Evidence of Endogenous Mu Opioid Receptor Regulation by Epigenetic Control of the Promoters

Abstract

The pharmacological effect of morphine as a painkiller is mediated mainly via the mu opioid receptor (MOR) and is dependent on the number of MORs in the cell surface membrane. While several studies have reported that the MOR gene is regulated by various cis-and trans-acting factors, many questions remain unanswered regarding in vivo regulation. The present study shows that epigenetic silencing and activation of the MOR gene are achieved through coordinated regulation at both the histone and DNA levels. In P19 mouse embryonal carcinoma cells, expression of the MOR was greatly increased after neuronal differentiation. MOR expression could also be induced by a demethylating agent (5-aza-2-deoxycytidine) or histone deacetylase inhibitors in the P19 cells, suggesting involvement of DNA methylation and histone deacetylation for MOR gene silencing. Analysis of CpG DNA methylation revealed that the proximal promoter region was unmethylated in differentiated cells compared to its hypermethylation in undifferentiated cells. In contrast, the methylation of other regions was not changed in either cell type. Similar methylation patterns were observed in the mouse brain. In vitro methylation of the MOR promoters suppressed promoter activity in the reporter assay. Upon differentiation, the in vivo interaction of MeCP2 was reduced in the MOR promoter region, coincident with histone modifications that are relevant to active transcription. When MeCP2 was disrupted using MeCP2 small interfering RNA, the endogenous MOR gene was increased. These data suggest that DNA methylation is closely linked to the MeCP2-mediated chromatin structure of the MOR gene. Here, we propose that an epigenetic mechanism consisting of DNA methylation and chromatin modification underlies the cell stage-specific mechanism of MOR gene expression.Opioids exert their pharmacological and physiological effects through binding to their endogenous receptors. Three types of opioid receptors, mu (), delta (␦), and kappa (), all belonging to the G-protein-coupled receptor superfamily, have been cloned. Upon agonist binding, these receptors couple to G proteins and affect several signal transduction pathways thought to mediate a broad range of functions and pharmacological effects of endogenous and exogenous opioids (51). Previous studies suggested that the opioid receptor (MOR) plays a key role in mediating the major clinical effects of analgesics, such as morphine, as well as the development of tolerance and physical dependence upon prolonged administration (39). MOR is mainly expressed in the central nervous system, with densities varying greatly in different regions, which can display different functional roles (55). During mouse embryonic development, the MOR message was specifically observed as early as embryonic day 8.5 (E8.5) using the reverse transcription (RT)-PCR method (44). In contrast, MOR transcripts were detected only beginning at E12 using the radioligand binding method (70) and at E10.5 by in situ hybridization (85). Transcript levels gradually incr...

Epigenetics in the perioperative period

“…Opiate receptors ↔ development (Hwang et al, 2007) Local anaesthetics ↓ methylation (Lirk et al, 2012(Lirk et al, , 2014 Opiates ↑ methylation (Doehring et al, 2013) Opiate-induced hyperalgesia (Doehring et al, 2013) ↓ MCP3 expression → pain (Imai et al, 2013) ↓ GAD65 expression → pain (Zhang et al, 2013) Prefrontal cortex methylation (Tajerian et al, 2013) TRPA1 methylation ↔ pain (Bell et al, 2014) Transcriptome in inflammation (Bais et al, 2009) Transcriptome in healing (Kruidenier et al, 2012;Barnes et al, 2013;Belkina et al, 2013) Cell migration/prolif, angiogenesis (Rafehi et al, 2011) Heritable response to injury (Zeybel et al, 2012) Wound healing inflammation…”

“…Specifically, P19 embryonic carcinoma cells showed hypermethylation in the proximal promoter region of the gene encoding for this receptor, associated with gene silencing. In contrast, the promoter was unmethylated, and the gene stably expressed when differentiation into mature neuronal/astrocyte cells was induced with retinoic acid (Hwang et al, 2007). At the same time, differentiation led to decreased methyl-CpG-binding protein 2 (MeCP2) binding in the promoter region of the gene for μ opioid receptors (Hwang et al, 2007).…”

“…In contrast, the promoter was unmethylated, and the gene stably expressed when differentiation into mature neuronal/astrocyte cells was induced with retinoic acid (Hwang et al, 2007). At the same time, differentiation led to decreased methyl-CpG-binding protein 2 (MeCP2) binding in the promoter region of the gene for μ opioid receptors (Hwang et al, 2007). MeCP2 binds to methylated DNA regions and causes histone deacetylation, with subsequent compacting of the histone complex and decreased transcription (Nan et al, 1998).…”

Epigenetic Hierarchy Governing Nestin Expression

“…DNA demethylation and histone modifications have been shown to play a synergic role in the activation of gene transcription [16][17][18][19]. Despite the intense characterization and the widespread use of Nestin as a marker for NSCs, relatively little is known about the epigenetic mechanism governing Nestin transcription.…”

Epigenetic control of the expression of opioid receptor genes

“…19,20 For the MOR gene, siRNA experiment provided the evidence for a role of MeCP2 in methylating CG sites of this promoter. 21 The chromatin of MOR regulatory region in P19 appeared to maintain a stable, and regularly spaced nucleosome array even in later stages of differentiation when MOR transcription was highly active. Through the action of an artificial demethylation agent, 5-Aza-C, its DNA methylation could be reduced; however, it is unclear what kind of endogenous signals could trigger DNA demethylation of this promoter in differentiated neurons.…”