Demethylation of G-Protein-Coupled Receptor 151 Promoter Facilitates the Binding of Krüppel-Like Factor 5 and Enhances Neuropathic Pain after Nerve Injury in Mice

Abstract

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.

Keywords: DNA methylation; DNMT3b; GPR151; KLF5; epigenetic; neuropathic pain.

Figure 1.

Generation of TALEN-mediated GPR151 mutant mice. A, A schematic shows the binding sites of TALENs (yellow) and space region (green) on Exon 1 of GPR151. FokI, An endonuclease. B, DNA sequencing shows the deletion of seven bases (ATGGCCG) in the chimera mice (F0 generation).

Figure 2.

GPR151 expression is increased in the spinal cord after SNL. A, Gene chip shows the upregulation of several GPCRs genes after SNL. B, There are eight GPCRs whose expression was increased >1.5-fold after SNL. C, The time course of GPR151 mRNA expression in the ipsilateral dorsal horn in naive, sham-, and SNL-operated mice. *p < 0.05, **p < 0.01, ***p < 0.001, versus sham. Student's t test. n = 6 mice/group. SC, Spinal cord. N, naive. D, E, In situ hybridization of GPR151 mRNA shows that no signal was found in spinal sections incubated with GPR151 sense probe (D), and positive signals were shown in spinal sections incubated with GPR151 antisense probe (E). F–H, In situ hybridization of GPR151 mRNA and immunofluorescence staining with NeuN (F), GFAP (G), and IBA-1 (H). I, Single-cell PCR shows the coexpression of GPR151 with neuronal marker NeuN. J, K, In situ hybridization of GPR151 mRNA on the spinal cord from SST-GFP (J) and GAD67-GFP (K) mice 10 d after SNL. L, The time course of GPR151 mRNA expression in L5 DRG. **p < 0.01, ***p < 0.001, versus sham. Student's t test. n = 4–6 mice/group. N, naive. M–O, The images of in situ hybridization of GPR151 mRNA in DRG sections incubated with GPR151 sense probe (M) or GPR151 antisense probe from naive (N) or SNL (O) animals.

Figure 3.

GPR151 KO mice are normal in the expression of cellular markers and neurochemical markers. A, PCR-based genotyping of WT and GPR151^−/− mice. ^+/+, ^+/−, and ^−/− indicate WT, heterozygote, and homozygote, respectively. B, Photographs of WT and GPR151^−/− mice show no changes in the gross anatomy of the GPR151^−/− mice. C, GPR151^−/− mice show normal distribution patterns in the spinal dorsal horn of the neurochemical marker NeuN, astrocytic marker GFAP, and microglial marker Iba-1. D, The expression of IB4⁺ non-peptidergic primary afferents, CGRP⁺ peptidergic primary afferents, and neurochemical marker PKCγ is normal in GPR151^−/− mice.

Figure 4.

Mutation or inhibition of GPR151 alleviates SNL-induced neuropathic pain. A, Acute pain threshold measured by tail immersion, Hargreaves test, von Frey test and motor function assessed by the rotarod test were comparable in WT and GPR151^−/− mice. n = 8–9 mice/group. Student's t test. B, C, SNL-induced mechanical allodynia (B) and heat hyperalgesia (C) were markedly alleviated in GPR151^−/− mice compared with WT mice. n = 6–7 mice/group. **p < 0.01, ***p < 0.001, versus WT. Two-way RM ANOVA followed by Bonferroni's tests. D, E, Intraspinal infusion of LV-GPR151 shRNA in the spinal cord alleviated SNL-induced mechanical allodynia 7 d after SNL (D) and blocked heat hyperalgesia (E). *p < 0.05, **p < 0.01, ***p < 0.001, versus LV-NC. Two-way RM ANOVA followed by Bonferroni's tests. n = 5–6 mice/group.

Figure 5.

Demethylation of GPR151 gene promoter region after SNL. A, The schematic shows the location of 10 CpG sites (red) within a CpG island of the GPR151 gene promoter region. B, Representative PCR shows that the ratio of methylated (M) to unmethylated (U) amplification products was reduced after SNL. *p < 0.05, versus sham. Student's t test. n = 4 mice/group. C, BSP of GPR151 promoter region of the spinal dorsal horn in sham- or SNL-operated mice. n = 3 mice/group. Ten clones were randomly selected from each mouse. Filled circles, Methylated CpG sites; unfilled circles, unmethylated CpG sites. D, The total methylation of GPR151 promoter was decreased after SNL. *p < 0.05, ***p < 0.001, SNL versus sham. Two-way RM ANOVA followed by Bonferroni's tests. n = 3 mice/group. E, Coelenterazine-using luciferase assay shows that the luciferase activity was increased when using HEK-293 cells transfected with the unmethylated pCpG-free-GPR151-promoter-Lucia vector. ***p < 0.001. Student's t test. n = 4 /group. F, Western blot shows that DNMT3b protein level was decreased after SNL. *p < 0.05; **p < 0.01, versus naive. n = 3 mice/group. G, ChIP-PCR shows that the binding of DNMT3b with GPR151 in the spinal dorsal horn was decreased after SNL. *p < 0.05, Student's t test. n = 5 mice/group. H, The mRNA expression of DNMT3b was decreased and GPR151 was increased 2 d after intrathecal injection of DNMT3b siRNA. **p < 0.01, versus NC. Student's t test. n = 6–7 mice/group. I, The ratio of methylated to unmethylated products of GPR151 promoter was decreased after intrathecal injection of DNMT3b siRNA. *p < 0.05, versus NC. Student's t test. n = 4 mice/group. J, Intrathecal injection of DNMT3b siRNA induced mechanical allodynia. *p < 0.05, **p < 0.01, versus NC. Two-way RM ANOVA followed by Bonferroni's tests. n = 7–8 mice/group. K, The mRNA expression of DNMT3b was increased and GPR151 was decreased after intraspinal infusion of LV-DNMT3b. ***p < 0.001, versus LV-NC. Student's t test. n = 6–7 mice/group. L, Pretreatment with LV-DNMT3b increased the methylation of GPR151 promoter in the spinal dorsal horn 10 d after SNL. *p < 0.05, versus LV-NC. Student's t test. n = 4 mice/group. M, Intraspinal infusion of LV-DNMT3b, 3 d before SNL, alleviated SNL-induced mechanical allodynia. *p < 0.05, **p < 0.01, versus LV-NC. Two-way RM ANOVA followed by Bonferroni's tests. n = 6–7 mice/group.

Figure 6.

The transcription factor KLF5 promotes the expression of GPR151. A, Schematic representation of GPR151 promoter region. Putative binding sites for KLF5, STAT3, and NFATC2 transcription factors are shown. B, The schematic shows the location of potential binding sites (red) of KLF5 with the GPR151 promoter region within the CpG sites. ECR, evolutionarily conserved regions. C, The logos of the standard KLF5 motif and five potential binding sites of KLF5 with GPR151 promoter (BS1–5). D, ChIP–PCR shows that the binding of KLF5 with GPR151 was increased after SNL. *p < 0.05, versus sham. Student's t test. n = 4 mice/group. E, Coelenterazine-using luciferase assay shows that the luciferase activity was dramatically increased when KLF5-expressing vector was transfected with unmethylated pCpG-free-GPR151-promoter-Lucia vector in HEK293 cells. ***p < 0.001. Student's t test. n = 4 /group. F, The luciferase reporter assay shows that the luciferase activity was decreased when cotransfection of KLF5-expressing vector with mutant KLF5 BS3 or BS4. ***p < 0.001, versus basic vector. ###p < 0.001, versus GPR151 vector. One-way ANOVA. n = 4/group.

Figure 7.

KLF5 is increased in the spinal cord after SNL and contributes to SNL-induced neuropathic pain. A, The time course of KLF5 mRNA expression in the spinal cord from naive and SNL-operated mice. The mRNA expression of KLF5 was increased at Days 1, 3, 10, and 21 after SNL. *p < 0.05, ***p < 0.001, SNL versus naive. One-way ANOVA. n = 5–6 mice/group. B, C, Representative images of KLF5 immunofluorescence in the spinal cord from naive and SNL mice. KLF5 was constitutively expressed in naive mice (B), and increased in SNL-operated mice (C). D–F, Double-immunofluorescence staining shows that KLF5 was mainly colocalized with the neuronal marker NeuN (D), a few with astrocyte marker GFAP (E), none with microglia marker CD11b (F) in the dorsal horn of spinal cord 10 d after SNL. G, In situ hybridization of GPR151 and immunostaining with KLF5 in the spinal cord 10 d after SNL. H, I, Immunostaining of KLF5 on the spinal cord from SST-GFP (H) and GAD67-GFP (I) mice 10 d after SNL. Arrows show typical double-staining neurons. Filled triangles show typical KLF5-single-labeled neurons. Blank triangles show SST- (in H) or GAD67-single-labeled (in I) neurons. J, K, Intrathecal injection of KLF5 siRNA alleviated SNL-induced mechanical allodynia (J) and heat hyperalgesia (K). *p < 0.05, versus NC siRNA. Two-way RM ANOVA followed by Bonferroni's tests. n = 6–7 mice/group. L, The mRNA expression of KLF5 and GPR151 were decreased after intrathecal injection of KLF5 siRNA. *p < 0.05, **p < 0.01, versus NC siRNA. Student's t test. n = 6–8 mice/group. M, N, Intrathecal injection of KLF5 inhibitor, ML264, 10 d after SNL alleviated SNL-induced mechanical allodynia (M) and thermal hyperalgesia (N). ***p < 0.001, versus Vehicle. Two-way RM ANOVA followed by Bonferroni's tests. n = 5–9 mice/group. O, The mRNA expression of KLF5 and GPR151 was decreased after intrathecal injection of ML264. *p < 0.05, versus vehicle, Student's t test. n = 7–9 mice/group.

Figure 8.

Gene expression profiles in the spinal cord of WT and GPR151 deficient mice after SNL. A, A scatter plot shows the gene expression in WT and GPR151^−/− mice. Red dots represent upregulated genes, and green dots represent downregulated genes. B, Heat map of expression of differentially expressed genes whose expression changes were >2-fold in SNL group compared with the sham group. A dendrogram (top) shows the clustering of the samples regarding these expression values (n = 2/group). C, Heat map representing expression values of 41 pain-related genes from Pain Gene database that were upregulated by nerve injury and normalized to the sham control after GPR151 mutation. D, Heat map representing expression values of 39 pain-related genes from the Pain Gene database that were upregulated in SNL GPR151^−/− group compared with the WT-SNL group. E, The significant pathways for downregulated genes in GPR151^−/−-SNL group. The MAPK pathway-associated genes were downregulated dramatically in GPR151^−/−-SNL mice compared with WT-SNL mice. F, The significant pathways for upregulated genes in GPR151 mutation group. G, RT-PCR for FGF14, PRKACA, CRK, MAP3K13, and GRB2 gene expression levels in WT and GPR151^−/− mice 10 d after SNL. **p < 0.01. Student's t test, n = 4 mice/group. H, I, Western blots for pERK, pp38, and pJNK in the spinal cord from WT and GPR151^−/− mice 10 d after sham or SNL operation. *p < 0.05, Student's t test, n = 3 mice/group.

Figure 9.

Schematic shows the epigenetic regulation of GPR151 expression and the mechanism of GPR151 underlying neuropathic pain. A, In healthy spinal cord, DNMT3b binds to the GPR151 promoter and silences its expression through DNA methylation. B, After nerve injury, DNMT3b dissociates from the GPR151 gene promoter following by active DNA demethylation, which induces chromatin accessibility, thereby promotes the recruitment of the transcriptional machinery. The transcription factor KLF5 is recruited onto the GPR151 gene promoter, facilitates GPR151 transcription, and further increases the expression of GPR151 protein. The increased GPR151 on the membrane is activated by extracellular signals and induce the activation of ERK. The pERK translocates into the nucleus and induces the expression of multiple algogenic genes that participate in pain processing, leading to the pathogenesis of neuropathic pain.