Smoke-induced AP-1 controls mucous vs squamous phenotype

描述(申请人提供)：烟草烟雾会在肺部引发多种反应，包括粘液和鳞状化生。因为鳞状上皮化生，而不是粘液性化生，可能是癌前病变，因此了解细胞如何将其转化为一种或另一种结果很重要。通过监测粘液基因MUC5AC和鳞状细胞癌基因SPRRI的表达，可以对粘液和鳞状细胞病变进行机械性研究。在特定的目标1中，基于初步数据显示TrE和Rare位点的突变都会降低MUC 5AC对烟雾的转录反应，我们将用以下三个位点的连接物驱动的荧光素酶构建物转染肺上皮细胞：Tre，Rare和Tre-Rare的组合。为了确定假定结合蛋白的内在反式激活潜力，我们将过度表达/删除已知的与每个结合位点相结合的共同因子。我们将进行超级位移和芯片分析，以确定在细胞中相关的蛋白质结合，其中tre和稀有结合蛋白已经被实验操纵。在特定目标2中，基于显示烟雾暴露大鼠鼻部鳞状化生和呼吸道粘液化生的数据，我们将解剖烟雾暴露和空气暴露大鼠的鼻部和呼吸道组织，并进行激光捕获显微解剖、TaqMan聚合酶链式反应、超级位移和芯片分析，以确定在烟雾诱导的鳞状细胞和粘液细胞中上调的tre和稀有结合蛋白。在特定的目标3，我们将检查人类吸烟者肺部的鳞状和粘液性病变，使用激光捕获显微切割和TaqMan聚合酶链式反应来定量相关转录因子的表达。我们还将用MUC 5AC-DS Red 2和SPRR1-E-YFP逆转录病毒感染尸检获得的原代人呼吸道细胞，然后再将其暴露在烟雾中。由于以这种方式获得的原代细胞具有文献记载的异质性，我们期望一些细胞表达一个荧光团，另一些细胞表达另一个。我们将FACS对这些细胞进行分类，并使用TaqMan PCR、CHIP和Gel Super Shift对它们进行检测，以确定暴露于MUC 5 AC和SPRRI的细胞正在产生和使用哪些TrE和稀有转录因子来响应烟雾。最后，我们将通过在原代呼吸道上皮细胞或CHO细胞等模型系统中过度表达鳞状细胞和粘液细胞特有的转录因子来检验这一假设，即可用的tre和稀有结合蛋白的特性决定了鳞状细胞和粘液细胞的表型。

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.