A novel signaling pathway of nitric oxide on transcriptional regulation of mouse κ opioid receptor gene

Nitric oxide (NO) suppressed the transcription of the mouse κ opioid receptor (KOR) gene, mediated by a rapid downregulation of c-myc gene expression. KOR was constitutively expressed in postnatal day 19 (P19) embryonal carcinoma stem cells and is suppressed by NO donors [sodium nitroprusside (SNP), 3-morpholinosydnonimine-1, and S-nitrosoglutathione] in P19 stem cells within 4 hr. The suppression was reversed by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO scavenger, but could not be blocked by dithiothreitol, ruling out S-nitrosylation as the underlying mechanism. The suppressive effect of NO on KOR occurred at the level of gene transcription, mediated by E boxes located in promoters I and II of this gene. Protein-DNA complexes that formed on these E boxes contained c-myc; c-myc expression was suppressed by NO in P19 stem cells within 2 hr of treatment. Furthermore, chromatin immunoprecipitation demonstrated reduced c-myc binding to the E boxes and hypoacetylation of histone H3 on the chromatin of endogenous KOR promoters in P19 stem cells treated with SNP. It is proposed that NO regulates KOR at the level of gene transcription, mediated by a rapid suppression of c-myc gene expression and its binding to KOR promoters, and followed by chromatin hypoacetylation of and reduced transcription from KOR promoters in P19 stem cells. A novel pathway mediating the potential interplay between NO and opioid systems is discussed.