Kim Y M, Lee B S, Yi K Y, Paik S G
Department of Biology, Chungnam National University, Taejon, South Korea.
Biochem Biophys Res Commun. 1997 Jul 30;236(3):655-60. doi: 10.1006/bbrc.1997.7031.
Transient transfection assays with various deletion mutants of the mouse inducible nitric oxide synthase (iNOS) promoter linked to a CAT reporter gene demonstrated that, besides the downstream NF-kappaB site, the region from -973 to -925 which contains a potential binding site for NF-kappaB (upstream NF-kappaB site) also mediated lipopolysaccharide (LPS)-inducibility in mouse macrophage cell line RAW 264.7. Site-specific mutation of three conserved nucleotides within the upstream NF-kappaB site abolished additional induction by LPS as well as maximal expression of iNOS by IFN-gamma plus LPS. In contrast, site-specific mutation of the downstream NF-kappaB site caused almost all reduction in expression of the reporter gene by LPS or LPS plus IFN-gamma. Electrophoretic mobility shift assays with the two NF-kappaB sites showed LPS-induced NF-kappaB binding to both probes and its higher affinity to the upstream NF-kappaB site. Taken together, these suggest that the upstream NF-kappaB site having enhancer function, besides the downstream NF-kappaB site as a core promoter, is essential for maximal expression of the iNOS gene.
将小鼠诱导型一氧化氮合酶（iNOS）启动子的各种缺失突变体与CAT报告基因进行瞬时转染分析表明，除了下游的NF-κB位点外，-973至-925区域包含一个潜在的NF-κB结合位点（上游NF-κB位点），该区域也介导了小鼠巨噬细胞系RAW 264.7中的脂多糖（LPS）诱导性。上游NF-κB位点内三个保守核苷酸的位点特异性突变消除了LPS的额外诱导作用以及IFN-γ加LPS对iNOS的最大表达。相反，下游NF-κB位点的位点特异性突变几乎导致LPS或LPS加IFN-γ引起的报告基因表达全部降低。对两个NF-κB位点进行电泳迁移率变动分析表明，LPS诱导NF-κB与两种探针结合，并且其对上游NF-κB位点具有更高的亲和力。综上所述，这些结果表明，除了作为核心启动子的下游NF-κB位点外，具有增强子功能的上游NF-κB位点对于iNOS基因的最大表达至关重要。
