Media type: E-Article Title: Oxidative stress enhances AP‐1 and NF‐κB‐mediated regulation of β2‐Glycoprotein I gene expression in hepatoma cells Contributor: Chiu, Wen‐Chin; Chen, Chun‐Jung; Lee, Tzong‐Shyuan; Chen, Zit‐Jie; Ke, Pei‐Hsin; Chiang, An‐Na imprint: Wiley, 2010 Published in: Journal of Cellular Biochemistry Language: English DOI: 10.1002/jcb.22787 ISSN: 0730-2312; 1097-4644 Keywords: Cell Biology ; Molecular Biology ; Biochemistry Origination: Footnote: Description: <jats:title>Abstract</jats:title><jats:p>β<jats:sub>2</jats:sub>‐Glycoprotein I (β<jats:sub>2</jats:sub>‐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 <jats:italic>β</jats:italic><jats:sub><jats:italic>2</jats:italic></jats:sub><jats:italic>‐GPI</jats:italic> gene regulation and determine the essential transcription element regulating <jats:italic>β</jats:italic><jats:sub><jats:italic>2</jats:italic></jats:sub><jats:italic>‐GPI</jats:italic> expression. We demonstrate that expression of β<jats:sub>2</jats:sub>‐GPI at the protein and mRNA levels was significantly elevated in Huh7 and HepG2 cells treated with 100 µM hydrogen peroxide (H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>). To address the transcriptional mechanism of H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>‐mediated <jats:italic>β</jats:italic><jats:sub><jats:italic>2</jats:italic></jats:sub><jats:italic>‐GPI</jats:italic> 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 <jats:italic>β</jats:italic><jats:sub><jats:italic>2</jats:italic></jats:sub><jats:italic>‐GPI</jats:italic> promoter activity by oxidative stress. In addition, we found that H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> 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<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>. Knockdown of the <jats:italic>c‐Jun</jats:italic>, <jats:italic>c‐Fos</jats:italic>, <jats:italic>p65</jats:italic>, and <jats:italic>p50</jats:italic> genes using small interfering RNAs (siRNAs) further confirmed that AP‐1 and NF‐κB play an essential role in the H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>‐induced β<jats:sub>2</jats:sub>‐GPI expression. Overall, these findings provide new insight suggesting that multiple <jats:italic>cis</jats:italic>‐elements in the <jats:italic>β</jats:italic><jats:sub><jats:italic>2</jats:italic></jats:sub><jats:italic>‐GPI</jats:italic> promoter work cooperatively to regulate β<jats:sub>2</jats:sub>‐GPI expression in cells under oxidative stress. J. Cell. Biochem. 111: 988–998, 2010. © 2010 Wiley‐Liss, Inc.</jats:p>