Calcitonin gene-related peptide regulates spinal microglial activation through the histone H3 lysine 27 trimethylation via enhancer of zeste homolog-2 in rats with neuropathic pain

Abstract

Background Calcitonin gene-related peptide (CGRP) as a mediator of microglial activation at the transcriptional level may facilitate nociceptive signaling. Trimethylation of H3 lysine 27 (H3K27me3) by enhancer of zeste homolog 2 (EZH2) is an epigenetic mark that regulates inflammatory-related gene expression after peripheral nerve injury. In this study, we explored the relationship between CGRP and H3K27me3 in microglial activation after nerve injury, and elucidated the underlying mechanisms in the pathogenesis of chronic neuropathic pain. Methods Microglial cells (BV2) were treated with CGRP and differentially enrichments of H3K27me3 on gene promoters were examined using ChIP-seq. A chronic constriction injury (CCI) rat model was used to evaluate the role of CGRP on microglial activation and EZH2/H3K27me3 signaling in CCI-induced neuropathic pain. Results Overexpressions of EZH2 and H3K27me3 were confirmed in spinal microglia of CCI rats by immunofluorescence. CGRP treatment induced the increased of H3K27me3 expression in the spinal dorsal horn and cultured microglial cells (BV2) through EZH2. ChIP-seq data indicated that CGRP significantly altered H3K27me3 enrichments on gene promoters in microglia following CGRP treatment, including 173 gaining H3K27me3 and 75 losing this mark, which mostly enriched in regulation of cell growth, phagosome, and inflammation. qRT-PCR verified expressions of representative candidate genes (TRAF3IP2, BCL2L11, ITGAM, DAB2, NLRP12, WNT3, ADAM10) and real-time cell analysis (RTCA) verified microglial proliferation. Additionally, CGRP treatment and CCI increased expressions of ITGAM, ADAM10, MCP-1, and CX3CR1, key mediators of microglial activation in spinal dorsal horn and cultured microglial cells. Such increased effects induced by CCI were suppressed by CGRP antagonist and EZH2 inhibitor, which were concurrently associated with the attenuated mechanical and thermal hyperalgesia in CCI rats. Conclusion Our findings highly indicate that CGRP is implicated in the genesis of neuropathic pain through regulating microglial activation via EZH2-mediated H3K27me3 in the spinal dorsal horn.

Evidence of the Involvement of Spinal EZH2 in the Development of Bone Cancer Pain in Rats

“…Similarly, in a rat chronic constriction injury (CCI) model, intrathecal administration of CGRP8-37 (a CGRP antagonist) and GSK126 (an inhibitor of EZH2) attenuated pain hypersensitivity and decreased the levels of EZH2 and H3K27me3 in the spinal dorsal horn. 28 Yadav et al 8 found that an intrathecal injection of DZNep prevents the development of neuropathic pain, and its analgesic effects are associated with suppression of glial activation and the production of TNF-α, IL-1β, and MCP-1. Zhang et al 29 reported that EZH2 overexpression abrogates the effects of miR-124-3p on neuroinflammation and neuropathic pain.…”

Mitogen-activating protein kinase kinase kinase kinase-3, inhibited by Astragaloside IV through H3 lysine 4 monomethylation, promotes the progression of diabetic nephropathy by inducing apoptosis

“…ChIP-seq analysis was performed as previously described [ 22 ]. The sequencing images were generated by the sequencing platform and transformed into Raw Reads or Raw Data using bcl2fastq conversion software.…”

Epigenetic Connection of the Calcitonin Gene-Related Peptide and Its Potential in Migraine

“…Although neuropathic pain is not directly related to migraine, inflammatory mediators can play a role in its pathogenesis. Overexpression of the EZH2 gene and increased H3K27me3 levels in rat spinal microglia was observed [ 84 ]. Treatment with CGRP caused the upregulation of H3K27me3 in the spinal dorsal horn and cultured microglial cells through the induction of EZH2.…”