REGULATION OF CILIATED CELL DIFFERENTIATION

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Summary of Work: Ciliated cells line the surface of the airways, and by the coordinated beating of their cilia provide the force necessary for mucociliary clearance. Patients with primary ciliary dyskinesia (PCD) suffer from repeated respiratory infections. Ciliated cells are easily damaged by a wide range of air pollutants and pathogens. Once damaged, it is essential that ciliated cells be regenerated and mucociliary clearance restored. The main goal of our research is to understand the regulation of ciliogenesis. We have begun several projects to investigate the mechanisms regulating the expression of ciliated cell-specific genes. We have identified and cloned partial cDNAs for seven unique axonemal dyneins from rat tracheal epithelial cells (RTE). The expression of these dyneins correlates with the development of ciliated cells. Because these dyneins are very large, we are using a PCR based strategy to clone larger pieces of these genes in the 5' direction. We have successfully "walked" 4.5 kb (out of the expected 6-7 kb) on one of the more abundant dyneins, and have made progress on several others. In addition, we have identified two novel genes, KPL1 and KPL2, which are upregulated during ciliogenesis using differential display. The complete cDNA for KPL1 has been cloned and sequenced and codes for a predicted protein of 24 kD. This unique protein contains a pleckstrin homology domain, which indicates that KPL1 probably plays a role in a signal transduction pathway. We have sequenced 3.7 kb of the 7 kb KPL2 message, and so far identified an ATP binding site in the predicted open reading frame. This message is only expressed in tissues that contain ciliated cells.