Oxidative stress enhances AP-1 and NF-κB-mediated regulation of β(2)-glycoprotein I gene expression in hepatoma cells.
β(2)-Glycoprotein I (β(2)-GPI), also known as apolipoprotein H, is a plasma glycoprotein with poorly defined gene regulation. The aim of this study was to clarify the role of oxidative stress in β(2)-GPI gene regulation and determine the essential transcription element regulating β(2)-GPI expression. We demonstrate that expression of β(2)-GPI at the protein and mRNA levels was significantly elevated in Huh7 and HepG2 cells treated with 100 µM hydrogen peroxide (H(2)O(2)). To address the transcriptional mechanism of H(2)O(2)-mediated β(2)-GPI gene regulation, several promoter constructs were cloned and characterized by deletion assays. A region spanning from -2141 to -1419 (relative to the transcription start site), which contains two activator protein-1 (AP-1) sites (AP1-2 and AP1-3) and one nuclear factor-kappaB (NF-κB) site was found to be the main target site for up-regulation of β(2)-GPI promoter activity by oxidative stress. In addition, we found that H(2)O(2) stimulation enhanced the nuclear translocation of AP-1 and NF-κB subunits. Using an electrophoretic mobility shift assay, it was confirmed that nuclear protein binding to the AP1-2, AP1-3, and NF-κB sites was increased in Huh7 cells treated with H(2)O(2). Knockdown of the c-Jun, c-Fos, p65, and p50 genes using small interfering RNAs (siRNAs) further confirmed that AP-1 and NF-κB play an essential role in the H(2)O(2)-induced β(2)-GPI expression. Overall, these findings provide new insight suggesting that multiple cis-elements in the β(2)-GPI promoter work cooperatively to regulate β(2)-GPI expression in cells under oxidative stress.
Chiu
Wen-Chin
WC
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan, ROC.
Chen
Chun-Jung
CJ
Lee
Tzong-Shyuan
TS
Chen
Zit-Jie
ZJ
Ke
Pei-Hsin
PH
Chiang
An-Na
AN
D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D053482:beta 2-Glycoprotein I / Q000235:genetics* / Q000378:metabolism
D016328:NF-kappa B; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018808:Transcription Factor AP-1; D053482:beta 2-Glycoprotein I; D006861:Hydrogen Peroxide