Primary cilia are sensory organelles present on most differentiated cells. Their assembly or function is impaired in several human diseases, including polycystic kidney disease (PKD). However, the mechanism by which ciliary dysfunction contributes to these pathologies is incompletely understood. PKD is characterized by altered trans-epithelial fluid transport, due to the mislocalization of ion pumps and changes in tight junctions. How ciliary dysregulation might lead to such changes in cell polarity and tight-junction function has not been shown. The overall hypothesis of this thesis is that ciliary dysfunction promotes specific changes in epithelial junctions and cell polarity that contribute to PKD. We developed an experimental model in which to test whether ciliary disruption indeed causes changes in cell polarity and tight junctions, and used it in conjunction with well-established methods to chemically induce ciliary shedding from cells, and with assays measuring the distribution of surface and secretory proteins in Madin-Darby canine kidney (MDCK) cells. This analysis revealed that several proteins, including Na/K-ATPase, were mislocalized following deciliation and that tight junctions became less permeable to paracellular ion flux. We infer that the combined effects of mislocalized ion transporters and increased tight-junction barrier function contribute to cyst formation in PKD. The functional specificity of tight junctions is determined by their expression of claudins, a protein family consisting of 24 members. Of these, claudin 2 expression has been shown to lead to an increase in junctional leakiness. We found that upon deciliation, claudin 2 expression was lost and trans-epithelial resistance increased 3-fold. The signaling pathway connecting deciliation to claudin 2 repression required ERK activation; phospho-ERK levels were elevated after exposure to deciliation stimuli, and an inhibitor of ERK activation blocked claudin 2 loss. The down-regulation of claudin 2 did not involve post-translational regulation, because protein half-life was not altered following deciliation. In contrast, the half-life of claudin 2 mRNA was greatly reduced in deciliated cells. Finally, when murine claudin 2 was ectopically expressed in MDCK cells, its expression was also suppressed upon deciliation. Collectively, these results support a model in which deciliation causes an ERK-dependent decrease in claudin 2 mRNA stability, and highlight a mechanism whereby ciliary dysregulation might contribute to altered trans-epithelial fluid transport associated with PKD.
| Identifer | oai:union.ndltd.org:uiowa.edu/oai:ir.uiowa.edu:etd-1601 |
| Date | 01 January 2009 |
| Creators | Overgaard, Christian Edmund |
| Contributors | Yeaman, Charles |
| Publisher | University of Iowa |
| Source Sets | University of Iowa |
| Language | English |
| Detected Language | English |
| Type | dissertation |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | Copyright 2009 Christian Edmund Overgaard |
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