Novel transcription factor to regulate differentiation of tracheal epithelial cells.

我们先前克隆并鉴定了牛溶菌酶5A(Lys 5A)启动子，目的是确定控制呼吸道上皮细胞特异性表达的顺式和反式作用元件。我们发现，这种表达是由蛋白质与位于转录起始点-46/-40处的ETS共识序列结合控制的。负责基因反式激活的ETS相关蛋白的身份尚不清楚。在本研究中，我们通过瞬时转染上皮细胞和成纤维细胞筛选了ETS相关蛋白：MEF、ESE-1、Elf-1、Ets-1、Ets-2和PEA 3。结果表明，在这些因子中，MEF对肺上皮细胞(A549)、结肠癌细胞(Caco 2)和皮肤成纤维细胞(NIH3T3)的转录刺激作用最强。使用含有ETS结合位点(-50/-31)的Lys 5A探针对上皮细胞核提取物进行凝胶位移分析，得到一条迁移率缓慢的单一条带。这条带被针对MEF的抗体过度移动，这表明MEF存在于肺上皮细胞A549中，并与Lys 5A启动子中的Ets结合位点结合。支持这一点，我们发现反义MEF mRNA降低了Lys 5A启动子的活性。此外，在稳定的转染体中过表达MEF增加了溶菌酶的mRNA和蛋白的表达。综上所述，这些研究首次提供了确定控制上皮细胞中溶菌酶表达的转录因子的信息。随着细菌对外源性抗生素的耐药性逐渐增强，有关天然免疫控制机制的信息，如上皮溶菌酶提供的机制，可能会提供重要的新治疗方法。

We previously cloned and characterized the bovine lysozyme 5A (lys 5A) promoter with the purpose of determining cis-and trans-acting elements controlling airway epithelial cell-specific expression. We found that such expression is controlled by protein binding to an ets consensus sequence located-at-46/-40 from the transcription start site. The identity of the ets-related protein responsible for gene transactivation was unknown. In the present study, we screened ets-related proteins : MEF, ESE-1, Elf-1, Ets-1, Ets-2, and PEA 3 by transient transfection into epithelial cells and fibroblasts. Results showed that among these factors, MEF most strongly stimulated transcription in lung epithelial cells (A549), colon carcinoma cells (Caco2), and skin fibrobalsts (NIH3T3). Gel shift analysis of epithelial cell nuclear extracts using a lys 5A probe including the ets binding site (-50/-31) yielded a single band with retarded mobility. This band was supershifted by an antibody directed against MEF.This indicated that MEF is present in lung epithelial cells A549 and binds to the ets binding site in the lys 5A promoter. Supporting this, we found that anti-sense MEF mRNA decreased lys 5A promoter activity. Moreover, MEF overexpression in stable transfectants increased lysozyme mRNA and protein expression. In summary, these studies provide the first information identifying a transcription factor controlling lysozyme expression in epithelial cells. As bacteria become progressively more resistant to exogenously applied antibiotics, information regarding mechanisms controlling innate immunity, such as that provided by epithelial lysozyme, may offer important new therapeutic approaches.