siRNA knockdown of GPR18 receptors in BV-2 microglia attenuates N-arachidonoyl glycine-induced cell migration.
siRNA knockdown of GPR18 receptors in BV-2 microglia attenuates N-arachidonoyl glycine-induced cell migration.
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DOI：
10.1186/1750-2187-7-10
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发表时间：
2012-07-26
影响因子：
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通讯作者：
Bradshaw, Heather B
中科院分区：
文献类型：
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作者：
McHugh, Douglas;Wager-Miller, James;Bradshaw, Heather B
背景：已知神经元利用内源性大麻素系统与CNS的其他细胞进行通讯。内源性大麻素信号通过接合大麻素CB 2和异常大麻二酚（Abn-CBD）受体来招募小胶质细胞朝向神经元。Abn-CBD受体是一种突出的非典型大麻素受体，已通过各种药理学和遗传学工具进行了区分，但仍有待在分子水平上进行鉴定。我们最近介绍了N-花生四烯酰甘氨酸（NAGly）信号通过GPR 18受体作为一个重要的新的小胶质细胞神经元通信的信号机制。NAGly是内源性大麻素N-花生四烯酸乙醇酰胺（AEA）的内源性酶促氧化代谢物。我们最近的研究强烈支持两个假设;首先，NAGly通过激活GPR 18启动CNS中的定向小胶质细胞迁移，其次，GPR 18是Abn-CBD受体。在这里，我们提出了siRNA敲低数据，进一步支持这些hypothesis.FINDINGS：GPR 18靶向siRNA pSUPER G418 GFP cDNA质粒被创建并转染到BV-2小胶质细胞。成功转染GFP+ GPR 18 siRNA的BV-2小胶质细胞显示GPR 18 mRNA水平和免疫细胞化学染色降低。在GFP+细胞中，1 μ M浓度的NAGly、O-1602和Abn-CBD诱导的细胞迁移显着减弱。结论：我们的数据提供了明确的证据，表明这些化合物（Abn-CBD受体药理学特征）通过BV-2小胶质细胞中的GPR 18发挥作用。更全面地了解迄今尚未确定的大麻素受体，如GPR 18;它们与内源性配体的分子相互作用;以及植物大麻素如何影响它们的信号传导，如果我们要全面评估内源性大麻素信号传导系统在人类健康和疾病中的功能，就至关重要。
UNLABELLED: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 muM 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.