Nuclear Factor-κB Activated by Capacitative Ca2+ Entry Enhances Muscarinic Receptor-mediated Soluble Amyloid Precursor Protein (sAPPα) Release in SH-SY5Y Cells

Shinkyu Choi,Jin Hyoung Kim,Eun-Jihn Roh,Moon-Jeong Ko,Ji-Eun Jung,Hwa-Jung Kim

doi:10.1074/jbc.m601018200

Abstract

G(q/11) protein-coupled muscarinic receptors are known to regulate the release of soluble amyloid precursor protein (sAPPalpha) produced by alpha-secretase processing; however, their signaling mechanisms remain to be elucidated. It has been reported that a muscarinic agonist activates nuclear factor (NF)-kappaB, a transcription factor that has been shown to play an important role in the Alzheimer disease brain, and that NF-kappaB activation is regulated by intracellular Ca2+ level. In the present study, we investigated whether NF-kappaB activation plays a role in muscarinic receptor-mediated sAPPalpha release enhancement and contributes to a changed capacitative Ca2+ entry (CCE), which was suggested to be involved in the muscarinic receptor-mediated stimulation of sAPPalpha release. Muscarinic receptor-mediated NF-kappaB activation was confirmed by observing the translocation of the active subunit (p65) of NF-kappaB to the nucleus by the muscarinic agonist, oxotremorine M (oxoM), in SH-SY5Y neuroblastoma cells expressing muscarinic receptors that are predominantly of the M3 subtype. NF-kappaB activation and sAPPalpha release enhancement induced by oxoM were inhibited by NF-kappaB inhibitors, such as an NF-kappaB peptide inhibitor (SN50), an IkappaB alpha kinase inhibitor (BAY11-7085), a proteasome inhibitor (MG132), the inhibitor of proteasome activity and IkappaB phosphorylation, pyrrolidine dithiocarbamate, the novel NF-kappaB activation inhibitor (6-amino-4-(4-phenoxyphenylethylamino) quinazoline), and by an intracellular Ca2+ chelator (TMB-8). Furthermore, both oxoM-induced NF-kappaB activation and sAPPalpha release were antagonized by CCE inhibitors (gadolinium or SKF96365) but not by voltage-gated Ca2+-channel blockers. On the other hand, treatment of cells with NF-kappaB inhibitors (SN50, BAY11-7085, MG132, or pyrrolidine dithiocarbamate) did not inhibit muscarinic receptor-mediated CCE. These findings provide evidence for the involvement of NF-kappaB regulated by CCE in muscarinic receptor-mediated sAPPalpha release enhancement.

Highlights

The nuclear factor (NF)-␬B/Rel family of dimeric transcriptional factors is involved in the immediate early transcription of a large array of genes induced by mitogenic and antiapoptotic pathogen-associated stimuli [7, 8]
NF-␬B was detected as two major bands, which increased after treating the cells with the muscarinic receptor agonists, carbachol or oxotremorine M (oxoM), as reported previously in SH-SY5Y cells [31]
To obtain information on the mechanism of NF-␬B activation mediated by muscarinic receptors in SH-SY5Y neuroblastoma cells, we carried out immunocytochemistry for the p65 subunit of NF-␬B in cells pretreated with muscarinic receptor agonist and/or antagonist

Summary

Introduction

The NF-␬B/Rel family of dimeric transcriptional factors is involved in the immediate early transcription of a large array of genes induced by mitogenic and antiapoptotic pathogen-associated stimuli [7, 8]. In response to various stimuli, NF-␬B dimers are released from cytoplasmic I␬B proteins by a process involving site-specific phosphorylation of I␬B by I␬B kinase, ubiquitination, and subsequent proteolytic degradation via the 26 S proteasome pathway [6, 17, 18]. Role of NF-␬B in Muscarinic Receptor-mediated sAPP␣ Release phatidylinositol-specific phospholipase C␥, whereas G-protein-coupled receptor activates phospholipase C␤ followed by CCE [24]. In neuronal cells, the involvement of CCE in muscarinic receptor-mediated NF-␬B activation or vice versa is little understood. In the present study, we examined how NF-␬B and CCE are involved in muscarinic receptor-mediated sAPP␣ release in SH-SY5Y human neuroblastoma cells

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