Sustained activation of phosphatidylinositol 3-kinase/Akt/nuclear factor kappaB signaling mediates G protein-coupled delta-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-kappaB survival cascade mediates dor expression during nerve growth factor (NGF)-induced differentiation of PC12h cells. Biochemical experiments showed that constitutive phosphorylation of Akt and IkappaBalpha correlates with NGF-induced dor expression. Overexpression of the transcriptional activator NF-kappaB/p65 increased dor promoter activity. Overexpression of the NF-kappaB signaling super inhibitor mutant IkappaBalpha (S32A/S36A) abolished the effect of p65 and blocked NGF-induced activation of NF-kappaB signaling, resulting in a significant reduction in dor promoter activity. Treatment with SN50, an NF-kappaB-specific nuclear translocation peptide inhibitor, inhibited the translocation of NF-kappaB, resulting in a reduction of dor mRNA. The gel shift assay supported the fact that there exists an NF-kappaB-binding site on the dor promoter. RNA interference experiments using NF-kappaB/p65 small interfering RNA confirmed that NF-kappaB 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.
Chen
Yulong L
YL
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA. chenx112@tc.umn.edu
Law
Ping-Yee
PY
Loh
Horace H
HH
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D020672:I-kappa B Proteins / Q000378:metabolism; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D020932:Nerve Growth Factor / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000737:chemistry; D016716:PC12 Cells; D010455:Peptides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis*; D017465:Receptors, Opioid, delta / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D051996:Transcription Factor RelA / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D015345:Oligonucleotide Probes; D010455:Peptides; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; D017465:Receptors, Opioid, delta; C118258:SN50 peptide; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D020932:Nerve Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt