Inhibition of TNF-α-induced NF-κB activation and IL-8 release in A549 cells with the proteasome inhibitor MG-132
Inhibition of TNF-α-induced NF-κB activation and IL-8 release in A549 cells with the proteasome inhibitor MG-132
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DOI：
10.1165/ajrcmb.19.2.3149
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发表时间：
1998-08-01
影响因子：
6.4
通讯作者：
Stark, JM
中科院分区：
文献类型：
--
作者：
Fiedler, MA;Wernke-Dollries, K;Stark, JM
本文所述研究的工作假设是，抑制蛋白酶体介导的I κ B降解将抑制TNF-α诱导的核因子-κ B（NF-κ B）活化、白细胞介素-8（IL-8）基因转录和A549细胞中IL-8蛋白的释放。IL-8基因5'侧翼区的突变分析证实，完整的NF-κ B位点对于TNF-α诱导的IL-8基因转录是必需的。向A549细胞中加入TNF-α导致I κ B从细胞质中快速丢失，与细胞核提取物中NF-κ B结合活性的相应增加相关。然而，用蛋白酶体抑制剂N-cbz-Leu-Leu-leucinal（MG-132，10 μ M）预处理细胞，逆转了TNF-α对A549细胞释放IL-8（用酶联免疫吸附试验[ELISA]测定）和IL-8基因转录（用转录因子基因试验测定）的影响。MG-132逆转TNF-α对I κ B降解的影响，如通过蛋白质印迹分析所确定的。I κ B磷酸化和泛素化未被MG-132改变，这意味着MG-132的作用继发于蛋白酶体抑制。MG-132还逆转了TNF-α处理细胞的核提取物中NF-κ B结合的增加。这些研究表明，蛋白酶体介导的I κ B降解的抑制通过限制NF-κ B介导的基因转录导致TNF-α诱导的A549细胞中IL-8产生的抑制。
The working hypothesis of the studies described herein was that inhibition of proteasome-mediated I kappa B degradation would inhibit TNF-alpha-induced nuclear factor-kappa B (NF-kappa B) activation, interleukin-8 (IL-8) gene transcription, and IL-8 protein release in A549 cells. Mutational analysis of the 5' flanking region of the IL-8 gene confirmed that an intact NF-kappa B Site is necessary for TNF-alpha-induced IL-8 gene transcription. The addition of TNF-alpha to A549 cells resulted in rapid loss of I kappa B from the cytoplasm of cells, associated with a corresponding increase in NF-kappa B-binding activity in nuclear extracts from the cells. However, pretreatment of the cells with the proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132, 10 mu M) reversed the effects of TNF-alpha on IL-8 release from A549 cells (as determined with an enzyme-linked immunosorbent assay [ELISA]) and on IL-8 gene transcription (as determined with reporter-gene assays). MG-132 reversed the effects of TNF-alpha on I kappa B degradation as determined by Western blot analysis. I kappa B phosphorylation and ubiquination were not altered by MG-132, which implies that the effects of MG-132 were secondary to proteasome inhibition. MG-132 also reversed the increase in NF-kappa B binding in nuclear extracts from TNF-alpha-treated cells. These studies show that inhibition of proteasome-mediated I kappa B degradation results in inhibition of TNF-alpha induced IL-8 production in A549 cells by limiting NF-kappa B-mediated gene transcription.