Smoke-induced AP-1 controls mucous vs squamous phenotype

烟雾诱导的 AP-1 控制粘液与鳞状细胞表型

• 批准号: 6999690
• 负责人: ZENA WERB
• 金额: $ 33.29万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-20 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6999690
• 关键词: binding proteins; chromatin immunoprecipitation; clinical research; flow cytometry; gene expression; gene mutation; genetic transcription; human subject; laboratory rat; lung; metaplasia; polymerase chain reaction; questionnaires; respiratory epithelium; smoking; spirometry; squamous cell carcinoma; transcription factor

DESCRIPTION (provided by applicant): Tobacco smoke triggers multiple responses in the lung, including mucous and squamous metaplasia. Because squamous, but not mucous metaplasia can be pre-neoplastic, it is important to understand how cells commit themselves to one outcome or the other. Mucous and squamous lesions can be studied mechanistically by monitoring expression of the mucous gene MUC 5 AC and the squamous gene SPRRI. In specific aim 1, based on preliminary data showing that mutagenesis of both TRE and RARE sites diminish MUC 5ACs transcriptional response to smoke, we will transfect lung epithelial cells with luciferase constructs driven by concatamers of the following sites: TRE, RARE and TRE-RARE combined. To determine the intrinsic transactivation potential of putative binding proteins, we will overexpress/delete common factors known to bind each site. We will perform super shift and ChIP assays to identify relevant protein binding in cells in which TRE- and RARE-binding proteins have been experimentally manipulated. In specific aim 2, based on data showing that smoke-exposed rats develop squamous metaplasia in the nose and mucous metaplasia in the airways, we will dissect nasal and airway tissues from smoke- vs. air-exposed rats and perform laser capture microdissection, TaqMan PCR, super shift and ChIP assays to identify TRE and RARE-binding proteins upregulated in smoke-induced squamous vs. mucous cells. In specific aim 3, we will examine squamous and mucous lesions in the lungs of human smokers, using laser capture microdissection followed by TaqMan PCR to quantify expression of relevant transcription factors. We will also retrovirally infect primary human airway cells obtained at autopsy with MUC 5AC-Ds Red 2 and SPRR1-E-YFP prior to exposing them to smoke. Because of the documented heterogeneity of primary cells obtained in this way, we expect some cells to express one fluorophore and others to express the other. We will FACS sort these cells and assay them using TaqMan PCR, ChIP and gel super shift, to determine which TRE and RARE transcription factors are being produced and used in response to smoke by cells exposing MUC 5 AC vs. SPRRI. Finally, we will test the hypothesis that the identity of available TRE- and RARE-binding proteins dictates squamous vs. mucous phenotype by overexpressing panels of transcription factors characteristic of squamous and mucous cells in primary airway epithelial cells or a model system such as CHO cells.

描述（由申请方提供）：烟草烟雾在肺中引发多种反应，包括粘液和鳞状化生。 因为鳞状上皮化生（而非粘液化生）可以是癌前病变，所以了解细胞如何使自己产生这种或那种结果是很重要的。 粘液和鳞状病变可以通过监测粘液基因MUC 5 AC和鳞状基因SPRRI的表达进行机制研究。 在具体的目标1中，基于显示TRE和RARE位点的诱变都会降低MUC 5AC对烟雾的转录应答的初步数据，我们将用由以下位点的多联体驱动的荧光素酶构建体转染肺上皮细胞：TRE、RARE和组合的TRE-RARE。 为了确定假定结合蛋白的内在反式激活潜力，我们将过表达/删除已知结合每个位点的共同因子。 我们将进行超级位移和ChIP测定，以确定相关的蛋白质结合细胞中的TRE和RARE结合蛋白已被实验操纵。 在具体目标2中，基于显示烟雾暴露大鼠在鼻中发生鳞状上皮化生和在气道中发生粘液化生的数据，我们将解剖来自烟雾与空气暴露大鼠的鼻和气道组织，并进行激光捕获显微切割、TaqMan PCR、超级移位和ChIP测定，以鉴定在烟雾诱导的鳞状细胞与粘液细胞中上调的TRE和RARE结合蛋白。 在具体目标3中，我们将使用激光捕获显微切割，然后使用TaqMan PCR来定量相关转录因子的表达，从而检查人类吸烟者肺部的鳞状和粘膜病变。 我们还将用MUC 5AC-Ds Red 2和SPRR 1-E-YFP逆转录病毒感染在尸检中获得的原代人气道细胞，然后将其暴露于烟雾。 由于以这种方式获得的原代细胞的记录异质性，我们预计一些细胞表达一种荧光团，而其他细胞表达另一种荧光团。 我们将对这些细胞进行FACS分选，并使用TaqMan PCR、ChIP和凝胶超位移对其进行分析，以确定哪些TRE和RARE转录因子正在产生，并通过暴露MUC 5 AC与SPRRI的细胞响应烟雾而使用。 最后，我们将通过在原代气道上皮细胞或模型系统（如CHO细胞）中过表达鳞状细胞和粘液细胞特征性转录因子组来检验以下假设：可用的TRE和RARE结合蛋白的身份决定鳞状细胞与粘液细胞的表型。