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. ? ? ?
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|Horani, Amjad; Brody, Steven L; Ferkol, Thomas W (2014) Picking up speed: advances in the genetics of primary ciliary dyskinesia. Pediatr Res 75:158-64|
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|Horani, Amjad; Brody, Steven L; Ferkol, Thomas W et al. (2013) CCDC65 mutation causes primary ciliary dyskinesia with normal ultrastructure and hyperkinetic cilia. PLoS One 8:e72299|
|Vladar, Eszter K; Brody, Steven L (2013) Analysis of ciliogenesis in primary culture mouse tracheal epithelial cells. Methods Enzymol 525:285-309|
|Horani, Amjad; Nath, Aditya; Wasserman, Mollie G et al. (2013) Rho-associated protein kinase inhibition enhances airway epithelial Basal-cell proliferation and lentivirus transduction. Am J Respir Cell Mol Biol 49:341-7|
|Horani, Amjad; Ferkol, Thomas W; Shoseyov, David et al. (2013) LRRC6 mutation causes primary ciliary dyskinesia with dynein arm defects. PLoS One 8:e59436|
|Jain, Raksha; Ray, Jennifer M; Pan, Jie-hong et al. (2012) Sex hormone-dependent regulation of cilia beat frequency in airway epithelium. Am J Respir Cell Mol Biol 46:446-53|
|Jain, Raksha; Javidan-Nejad, Cylen; Alexander-Brett, Jennifer et al. (2012) Sensory functions of motile cilia and implication for bronchiectasis. Front Biosci (Schol Ed) 4:1088-98|
|Horani, Amjad; Druley, Todd E; Zariwala, Maimoona A et al. (2012) Whole-exome capture and sequencing identifies HEATR2 mutation as a cause of primary ciliary dyskinesia. Am J Hum Genet 91:685-93|
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