title: Identification of hamster inducible nitric oxide synthase (iNOS) promoter sequences that influence basal and inducible iNOS expression.
abstract: IFN-γ/LPS-activated hamster (Mesocricetus auratus) macrophages express significantly less iNOS (NOS2) than activated mouse macrophages, which contributes to the hamster's susceptibility to intracellular pathogens. We determined a mechanism responsible for differences in iNOS promoter activity in hamsters and mice. The HtPP (1.2 kb) showed low basal and inducible promoter activity when compared with the mouse, and sequences within a 100-bp region (-233 to -133) of the mouse and hamster promoters influenced this activity. Moreover, within this 100 bp, we identified a smaller region (44 bp) in the mouse promoter, which recovered basal promoter activity when swapped into the hamster promoter. The mouse homolog (100-bp region) contained a cis-element for NF-IL-6 (-153/-142), which was absent in the hamster counterpart. EMSA and supershift assays revealed that the hamster sequence did not support the binding of NF-IL-6. Introduction of a functional NF-IL-6 binding sequence into the hamster promoter or its alteration in the mouse promoter revealed the critical importance of this transcription factor for full iNOS promoter activity. Furthermore, the binding of NF-IL-6 to the iNOS promoter (-153/-142) in vivo was increased in mouse cells but was reduced in hamster cells after IFN-γ/LPS stimulation. Differences in the activity of the iNOS promoters were evident in mouse and hamster cells, so they were not merely a result of species-specific differences in transcription factors. Thus, we have identified unique DNA sequences and a critical transcription factor, NF-IL-6, which contribute to the overall basal and inducible expression of hamster iNOS.
authors.affiliation: Research Service, Department of Veterans Affairs Medical Center, South Texas Veterans Health Care System, San Antonio, TX, USA.
authors.forename: Omar A
authors.initials: OA
authors.lastname: Saldarriaga
authors.forename: Bruno L
authors.initials: BL
authors.lastname: Travi
authors.forename: Goutam Ghosh
authors.initials: GG
authors.lastname: Choudhury
authors.forename: Peter C
authors.initials: PC
authors.lastname: Melby
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D006224:Cricetinae; D016207:Cytokines / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D047468:Immunoprecipitation; D007371:Interferon-gamma / Q000494:pharmacology; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D016207:Cytokines; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D007371:Interferon-gamma; D008156:Luciferases; D052247:Nitric Oxide Synthase Type II