Sustained activation of phosphatidylinositol 3-kinase/Akt/nuclear factor κB signaling mediates G protein-coupled δ-opioid receptor gene expression

Expression of the delta-opioid receptor gene ( dor) is tightly controlled during neuronal differentiation and developmental stages. Such distinct temporal and spatial expression of dor during development suggests a role for the delta-opioid receptor in early developmental events. However, little is known about intracellular signaling pathways that control dor expression. A well established cell line model for the study of gene expression during neuronal differentiation is the rat adrenal pheochromocytoma PC12 cell line. Here we found that the constitutively activated TrkA/phosphatidylinositol 3-kinase/Akt ( protein kinase B)/NF-kappa B survival cascade mediates dor expression during nerve growth factor ( NGF)-induced differentiation of PC12h cells. Biochemical experiments showed that constitutive phosphorylation of Akt and I kappa B alpha correlates with NGF-induced dor expression. Overexpression of the transcriptional activator NF-kappa B/p65 increased dor promoter activity. Overexpression of the NF-kappa B signaling super inhibitor mutant I kappa B alpha ( S32A/ S36A) abolished the effect of p65 and blocked NGF-induced activation of NF-kappa B signaling, resulting in a significant reduction in dor promoter activity. Treatment with SN50, an NF-kappa B-specific nuclear translocation peptide inhibitor, inhibited the translocation of NF-kappa B, resulting in a reduction of dor mRNA. The gel shift assay supported the fact that there exists an NF-kappa B-binding site on the dor promoter. RNA interference experiments using NF-kappa B/p65 small interfering RNA confirmed that NF-kappa B signaling is required for dor expression. Our findings not only provide a new mechanistic explanation for NGF-induced dor expression but also shed some light on the molecular mechanism of the temporal and spatial expression of dor and the roles of the delta-opioid receptor during neuronal differentiation.