Neutrophil elastase stimulates MUC1 gene expression through increased Sp1 binding to the MUC1 promoter
Neutrophil elastase stimulates MUC1 gene expression through increased Sp1 binding to the MUC1 promoter
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DOI：
10.1152/ajplung.00040.2005
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发表时间：
2005-08-01
影响因子：
4.9
通讯作者：
Kim, KC
中科院分区：
文献类型：
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作者：
Kuwahara, I;Lillehoj, EP;Kim, KC
我们先前报道MUC 1是铜绿假单胞菌的细胞表面受体，并且通过用中性粒细胞弹性蛋白酶（NE）预处理显著降低了细菌与细胞的结合（Lillehoj EP，Hyun SW，Kim BT，Zhang XG，Lee DI，Rowland S，and Kim KC. Am J Physiol Lung Cell Mol Physiol 280：L181-L187，2001）。本研究旨在探讨NE对MUC 1基因转录、MUC 1蛋白合成和降解的影响。用NE处理的A549人肺癌细胞与仅用载体处理的细胞相比，在去污剂裂解物中表现出显著更高的MUC 1蛋白水平。此外，脱落到细胞条件培养基中的MUC 1蛋白被NE迅速完全降解。放线菌素D阻断NE刺激的MUC 1蛋白表达的增加，这表明增加基因转录的机制，通过测量NE处理的细胞与对照组相比MUC 1 mRNA水平的定量增加来证实。然而，NE没有改变MUC 1 mRNA的稳定性，这意味着增加从头转录诱导的蛋白酶。NE可增强MUC 1基因启动子-荧光素酶报告质粒转染的A549细胞的启动子活性。NE的这一作用可被Sp1抑制剂光辉霉素A完全阻断，同时也可被MUC 1启动子中位于转录起始位点-99/-90的Sp1结合位点突变所阻断。EMSA显示NE以时间依赖性方式增强Sp1与该10-bp片段的结合。这些结果表明NE通过增加Sp1与MUC 1启动子-99/-90片段的结合来介导MUC 1基因转录的增加。
We previously reported MUC1 was a cell surface receptor for Pseudomonas aeruginosa, and binding of bacteria to cells was significantly reduced by pretreatment with neutrophil elastase (NE)(Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, and Kim KC. Am J Physiol Lung Cell Mol Physiol 280: L181-L187, 2001). The current study was conducted to ascertain NE effects on MUC1 gene transcription, and MUC1 protein synthesis and degradation. A549 human lung carcinoma cells treated with NE exhibited significantly higher MUC1 protein levels in detergent lysates compared with cells treated with vehicle alone. Also, MUC1 protein shed into cell-conditioned medium was rapidly and completely degraded by NE. Actinomycin D blocked NE-stimulated increase in MUC1 protein expression, suggesting a mechanism of increased gene transcription that was confirmed by measurement of quantitatively greater MUC1 mRNA levels in NE-treated cells compared with controls. However, NE did not alter MUC1 mRNA stability, implying increased de novo transcription induced by the protease. NE increased promoter activity in A549 cells transfected with MUC1 gene promoter-luciferase reporter plasmid. This effect of NE was completely blocked by mithramycin A, an inhibitor of Sp1, as well as mutation of one of the putative Sp1 binding sites in MUC1 promoter located at -99/-90 relative to transcription initiation site. EMSA revealed NE enhanced binding of Sp1 to this 10-bp segment in a time-dependent manner. These results indicate the increase in MUC1 gene transcription by NE is mediated through increase in Sp1 binding to -99/-90 segment of MUC1 promoter.