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

Abstract

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.

Figure 1. Comparison of the TNFα-induced up-regulation of PHGPx mRNA expression between neutrophil-like differentiated and non-differentiated HL60 cells

(A) Non-differentiated HL60 (black bars) and neutrophil-like differentiated HL60 cells (grey bars) were incubated for 24 h in RPMI 1640 medium containing 10% (v/v) FBS with TNFα at the concentrations indicated. Total cellular RNA was isolated from the cells, and the mRNA levels of PHGPx and 18S rRNA were detected by quantitative PCR. The relative quantity of PHGPx mRNA was normalized with that of 18S rRNA. Results are means±S.D. of the percentage of the medium control. Data were analysed statistically by Dunnett's test (n=4, *P<0.05 significant increase against the medium control). Reproducibility of these results was confirmed in three experiments. (B) PMNs, non-differentiated, neutrophil-like (neutro-like) differentiated and macrophage-like (MΦ-like) differentiated HL60 cells, ECV304, A498 and HEK-293 cells were cultured for 24 h in RPMI 1640 medium containing 10% (v/v) FBS with or without 50 ng/ml TNFα. The RNA isolation and quantitative PCR were performed using the same methods described in (A). Reproducibility of these results was confirmed in three experiments. (C) Non-differentiated HL60 (black bars) and neutrophil-like HL60 cells (grey bars) were cultured for 24 h in the presence or absence of 50 ng/ml TNFα with or without 3 μM Bay 11-7082 (Bay), 1 nM PP2, 30 μM PD98059 (PD), 10 μM SB20358 (SB), 300 nM SP600125 (SP), 50 mM NAC or 50 μM PDTC. RNA isolation, quantitative PCR and data analysis were performed as described in (A). Reproducibility of these results was confirmed in three experiments. (D) Non-differentiated HL60 cells were cultured for 24 h in RPMI 1640 medium containing 10% (v/v) FBS with (black bars) or without (grey bars) 50 ng/ml TNFα. The RNA isolation was performed as described in (A). The mRNA level of non-mitochondrial and mitochondrial PHGPx was detected by TaqMan PCR with the relevant specific primer and probe. The relative quantity of each PHGPx mRNA was normalized with that of 18S rRNA. Results are means±S.D. of the percentage of the medium control. Data were analysed statistically by Student's t test (n=4, *P<0.05 significant increase compared with the medium control). Reproducibility of these results was confirmed in three experiments.

Figure 2. Functional analysis of the PHGPx promoter region in mice and humans

HL60 cells were transfected by electroporation with the constructed reporter vectors encoding the deletion mutants of the promoter region of the mouse (A) or human (B) PHGPx gene. Firefly luciferase activity of HL60 cells was measured after cultivating with (black bars) or without (grey bars) 50 ng/ml TNFα for 16 h. The phRL-TK vector was used as the internal control to determine transfection efficiency. Firefly luciferase activity from the luciferase reporter vector was normalized to the renilla luciferase activity from the phRL-TK vector, and this was expressed as a ratio of the activity of the pGL3-Basic vector without the PHGPx promoter region. Results are means±S.D. The transcriptional start sites for mitochondrial and non-mitochondrial PHGPx are shown as inverted open and closed triangles respectively. Reproducibility of these results was confirmed in three experiments.

Figure 3. Mutational analysis of C/EBP- and NF-Y-binding sequences in the promoter region of the human PHGPx gene

(A) Sequences that were highly conserved between human and mouse promoter regions are represented as closed circles. When the sequence of the responsible promoter region from −158 to −137 in humans (black square) was compared with that from −113 to −91 in mice (grey square), highly homologous sequences and binding domains for C/EBP and NF-Y (CCAAT box) were found in the regions. Asteriks indicate identical bases between mouse and human PHGPx. (B) HL60 cells were transfected with the human H3 reporter vector with a mutation in the region responsible for the TNFα-induced up-regulation of PHGPx promoter activity (bold and underlined). Firefly luciferase activity of HL60 cells was measured after incubation for 16 h with (black bars) or without (grey bars) 50 ng/ml TNFα. The phRL-TK vector was used as the internal control to determine transfection efficiency. Firefly luciferase activity from the reporter vector was normalized to the renilla luciferase activity from the phRL-TK vector. Results are means±S.D. of the percentage of the non-stimulated control. Reproducibility of these results was confirmed in three experiments.

Figure 4. Effects of signalling pathway inhibitors on TNFα-induced up-regulation of PHGPx promoter activity

HL60 cells were transfected with the human H3 reporter vector. Firefly luciferase activity was measured in HL60 cells cultured for 16 h with (black bars) or without (grey bars) 50 ng/ml TNFα in the presence or absence of NAC (50 mM), Bay 11-7082 (1 μM) or PP2 (10 nM). The phRL-TK vector was used as the internal control to determine transfection efficiency. Firefly luciferase activity from the reporter vector was normalized to the renilla luciferase activity from the phRL-TK vector. Results are means±S.D. of the percentage of the non-stimulated control. Reproducibility of these results was confirmed in three experiments.

Figure 5. Binding of C/EBPϵ to the promoter region of the human PHGPx gene

(A) HL60 cells were incubated with or without 50 ng/ml TNFα for 8 h, and nuclear protein was then extracted. Proteins binding to the DNA probe containing the C/EBP-binding sequence were detected by EMSA. To identify a protein binding to the probe, anti-C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ or C/EBPϵ antibody or the non-radiolabelled probe was mixed and incubated for 1 h at 4 °C with the nuclear protein extract before performing the binding reaction with the radiolabelled probe as a competition experiment. The arrowhead indicates DNA–protein complex. Reproducibility of these results was confirmed in two experiments. (B) HL60 cells incubated for 8 h with or without 50 ng/ml TNFα, fixed with 1% (v/v) formaldehyde, lysed and subjected to immunoprecipitation using the anti-C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ or C/EBPϵ antibody. DNA was released from immunoprecipitates by proteinase K treatment, and the DNA was amplified by PCR using primers corresponding to the promoter region from −247 to −34 of the PHGPx gene. Reproducibility of these results was confirmed in two experiments.

Figure 6. Expression level of C/EBPϵ mRNA in various cell lines, and TNFα-induced up-regulation of PHGPx mRNA expression in HEK-293 cells overexpressing C/EBPϵ

(A) Total cellular RNA was isolated from the cells, and the mRNA levels of C/EBPϵ, CREB and NF-Y were detected by RT-PCR. After PCR, 10 μl of the PCR products were subjected to electrophoresis and the DNA was visualized by ethidium bromide staining. Reproducibility of these results was confirmed in three experiments. (B) HEK-293 cells transiently overexpressing a transcription factor (NF-Y, CREB, NF-κB, C/EBPϵ, C/EBPδ or C/EBPγ) were incubated for 24 h with (black bars) or without (grey bars) 50 ng/ml TNFα. Total cellular RNA was isolated from the cells, and mRNA levels of PHGPx and 18S rRNA as an internal control, were detected by quantitative PCR. Results are means±S.D. of the percentage of the vector control incubated without TNFα. Reproducibility of these results was confirmed in two experiments.