| Summary: | 博士 === 國立陽明大學 === 生化暨分子生物研究所 === 100 === β2-glycoprotein I (β2-GPI) is a plasma glycoprotein with diverse biological functions; however, the molecular mechanisms of its biological action remain unknown. The aim of this study was to assess the contribution of β2-GPI to human aortic endothelial cell (HAEC) migration and the details of its underlying mechanism. Using wound healing and Boyden chamber assays, we found that β2-GPI inhibited endothelial cell migration, which was restored by neutralizing antibody. Nuclear factor-kB (NF-kB) inhibitors (QNZ, PDTC, BAY11-7082) and lentiviral small interfering RNAs (siRNAs) silencing of NF-kB were used to address the molecular mechanism by which β2-GPI reduced cell migration. NF-kB inhibitors and p65 or p50 siRNAs significantly attenuated the inhibitory effect of β2-GPI on cell migration. Moreover, β2-GPI was found to induce phosphorylation of inhibitor of NF-kB (IkBa), IkBa degradation, and translocation of p65 and p50. We also demonstrated that mRNA and protein levels of endothelial nitric oxide synthase (eNOS) as well as nitric oxide (NO) production were all increased by β2-GPI in HAECs. The increase in eNOS expression and NO generation was remarkably inhibited by NF-kB inhibitors and siRNAs of p65 and p50. Furthermore, β2-GPI-mediated inhibition of cell migration was reversed by eNOS inhibitors (L-NAME, L-NMMA) and eNOS siRNAs. Our findings provide a novel insight that β2-GPI could inhibit endothelial cell migration predominantly through NF-kB/eNOS/NO signaling pathway. These results suggest that β2-GPI may be a potential component for clinical therapy in vascular diseases.
β2-GPI is primarily synthesized in the liver, however, the regulation of β2-GPI gene expression in cell remains unknown. Another aim of this study was to clarify the role of oxidative stress in β2-GPI gene regulation and determine the essential transcription element regulating β2-GPI expression. We demonstrate that expression of β2-GPI was significantly elevated in Huh7 and HepG2 cells treated with 100 µM hydrogen peroxide (H2O2). A region spanning from –2141 to –1419 (relative to the transcription start site) containing two activator protein-1 (AP-1) sites (AP1-2 and AP1-3) and one NF-kB site was found to be the main target sites for up-regulation of β2-GPI expression by oxidative stress. In addition, we found that H2O2 stimulation enhanced the nuclear translocation of AP-1 and NF-kB subunits. Using an electrophoretic mobility shift assay, it was confirmed that nuclear protein binding to the AP1-2, AP1-3, and NF-kB sites was increased in Huh7 cells treated with H2O2. Knockdown of the c-Jun, c-Fos, p65, and p50 genes using siRNAs further confirmed that AP-1 and NF-kB play an essential role in the H2O2-induced β2-GPI expression. Overall, these findings demonstrate the involvement of oxidative stress in β2-GPI regulation and explore several regulatory elements in the promoter region of β2-GPI gene.
|
|---|
