Nitric oxide synthase inhibitors modulate nerve growth factor‐mediated regulation of amyloid precursor protein expression in PC12 cells

Abstract

Nerve growth factor (NGF) can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF-mediated neurotrophic responses. In this study, the role of NO in NGF-stimulated amyloid precursor protein (APP) levels was studied. PC12 cells were treated with either the non-selective NOS inhibitor N(omega)-nitro-L-arginine methylester (L-NAME) or the inducible NOS selective inhibitor s-methylisothiourea (S-MIU), and the effect on NGF-mediated increases in APP expression was determined. NGF significantly increased total APP protein levels following 96 h of treatment and this increase was prevented in cells pre-treated with S-MIU. Pre-treatment of cells with actinomycin D also blocked this NGF-mediated induction of APP, indicating de novo protein synthesis is necessary. Treatment with NGF increased APP promoter activity; however, this increase was only partially inhibited by pre-treatment with S-MIU and was increased in the presence of L-NAME. This suggests that NO may be modulating other aspects of APP expression in addition to transcription. Inhibition of NGF signaling pathways was also investigated using inhibitors of mitogen-activated protein (MAP) kinase (U0126), Akt (LY294002) and protein kinase C (PKC; U73122 and bisindolylmaleimide 1 (BIS-1)) activation. Inhibition of each of these pathways prevented NGF-mediated increases in APP protein expression; however, only BIS-1 attenuated NGF-mediated increases in promoter activation. This study indicates that NO is involved in the NGF-mediated regulation of APP, in part at the level of APP transcription and could involve the modulation of NGF signal transduction pathways.