Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is transcribed into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar types, from one gene by alternative transcription using different first exons, Ia and Ib. We investigated the regulatory mechanisms of the expressions of the three types of PHGPx using promoter analysis with luciferase as the reporter gene and electrophoretical mobility shift analysis. Here we report a draft of the positive regulatory region and the core promoter regions of PHGPx in several cell lines. From promoter deletion analysis we identified the three distinct core regions of mitochondrial PHGPx, non-mitochondrial PHGPx and nucleolar PHGPx. The core promoter activity of non-mitochondrial PHGPx was high in L929 cells, but relatively low for mitochondrial and nucleolar PHGPx. We also identified the positive regulatory region of mitochondrial PHGPx by deletion and mutation analysis of 5'-flanking regions of mitochondrial PHGPx. This region could regulate the promoter activity of non-mitochondrial PHGPx; however, up-regulation by this region was normally suppressed by the upstream region in somatic cells. Electrophoretical mobility shift analysis demonstrated that a specific transcription factor complex bound to this region in adult testis, but not in young testis and different sizes of complexes bound to this region between testis and brain.

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.