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RNA Interference Screen to Identify Kinases That Suppress Rescue of ?F508-CFTR.

ABSTRACT: Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the Cystic fibrosis transmembrane conductance regulator (CFTR). ?F508-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 ?F508-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 ?F508-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) ?F508-CFTR and measuring the amount of surface ?F508-CFTR by ELISA. The role of FGFR signaling on ?F508-CFTR trafficking was further elucidated by knocking down FGFRs and their downstream signaling proteins: Erk1/2, Akt, PLC?-1, and FRS2. Interestingly, inhibition of FGFR1 with SU5402 administered to intestinal organoids (mini-guts) generated from the ileum of ?F508-CFTR homozygous mice resulted in a robust ?F508-CFTR rescue. Moreover, combination of SU5402 and VX-809 treatments in cells led to an additive enhancement of ?F508-CFTR rescue, suggesting these compounds operate by different mechanisms. Chaperone array analysis on human bronchial epithelial cells harvested from ?F508/?F508-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 ?F508-CFTR maturation, and suggest that FGFRs may serve as important targets for therapeutic intervention for the treatment of CF.

SUBMITTER: Trzcinska-Daneluti AM

PROVIDER: S-EPMC4458721 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR.

Trzcińska-Daneluti Agata M AM Chen Anthony A Nguyen Leo L Murchie Ryan R Jiang Chong C Moffat Jason J Pelletier Lawrence L Rotin Daniela D

Molecular & cellular proteomics : MCP 20150329 6

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) t ...[more]

PMID: 25825526

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Project description:BackgroundWe previously reported that expression of a miR-138 mimic or knockdown of SIN3A in primary cultures of cystic fibrosis (CF) airway epithelia increased ΔF508-CFTR mRNA and protein levels, and partially restored CFTR-dependent chloride transport. Global mRNA transcript profiling in ΔF508-CFBE cells treated with miR-138 mimic or SIN3A siRNA identified two genes, SYVN1 and NEDD8, whose inhibition significantly increased ΔF508-CFTR trafficking, maturation, and function. Little is known regarding the dynamic changes in the CFTR gene network during such rescue events. We hypothesized that analysis of condition-specific gene networks from transcriptomic data characterizing ΔF508-CFTR rescue could help identify dynamic gene modules associated with CFTR biogenesis.MethodsWe applied a computational method, termed M-module, to analyze multiple gene networks, each of which exhibited differential activity compared to a baseline condition. In doing so, we identified both unique and shared gene pathways across multiple differential networks. To construct differential networks, gene expression data from CFBE cells were divided into three groups: (1) siRNA inhibition of NEDD8 and SYVN1; (2) miR-138 mimic and SIN3A siRNA; and (3) temperature (27 °C for 24 h, 40 °C for 24 h, and 27 °C for 24 h followed by 40 °C for 24 h).ResultsInterrogation of individual networks (e.g., NEDD8/SYVN1 network), combinations of two networks (e.g., NEDD8/SYVN1 + temperature networks), and all three networks yielded sets of 1-modules, 2-modules, and 3-modules, respectively. Gene ontology analysis revealed significant enrichment of dynamic modules in pathways including translation, protein metabolic/catabolic processes, protein complex assembly, and endocytosis. Candidate CFTR effectors identified in the analysis included CHURC1, GZF1, and RPL15, and siRNA-mediated knockdown of these genes partially restored CFTR-dependent transepithelial chloride current to ΔF508-CFBE cells.ConclusionsThe ability of the M-module to identify dynamic modules involved in ΔF508 rescue provides a novel approach for studying CFTR biogenesis and identifying candidate suppressors of ΔF508.

Dataset Information

RNA Interference Screen to Identify Kinases That Suppress Rescue of ?F508-CFTR.

Publications

RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR.

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