An acute phase response factor NF-kappa B site downstream of the junB gene that mediates responsiveness to interleukin-6 in a murine plasmacytoma
An acute phase response factor NF-kappa B site downstream of the junB gene that mediates responsiveness to interleukin-6 in a murine plasmacytoma
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DOI：
10.1074/jbc.270.52.31129
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发表时间：
1995-12-29
影响因子：
4.8
通讯作者：
Nordan, RP
中科院分区：
文献类型：
--
作者：
Brown, RT;Ades, IZ;Nordan, RP
即早基因junB在浆细胞瘤中由白细胞介素-6（IL-6）诱导。为了鉴定介导这种作用的增强子，我们将该基因的上游和下游侧翼序列克隆到含有junB启动子的荧光素酶报告基因载体中，并通过在IL-6依赖性浆细胞瘤细胞系中瞬时表达来评估这些序列的IL-6诱导作用。尽管上游侧翼序列的6.5内切酶片段不增加junB启动子的IL-6诱导作用，在下游侧翼序列的2.1个碱基对的片段中鉴定出既上调启动子又赋予IL-6诱导的222个碱基对片段。该区域内的急性时相反应因子（APRF）位点的点突变显著降低了在IL-6中连续生长的细胞中启动子的上调，以及在退出生长因子后用IL-6再刺激细胞时的诱导。与APRF位点共享5个核苷酸的NF-κ B位点的点突变降低了启动子的上调，但不受IL-6的诱导，而222个碱基对片段中的另外两个NF-κ B位点的突变对表达没有影响。通过DNA亲和层析纯化的核蛋白的Western印迹显示Stat 3的诱导性结合和NF-κ B p65与APRF/NF-κ B位点的组成性结合。
The immediate early gene, junB, is induced by interleukin-6 (IL-6) in plasmacytomas. In order to identify enhancers that mediate this effect, we cloned upstream and downstream sequences flanking the gene into a luciferase reporter gene vector containing the junB promoter and evaluated the IL-6 inducibility of these se quences by transient expression in an IL-6 dependent plasmacytoma cell line, Although a 6.5 kilobase fragment of upstream flanking sequence did not increase the IL-6 inducibility of the junB promoter, a 222-base pair fragment was identified in 2.1 kilobases of down stream flanking sequence that both up regulates the promoter and confers inducibility by IL-6. Point mutation of an acute phase response factor (APRF) site within this region significantly reduced up-regulation of the promoter in cells grown continuously in IL-6, as well as inducibility upon restimulation of cells with IL-6 after withdrawal from the growth factor. Point mutation of an NF-kappa B site sharing five nucleotides with the APRF site reduced up-regulation of the promoter but not inducibility by IL-6, whereas mutation of two other NF-kappa B sites in the 222-base pair fragment had no effect on expression. Western blotting of nuclear proteins purified by DNA affinity chromatography revealed inducible binding of Stat3 and constitutive binding of NF-kappa B p65 to the APRF/NF-kappa B site.