P38 Mitogen-activated protein kinase and PI3-kinase are involved in up-regulation of mu opioid receptor transcription induced by cycloheximide

Despite several decades of efforts to develop safer, efficacious, and non-addictive opioids for pain treatment, morphine remains the most valuable painkiller in contemporary medicine. Morphine and endogenous mu opioid peptides exert their pharmacological actions mainly through the mu opioid receptor (MOR). Analgesic effects of opioids in animals are dependent on the MOR expression levels, as demonstrated by studies of MOR-knockout mice (homo/heterozygotes) and MOR-less expressing mice. Surprisingly, in the course of our investigation to understand the mechanisms involved in the regulation of MOR gene expression, cycloheximide (CHX), a known protein synthesis inhibitor, markedly induced accumulation of MOR mRNAs in both MOR-negative and -positive cells. This induction was blocked by inhibitors of phosphoinositide 3-kinase (PI3-K) and p38 MAPK, but not by a p42/44 MAPK inhibitor. In vitro, CHX was found to activate the MOR promoter and this activation was suppressed by inhibition of PI3-K. The transcriptional activator Sox18 was recruited to the MOR promoter in CHX-treated cells and this recruitment was also inhibited by the PI3-K and p38 MAPK inhibitors, Ly294002 and SB203580, respectively. Consistently, acetylation of histone H3 and induction of H3-K4 methylation were detected while reductions of histone deacetylase 2 binding and H3-K9 methylation were observed on the promoter. Furthermore, the MOR mRNA accumulation was almost completely inhibited in the presence of actinomycin-D, indicating that this effect occurs mainly through activation of the transcriptional machinery. These observations suggest that CHX directly induces MOR gene transcription by recruiting the active transcription factor Sox18 to the MOR promoter through PI3- and/or p38 MAPK pathways.