Identification of a responsible promoter region and a key transcription factor, CCAAT/enhancer-binding protein epsilon, for up-regulation of PHGPx in HL60 cells stimulated with TNF alpha.
In the present study we investigated promoter regions of the PHGPx [phospholipid hydroperoxide GPx (glutathione peroxidase)] gene and transcription factors involved in TNFalpha (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 TNFalpha stimulation. The promoter activity was up-regulated by TNFalpha 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/EBPepsilon bound to the -247 to -34 region in HL60 cells, but C/EBPalpha, beta, gamma and delta did not. The binding of C/EBPepsilon to the promoter region was increased in HL60 cells stimulated with TNFalpha 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 TNFalpha, and it was inhibited by pre-treatment with an anti-C/EBPepsilon antibody, but not with other antibodies. The C/EBPepsilon mRNA was expressed in PMNs (polymorphonuclear cells), non-differentiated HL60 cells and neutrophil-like differentiated HL60 cells displaying TNFalpha-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/EBPepsilon as a result of TNFalpha stimulation. These results indicate that C/EBPepsilon is a critical transcription factor in TNFalpha-induced up-regulation of PHGPx expression.
Hattori
Hiroyuki
H
School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
Imai
Hirotaka
H
Kirai
Nozomu
N
Furuhama
Kazuhisa
K
Sato
Osamu
O
Konishi
Kumiko
K
Nakagawa
Yasuhito
Y
D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D005979:Glutathione Peroxidase / Q000096:biosynthesis* / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D011401:Promoter Regions, Genetic / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D015854:Up-Regulation / Q000235:genetics / Q000502:physiology*
D022762:CCAAT-Enhancer-Binding Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C070790:CEBPE protein, human; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase
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