euk-134 and Cystic-Fibrosis

Channel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded ΔF508-CFTR and are poorly responsive to potentiators, because ΔF508-CFTR is intrinsically unstable at the plasma membrane (PM) even if rescued by pharmacological correctors. We have demonstrated that human and mouse CF airways are autophagy deficient due to functional sequestration of BECN1 and that the tissue transglutaminase-2 inhibitor, cystamine, or antioxidants restore BECN1-dependent autophagy and reduce SQSTM1/p62 levels, thus favoring ΔF508-CFTR trafficking to the epithelial surface. Here, we investigated whether these treatments could facilitate the beneficial action of potentiators on ΔF508-CFTR homozygous airways. Cystamine or the superoxide dismutase (SOD)/catalase-mimetic EUK-134 stabilized ΔF508-CFTR at the plasma membrane of airway epithelial cells and sustained the expression of CFTR at the epithelial surface well beyond drug withdrawal, overexpressing BECN1 and depleting SQSTM1. This facilitates the beneficial action of potentiators in controlling inflammation in ex vivo ΔF508-CFTR homozygous human nasal biopsies and in vivo in mouse ΔF508-CFTR lungs. Direct depletion of Sqstm1 by shRNAs in vivo in ΔF508-CFTR mice synergized with potentiators in sustaining surface CFTR expression and suppressing inflammation. Cystamine pre-treatment restored ΔF508-CFTR response to the CFTR potentiators genistein, Vrx-532 or Vrx-770 in freshly isolated brushed nasal epithelial cells from ΔF508-CFTR homozygous patients. These findings delineate a novel therapeutic strategy for the treatment of CF patients with the ΔF508-CFTR mutation in which patients are first treated with cystamine and subsequently pulsed with CFTR potentiators.

Topics: Adaptor Proteins, Signal Transducing; Adolescent; Animals; Antioxidants; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Membrane; Child; Cystamine; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Epithelium; Female; Genistein; Heat-Shock Proteins; Humans; Inflammation; Lipopolysaccharides; Lung; Male; Membrane Proteins; Mice; Molecular Targeted Therapy; Nasal Mucosa; Nasal Polyps; Organometallic Compounds; Protein Glutamine gamma Glutamyltransferase 2; Salicylates; Sequestosome-1 Protein

Accumulation of unwanted/misfolded proteins in aggregates has been observed in airways of patients with cystic fibrosis (CF), a life-threatening genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show how the defective CFTR results in defective autophagy and decreases the clearance of aggresomes. Defective CFTR-induced upregulation of reactive oxygen species (ROS) and tissue transglutaminase (TG2) drive the crosslinking of beclin 1, leading to sequestration of phosphatidylinositol-3-kinase (PI(3)K) complex III and accumulation of p62, which regulates aggresome formation. Both CFTR knockdown and the overexpression of green fluorescent protein (GFP)-tagged-CFTR(F508del) induce beclin 1 downregulation and defective autophagy in non-CF airway epithelia through the ROS-TG2 pathway. Restoration of beclin 1 and autophagy by either beclin 1 overexpression, cystamine or antioxidants rescues the localization of the beclin 1 interactome to the endoplasmic reticulum and reverts the CF airway phenotype in vitro, in vivo in Scnn1b-transgenic and Cftr(F508del) homozygous mice, and in human CF nasal biopsies. Restoring beclin 1 or knocking down p62 rescued the trafficking of CFTR(F508del) to the cell surface. These data link the CFTR defect to autophagy deficiency, leading to the accumulation of protein aggregates and to lung inflammation.

Topics: Acetylcysteine; Adaptor Proteins, Signal Transducing; Adolescent; Adult; Animals; Antioxidants; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Line; Cystamine; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Sodium Channels; GTP-Binding Proteins; Heat-Shock Proteins; Humans; Inflammation; Membrane Proteins; Mice; Mice, Inbred CFTR; Mice, Inbred Strains; Mice, Transgenic; Microtubule-Associated Proteins; Models, Biological; Nasal Polyps; Organometallic Compounds; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Binding; Protein Glutamine gamma Glutamyltransferase 2; Protein Transport; Reactive Oxygen Species; Respiratory Mucosa; Salicylates; Sequestosome-1 Protein; Small Ubiquitin-Related Modifier Proteins; Transglutaminases; Young Adult