title: Localization of cystic fibrosis transmembrane conductance regulator to lipid rafts of epithelial cells is required for Pseudomonas aeruginosa-induced cellular activation.
abstract: The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein is an epithelial cell receptor for the outer core oligosaccharide of the Pseudomonas aeruginosa LPS. Bacterial binding leads to CFTR-dependent bacterial internalization, initiation of NF-kappaB nuclear translocation, cellular desquamation, and eventual apoptosis of the infected cells, all of which are critical for innate immune resistance to infection with this pathogen. Lack of this reaction in CF patients underlies their hypersusceptibility to chronic P. aeruginosa infection. In this study we tested whether these epithelial cell responses are dependent upon the localization of CFTR to lipid rafts. Confocal microscopy showed that green fluorescent protein-tagged CFTR (GFP-CFTR) and the lipid raft marker ganglioside GM1 colocalized at sites of P. aeruginosa contact and internalization. GFP-CFTR localized to low density Triton X-100-insoluble fractions in lysates of Madin-Darby canine kidney GFP-CFTR cells, and P. aeruginosa infection increased the levels of GFP-CFTR in these fractions as determined by Western blot. Cells expressing GFP-DeltaF508-CFTR did not have rafts with detectable CFTR protein. Extraction of cell surface cholesterol via cyclodextrin treatment of the cells inhibited CFTR entry into rafts. In addition, cyclodextrin treatment of both human and canine epithelial cells inhibited cellular ingestion of P. aeruginosa, NF-kappaB nuclear translocation, and apoptosis. These results indicate that lipid raft localization of CFTR is required for signaling in response to P. aeruginosa infection. Such signaling is needed for the coordination of innate immunity to P. aeruginosa lung infection, a process that is defective in CF.
authors.affiliation: Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Kowalski
authors.forename: Gerald B
authors.initials: GB
authors.lastname: Pier
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002461:Cell Line, Transformed; D003505:Cyclodextrins / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology; D005677:G(M1) Ganglioside / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology; D009154:Mutation; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D017830:Octoxynol; D011552:Pseudomonas Infections / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D012995:Solubility; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D047392:beta-Cyclodextrins
chemical_list: C505032:CFTR protein, human; D003505:Cyclodextrins; D016328:NF-kappa B; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005677:G(M1) Ganglioside; D017830:Octoxynol