FOXJ1 EXPRESSION IN PULMONARY EPITHELIUM

描述(由申请人提供)：纤毛的遗传和后天缺陷越来越多地被认为是疾病的基础。运动纤毛是正常呼吸道清除所必需的，而这些纤毛功能障碍发生在遗传综合征、呼吸道感染、炎症和吸烟中。因此，我们研究的目标是确定呼吸道上皮细胞纤毛分化和纤毛发生的程序。我们前期的研究表明，叉头转录因子Foxj1是纤毛发生所必需的一种纤毛细胞特异性转录因子。Foxj1功能是纤毛组装的关键，也是纤毛细胞顶膜上多蛋白复合体的组织。Foxj1仍然是唯一已知的指导纤毛运动发生的转录因子。为了剖析Foxj1功能的分子组成并发现新的纤毛发生途径，我们通过检测野生型和Foxj1-/-小鼠呼吸道上皮细胞分化过程中的基因表达，构建了一个具有功能的纤毛发生转录组。对转录组的分析表明，在运动性纤毛遗传病中，依赖Foxj1的基因与那些突变的基因聚集在一起。然而，在呼吸道细胞中表达的不依赖于Foxj1的基因与一类编码感觉功能的基因聚集在一起，包括机械感觉血流检测。这些基因已被确定为非运动性原生纤毛遗传病的突变。这组纤毛相关的感觉基因在呼吸道上皮细胞中的表达表明，在肺中存在以前未被识别的程序。基于这些观察，我们首先描述了一组依赖Foxj1的基因。这一分析表明，Foxj1直接激活了运动纤毛基因的表达，如Spag6和一组独特的调节基因。这些调控基因包括一种名为Mlf1的新转录因子，我们发现它也是运动纤毛发生所必需的。其次，对Foxj1非依赖基因的研究发现，感觉相关基因在呼吸道细胞的原代纤毛中表达。流经这些细胞顶端表面的液体诱导细胞内钙信号，暗示感觉功能。有趣的是，Foxj1不依赖于感觉纤毛的基因也在运动纤毛中表达，这表明这些蛋白在运动纤毛的组装和功能中具有功能。综上所述，我们假设运动纤毛的形成和功能需要依赖于Foxj1的程序的顺序激活，并且与第二类与感觉纤毛相关的基因提供的功能相关联，这些基因不依赖于Foxj1。验证这一假说的具体目的将涉及：(1)依赖于Foxj1的基因来表征Foxj1激活运动组装和功能的机制，以及(2)Foxj1不依赖于感觉纤毛的基因来研究它们在运动纤毛组装和功能中的作用。这项拟议研究的完成将提供与哺乳动物纤毛发生调控相关的信息，并为研究感觉蛋白在呼吸道上皮细胞中的进化作用开辟新的途径。纤毛细胞功能的研究将为呼吸道疾病的发生、分化和发病机制提供新的见解。项目简介：呼吸道内排列着含有毛发状纤毛的细胞，这些纤毛对呼吸道的防御和清除至关重要，但这些纤毛是如何组装和维护的还没有很好的定义。我们感兴趣的是识别新的基因和分子程序，这些基因和分子程序对于纤毛细胞在健康和疾病中的功能至关重要。

DESCRIPTION (provided by applicant): Genetic and acquired defects of cilia are increasingly implicated as the basis of disease. Motile cilia are required for normal airway clearance, while dysfunction of these cilia occurs in genetic syndromes, airway infection, inflammation, and cigarette smoking. Thus, the goal of our studies is to identify programs of ciliated cell differentiation and ciliogenesis in airway epithelial cells. Our prior studies demonstrated that forkhead transcription factor Foxj1 is a ciliated cell specific transcription factor that is required for ciliogenesis. Foxj1 function is pivotal, not only for cilia assembly, but for the organization of multiprotein complex on the apical membrane of ciliated cells. Foxj1 remains the only known transcription factor that directs motile ciliogenesis. To dissect the molecular components for Foxj1 function and discover new ciliogenesis pathways, we generated a functional ciliogenesis transcriptome by examining gene expression during differentiation of airway epithelial cells from wild type and Foxj1-/- mice. Analysis of the transcriptome revealed that Foxj1-dependent genes clustered with those mutant in genetic diseases of motile cilia. However, the Foxj1-independent genes that were expressed in airway cells clustered with a class of genes coding for sensory functions, including mechanosensory flow detection. These genes have been identified as mutant in genetic diseases of non- motile, primary cilia. The expression of this sensory group of cilia-related genes in airway epithelial cells suggested the presence of previously unrecognized programs in the lung. Based on these observations, we first characterized a set of Foxj1-dependent genes. This analysis demonstrated Foxj1 directly activated expression of motile cilia genes such as Spag6 and a unique group of regulatory genes. These regulatory genes included a novel transcription factor called Mlf1 that we found is also required for motile ciliogenesis. Second, investigation of the Foxj1-independent genes revealed expression of sensory associated genes in primary cilia of airway cells. Fluid flow over the apical surface of these cells induces an intracellular calcium signal, suggesting a sensory function. Interestingly, Foxj1-independent sensory cilia genes are also expressed in motile cilia, suggesting a function for these proteins in motile cilia assembly and function. Taken together, we hypothesize that motile cilia formation and function require the sequential activation of Foxj1-dependent programs, and are linked to functions provided by a second class of genes associated with sensory cilia, that are Foxj1-independent. Specific Aims to test this hypothesis will address: (1) Foxj1-dependent genes to characterize mechanisms of Foxj1 activation for motile assembly and function, and (2) Foxj1-independent sensory cilia genes to investigate their roles in motile cilia assembly and function. Completion of the proposed studies will provide information related to the regulation of mammalian ciliogenesis and open new avenues for examining the evolving role of sensory proteins in airway epithelial cells. Investigation of functions of the ciliated cell will provide new insights into development, differentiation and pathogenesis of airways diseases. Project Narrative: The respiratory tract is lined with cells containing hair-like cilia that are critical for defense and clearance of the airways but how these cilia are assembled and maintained is not well defined. We are interested in the identification of new genes and molecular programs that are critical for function of cilia-containing cells in health and disease.