FOXJ1 EXPRESSION IN PULMONARY EPITHELIUM

描述（由申请人提供）：遗传性和获得性纤毛缺陷越来越多地被认为是疾病的基础。正常的气道清除需要活动纤毛，而这些纤毛功能障碍发生在遗传综合征、气道感染、炎症和吸烟中。因此，我们研究的目的是确定纤毛细胞在气道上皮细胞中的分化和纤毛发生程序。我们之前的研究表明叉头转录因子Foxj1是纤毛细胞特异性转录因子，是纤毛发生所必需的。Foxj1的功能不仅对纤毛的组装起关键作用，而且对纤毛细胞顶膜上多蛋白复合物的组织起关键作用。Foxj1仍然是唯一已知的指导运动纤毛发生的转录因子。为了解剖Foxj1功能的分子成分并发现新的纤毛发生途径，我们通过检测野生型和Foxj1-/-小鼠气道上皮细胞分化过程中的基因表达，构建了一个功能性纤毛发生转录组。转录组分析显示，foxj1依赖性基因与运动性纤毛遗传病的突变基因聚集在一起。然而，在气道细胞中表达的foxj1独立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。这些基因已被鉴定为突变的遗传性疾病的非运动性，初级纤毛。气道上皮细胞中这组感觉纤毛相关基因的表达表明，肺部存在以前未被识别的程序。基于这些观察，我们首先确定了一组foxj1依赖基因。该分析表明Foxj1直接激活了运动纤毛基因如Spag6和一组独特的调控基因的表达。这些调节基因包括一种名为Mlf1的新型转录因子，我们发现它也是运动纤毛发生所必需的。其次，对foxj1独立基因的研究揭示了气道细胞原毛中感觉相关基因的表达。这些细胞顶端表面的液体流动诱导细胞内钙信号，提示有感觉功能。有趣的是，与foxj1无关的感觉纤毛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起作用。综上所述，我们假设运动纤毛的形成和功能需要foxj1依赖程序的顺序激活，并且与与感觉纤毛相关的第二类基因提供的功能相关，这些基因与foxj1无关。验证这一假设的具体目的将包括：(1)Foxj1依赖基因，以表征Foxj1激活运动组装和功能的机制；(2)Foxj1独立的感觉纤毛基因，以研究其在运动纤毛组装和功能中的作用。这些研究的完成将为哺乳动物纤毛发生的调控提供相关信息，并为研究感觉蛋白在气道上皮细胞中的进化作用开辟新的途径。研究纤毛细胞的功能将为气道疾病的发生、分化和发病机制提供新的认识。项目描述：呼吸道由含有毛发状纤毛的细胞组成，纤毛对气道的防御和清除至关重要，但这些纤毛是如何组装和维持的尚不清楚。我们对鉴定新的基因和分子程序感兴趣，这些基因和分子程序对健康和疾病中含有纤毛的细胞的功能至关重要。

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.