title: RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR.
abstract: Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the Cystic fibrosis transmembrane conductance regulator (CFTR). ΔF508-CFTR, the most common disease-causing CF mutant, exhibits folding and trafficking defects and is retained in the endoplasmic reticulum, where it is targeted for proteasomal degradation. To identify signaling pathways involved in ΔF508-CFTR rescue, we screened a library of endoribonuclease-prepared short interfering RNAs (esiRNAs) that target ∼750 different kinases and associated signaling proteins. We identified 20 novel suppressors of ΔF508-CFTR maturation, including the FGFR1. These were subsequently validated by measuring channel activity by the YFP halide-sensitive assay following shRNA-mediated knockdown, immunoblotting for the mature (band C) ΔF508-CFTR and measuring the amount of surface ΔF508-CFTR by ELISA. The role of FGFR signaling on ΔF508-CFTR trafficking was further elucidated by knocking down FGFRs and their downstream signaling proteins: Erk1/2, Akt, PLCγ-1, and FRS2. Interestingly, inhibition of FGFR1 with SU5402 administered to intestinal organoids (mini-guts) generated from the ileum of ΔF508-CFTR homozygous mice resulted in a robust ΔF508-CFTR rescue. Moreover, combination of SU5402 and VX-809 treatments in cells led to an additive enhancement of ΔF508-CFTR rescue, suggesting these compounds operate by different mechanisms. Chaperone array analysis on human bronchial epithelial cells harvested from ΔF508/ΔF508-CFTR transplant patients treated with SU5402 identified altered expression of several chaperones, an effect validated by their overexpression or knockdown experiments. We propose that FGFR signaling regulates specific chaperones that control ΔF508-CFTR maturation, and suggest that FGFRs may serve as important targets for therapeutic intervention for the treatment of CF.
authors.affiliation: From the ‡Program in Cell Biology, The Hospital for Sick Children, Toronto, and Biochemistry Department, University of Toronto; PGCRL, 19-9715, 686 Bay St., Toronto, Ont., Canada, M5G 0A4.
authors.forename: Agata M
authors.initials: AM
authors.lastname: Trzcińska-Daneluti
authors.affiliation: From the ‡Program in Cell Biology, The Hospital for Sick Children, Toronto, and Biochemistry Department, University of Toronto; PGCRL, 19-9715, 686 Bay St., Toronto, Ont., Canada, M5G 0A4.
authors.forename: Anthony
authors.initials: A
authors.lastname: Chen
authors.affiliation: From the ‡Program in Cell Biology, The Hospital for Sick Children, Toronto, and Biochemistry Department, University of Toronto; PGCRL, 19-9715, 686 Bay St., Toronto, Ont., Canada, M5G 0A4.
authors.forename: Leo
authors.initials: L
authors.lastname: Nguyen
authors.affiliation: From the ‡Program in Cell Biology, The Hospital for Sick Children, Toronto, and Biochemistry Department, University of Toronto; PGCRL, 19-9715, 686 Bay St., Toronto, Ont., Canada, M5G 0A4.
authors.forename: Ryan
authors.initials: R
authors.lastname: Murchie
authors.affiliation: From the ‡Program in Cell Biology, The Hospital for Sick Children, Toronto, and Biochemistry Department, University of Toronto; PGCRL, 19-9715, 686 Bay St., Toronto, Ont., Canada, M5G 0A4.
authors.forename: Chong
authors.initials: C
authors.lastname: Jiang
authors.affiliation: §CCBR, University of Toronto;
authors.forename: Jason
authors.initials: J
authors.lastname: Moffat
authors.affiliation: ¶The Samuel Lunenfeld Research Institute, University of Toronto.
authors.forename: Lawrence
authors.initials: L
authors.lastname: Pelletier
authors.affiliation: From the ‡Program in Cell Biology, The Hospital for Sick Children, Toronto, and Biochemistry Department, University of Toronto; PGCRL, 19-9715, 686 Bay St., Toronto, Ont., Canada, M5G 0A4 drotin@sickkids.ca.
authors.forename: Daniela
authors.initials: D
authors.lastname: Rotin
mesh_terms: D000818:Animals; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D008822:Mice, Transgenic; D009940:Organoids / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D034622:RNA Interference
chemical_list: C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011494:Protein Kinases
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