G-protein-coupled receptors are considered to be cell-surface sensors of extracellular signals, thereby having a crucial role in signal transduction and being the most fruitful targets for drug discovery. G-protein-coupled receptor 151 (GPR151) was reported to be expressed specifically in the habenular area. Here we report the expression and the epigenetic regulation of GRP151 in the spinal cord after spinal nerve ligation (SNL) and the contribution of GPR151 to neuropathic pain in male mice. SNL dramatically increased GPR151 expression in spinal neurons. GPR151 mutation or spinal inhibition by shRNA alleviated SNL-induced mechanical allodynia and heat hyperalgesia. Interestingly, the CpG island in the GPR151 gene promoter region was demethylated, the expression of DNA methyltransferase 3b (DNMT3b) was decreased, and the binding of DNMT3b with GPR151 promoter was reduced after SNL. Overexpression of DNMT3b in the spinal cord decreased GPR151 expression and attenuated SNL-induced neuropathic pain. Furthermore, Krüppel-like factor 5 (KLF5), a transcriptional factor of the KLF family, was upregulated in spinal neurons, and the binding affinity of KLF5 with GPR151 promoter was increased after SNL. Inhibition of KLF5 reduced GPR151 expression and attenuated SNL-induced pain hypersensitivity. Further mRNA microarray analysis revealed that mutation of GPR151 reduced the expression of a variety of pain-related genes in response to SNL, especially mitogen-activated protein kinase (MAPK) signaling pathway-associated genes. This study reveals that GPR151, increased by DNA demethylation and the enhanced interaction with KLF5, contributes to the maintenance of neuropathic pain via increasing MAPK pathway-related gene expression.SIGNIFICANCE STATEMENT G-protein-coupled receptors (GPCRs) are targets of various clinically approved drugs. Here we report that SNL increased GPR151 expression in the spinal cord, and mutation or inhibition of GPR151 alleviated SNL-induced neuropathic pain. In addition, SNL downregulated the expression of DNMT3b, which caused demethylation of GPR151 gene promoter, facilitated the binding of transcriptional factor KLF5 with the GPR151 promoter, and further increased GPR151 expression in spinal neurons. The increased GPR151 may contribute to the pathogenesis of neuropathic pain via activating MAPK signaling and increasing pain-related gene expression. Our study reveals an epigenetic mechanism underlying GPR151 expression and suggests that targeting GPR151 may offer a new strategy for the treatment of neuropathic pain.

Peripheral nerve injury-induced spinal microglial proliferation plays a pivotal role in neuropathic pain. So far, key intracellular druggable molecules involved in this process are not identified. The nuclear factor of activated T-cells (NFAT1) is a master regulator of immune cell proliferation. Whether and how NFAT1 modulates spinal microglial proliferation during neuropathic pain remain unknown. Here it is reported that NFAT1 is persistently upregulated in microglia after spinal nerve ligation (SNL), which is regulated by TET2-mediated DNA demethylation. Global or microglia-specific deletion of Nfat1 attenuates SNL-induced pain and decreases excitatory synaptic transmission of lamina II neurons. Furthermore, deletion of Nfat1 decreases microglial proliferation and the expression of multiple microglia-related genes, such as cytokines, transmembrane signaling receptors, and transcription factors. Particularly, SNL increases the binding of NFAT1 with the promoter of Itgam, Tnf, Il-1b, and c-Myc in the spinal cord. Microglia-specific overexpression of c-MYC induces pain hypersensitivity and microglial proliferation. Finally, inhibiting NFAT1 and c-MYC by intrathecal injection of inhibitor or siRNA alleviates SNL-induced neuropathic pain. Collectively, NFAT1 is a hub transcription factor that regulates microglial proliferation via c-MYC and guides the expression of the activated microglia genome. Thus, NFAT1 may be an effective target for treating neuropathic pain.