The abnormal structure and amount of mucus in CF patients indicates a direct link between mucin synthesis and secretion, and CFTR function in epithelial cells. The nature of this link is not well-defined. Furthermore, the mechanisms of mucin synthesis and secretion in epithelial cells is itself not clearly understood. In this proposal, we test the hypothesis that the CFTR chloride channel plays a regulatory role in mucin synthesis and secretion by the columnar epithelium. Recently, we transduced a normal well-differentiated biliary epithelial cell line with a retroviral vector carrying the cDNA for human CFTR. Overexpression of the CFTR protein by these cells was linked to an increase in constitutive mucin synthesis and secretion, suggesting a """"""""gain-of-function"""""""" result. To explain these findings, one can postulate a regulatory role for CFTR in the process of mucin granule exocytosis and mucin precursor uptake. An extension of this line of reasoning is a direct role for the CFTR ion channel on mucin granule membranes; the presence of CFTR at this location could act as the trigger for the exceedingly rapid event that signals the expulsion of mucin from granules to the extracellular milieu. Both scenarios imply a wider regulatory role for CFTR than its previously acknowledged function as an apical membrane low conductance CI channel. By modifying CFTR ion channel activities and correlating this with mucin synthesis and secretion in a well-characterized cell line, we will then be able to corroborate the """"""""gain of function"""""""" observations from gene transfer and over-expression of CFTR with """"""""loss of function"""""""" from inhibition of CFTR with molecular probes. These include an antibody, which we have synthesized and antisense oligodeoxynucleotides against CFTR, as well as a selected spectrum of chloride channel inhibitors. We will also use gallbladder epithelial cells from CFTR knock-out mice, which we have successfully maintained as a cell line. We will perform """"""""regain of function"""""""" experiments by transducing the cDNA for human CFTR into mouse wild-type and homozygous (cf-I-) epithelial cells. In addition, we will conduct parallel experiments examining chloride efflux. This will address the possibility that in CF there is a dissociated or uncoupled functional impairment of mucin secretion with chloride (and fluid) secretion, leading to excessively condensed and viscous mucin gels. The planned studies will add understanding and insight into the role of CFTR in mucin exocytosis and the cellular and molecular defect of cystic fibrosis, and may provide information leading to novel benefits for CF patients.
