Xu Yong, Porntadavity Sureerut, St Clair Daret K
Graduate Center for Toxicology, University of Kentucky, 361 Health Sciences Research Building, Lexington KY 40536, USA.
Biochem J. 2002 Mar 1;362(Pt 2):401-12. doi: 10.1042/0264-6021:3620401.
Manganese superoxide dismutase (MnSOD) plays an important role in regulating cellular redox conditions. Expression of MnSOD has been shown to protect against damage by oxidative stress and to suppress the malignant phenotype of human cancer cells. We have previously cloned the human MnSOD (SOD2) gene and analysed its 5' proximal promoter, which has been characterized by a lack of a TATA or CAAT box and the presence of multiple GC boxes. To define further the molecular mechanisms for the regulation of MnSOD expression, multiple transcription factor-binding motifs containing overlapping specificity protein 1 (Sp1)- and activator protein (AP)-2-binding sites were identified by DNase I footprinting analysis. Functional studies in three cell lines with different levels of Sp1 and AP-2 proteins suggested that the cellular levels of these proteins may differentially regulate transcription via GC-binding motifs in the human SOD2 promoter. Co-transfection of an Sp1 expression vector resulted in an increase in the transcription of the promoter-driven reporter gene. In contrast, co-transfection of the AP-2 expression vector caused a decrease in transcription. Direct mutagenesis analysis of Sp1- and AP-2-binding sites showed that Sp1 is essential for transcription of the human SOD2 gene, whereas AP-2 plays a negative role in the transcription. Immunoprecipitation of Sp1 and AP-2 proteins demonstrated that Sp1 interacts with AP-2 in vivo. Two-hybrid analysis revealed that interaction between Sp1 and AP-2 plays both a positive and negative role in the transcription of the reporter gene in vivo. Taken together, our data indicate that AP-2 down-regulates transcription of the human SOD2 gene via its interaction with Sp1 within the promoter region. These findings, coupled with our previous observation that several cancer cell lines have mutations in the promoter region of the human MnSOD gene, which lead to an increase in an AP-2-binding site and a decrease in the promoter activity, signal the importance of understanding the promoter structure and the regulation of the human SOD2 gene by Sp1 and AP-2.
锰超氧化物歧化酶（MnSOD）在调节细胞氧化还原状态中起重要作用。已表明MnSOD的表达可保护细胞免受氧化应激损伤，并抑制人类癌细胞的恶性表型。我们之前克隆了人类MnSOD（SOD2）基因并分析了其5'近端启动子，该启动子的特点是缺乏TATA或CAAT框以及存在多个GC框。为了进一步确定MnSOD表达调控的分子机制，通过DNA酶I足迹分析鉴定了多个包含重叠特异性蛋白1（Sp1）和激活蛋白（AP）-2结合位点的转录因子结合基序。在三种具有不同水平Sp1和AP-2蛋白的细胞系中进行的功能研究表明，这些蛋白的细胞水平可能通过人类SOD2启动子中的GC结合基序差异调节转录。Sp1表达载体的共转染导致启动子驱动的报告基因转录增加。相反，AP-2表达载体的共转染导致转录减少。对Sp1和AP-2结合位点的直接诱变分析表明，Sp1对人类SOD2基因的转录至关重要，而AP-2在转录中起负作用。Sp1和AP-2蛋白的免疫沉淀表明Sp1在体内与AP-2相互作用。双杂交分析显示，Sp1和AP-2之间的相互作用在体内报告基因的转录中起正负双重作用。综上所述，我们的数据表明AP-2通过其与启动子区域内的Sp1相互作用下调人类SOD2基因的转录。这些发现，再加上我们之前的观察结果，即几种癌细胞系在人类MnSOD基因的启动子区域存在突变，导致AP-2结合位点增加和启动子活性降低，表明了解启动子结构以及Sp1和AP-2对人类SOD2基因的调控的重要性。
