Lin Sui, Perl Anne-Karina T, Shannon John M
Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center and the University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA.
J Biol Chem. 2006 Jun 16;281(24):16716-26. doi: 10.1074/jbc.M602221200. Epub 2006 Apr 13.
Expression of surfactant protein C (SP-C), which is restricted to alveolar type II epithelial cells of the adult lung, is critically dependent on thyroid transcription factor 1 (TTF-1). In the present study we have demonstrated that Erm, a member of the Ets family of transcription factors, is expressed in the distal lung epithelium during development and is also restricted to alveolar type II cells in the adult. Erm was up-regulated by fibroblast growth factors (FGFs) in culture, and blocking FGF signaling inhibited Erm expression both in vivo and in vitro. The SP-C minimal promoter was found to contain two potential Ets binding sites, and electrophoretic mobility shift assays showed that two 20-bp wild-type oligonucleotides containing the 5'-GGA(A/T)-3' Ets consensus binding motif were shifted by nuclear extracts from MLE15 cells. Co-transfection assays showed that Erm by itself had little effect on SP-C promoter activity but that Erm significantly enhanced TTF-1-mediated SP-C transcription. Mutation of one of the Ets binding sites reduced SP-C transcription to background levels, whereas mutation of the other site resulted in increased SP-C transcription. Protein-protein interactions between Erm and TTF-1 were demonstrated by mammalian two-hybrid assays and by co-immunoprecipitation assays. Mapping studies showed that the Ets domain of Erm and the combined N terminus and homeodomain of TTF-1 were critical for this interaction. Treatment of primary cultures of adult alveolar type II cells with siRNA targeting Erm diminished expression of both Erm and SP-C but had no effect on beta-actin or GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Taken together, these results demonstrate that Erm is involved in SP-C regulation, which results from an interaction with TTF-1.
表面活性蛋白C（SP-C）的表达仅限于成年肺的II型肺泡上皮细胞，其表达严重依赖于甲状腺转录因子1（TTF-1）。在本研究中，我们证明了Ets转录因子家族成员Erm在发育过程中在远端肺上皮中表达，在成体中也仅限于II型肺泡细胞。在培养中，Erm被成纤维细胞生长因子（FGFs）上调，阻断FGF信号传导在体内和体外均抑制Erm表达。发现SP-C最小启动子包含两个潜在的Ets结合位点，电泳迁移率变动分析表明，来自MLE15细胞的核提取物使两个含有5'-GGA(A/T)-3'Ets共有结合基序的20bp野生型寡核苷酸发生迁移。共转染分析表明，Erm本身对SP-C启动子活性影响很小，但Erm显著增强了TTF-1介导的SP-C转录。其中一个Ets结合位点的突变将SP-C转录降低到背景水平，而另一个位点的突变导致SP-C转录增加。通过哺乳动物双杂交分析和共免疫沉淀分析证明了Erm与TTF-1之间的蛋白质-蛋白质相互作用。定位研究表明，Erm的Ets结构域以及TTF-1的N末端和同源结构域的组合对于这种相互作用至关重要。用靶向Erm的siRNA处理成年II型肺泡细胞原代培养物可减少Erm和SP-C的表达，但对β-肌动蛋白或甘油醛-3-磷酸脱氢酶（GAPDH）没有影响。综上所述，这些结果表明Erm参与了SP-C的调控，这是由与TTF-1的相互作用导致的。
