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
基本信息
- 批准号:6718687
- 负责人:
- 金额:$ 34.09万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2004
- 资助国家:美国
- 起止时间:2004-01-20 至 2008-12-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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(由申请人提供):烟草烟雾引发肺部多种反应,包括粘膜和鳞状皮化生。因为鳞状化生,而不是粘液化生,可能是肿瘤前病变,所以了解细胞如何导致一种或另一种结果是很重要的。粘膜和鳞状病变可以通过监测粘膜基因muc5ac和鳞状基因SPRRI的表达来进行机制研究。在具体目标1中,基于初步数据显示,TRE和RARE位点的突变都会降低MUC 5ACs对烟雾的转录反应,我们将用以下位点的连接物驱动的荧光素酶构建体转染肺上皮细胞:TRE、RARE和TRE-RARE组合。为了确定假定结合蛋白的内在转激活电位,我们将过表达/删除已知与每个位点结合的共同因子。我们将进行超移位和ChIP分析,以鉴定细胞中相关的蛋白质结合,其中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鼻子鳞状化生和气道粘膜化生的数据,我们将解剖烟雾暴露大鼠的鼻腔和气道组织,并进行激光捕获显微解剖、TaqMan PCR、super shift和ChIP检测,以鉴定烟雾诱导的鳞状细胞和粘膜细胞中上调的TRE和罕见结合蛋白。在具体目标3中,我们将检查人类吸烟者肺部的鳞状和粘膜病变,使用激光捕获显微解剖和TaqMan PCR来量化相关转录因子的表达。我们还将在将人体解剖获得的原代人气道细胞暴露于烟雾之前,用MUC 5AC-Ds Red 2和SPRR1-E-YFP进行逆转录病毒感染。由于以这种方式获得的原代细胞的异质性,我们预计一些细胞表达一种荧光团,而另一些细胞表达另一种。我们将对这些细胞进行FACS分选,并使用TaqMan PCR、ChIP和gel super shift进行分析,以确定暴露于MUC 5 AC和SPRRI的细胞对烟雾的反应中产生并使用哪些TRE和RARE转录因子。最后,我们将通过在原代气道上皮细胞或CHO细胞等模型系统中过表达鳞状细胞和黏液细胞特征的转录因子,验证可用的TRE-和罕见结合蛋白的身份决定鳞状细胞和黏液细胞表型的假设。
项目成果
期刊论文数量(0)
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{{ truncateString('CAROL B BASBAUM', 18)}}的其他基金
Role of Airway Epithelium in Mycoplasma Pathogenesis
气道上皮在支原体发病机制中的作用
- 批准号:
6955248 - 财政年份:2004
- 资助金额:
$ 34.09万 - 项目类别:
LYMPHOCYTE/EPITHELIAL INTERACTIONS IN MUCOSAL REMODELING
粘膜重塑中的淋巴细胞/上皮相互作用
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6781168 - 财政年份:2003
- 资助金额:
$ 34.09万 - 项目类别:
Role of Chloride Channels in Mucin Production
氯离子通道在粘蛋白生产中的作用
- 批准号:
6606386 - 财政年份:2003
- 资助金额:
$ 34.09万 - 项目类别:
Role of Chloride Channels in Mucin Production
氯离子通道在粘蛋白生产中的作用
- 批准号:
6723666 - 财政年份:2003
- 资助金额:
$ 34.09万 - 项目类别:
LYMPHOCYTE/EPITHELIAL INTERACTIONS IN MUCOSAL REMODELING
粘膜重塑中的淋巴细胞/上皮相互作用
- 批准号:
6616334 - 财政年份:2002
- 资助金额:
$ 34.09万 - 项目类别:
LYMPHOCYTE/EPITHELIAL INTERACTIONS IN MUCOSAL REMODELING
粘膜重塑中的淋巴细胞/上皮相互作用
- 批准号:
6491087 - 财政年份:2001
- 资助金额:
$ 34.09万 - 项目类别:
LYMPHOCYTE/EPITHELIAL INTERACTIONS IN MUCOSAL REMODELING
粘膜重塑中的淋巴细胞/上皮相互作用
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6325905 - 财政年份:2000
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$ 34.09万 - 项目类别:
LYMPHOCYTE/EPITHELIAL INTERACTIONS IN MUCOSAL REMODELING
粘膜重塑中的淋巴细胞/上皮相互作用
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6109555 - 财政年份:1999
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