Attard F A, Wang L, Potter J J, Rennie-Tankersley L, Mezey E
Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Arch Biochem Biophys. 2000 Jun 1;378(1):57-64. doi: 10.1006/abbi.2000.1803.
Acetaldehyde was previously shown to activate the alpha1(I) and alpha2(I) collagen promoters and to increase collagen production in activated stellate cells. Also, CCAAT/enhancer binding protein beta (C/EBPbeta) binds and activates the mouse alpha1(I) collagen promoter. This study investigates the role of C/EBPbeta in mediating the activation of the alpha1(I) collagen promoter by acetaldehyde. Nuclear extracts isolated from cultured activated rat hepatic stellate cells formed four protein-DNA complexes on electrophoretic mobility shift assay with an oligonucleotide including the C/EBP binding site between -365 and -335 in the alpha1(I) collagen promoter. The four complexes were identified to represent C/EBPbeta binding to the oligonucleotide by supershift with C/EBPbeta antibody. The principal C/EBP isoform found in the nuclear extracts from stellate cells was C/EBPbeta, with very low amounts of C/EBPalpha detected. Acetaldehyde (200 microM) increased C/EBPbeta protein in stellate nuclear extracts, increased its binding to the promoter, and activated the alpha1(I) collagen promoter in transfected stellate cells. Mutation of the C/EBPbeta binding site markedly decreased nuclear protein binding. A transfected promoter, mutated at the C/EBP binding site, had decreased basal activity, was not activated by acetaldehyde, and was not activated when cotransfected with a C/EBPbeta expression vector. This study shows that C/EBPbeta is the predominant C/EBP isoform found in activated stellate cells and that increased C/EBPbeta protein and C/EBPbeta binding to a proximal C/EBP binding site in the promoter mediates the activating effect of acetaldehyde.
先前的研究表明，乙醛可激活α1（I）和α2（I）胶原蛋白启动子，并增加活化星状细胞中的胶原蛋白生成。此外，CCAAT/增强子结合蛋白β（C/EBPβ）可结合并激活小鼠α1（I）胶原蛋白启动子。本研究旨在探讨C/EBPβ在介导乙醛对α1（I）胶原蛋白启动子激活作用中的作用。从培养的活化大鼠肝星状细胞中分离得到的核提取物，在电泳迁移率变动分析中，与包含α1（I）胶原蛋白启动子中-365至-335之间C/EBP结合位点的寡核苷酸形成了四种蛋白质-DNA复合物。通过用C/EBPβ抗体进行超迁移，确定这四种复合物代表C/EBPβ与寡核苷酸的结合。在星状细胞核提取物中发现的主要C/EBP同工型是C/EBPβ，仅检测到极少量的C/EBPα。乙醛（200μM）增加了星状细胞核提取物中的C/EBPβ蛋白，增加了其与启动子的结合，并在转染的星状细胞中激活了α1（I）胶原蛋白启动子。C/EBPβ结合位点的突变显著降低了核蛋白结合。在C/EBP结合位点发生突变的转染启动子，其基础活性降低，未被乙醛激活，与C/EBPβ表达载体共转染时也未被激活。本研究表明，C/EBPβ是活化星状细胞中发现的主要C/EBP同工型，并且增加的C/EBPβ蛋白以及C/EBPβ与启动子近端C/EBP结合位点的结合介导了乙醛的激活作用。
