MOLECULAR BASIS OF AIRWAY SQUAMOUS CELL DIFFERENTIATION

气道鳞状细胞分化的分子基础

• 批准号: 2638558
• 负责人: Sekhar P. Reddy
• 金额: $ 1.6万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 1998-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2638558
• 关键词: DNA footprinting; animal tissue; biomarker; cell differentiation; cell type; developmental genetics; environmental toxicology; fibroblasts; gel mobility shift assay; gene expression; genetic regulatory element; human tissue; lung injury; phorbols; respiratory epithelium; site directed mutagenesis; tissue /cell culture; tobacco; transcription factor

DESCRIPTION (applicant's abstract): My long-term research goal is to elucidate the molecular mechanisms regulating epithelial cell type-specific gene expression that are responsible for the diverse roles of epithelial cells in airway function and disease. The plasticity of cell differentiation is the major biological thrust of airway epithelium, maintaining mucociliary function under normal conditions and expressing squamous and keratinizing properties after injury. The nature of this plasticity is not known. It has been recognized that a similar "squamous cell differentiation" is involved in the pre-neoplastic lesion of developing bronchogenic cancer. Our central hypothesi of this first award application is that the expression of SPR1 gene is essential for squamous cell differentiation in airway epithelium. The expression of small proline-rich protein (SPR1) gene largely occurs in squamou tissues and is associated with the suprabasal cell layer. In vivo, airway epithelium normally does not express any significant level of SPR1 gene products. However, a rapid induction of the expression of this gene occurs immediately after epithelial injury caused by various environmental pollutants Therefore, SPR1 can be used as a squamous cell biomarker for airway epithelium injury. We have demonstrated in our preliminary studies (in vitro and in vivo) that the 622 bp SPR1 promoter region contains the necessary information to direct high-level cell type-specific expression in squamous tissues and a basal-level expression in airway epithelium. We have also observed a strong correlation between tobacco smoke and TPA-induced squamous cell differentiatio and SPR1 gene expression in airway epithelium, mainly at the transcriptional level. Based on our preliminary data, we hypothesize that SPR1 promoter region contains regulatory elements for the binding of cell type-specific transcription factor(s) that are activated or induced during airway squamous cell differentiation. To test our hypothesis, we will use an in vitro cell culture system such as SPR1-expression airway epithelial cells and SPR1-nonexpressing fibroblast cells to characterize airway epithelial cell-specific molecular mechanisms. Specifically, we will focus on the mode of molecular mechanisms that are involved as follows: (1) cell type-specific basa expression of the human SPR1 gene in airway epithelium and (2) inducible expression of human SPR1 gene by TPA and tobacco smoke. We will use a combination of in vivo footprinting, deletion, site-directed mutagenesis, and gel mobility shift assays to identify cis-acting elements that are responsible for both the basal and inducible gene expression. Based on the gel mobility shift assay, we will use DNA affinity column and/or expression screening technique to isolate trans-acting protein factor(s) that are responsible for both basal and inducible gene expression. cDNA and immunological probes of these isolated trans-acting factor(s) will be characterized in vivo to elucidate tissue-specific and cell type-specific gene expression. This study will help us better understand the plasticity of airway epithelial cell differentiation and the response of airway epithelial cells to environmental insults.

描述（申请人的摘要）：我的长期研究目标是 阐明调节上皮细胞类型特异性的分子机制 基因表达，负责上皮的各种作用 气道功能和疾病中的细胞。 细胞的可塑性 分化是气道上皮的主要生物学推力， 在正常条件下维持粘纤毛功能并表达 受伤后的鳞状和角化特性。 这个的本质 可塑性尚不清楚。 已经认识到类似的“鳞 细胞分化”参与发展前的塑性病变 支气管癌。 我们对第一个奖项申请的中心假设 是Spr1基因的表达对于鳞状细胞至关重要 气道上皮的分化。 小脯氨酸的表达 蛋白质（SPR1）基因在很大程度上发生在Squamou组织中，并且与 上胸腔细胞层。 在体内，气道上皮通常不 表达任何显着水平的SPR1基因产品。 但是，很快 该基因表达的诱导立即发生在上皮之后 因此，由各种环境污染物造成的伤害，SPR1可能是 用作气道上皮损伤的鳞状细胞生物标志物。 我们有 在我们的初步研究（体外和体内）中证明了622 BP SPR1启动子区域包含必要的信息来指导 鳞状组织中的高级细胞类型特异性表达和A 气道上皮的基础水平表达。 我们还观察到了一个强大的 烟草烟与TPA诱导的鳞状细胞之间的相关性 气道上皮中的区分和SPR1基因表达，主要是在 转录级别。 根据我们的初步数据，我们假设 SPR1启动子区域包含用于结合细胞的调节元素 特异性转录因子被激活或诱导 气道鳞状细胞分化。 为了检验我们的假设，我们将使用 体外细胞培养系统，例如SPR1表达气道上皮 细胞和spr1-nonexpress成纤维细胞以表征气道 上皮细胞特异性分子机制。 具体来说，我们将集中精力 在所涉及的分子机制模式下：（1）细胞 人类SPR1基因在气道上皮的类型BASA表达 （2）TPA和烟草烟雾对人类SPR1基因的诱导表达。 我们将使用体内足迹，删除，定位的组合 诱变和凝胶迁移率分析以识别顺式作用元件 负责基础基因表达和诱导基因表达。 根据凝胶迁移率分析，我们将使用DNA亲和力柱 和/或表达筛选技术以分离反式作用蛋白 负责基础基因表达和诱导基因表达的因子。 这些分离的跨作用因子的cDNA和免疫学探针（S）将 在体内表征以阐明组织特异性和细胞类型特异性 基因表达。 这项研究将帮助我们更好地了解可塑性 气道上皮细胞分化和气道响应 对环境侮辱的上皮细胞。