siRNA knockdown of GPR18 receptors in BV-2 microglia attenuates N-arachidonoyl glycine-induced cell migration
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.
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.
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.
KeywordsGPR18 NAGly Abnormal cannabidiol receptor Cellular migration Endocannabinoid Microglia
Analysis of variance
Cannabinoid receptor 1
Cannabinoid receptor 2
Central nervous system
Dulbecco’s Minimum Essential Medium
Endogenous cannabinoid system
Fatty acid amide hydrolase
Fluorescence activated cell sorting
Fetal bovine serum
Formyl peptide receptor 1
Formyl peptide receptor-like 1
Green fluorescence protein
G protein-coupled receptor
- MAP kinase
Mitogen activated protein kinase
Reverse transcriptase polymerase chain reaction
Short interfering messenger ribonucleic acid.
Background and significance
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.
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.
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  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 . 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 5 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.
BV-2 cells (a gift from Dr. N. Stella; University of Washington, Seattle), an immortalized mouse microglial cell line, were grown in high glucose DMEM (Gibco, USA) with FBS (5%; J R Scientific, USA), penicillin (100 units/ml; Sigma, USA), streptomycin (100 μg/ml; Sigma, USA) and L-glutamine (0.292 mg/ml; Gibco, USA), and passaged every 4–5 days for a maximum of 30 passages. Twenty-four hours prior to experimentation, the culture media was replaced by serum-free high-glucose DMEM supplemented with penicillin (100 units/ml), and streptomycin (100 μg/ml). Test compounds were dissolved in DMSO to a final concentration of 0.1%
siRNA knockdown of GPR18
Initially, custom double-stranded GPR18 Stealth RNAi™ siRNA primers were purchased from Invitrogen. Three primer pairs were ordered to maximize the probability of achieving successful GPR18 knockdown. The double-stranded siRNA pairs were introduced into BV-2 microglial cells in order to activate the cell’s RNAi pathway and interfere with the expression of GPR18 (see Additional file 1 for further details). The cells were evaluated for GPR18 silencing and transfection efficiency was determined to be less than 10%. Therefore we next employed the pSUPER vector system, which is designed specifically for the expression of short interfering RNA (siRNA).
Transfection of an exogenous siRNA can be problematic because the gene knockdown effect is only transient, particularly in rapidly dividing cells. One way of overcoming this challenge is to modify the siRNA in such a way as to allow it to be expressed by an appropriate vector, e.g., a plasmid. This is done by the introduction of a loop between the two strands, thus producing a single transcript, which can be processed into a functional siRNA. Such transcription cassettes typically use an RNA polymerase III promoter (e.g., H1), which directs the transcription of small nuclear RNAs (snRNAs) (H1 is the RNase component of human RNase P). The resulting siRNA transcript would then processed by the enzyme, Dicer.
To effect the silencing of murine GPR18, the pSUPER G418 GFP vector was used in concert with 3 pairs of custom oligonucleotides that contain a unique 19-nt sequence (the “N-19 target sequence”) derived from the mRNA transcript of the murine GPR18 gene. The N-19 target sequence, found using Ambion siRNA Target Finder (Applied Biosystems), corresponds to the sense strand of the pSUPER-generated siRNA, which in turn corresponds to a 19-nt sequence within the murine GPR18 mRNA.
GPR18 RNAi sequence 1
Sense GATCCCCtcacaaccagcttgatcttttTTCAAGAGAaaaa gatcaagctggttgtgaTTTTT
GPR18 RNAi sequence 2
Sense GATCCCCtggctcacacccagaggaattTTCAAGAGA aattcctctgggtgtgagccaTTTTT
GPR18 RNAi sequence 3
In the mechanism of RNAi, the antisense strand of the siRNA duplex hybridizes to this region of the mRNA to mediate cleavage of the molecule. These forward and reverse oligos were annealed and cloned into the vector, between the unique BgIII and HindIII enzyme sites. This positions the forward oligo at the correct position downstream from the H1 promoter’s TATA box to generate the desired siRNA duplex (see Additional file 1 for further details).
Lipofectamine 2000 (Invitrogen; USA) was employed to transfect BV-2 microglia with this custom plasmid (see Additional file 1 for further details). BV-2 microglia were then monitored for GFP fluorescence to indicate successful transfection and processing of the GPR18-containing pSUPER GFP G418 cDNA plasmid. Only BV-2 microglia transfected with GPR18 RNAi sequence 1 produced such GFP fluorescence. We tried to select the successfully transfected BV-2 microglia with G418 antibiotic in order to establish a stable cell line. This was unsuccessful; BV-2 microglia do not tolerate G418 antibiotic selection well. Therefore, using a fluorescence-activated cell sorting (FACS) Aria II, the GFP+ subpopulation of BV-2 microglia were aseptically sorted from the non-transfected GFP- subpopulation based on the fluorescent characteristics of each cell. The FACS Aria II additionally discarded the <1% of dead BV-2 cells. Cells were allowed to recover for 3 h in serum-free DMEM before being used for experimentation. A control population of BV-2 microglia were similarly transfected with the original pSUPER G418 GFP vector (i.e., the plasmid encoded GFP but not a GPR18 RNAi sequence) and sorted via FACS.
Reverse transcriptase PCR
Reverse-transcriptase PCR was employed to assess the knockdown of GPR18 mRNA in the GFP+ BV-2 cells. Standard PCR techniques were performed with optimized GPR18 primers that had been verified in BV-2 microglia and GPR18-transfected HEK293 cells as previously described .
Briefly (see Additional file 1 for further details), cells were fixed with 3.7% paraformaldehyde, blocked, and stained as follows: polyclonal rabbit anti-C-terminal GPR18 (1:150) (generated in a previous study)  and Texas Red-conjugated phalloidin (1:40; Molecular Probes, Eugene, OR). Secondary IgG antibodies were FITC-conjugated donkey anti-rabbit (1:150; Jackson ImmunoResearch, USA). Images were acquired with a Nikon Eclipse TE2000-E confocal microscope (Nikon, USA).
In vitro cell migration assays
Were performed using a modified 96-well Boyden chamber. BV-2 cells, 1×106 cells/ml, were loaded into the upper wells; lower wells contained test compound. The chamber was incubated in a 5% CO2 atmosphere at 37°C for 3 h. Following incubation, migrated cells were stained with Diff-Quik® then counted in ten non-overlapping fields (×40) by multiple investigators. BV-2 cell migration in response to test compounds were expressed as the mean number of cells migrated.
Analysis of data
All data are expressed as means ± s.e.mean and n = number of independent experiments. Statistical analyses were performed with GraphPad Prism 4.
This work was supported by NIH DA032150.
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