Miralles F, Ibáñez-Tallon I, Parra M, Crippa M, Blasi F, Besser D, Nagamine Y, Muñoz-Cánoves P
Institut de Recerca Oncològica, Barcelona, Spain.
Thromb Haemost. 1999 May;81(5):767-74.
We have previously shown that urokinase-type plasminogen activator (uPA) is highly expressed in murine C2C12 myoblasts and that antibodies against uPA are able to block both myoblast fusion and differentiation. Here we show the characterization of cis-acting elements in the mouse uPA promoter in vitro which are involved in uPA gene expression in C2C 12 myoblast cells. DNase I hypersensitive (HS) site analysis revealed the presence of three HS sites in myoblasts. Deletion analysis of stably transfected uPA-promoter constructs revealed that at least two of the three HS sites accounted for the high transcriptional expression in C2C12 cells. One was located at -2.4 kb and corresponded to a known PEA3/AP1A element and the other one was located at -4.9 kb and contained a CArG box and a CRE element. So far, no regulatory function had been assigned to this CRE/CArG element. Both HS sites alone were able to activate transcription of a heterologous promoter and showed a cooperative effect when placed together. Electrophoretic mobility-shift assays using myoblast nuclear extracts and specific antibodies demonstrated that cJun, JunD and ATF2 bound to the PEA3/AP1A element, whereas the CRE/CArG element bound SRF. Altogether, these results suggest that high uPA expression in myoblasts is dependent on the cooperation of two regulatory sites in the uPA promoter.
我们之前已经表明，尿激酶型纤溶酶原激活剂（uPA）在小鼠C2C12成肌细胞中高度表达，并且抗uPA抗体能够阻断成肌细胞融合和分化。在此我们展示了体外小鼠uPA启动子中顺式作用元件的特征，这些元件参与了C2C12成肌细胞中uPA基因的表达。DNA酶I超敏（HS）位点分析揭示了成肌细胞中存在三个HS位点。对稳定转染的uPA启动子构建体的缺失分析表明，三个HS位点中的至少两个是C2C12细胞中高转录表达的原因。一个位于-2.4 kb处，对应于一个已知的PEA3/AP1A元件，另一个位于-4.9 kb处，包含一个CArG框和一个CRE元件。到目前为止，尚未赋予这个CRE/CArG元件任何调控功能。单独的两个HS位点都能够激活异源启动子的转录，并且放在一起时显示出协同效应。使用成肌细胞核提取物和特异性抗体进行的电泳迁移率变动分析表明，cJun、JunD和ATF2与PEA3/AP1A元件结合，而CRE/CArG元件结合SRF。总之，这些结果表明成肌细胞中uPA的高表达依赖于uPA启动子中两个调控位点的协同作用。
