ROS Regulates Inflammation-related Genes Through ZNF580 In Human Endothelial Cells

Objective: To investigated the potential mechanisms of ZNF580 in H₂O₂ regulated inflammation-related signaling pathway in human endothelial cells.Methods:1 The human endothelial cell hybridoma line EAhy926 cells were cultured with high glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin–streptomycin in 5% CO₂ at 37°C.2 LDH assay quantifies cell death based on measuring the release of LDH in supernatant, which indicates the loss of membrane integrity. LDH is the most important inducer for endothelial cell in inflammation.Cells were plated in a 24-well plate and incubated with different concentrations of H₂O₂ (0-600μM) and cells were incubated further for 6 h. The concentration of LDH in the supernatant was measured.3 The expression of ZNF580 in response to different concentrations of H₂O₂ (0μM,50μM,100μM,200μM,300μM,400μM) were detected by PCR and Western blot.4 The expression of ZNF580 in response to same concentration of H₂O₂ (100μM) were detected by RT-PCR and Western blot at different time points(0h,0.5h,1h,2h,4h,6h).5 To evaluate the effect of H₂O₂ on the nuclear translocation of p65 was examined using an immunocytochemistry, the role of H₂O₂ in the activation of NF-κB, a NF-κB well-known chemical inhibitor, pyrrolidine dithiocarbamate (PDTC) was used.6 To determine whether the NF-κB pathway is implicated in the upregulation of ZNF580 expression by H₂O₂, EAhy926 cells were pretreated with or without PDTC, the expression levels of ZNF580 were determined by RT-PCR and Western blot.7 To examine whether ZNF580 is necessary for inflammation, the EAhy926 endothelial cells were transiently transfected with empty vector pEGFP-C1 or pEGFP-ZNF580 by Lipofectamine 2000. Overexpression of ZNF580 was confirmed by fluorescence microscope, RT-PCR and Western blot.8 To observe the different expression of IL-8 in cells that overexpressed ZNF580,The RNA was isolated from the transfected cells and IL-8 was assessed by real-time PCR. Supernatants from cells were analyzed to measure secreted IL-8 by ELISA.9 THP-1 cells were cultured with RPMI 1640 medium. All the cells were cultured containing 10% fetal bovine serum and 1% penicillin–streptomycin in 5% CO₂ at 37°C. THP-1 monocytic cell migration in response to EAhy926 cells and pEGFP-ZNF580 cells was analyzed by transwell assasy.Results:1 After treatment with H₂O₂ at 0–600μM, cells were tested in the LDH release assay. The release of LDH was correlated with H₂O₂. The release of LDH peaked with concentrations of H₂O₂ up to 400μM.2 PCR and Western blot detections revealed that ZNF580 was highly expression induced by H₂O₂ in different concentrations (50–400μM).3 ZNF580 was upregulated by H₂O₂ at different time points. Expression was enhanced after 30 minutes treatment with H₂O₂, it peaked at 1 h in a concentration of 100μM and was sustained at a high level for 6 h (P<0.05).4 In untreated cells, p65 primarily existed in the cytoplasm and not in the nucleus. Following treatment with H₂O₂, there was an increase in the accumulation of p65 in the nucleus. After treament with H₂O₂, the translocation of p65 was blocked by PDTC(100μM). the treatment with PDTC and DMSO did not markedly induce NF-κB activation after 1 h of stimulation.5 Results showed that PDTC significantly suppressed H₂O₂ -induced upregulation of ZNF580 expression both in mRNA and protein levels6 The plasmid transfected with ZNF580 caused a significant increase in ZNF580 expression(P<0.01)。7 Transient transfection of ZNF580 results in the increase of the pro-inflammatory cytokines IL-8, along with a enhancing the motility of THP-1 cells significantly (P<0.01)。Conclusions:ZNF580, a new found C2H2 zinc finger transcription factor, was first described by our laboratory. Emerging evidence suggests that H₂O₂ plays an important role in redox sensitive signal transduction. Here, we investigated the potential mechanisms of ZNF580 in H₂O₂ regulated inflammation-related signaling pathway in human endothelial cells. Therefore, we concluded that H₂O₂ upregulated expression of ZNF580 via NF-κB signaling pathway and ZNF580 contributed to the production of inflammatory cytokine IL-8 .