Novel transcription factor to regulate differentiation of tracheal epithelial cells.

调节气管上皮细胞分化的新型转录因子。

• 批准号: 09672239
• 负责人: KAI Hirofumi
• 金额: $ 1.86万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672239/
• 关键词: LYSOZYME; MEF; ETS; EPITHELIUM; 気道上皮細胞; 分化; 転写因子; 肺胞上皮細胞; アポトーシス

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.

我们之前克隆并表征了牛溶菌酶 5A (lys 5A) 启动子，目的是确定控制气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通过与位于转录起始位点-46/-40的ets共有序列结合的蛋白质来控制的。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来筛选 ets 相关蛋白：MEF、ESE-1、Elf-1、Ets-1、Ets-2 和 PEA 3。结果显示，在这些因子中，MEF 最强烈地刺激肺上皮细胞 (A549)、结肠癌细胞 (Caco2) 和皮肤成纤维细胞 (NIH3T3) 中的转录。使用包含 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，产生迁移率迟缓的单条带。该条带被针对 MEF 的抗体超移。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。我们发现反义 MEF mRNA 降低了 lys 5A 启动子活性，这支持了这一点。此外，稳定转染子中 MEF 的过表达增加了溶菌酶 mRNA 和蛋白质的表达。总之，这些研究提供了识别控制上皮细胞中溶菌酶表达的转录因子的第一个信息。随着细菌对外源性抗生素的抵抗力逐渐增强，有关控制先天免疫机制（例如上皮溶菌酶提供的机制）的信息可能会提供重要的新治疗方法。

项目成果

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H.Kai 等人：“MEF 上调上皮细胞中的溶菌酶转录”J.Biol.Chem.（出版中）。

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M.Okumuraa、H.Kai、S.Shinozawaa、Y.Isohama 和 T.Miyata：“嗜酸性粒细胞颗粒主要碱性蛋白对大鼠 II 型肺细胞磷脂酰胆碱分泌的影响”Am.J，Phisiol.（出版中）。

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M.Okumura et al.: "Effects of eosinophil-granule major basic protein on phosphatidylcholine secretion in rat type II pneumocytes" Am.J.Physiol.(印刷中). (1999)

M.Okumura 等人：“嗜酸性粒细胞颗粒主要碱性蛋白对大鼠 II 型肺细胞磷脂酰胆碱分泌的影响”Am.J.Physiol.（出版中）。