trichostatin-a and 7-nitroindazole

The neuronal nitric oxide synthase (nNOS) is predominantly expressed in nervous tissues and subject to complex transcriptional controls. To determine the effect of acetylation on nNOS expression, human neuroblastoma SK-N-SH cells were treated with trichostatin A (TSA), a histone deacetylase inhibitor. As a consequence, total and exon 1f-specific nNOS mRNA, nNOS protein and nNOS-derived nitric oxide production were increased. Immunoprecipitation and western blot showed both nuclear factor-kappaB (NF-kappaB) subunits p65 and p50 were acetylated in the presence of TSA. The enhancement of the p65 and p50 acetylation was in accordance with their increased binding affinities to the NF-kappaB responsive element, which was identified at position -893 to -884 of the nNOS exon 1f promoter. Luciferase assays revealed that TSA up-regulated the transcriptional activity of the nNOS 1f promoter through NF-kappaB-mediated transactivation. Taken together, we demonstrate that acetylation plays a crucial role in nNOS expression and suggest that acetylation of NF-kappaB p65 and p50 subunits by TSA treatment may augment their DNA-binding affinities, thereby activating the nNOS exon 1f promoter. It may be one of the mechanisms by which acetylation modulates nNOS expression and nitric oxide output in SK-N-SH cells and may be the molecular basis for certain neurological disorders.

Topics: Acetylation; Cell Line, Tumor; Chromatin Immunoprecipitation; Dose-Response Relationship, Drug; Drug Interactions; Exons; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Indazoles; Luciferases; Neuroblastoma; NF-kappa B; Nitric Oxide Synthase Type I; Protein Synthesis Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transfection