Promoted Interaction of C/EBPα with DemethylatedCxcr3Gene Promoter Contributes to Neuropathic Pain in Mice

DNA methylation has been implicated in the pathogenesis of chronic pain. However, the specific genes regulated by DNA methylation under neuropathic pain condition remain largely unknown. Here we investigated how chemokine receptor CXCR3 is regulated by DNA methylation and how it contributes to neuropathic pain induced by spinal nerve ligation (SNL) in mice. SNL increasedCxcr3mRNA and protein expression in the neurons of the spinal cord. Meanwhile, the CpG (5′-cytosine-phosphate-guanine-3′) island in theCxcr3gene promoter region was demethylated, and the expression of DNA methyltransferase 3b (DNMT3b) was decreased. SNL also increased the binding of CCAAT (cytidine–cytidine–adenosine–adenosine–thymidine)/enhancer binding protein α (C/EBPα) withCxcr3promoter and decreased the binding of DNMT3b withCxcr3promoter in the spinal cord. C/EBPα expression was increased in spinal neurons after SNL, and inhibition of C/EBPα by intrathecal small interfering RNA attenuated SNL-induced pain hypersensitivity and reducedCxcr3expression. Furthermore, SNL-induced mechanical allodynia and heat hyperalgesia were markedly reduced inCxcr3−/−mice. Spinal inhibition ofCxcr3by shRNA or CXCR3 antagonist also attenuated established neuropathic pain. Moreover, CXCL10, the ligand of CXCR3, was increased in spinal neurons and astrocytes after SNL. Superfusing spinal cord slices with CXCL10 enhanced spontaneous EPSCs and potentiated NMDA-induced and AMPA-induced currents of lamina II neurons. Finally, intrathecal injection of CXCL10 induced CXCR3-dependent pain hypersensitivity in naive mice. Collectively, our results demonstrated that CXCR3, increased by DNA demethylation and the enhanced interaction with C/EBPα, can be activated by CXCL10 to facilitate excitatory synaptic transmission and contribute to the maintenance of neuropathic pain. SIGNIFICANCE STATEMENTPeripheral nerve injury induces changes of gene expression in the spinal cord that may contribute to the pathogenesis of neuropathic pain. CXCR3 is a chemokine receptor. Whether it is involved in neuropathic pain and how it is regulated after nerve injury remain largely unknown. Our study demonstrates that spinal nerve ligation downregulates the expression of DNMT3b, which may cause demethylation ofCxcr3gene promoter and facilitate the binding of CCAAT/enhancer binding protein α withCxcr3promoter and further increase CXCR3 expression in spinal neurons. The upregulated CXCR3 may contribute to neuropathic pain by facilitating central sensitization. Our study reveals an epigenetic mechanism underlying CXCR3 expression and also suggests that targeting the expression or activation of CXCR3 signaling may offer new therapeutics for neuropathic pain.