Takahashi T, Sugishita Y, Kinugawa K, Shimizu T, Yao A, Harada K, Matsui H, Nagai R
Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Japan.
Mol Cell Biochem. 2001 Oct;226(1-2):57-65. doi: 10.1023/a:1012781618109.
In order to elucidate roles of Ets family of transcription factors in transcriptional activation of inducible nitric oxide synthase (iNOS) genes, we analyzed the chick iNOS gene expression in cultured chick embryonic ventricular myocytes (CEVM). Deletional analysis and site-directed mutagenesis demonstrated that both the Ets/PEA3 site (-221 to -216 bp) and the kappaB site (-101 to -93 bp) of the 5'-flanking region of the chick iNOS gene were involved in the maximal activation of the lipopolysaccharide (LPS)-induced expression of the reporter (luciferase) gene, although the proximal kappaB site played the more essential role. Electrophoretic mobility shift assay revealed that LPS augmented the nuclear protein bindings to the Ets/PEA3 as well as kappaB motifs. Ets-1, one of the Ets proteins, was suggested to be bound to the Ets/PEA3 oligonucleotide. By Northern blot analysis, LPS was shown to induce iNOS mRNA in CEVM, along with a preceding increase in the levels of c-ets-1 mRNA. Ets-1 may be involved in the iNOS gene transcription in CEVM, presumably through interacting with the NF-kappaB.
为了阐明Ets转录因子家族在诱导型一氧化氮合酶（iNOS）基因转录激活中的作用，我们分析了培养的鸡胚心室肌细胞（CEVM）中鸡iNOS基因的表达。缺失分析和定点诱变表明，鸡iNOS基因5'侧翼区的Ets/PEA3位点（-221至-216 bp）和κB位点（-101至-93 bp）均参与脂多糖（LPS）诱导的报告基因（荧光素酶）表达的最大激活，尽管近端κB位点发挥了更重要的作用。电泳迁移率变动分析显示，LPS增强了核蛋白与Ets/PEA3以及κB基序的结合。Ets蛋白之一Ets-1被认为与Ets/PEA3寡核苷酸结合。通过Northern印迹分析，LPS被证明可在CEVM中诱导iNOS mRNA，同时c-ets-1 mRNA水平先升高。Ets-1可能参与CEVM中iNOS基因的转录，大概是通过与NF-κB相互作用实现的。
