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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Abstract
In the present study we investigated promoter regions of the PHGPx [phospholipid hydroperoxide GPx (glutathione peroxidase)] gene and transcription factors involved in TNFα (tumour necrosis factor α)-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 TNFα stimulation. The promoter activity was up-regulated by TNFα 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/EBPϵ bound to the −247 to −34 region in HL60 cells, but C/EBPα, β, γ and δ did not. The binding of C/EBPϵ to the promoter region was increased in HL60 cells stimulated with TNFα 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 TNFα, and it was inhibited by pre-treatment with an anti-C/EBPϵ antibody, but not with other antibodies. The C/EBPϵ mRNA was expressed in PMNs (polymorphonuclear cells), non-differentiated HL60 cells and neutrophil-like differentiated HL60 cells displaying TNFα-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/EBPϵ as a result of TNFα stimulation. These results indicate that C/EBPϵ is a critical transcription factor in TNFα-induced up-regulation of PHGPx expression.