Porntadavity S, Xu Y, Kiningham K, Rangnekar V M, Prachayasittikul V, St Clair D K
Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536, USA.
DNA Cell Biol. 2001 Aug;20(8):473-81. doi: 10.1089/104454901316976109.
Induction of manganese superoxide dismutase (MnSOD) in response to oxidative stress has been well established in animals, tissues, and cell culture. However, the role of the human MnSOD (hMnSOD) promoter in stimulus-dependent activation of transcription is unknown. The hMnSOD promoter lacks both a TATA and a CAAT box but possesses several GC motifs. In a previous study, we showed that the basal promoter contains multiple Sp1 and AP-2 binding sites and that Sp1 is essential for the constitutive expression of the hMnSOD gene. In this study, we identified an Egr-1 binding site in the basal promoter of hMnSOD. We also found that the basal promoter is responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated hMnSOD transcription in the human hepatocarcinoma cell line HepG2. The contributions of these binding sites and the roles of the transcription factors Egr-1, AP-2, and Sp1 in the activation of hMnSOD transcription by TPA were investigated by site-directed mutation analysis, Western blotting, and overexpression of transcription factors. The results showed that Sp1 plays a positive role for both basal and TPA-activated hMnSOD transcription, whereas overexpression of Egr-1 has a negative role in the basal promoter activity without any effect on TPA-mediated activation of hMnSOD transcription.
在动物、组织和细胞培养中，锰超氧化物歧化酶（MnSOD）对氧化应激的诱导作用已得到充分证实。然而，人类MnSOD（hMnSOD）启动子在刺激依赖性转录激活中的作用尚不清楚。hMnSOD启动子既缺乏TATA盒也缺乏CAAT盒，但含有几个GC基序。在先前的一项研究中，我们表明基础启动子包含多个Sp1和AP-2结合位点，并且Sp1对于hMnSOD基因的组成型表达至关重要。在本研究中，我们在hMnSOD的基础启动子中鉴定出一个Egr-1结合位点。我们还发现基础启动子对人肝癌细胞系HepG2中12-O-十四烷酰佛波醇-13-乙酸酯（TPA）激活的hMnSOD转录有反应。通过定点突变分析、蛋白质免疫印迹法和转录因子过表达，研究了这些结合位点的贡献以及转录因子Egr-1、AP-2和Sp1在TPA激活hMnSOD转录中的作用。结果表明，Sp1对基础和TPA激活的hMnSOD转录均起积极作用，而Egr-1的过表达对基础启动子活性起消极作用，对TPA介导的hMnSOD转录激活没有任何影响。
