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.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Calcium/metabolism , NF-kappa B/metabolism , Receptors, Muscarinic/metabolism , Alzheimer Disease/metabolism , Cell Line, Tumor , Cell Nucleus/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Humans , Leupeptins/pharmacology , Nitriles/pharmacology , Peptides/chemistry , Peptides/pharmacology , Proteasome Endopeptidase Complex/metabolism , Sulfones/pharmacologyABSTRACT
Collapsin response mediator protein-2 is highly expressed in the adult brain and it has been speculated to play roles in nervous system diseases. Studies determined for the first time whether collapsin response mediator protein-2 expression is altered in brain ischemia. We observed ischemia-modulated expression of different-sized collapsin response mediator protein-2. As the ischemic duration increased, the expression level of a known major 62 kDa collapsin response mediator protein-2 was decreased, whereas the expression of a newly-detected 58 kDa collapsin response mediator protein-2 was clearly increased in middle carotid artery-occluded rat brain tissues. Analysis of the two collapsin response mediator protein-2 bands revealed that the novel 58 kDa collapsin response mediator protein-2 observed in middle carotid artery-occluded rat brain tissues was a cleavage form; the predicted cleavage site is located at the carboxy-terminal of the collapsin response mediator protein-2. These data suggest that collapsin response mediator protein-2 is an important candidate controlling ischemic stroke.
