Abstract
An innate front-line defense mechanism termed mucociliary clearance (MCC) occurs in the airway epithelium and is critical for respiratory health. MCC is made possible by the coordinated efforts of goblet and multiciliated cells, two cell types that line the airway epithelium. Maintaining the relative populations of goblet and multiciliated cells is necessary for optimum MCC, however, decreases in the multiciliated cell population and dysfunctional MCC are seen in a variety of respiratory diseases. Despite the clinical relevance, there is still much to understand about the signaling pathways that control multiciliated cell fate. Previous data has shown multiciliated cell differentiation of primary human airway epithelial cells occurred in vitro if they were grown at an air:liquid interface (ALI), but multiciliated cell differentiation was inhibited if the cells were kept submerged in media. However, after two decades, the mechanism behind submersion dependent inhibition of multiciliated cell differentiation remained elusive. Published literature demonstrated submersion could create a low oxygen environment. Therefore, the hypotheses that submersion inhibits multiciliated cell differentiation by creating a low oxygen (hypoxic) environment and suppresses genes necessary for multiciliated cell differentiation were tested. The results showed both submersion and hypoxia repressed multicilin and FOXJ1 expression, genes necessary for multiciliated cell differentiation. It also showed that inhibition was dependent upon the Notch signaling pathway, as pharmacological and neutralizing antibody inhibition of Notch signaling restored multiciliated cell differentiation. Additionally, activation of Notch signaling via expression of activated Notch intracellular domain (NICD) resulted in repression of multiciliated cell differentiation. Furthermore, Notch neutralizing antibody data indicated that Notch2 but not Notch1 was important for this repression. These results implicated submersion and hypoxia activated Notch signaling to prevent the differentiation of multiciliated cells by blocking the expression of genes required for multiciliated cell differentiation. The finding that multiciliated cell differentiation does not require an ALI when Notch was inhibited lead to the discovery that it was possible to differentiate primary airway epithelial cells into multiciliated cells submerged in plastic wells. Moreover, transduction of airway epithelial cells with lentivirus containing the human FOXJ1 promoter driving the expression of a fluorescent protein was successful in detecting and quantifying multiciliated cell differentiation of airway epithelial cells differentiated in 96-wells. These data provided evidence that high-throughput approaches are possible to detect compounds and/or signaling pathways that regulate multiciliated cell differentiation. Since submersion created a low oxygen environment, the influence of hypoxia inducible factor (HIF) signaling on multiciliated cell differentiation was tested. The data showed the expression of a lentiviral transduced constitutively active form of HIF-2α but not HIF-1α repressed multiciliated cell differentiation in airway epithelial cells. These data suggested HIF signaling might be a cause of inhibition of multiciliated cell differentiation in low oxygen environments. In addition to submersion via Notch signaling and/or HIF-2α signaling, the inflammation related cytokine, IL-13, has been shown to reduce multiciliated cell differentiation and is elevated in respiratory diseases such as asthma. Given Notch signaling inhibits multiciliated cell differentiation; the hypothesis was tested whether IL-13 was activating Notch to inhibit multiciliated cells. The results showed IL-13 repressed multiciliated cell differentiation through a Notch independent mechanism. Pharmacological inhibitor experiments showed IL-13 utilized the JAK/STAT but not MEK signaling pathway to repress multiciliated cell differentiation. Additionally, the data indicated IL-13, like Notch, repressed multiciliated cell differentiation by suppressing the expression of multicilin and FOXJ1. The data in this thesis provide novel insights into the mechanisms that control human multiciliated cell differentiation. They show that low oxygen and/or chronically inflamed environments will hinder multiciliated cell differentiation through different mechanisms. They also reveal multicilin expression is regulated both by JAK/STAT and by Notch signaling pathways independently of each other. These insights have furthered our knowledge into the signals that control multiciliated cell fate and may lead to novel therapeutics to help those with respiratory disease. Additionally, these data demonstrate technical innovations for studying differentiating multiciliated cells, and show it is possible to use novel high-throughput assays that could one day reveal more aspects of multiciliated cell differentiation.