glycine-induced cell migration
Abstract
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 CB2and 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.
Findings
Background and significance
Neurons are known to use the endogenous cannabinoid system (eCBs) to communicate [1,2]. Walter et al. (2003) [3] reported the involvement of eCB signaling in recruiting microglia toward dying neurons in vitro. They demonstrated that pathological stimulation of neurons and microglia triggered microglial cell migration by engaging cannabinoid CB2 and abnormal cannabidiol (Abn-CBD) receptors. The Abn-CBD receptor is a prominent non-CB1/non-CB2 cannabinoid receptor that had been discriminated by means of various pharmacological and genetic tools but remained to be identified at the molecular level. It has been implicated in the modulation of microglial, endothelial and glioma cell migration, and a selection of cardiovascular responses [3–10]. We recently introduced N-arachidonoyl glycine (NAGly) signaling via GPR18 receptors as an important ‘new player’ in microglial-neuronal communication, providing a novel mechanism (both receptor and ligand) for directed migration and phenotypic switches in microglia [11,12]. NAGly is an endogenous, enzymatically oxygenated metabolite of the endocannabinoid N-arachidonoyl ethanolamide (Anandamide; AEA) (Figure ?(Figure1)1) with two distinct biosynthetic pathways, one of which is fatty acid amide hydrolase (FAAH) dependent. NAGly is produced throughout the body, like AEA, but has no activity at either CB1 or CB2[13–16]. Instead, NAGly is known to act as a high affinity ligand for the Gi/o-coupled GPCR GPR18 and a partial agonist of Gq/11-coupled GPR92 receptors [17,18]. Moreover, NAGly is the most potent pro-migratory lipid with regard to BV-2 microglia reported to date. It is able to elicit a migratory response double that of 1 μM
N-formyl-methionine-leucine-phenylalanine (fMLP), an established chemotactic peptide, at a concentration of 170 pM [11].
Our published studies [11,12] support strongly two hypotheses; first, NAGly initiates directed microglial migration in the CNS through activation of GPR18, and second, GPR18 is the Abn-CBD receptor reported to be present in microglia: BV-2 microglia show GPR18 receptor immunoreactivity and qPCR demonstrates that primary microglia, likewise, express abundant GPR18 mRNA. The pharmacological profile characteristic of the Gi/o-coupled Abn-CBD receptor includes activation by Abn-CBD and O-1602, which are inactive at CB1 and CB2, and antagonism by O-1918 and CBD [3,5–7,19]. Migration assays with wildtype HEK293 and HEK293 cells stably transfected with GPR18 support our hypothesis that NAGly is acting via Gi/o-coupled GPR18 receptors expressed by BV-2 microglia (Figure ?(Figure2;2; This figure represents data previously published [11] and is intended to illustrate the context of the novel findings reported here). Pertussis toxin (PTX) blocks NAGly’s effects in BV-2 microglia and HEK293-GPR18 cells. Abn-CBD and O-1602 drive migration in GPR18 transfected cells but not wildtype, suggesting that GPR18 is the receptor target for Abn-CBD (Figure ?(Figure2).2). Additionally, the migration induced by NAGly, Abn-CBD and O-1602 in BV-2 microglia and HEK293-GPR18 cells is attenuated by O-1918 and CBD [11]. Here we present siRNA knockdown data in further support of the hypotheses that GPR18 mediates the migratory effects of NAGly in BV-2 microglia, and that GPR18 is the Abn-CBD receptor present in microglia.
=3. This figure represents data …Results
In order to test our hypothesis that GPR18 receptors mediate the promigratory signaling of NAGly, O-1602 and Abn-CBD we first sought to silence GPR18 in BV-2 microglial cells.
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 were then sorted from non-transfected BV-2 cells via FACS analysis. Reverse-transcriptase PCR was employed to assess the knockdown of GPR18 mRNA in GFP+ BV-2 cells. HEK293 non-transfected, HEK293-GPR18 transfected and normal BV-2 cells were included as controls (Figure ?(Figure3).3). Figure ?Figure33 shows the definitive band at 163 bp that coincides with the primer product for GPR18 mRNA, and indicates that GPR18 mRNA levels in GFP+ BV-2 microglia were successfully attenuated. Immunocytochemical staining of GPR18 receptors revealed a similar observation that in GFP+ BV-2 cells GPR18 receptor expression is substantially reduced (Figure ?(Figure44).
Having verified effective knockdown of GPR18 in the GFP+ transfected BV-2 microglia, we next investigated the migration of these cells compared to normal BV-2 microglia.
Migration induced by 1 μM concentrations of NAGly, O-1602 and Abn-CBD was significantly attenuated in GFP+ cells compared to control, whereas migration to Vh (0.1% DMSO) and fMLP remained unchanged (Figure ?(Figure5;5; p<0.001, one-way ANOVA). fMLP is a tripeptide chemoattractant released from both bacteria and damaged mitochondria [20,21], and activates two formyl peptide receptors, designated FPR and FPRL-1; silencing of GPR18 would be expected to have no effect on fMLP signaling. A control population of BV-2 cells transfected with the original pSUPER G418 GFP vector (i.e., the plasmid encoded GFP but not a GPR18 RNAi sequence) behaved no differently from wildtype BV-2 microglia. Our data provide definitive evidence that these compounds, characteristic of Abn-CBD receptor pharmacology, are acting via GPR18 in BV-2 microglia.
Discussion
For years well-documented evidence has supported the existence of additional cannabinoid receptors, other than CB1 and CB2, contributing to the system [5,6,22]. Our McHugh et al. (2010) study [11] was the first to demonstrate that the Gi/o-coupled GPCR, GPR18, is one of these unknown receptors in that AEA and its metabolite NAGly exert potent control of central nervous system microglia. Since then we have further characterized GPR18 pharmacology via p44/42 mitogen activated kinase (MAP kinase) activation [12]. We proposed the working hypotheses: first, that NAGly initiates directed microglial migration in the CNS through activation of GPR18; and second, that GPR18 is the molecular identity of the Abn-CBD receptor present in microglia. Here, we provide definitive evidence in support of these. Namely, that NAGly, O-1602 and Abn-CBD – compounds characteristic of Abn-CBD receptor pharmacology – are acting via GPR18 in BV-2 microglia. Figure ?Figure55 shows the statistically significant and substantial attenuation of cell migration in GFP+ BV-2 microglia. The lack of a complete block of the NAGly, O-1602 and Abn-CBD effects may suggest an additional GPCR target for these ligands other than GPR18. However, it is important to note that achieving 100% siRNA knockdown efficiency is highly problematic, especially in cells where the gene in question is performing an essential function. The reduced but still detectable GPR18 amplicon band and immunocytochemical staining imply that enough GPR18 mRNA is being transcribed to allow these compounds to continue to signal. Future studies with GPR18 knockout animals will help clarify this.
An understanding of the expression, function, and regulation 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….
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493281/
siRNA knockdown of GPR18 receptors in BV-2 microglia attenuates N-arachidonoyl glycine-induced cell.
