Genomic cloning and promoter analysis of macrophage inflammatory protein (MIP)-2, MIP-1 alpha, and MIP-1 beta, members of the chemokine superfamily of proinflammatory cytokines.
Genomic cloning and promoter analysis of macrophage inflammatory protein (MIP)-2, MIP-1 alpha, and MIP-1 beta, members of the chemokine superfamily of proinflammatory cytokines.
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DOI：
10.4049/jimmunol.150.11.4996
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发表时间：
1993-06
影响因子：
4.4
通讯作者：
U. Widmer;K. Manogue;A. Cerami;B. Sherry
中科院分区：
文献类型：
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作者：
U. Widmer;K. Manogue;A. Cerami;B. Sherry
巨噬细胞炎性蛋白(MIP)-2、MIP-1α和MIP-1β都属于新发现的具有炎症和生长调节活性的结构相关、活化诱导的“趋化因子”超家族。我们报道了小鼠MIP-2和小鼠MIP-1β的基因组克隆的分离和测序，并分析了它们之间以及与趋化因子家族其他几个成员的调控序列。小鼠(Mu)MIP-2基因组克隆显示了典型的四外显子/三内含子结构，这与趋化因子α亚家族中的其他基因(如IL-8)一样。近端启动子区域潜在的顺式调控元件在muMIP-2与其最接近的三个人类同源物：人(Hu)Gro-α、huGRO-β和huGRO-Gamma之间高度保守。以小鼠巨噬细胞系RAW 264.7为实验材料，构建了由mMIP-2 5‘端启动子控制的生长激素报告基因，通过嵌套缺失突变分析，将内毒素反应元件定位于转录起始点51~70bp5’端含有保守的核因子kappaB共同基序的区域。与MIP-2不同，muMIP-1β基因组克隆具有趋化因子β家族成员(如MCP-1)的三个外显子/两个内含子的结构特征。MuMIP-1β启动子和MuMIP-1α启动子的比较揭示了一个保守的CK-1元件，但在RAW 264.7巨噬细胞中的瞬时表达研究表明，MuMIP-1β或muMIP-1α5‘启动子的近端片段与人生长激素报告基因融合在一起，两者的启动子片段都与一致的CK-1序列附近的启动子片段有关，但不完全相同。从MIP-1α和MIP-1β基因克隆的近端5‘端启动子片段意外地在巨噬细胞样细胞中的融合报告基因序列上获得了结构性表达，但最初的5’端缺失分析没有将这种反应与已知的序列基序联系起来。在B16小鼠黑色素瘤细胞中，muMIP-1β启动子具有结构性活性，而在骨髓单核细胞系WEHI 3B(A)d-中两种启动子均具有活性，其中muMIP-1α启动子的强度是前者的3倍。
Macrophage inflammatory protein (MIP)-2, MIP-1 alpha, and MIP-1 beta all belong to the newly recognized "chemokine" superfamily of structurally related, activation-inducible cytokines with inflammatory and growth regulatory activities. We report the isolation and sequencing of genomic clones for murine MIP-2 and murine MIP-1 beta, and analyze their regulatory sequences in comparison with each other and with several other members of the chemokine family. The murine (mu)MIP-2 genomic clone displays the canonical four exon/three intron structure typical of other genes in the chemokine alpha subfamily (e.g., IL-8). Potential cis regulatory elements in the proximal promoter region were highly conserved between muMIP-2 and its three most closely related human homologs: human (hu)GRO-alpha, huGRO-beta, and huGRO-gamma. A mouse macrophage cell line, RAW 264.7, was transfected with a growth hormone reporter construct driven by a proximal fragment of the muMIP-2 5' promoter, and nested deletion mutant analysis localized the LPS responsive element to a region that contains a conserved NF kappa B consensus motif and lies 51 to 70 bp 5' from the transcription start site. In contrast to that of MIP-2, the muMIP-1 beta genomic clone exhibited the three exon/two intron structure characteristic of the chemokine beta family members (e.g., MCP-1). A comparison of the promoters for muMIP-1 beta and muMIP-1 alpha reveals a conserved CK-1 element, but transient expression studies in RAW 264.7 macrophages with proximal fragments of either the muMIP-1 beta or the muMIP-1 alpha 5' promoter fused to a human growth hormone reporter gene link LPS-inducibility in both to promoter segments near to, but not identical with, the consensus CK-1 sequence. Proximal 5' promoter fragments cloned from both the MIP-1 alpha and MIP-1 beta genes unexpectedly conferred constitutive expression on the fused reporter gene sequences in macrophage-like cells, but initial 5' deletion analysis did not link this responsiveness to known sequence motifs. The muMIP-1 beta promoter, but not the muMIP-1 alpha promoter, was constitutively active in B16 mouse melanoma cells, and both promoters were active in the myelomonocytic cell line WEHI 3B(A)d-, the muMIP-1 alpha promoter being three times stronger.