Abstract
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.
Highlights
Spatially expressed in developmental stages [1, 2]
In this study, using specific signaling inhibitors and biological agents in combination with the small interfering RNA approach, we found that Nerve growth factor (NGF) receptor TrkA and the downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascade, not the mitogen-activated protein kinase (MAPK), phospholipase C␥, and protein kinase C signaling cascades, serves as the main pathway required for dor expression in the NGF-differentiated PC12h cell model
Knockdown of nuclear factor B (NF-B) p65 Protein by NF-B p65 small interfering RNA (siRNA) Resulted in a Reduction in dor Expression—To confirm that the NF-B pathway is required for NGF/Trk/PI3K/Akt and NF-B signaling-dependent dor expression, using the RNA interference method we examined the effect of the NF-B p65 expression level on NGF-induced dor expression
Summary
Cell Culture and Reagents—The PC12h cell line was a generous gift from Dr Hiroshi Hatanaka. After IKBM is transfected into cells, expression of super inhibitor IB␣ (S32A/S36A) mutant protein (IB␣M) results in blocking NF-B signaling in a particular cell line. Transfection of PC12h Cells and Reporter Gene Assay—PC12h cells (8 ϫ 105 cells/60-mm dish) were transiently cotransfected for 3 h with 2.5 pmol of individual DNA constructs and a 1:5 molar ratio of pCH110 plasmid (Amersham Biosciences) or 0.5 g of Renilla luciferase expression vector pRL-TK (Promega) using SuperFect or Effectene transfection reagent (Qiagen) unless otherwise noted. The cells were harvested, lysed, and assayed for luciferase activities using a luciferase reporter assay system (Promega) according to the manufacturer’s protocols. Western Blot Analysis of Phosphorylation of Akt and IB␣—The proteins from cell lysates were separated on 10% SDS-PAGE gels and blotted onto nitrocellular membranes according to the manufacturer’s protocols (Cell Signaling, Beverly, MA).
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