Miao K, Potter J J, Anania F A, Rennie-Tankersley L, Mezey E
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2195, USA.
Arch Biochem Biophys. 1997 May 1;341(1):140-52. doi: 10.1006/abbi.1997.9948.
Acetaldehyde activates the mouse alpha 2(I) collagen promoter and this effect is mediated in part by increased binding of nuclear factor I (NF-I). Additional mechanisms may exist since deletions in the promoter upstream to the NF-I binding site prevented enhancement by acetaldehyde. Three adjacent areas of binding by nuclear proteins from activated hepatic stellate cells were identified at -568 to -554 (region 1), -542 to -518 (region 2), and -473 to -453 (region 3) of the promoter using DNase I protection analyses. Multiple DNA-protein complexes were formed in electrophoretic mobility shift assays with oligonucleotide probes specifying the three regions. Sp1 and NF-1 bound to all three regions, while Sp3 bound to region 2. Acetaldehyde decreased nuclear protein binding to all three regions. Mutations of regions 1, 2, and 3 reduced basal activity of the promoter and inhibited acetaldehyde stimulation in transfected stellate cells. Acetaldehyde inhibited the stimulatory effect of the Sp1 vector pPacSp1 on the promoter in transfected Drosophila cells. In conclusion, three regions of Sp1 binding were identified and are required for optimal activity of the alpha 2(I) collagen promoter. Sp1 is required for basal activity of the alpha 2(I) collagen promoter; however, the enhancing effect of acetaldehyde on the promoter is not mediated by Sp1.
乙醛可激活小鼠α2（I）胶原启动子，且这一效应部分是由核因子I（NF-I）结合增加所介导的。由于NF-I结合位点上游启动子区域的缺失可阻止乙醛的增强作用，因此可能存在其他机制。利用DNA酶I保护分析，在启动子的-568至-554（区域1）、-542至-518（区域2）和-473至-453（区域3）处鉴定出了来自活化肝星状细胞的核蛋白的三个相邻结合区域。在电泳迁移率变动分析中，使用指定这三个区域的寡核苷酸探针形成了多个DNA-蛋白质复合物。Sp1和NF-1与所有三个区域结合，而Sp3与区域2结合。乙醛减少了核蛋白与所有三个区域的结合。区域1、2和3的突变降低了启动子的基础活性，并抑制了转染星状细胞中乙醛的刺激作用。乙醛抑制了转染果蝇细胞中Sp1载体pPacSp1对启动子的刺激作用。总之，已鉴定出Sp1结合的三个区域，它们是α2（I）胶原启动子最佳活性所必需的。Sp1是α2（I）胶原启动子基础活性所必需的；然而乙醛对启动子的增强作用不是由Sp1介导的。
