Identification of a responsible promoter region and a key transcription factor, CCAAT/enhancer-binding protein ε, for up-regulation of PHGPx in HL60 cells stimulated with TNFα
Identification of a responsible promoter region and a key transcription factor, CCAAT/enhancer-binding protein ε, for up-regulation of PHGPx in HL60 cells stimulated with TNFα
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DOI：
10.1042/bj20070245
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发表时间：
2007-12-01
影响因子：
4.1
通讯作者：
Nakagawa, Yasuhito
中科院分区：
文献类型：
--
作者：
Hattori, Hiroyuki;Imai, Hirotaka;Nakagawa, Yasuhito
在本研究中，我们研究了PHGPx[磷脂氢过氧化物GPx（谷胱甘肽过氧化物酶）]基因的启动子区域和参与TNF α（肿瘤坏死因子α）诱导的未分化HL60细胞中PHGPx上调的转录因子。未分化的HL60细胞在TNF α刺激下表现出非线粒体和线粒体PHGPx mRNA的上调。在转染了编码人PHGPx基因-282至-123区域的荧光素酶报告载体的细胞中，与未受刺激的对照相比，TNF - α刺激使启动子活性上调。该区域C/EBP （CCAAT/增强子结合蛋白）结合序列的突变有效地消除了上调的启动子活性。ChIP（染色质免疫沉淀）实验表明，C/EBP epsilon在HL60细胞中与-247至-34区结合，但C/EBP α、β、γ和δ不与-247至-34区结合。与未受TNF α刺激的对照组相比，受TNF α刺激的HL60细胞中C/EBP epsilon与启动子区域的结合增加。在TNF α刺激的细胞中，EMSA观察到核蛋白与C/EBP结合序列的结合增加，并且用抗C/EBP epsilon抗体预处理可以抑制这种结合，但其他抗体不能抑制。C/EBP epsilon mRNA在PMNs（多形核细胞）、未分化的HL60细胞和中性粒细胞样分化的HL60细胞中表达，显示TNF α诱导的PHGPx mRNA上调，但在巨噬细胞样分化的HL60细胞、HEK-293细胞（人胚胎肾-293细胞）和其他细胞系中不表达，未显示上调。然而，由于TNF α刺激，在过表达C/EBP epsilon的HEK-293细胞中检测到PHGPx mRNA的上调。这些结果表明，C/EBP epsilon是TNF α诱导的PHGPx表达上调的关键转录因子。
In the present study we investigated promoter regions of the PHGPx [phospholipid hydroperoxide GPx (glutathione peroxidase)] gene and transcription factors involved in TNF alpha (tumour necrosis factor alpha)-induced up-regulation of PHGPx in non-differentiated HL60 cells. Non-differentiated HL60 cells displayed up-regulation of non-mitochondrial and mitochondrial PHGPx mRNA in response to TNF alpha stimulation. The promoter activity was up-regulated by TNF alpha stimulation in cells transfected with a luciferase reporter vector encoding the region from -282 to -123 of the human PHGPx gene compared with the non-stimulated control. The up-regulated promoter activity was effectively abrogated by a mutation in the C/EBP (CCAAT/enhancer-binding protein)-binding sequence in this region. ChIP (chromatin immunoprecipitation) assays demonstrated that C/EBP epsilon bound to the -247 to -34 region in HL60 cells, but C/EBP alpha, beta, gamma and delta did not. The binding of C/EBP epsilon to the promoter region was increased in HL60 cells stimulated with TNF alpha compared with that of the non-stimulated control. An increased binding of nuclear protein to the C/EBP-binding sequence was observed by EMSA (electrophoretic mobility-shift assay) in cells stimulated with TNF alpha, and it was inhibited by pre-treatment with an anti-C/EBP epsilon antibody, but not with other antibodies. The C/EBP epsilon mRNA was expressed in PMNs (polymorphonuclear cells), non-differentiated HL60 cells and neutrophil-like differentiated HL60 cells displaying TNF alpha-induced up-regulation of PHGPx mRNA, but not in macrophage-like differentiated HL60 cells, HEK-293 cells (human embryonic kidney-293 cells) and other cell lines exhibiting no up-regulation. The up-regulation of PHGPx mRNA, however, was detected in HEK-293 cells overexpressing C/EBP epsilon as a result of TNF alpha stimulation. These results indicate that C/EBP epsilon is a critical transcription factor in TNF alpha -induced up-regulation of PHGPx expression.