Identification of cis-regulatory regions necessary for robust Nos2 promoter activity in glial cells: indirect role for NF-kappaB.
Previous reports suggest the nitric-oxide synthase 2 (Nos2) promoter contains negative and positive cis-regulatory regions. This study identified such regions using rat C6 glial cells. Activity of the serially deleted rat Nos2 promoter fused to a luciferase reporter gene was found to vary with construct size independent of stimuli, decreasing markedly from 160 to 130 bp then increasing significantly from 110 to 94 bp. In contrast, time to peak activity was stimulus-dependent but size-independent; 4-8 h for a cytokine mixture or lipopolysaccharide + interferon-gamma, and 8-16 h for lipopolysaccharide + phorbol 12-myristate 13-acetate. Peak activity with heterologous promoters also varied; 4 h for 3.7 kb of the human Nos2A promoter, and 36 h for 1.8 kb of the murine promoter. Electrophoretic mobility shift assays and in vivo DNA footprinting data confirmed nuclear protein binding to promoter regions suspected of containing important regulatory sites based on reporter gene data. A binding site for NF-kappaB was not required for Nos2 promoter activity. These findings provide significant new information on the relative importance of different regions of the rat Nos2 promoter for transcriptional activation and nitric oxide production by glial cells and support the existence of cell- and species-specific mechanisms for transcriptional regulation of Nos2 activation.
Sanchez
Alma C
AC
Alcohol and Brain Research Laboratory, Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.
Davis
Randall L
RL
Syapin
Peter J
PJ
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D024202:Electrophoretic Mobility Shift Assay; D004790:Enzyme Induction / Q000187:drug effects / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D017930:Genes, Reporter; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology*; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology*; D017384:Sequence Deletion; D013268:Stimulation, Chemical; D013329:Structure-Activity Relationship; D014162:Transfection
D016328:NF-kappa B; D009687:Nuclear Proteins; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat