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A reverse nuclear factor-kappaB element in the rat type II nitric oxide synthase promoter mediates the induction by interleukin-1beta and interferon-gamma in rat aortic smooth muscle cells.

Abstract

The rat type II nitric oxide synthase (iNOS) promoter contains two nuclear factor-kappaB (NF-kappaB)-binding sites, one upstream (-965 to -956 bp) and one downstream (-107 to -98 bp), which are important for iNOS induction. We have identified a third NF-kappaB site located at -901 to -892 bp whose sequence is identical to that of the upstream site but with the opposite orientation ("the reverse NF-kappaB site"). We hypothesized that the reverse NF-kappaB site, like the other two sites, is important for iNOS induction. With the use of a rat iNOS promoter fragment of -906 to -887 bp as probe, electrophoretic mobility shift assays were performed on nuclear proteins extracted from rat aortic smooth muscle cells (RASMCs) treated with interleukin-1beta (IL-1beta, 100 U/ml) +/- interferon-gamma (IFN-gamma, 250 U/ml) for 30 min. IL-1beta, but not IFN-gamma, induced a DNA-protein complex that was supershifted by either anti-NF-kappaB p50 or anti-NF-kappaB p65 antibody. The functionality of the reverse NF-kappaB site was evaluated by mutation experiments and transfection assays. The wild-type and mutated -1.4 kb rat iNOS promoter-luciferase constructs were transfected into RASMCs. Compared with the wild type, reverse-NF-kappaB site (-901 to -892 bp) deletion, substitution of T for C at -894 bp, and substitution TTT for CCC at -896 to -894 bp decreased the IL-1beta-induced promoter activity by 67% (p < 0.001), 45% (p < 0.001), and 56% (p < 0.001), respectively. These deletion/substitutions also decreased the IL-1beta- and IFN-gamma-induced promoter activity by 74% (p < 0.001), 53%(p < 0. 001), and 63% (p < 0.001), respectively. In conclusion, a p50 and p65 NF-kappaB heterodimer binds to a reverse-NF-kappaB site on the rat iNOS promoter and contributes to iNOS induction by IL-1beta and IFN-gamma in RASMCs.

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  • Authors+Show Affiliations

    ,

    Vascular Biology Center and Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2500, USA.

    , ,

    Source

    General pharmacology 34:1 2000 Jan pg 9-16

    MeSH

    Animals
    Antiviral Agents
    Aorta
    Interferon-gamma
    Interleukin-1
    Mice
    Muscle, Smooth
    NF-kappa B
    Nitric Oxide Synthase
    Nitric Oxide Synthase Type II
    Promoter Regions, Genetic
    Rats
    Rats, Wistar

    Pub Type(s)

    Journal Article
    Research Support, U.S. Gov't, P.H.S.

    Language

    eng

    PubMed ID

    10793263

    Citation

    Teng, X, et al. "A Reverse Nuclear factor-kappaB Element in the Rat Type II Nitric Oxide Synthase Promoter Mediates the Induction By Interleukin-1beta and Interferon-gamma in Rat Aortic Smooth Muscle Cells." General Pharmacology, vol. 34, no. 1, 2000, pp. 9-16.
    Teng X, Zhang H, Snead C, et al. A reverse nuclear factor-kappaB element in the rat type II nitric oxide synthase promoter mediates the induction by interleukin-1beta and interferon-gamma in rat aortic smooth muscle cells. Gen Pharmacol. 2000;34(1):9-16.
    Teng, X., Zhang, H., Snead, C., & Catravas, J. D. (2000). A reverse nuclear factor-kappaB element in the rat type II nitric oxide synthase promoter mediates the induction by interleukin-1beta and interferon-gamma in rat aortic smooth muscle cells. General Pharmacology, 34(1), pp. 9-16.
    Teng X, et al. A Reverse Nuclear factor-kappaB Element in the Rat Type II Nitric Oxide Synthase Promoter Mediates the Induction By Interleukin-1beta and Interferon-gamma in Rat Aortic Smooth Muscle Cells. Gen Pharmacol. 2000;34(1):9-16. PubMed PMID: 10793263.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - A reverse nuclear factor-kappaB element in the rat type II nitric oxide synthase promoter mediates the induction by interleukin-1beta and interferon-gamma in rat aortic smooth muscle cells. AU - Teng,X, AU - Zhang,H, AU - Snead,C, AU - Catravas,J D, PY - 2000/5/4/pubmed PY - 2000/6/17/medline PY - 2000/5/4/entrez SP - 9 EP - 16 JF - General pharmacology JO - Gen. Pharmacol. VL - 34 IS - 1 N2 - The rat type II nitric oxide synthase (iNOS) promoter contains two nuclear factor-kappaB (NF-kappaB)-binding sites, one upstream (-965 to -956 bp) and one downstream (-107 to -98 bp), which are important for iNOS induction. We have identified a third NF-kappaB site located at -901 to -892 bp whose sequence is identical to that of the upstream site but with the opposite orientation ("the reverse NF-kappaB site"). We hypothesized that the reverse NF-kappaB site, like the other two sites, is important for iNOS induction. With the use of a rat iNOS promoter fragment of -906 to -887 bp as probe, electrophoretic mobility shift assays were performed on nuclear proteins extracted from rat aortic smooth muscle cells (RASMCs) treated with interleukin-1beta (IL-1beta, 100 U/ml) +/- interferon-gamma (IFN-gamma, 250 U/ml) for 30 min. IL-1beta, but not IFN-gamma, induced a DNA-protein complex that was supershifted by either anti-NF-kappaB p50 or anti-NF-kappaB p65 antibody. The functionality of the reverse NF-kappaB site was evaluated by mutation experiments and transfection assays. The wild-type and mutated -1.4 kb rat iNOS promoter-luciferase constructs were transfected into RASMCs. Compared with the wild type, reverse-NF-kappaB site (-901 to -892 bp) deletion, substitution of T for C at -894 bp, and substitution TTT for CCC at -896 to -894 bp decreased the IL-1beta-induced promoter activity by 67% (p < 0.001), 45% (p < 0.001), and 56% (p < 0.001), respectively. These deletion/substitutions also decreased the IL-1beta- and IFN-gamma-induced promoter activity by 74% (p < 0.001), 53%(p < 0. 001), and 63% (p < 0.001), respectively. In conclusion, a p50 and p65 NF-kappaB heterodimer binds to a reverse-NF-kappaB site on the rat iNOS promoter and contributes to iNOS induction by IL-1beta and IFN-gamma in RASMCs. SN - 0306-3623 UR - https://www.unboundmedicine.com/medline/citation/10793263/A_reverse_nuclear_factor_kappaB_element_in_the_rat_type_II_nitric_oxide_synthase_promoter_mediates_the_induction_by_interleukin_1beta_and_interferon_gamma_in_rat_aortic_smooth_muscle_cells_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0306362399000476 DB - PRIME DP - Unbound Medicine ER -