RESUMO
We previously reported that delivery of a microRNA-138 mimic or siRNA against SIN3A to cultured cystic fibrosis (ΔF508/ΔF508) airway epithelia partially restored ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated cAMP-stimulated Cl- conductance. We hypothesized that dissecting this microRNA-138/SIN3A-regulated gene network would identify individual proteins contributing to the rescue of ΔF508-CFTR function. Among the genes in the network, we rigorously validated candidates using functional CFTR maturation and electrolyte transport assays in polarized airway epithelia. We found that depletion of the ubiquitin ligase SYVN1, the ubiquitin/proteasome system regulator NEDD8, or the F-box protein FBXO2 partially restored ΔF508-CFTR-mediated Cl- transport in primary cultures of human cystic fibrosis airway epithelia. Moreover, knockdown of SYVN1, NEDD8, or FBXO2 in combination with corrector compound 18 further potentiated rescue of ΔF508-CFTR-mediated Cl- conductance. This study provides new knowledge of the CFTR biosynthetic pathway. It suggests that SYVN1 and FBXO2 represent two distinct multiprotein complexes that may degrade ΔF508-CFTR in airway epithelia and identifies a new role for NEDD8 in regulating ΔF508-CFTR ubiquitination.
Assuntos
Sequência de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Proteínas F-Box/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Mucosa Respiratória/metabolismo , Deleção de Sequência , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo , Proteínas de Ciclo Celular/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/patologia , Proteínas F-Box/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Transporte de Íons/genética , Proteína NEDD8 , Proteínas do Tecido Nervoso/genética , Complexo de Endopeptidases do Proteassoma/genética , Mucosa Respiratória/fisiologia , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
The most common cystic fibrosis (CF) mutation, ΔF508, causes protein misfolding, leading to proteosomal degradation. We recently showed that expression of miR-138 enhances CF transmembrane conductance regulator (CFTR) biogenesis and partially rescues ΔF508-CFTR function in CF airway epithelia. We hypothesized that a genomic signature approach can be used to identify new bioactive small molecules affecting ΔF508-CFTR rescue. The Connectivity Map was used to identify 27 small molecules with potential to restore ΔF508-CFTR function in airway epithelia. The molecules were screened in vitro for efficacy in improving ΔF508-CFTR trafficking, maturation, and chloride current. We identified four small molecules that partially restore ΔF508-CFTR function in primary CF airway epithelia. Of these, pyridostigmine showed cooperativity with corrector compound 18 in improving ΔF508-CFTR function. There are few CF therapies based on new molecular insights. Querying the Connectivity Map with relevant genomic signatures offers a method to identify new candidates for rescuing ΔF508-CFTR function.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação da Expressão Gênica , Genômica , Biperideno/química , Brônquios/metabolismo , Cloretos/química , Biologia Computacional , Fibrose Cística/genética , Genoma Humano , Células HeLa , Humanos , Fenótipo , Pizotilina/química , Transporte Proteico , Brometo de Piridostigmina/química , Mucosa Respiratória/metabolismo , Software , Ácido Valproico/químicaRESUMO
MicroRNAs (miRNAs) are increasingly recognized as important posttranscriptional regulators of gene expression, and changes in their actions can contribute to disease states. Little is understood regarding miRNA functions in the airway epithelium under normal or diseased conditions. We profiled miRNA expression in well-differentiated primary cultures of human cystic fibrosis (CF) and non-CF airway epithelia, and discovered that miR-509-3p and miR-494 concentrations were increased in CF epithelia. Human non-CF airway epithelia, transfected with the mimics of miR-509-3p or miR-494, showed decreased cystic fibrosis transmembrane conductance regulator (CFTR) expression, whereas their respective anti-miRs exerted the opposite effect. Interestingly, the two miRNAs acted cooperatively in regulating CFTR expression. Upon infecting non-CF airway epithelial cells with Staphylococcus aureus, or upon stimulating them with the proinflammatory cytokines TNF-α or IL-1ß, we observed an increased expression of both miRNAs and a concurrent decrease in CFTR expression and function, suggesting that inflammatory mediators may regulate these miRNAs. Transfecting epithelia with anti-miRs for miR-509-3p and miR-494, or inhibiting NF-κB signaling before stimulating cells with TNFα or IL-1ß, suppressed these responses, suggesting that the expression of both miRNAs was responsive to NF-κB signaling. Thus, miR-509-3p and miR-494 are dynamic regulators of CFTR abundance and function in normal, non-CF airway epithelia.