The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, ⌬F508, causes retention of ⌬F508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl ؊ channels in the apical plasma membrane. Rescue of ⌬F508-CFTR by reduced temperature or chemical means reveals that the ⌬F508 mutation reduces the half-life of ⌬F508-CFTR in the apical plasma membrane. Because ⌬F508-CFTR retains some Cl ؊ channel activity, increased expression of ⌬F508-CFTR in the apical membrane could serve as a potential therapeutic approach for cystic fibrosis. However, little is known about the mechanisms responsible for the short apical membrane half-life of ⌬F508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued ⌬F508-CFTR that lead to the decreased apical membrane half-life of ⌬F508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o؊) the ⌬F508 mutation increased endocytosis of CFTR from the apical membrane without causing a global endocytic defect or affecting the endocytic recycling of CFTR in the Rab11a-specific apical recycling compartment.The cystic fibrosis transmembrane conductance regulator (CFTR) 2 is an ATP binding cassette (ABC) transporter and a cAMP-regulated Cl Ϫ channel that mediates transepithelial Cl Ϫ transport in the airways, intestine, pancreas, testis, and other tissues (1-3). Cystic fibrosis (CF), a lethal genetic disease, is caused by mutations in the CFTR gene (1, 2). The most common mutation in CFTR is ⌬F508 (4, 5). ⌬F508-CFTR does not fold properly, and most of the protein is retained within the endoplasmic reticulum (ER) where it is subsequently degraded (5, 6). Several studies suggest that the ER retention of ⌬F508-CFTR is not complete, and some ⌬F508-CFTR is constitutively expressed in the plasma membrane of primary epithelial cells from individuals homozygous for the ⌬F508 mutation (7-10). Because ⌬F508-CFTR retains some Cl Ϫ channel activity when expressed in the plasma membrane (5,6,(11)(12)(13)(14), it would be desirable to increase the expression of ⌬F508-CFTR in the plasma membrane to alleviate the symptoms in CF patients. The trafficking of ⌬F508-CFTR to the plasma membrane can be increased by chemical means or reduced temperature (15-21). Yet, functional and biochemical studies in heterologous cell lines demonstrate that rescued ⌬F508-CFTR has a greatly reduced stability or halflife in the post-ER compartments, including the plasma membrane (13,(22)(23)(24). Very little is known about the apical membrane half-life of rescued ⌬F508-CFTR in polarized human airway epithelial cells. A recent study demonstrates that the functional stability of ⌬F508-CFTR in the apical membrane of differentiated respiratory epithelial cells derived from nasal polyps from individuals homozygous for the ⌬F508 mutation is decreased compared with WT-CFTR (25). Furthermore, the bioc...

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, is a cyclic AMP-regulated chloride channel and a regulator of other ion channels and transporters. In epithelial cells CFTR is rapidly endocytosed from the apical plasma membrane and efficiently recycles back to the plasma membrane. Because ubiquitination targets endocytosed CFTR for degradation in the lysosome, deubiquitinating enzymes (DUBs) are likely to facilitate CFTR recycling. Accordingly, the aim of this study was to identify DUBs that regulate the post-endocytic sorting of CFTR. Using an activity-based chemical screen to identify active DUBs in human airway epithelial cells, we demonstrated that Ubiquitin Specific Protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and its trafficking in the post-endocytic compartment. small interference RNA-mediated knockdown of USP10 increased the amount of ubiquitinated CFTR and its degradation in lysosomes, and reduced both apical membrane CFTR and CFTR-mediated chloride secretion. Moreover, a dominant negative USP10 (USP10-C424A) increased the amount of ubiquitinated CFTR and its degradation, whereas overexpression of wt-USP10 decreased the amount of ubiquitinated CFTR and increased the abundance of CFTR. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl؊ secretion across fluid-transporting epithelia is regu-

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens.

The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), DeltaF508, leads to the absence of CFTR Cl(-) channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of DeltaF508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl(-) channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl(-) secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl(-) secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and DeltaF508-CFTR Cl(-) channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of DeltaF508-CFTR.

Role of glucocorticoid receptor in acclimation of killifish (Fundulus heteroclitus) to seawater and effects of arsenic.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated Cl ؊ channel expressed in the apical plasma membrane in fluid-transporting epithelia. Although CFTR is rapidly endocytosed from the apical membrane of polarized epithelial cells and efficiently recycled back to the plasma membrane, little is known about the molecular mechanisms regulating CFTR endocytosis and endocytic recycling. Myosin VI, an actin-dependent, minus-end directed mechanoenzyme, has been implicated in clathrin-mediated endocytosis in epithelial cells. The goal of this study was to determine whether myosin VI regulates CFTR endocytosis. Endogenous, apical membrane CFTR in polarized human airway epithelial cells (Calu-3) formed a complex with myosin VI, the myosin VI adaptor protein Disabled 2 (Dab2), and clathrin. The tail domain of myosin VI, a dominant-negative recombinant fragment, displaced endogenous myosin VI from interacting with Dab2 and CFTR and increased the expression of CFTR in the plasma membrane by reducing CFTR endocytosis. However, the myosin VI tail fragment had no effect on the recycling of endocytosed CFTR or on fluid-phase endocytosis. CFTR endocytosis was decreased by cytochalasin D, an actin-filament depolymerizing agent. Taken together, these data indicate that myosin VI and Dab2 facilitate CFTR endocytosis by a mechanism that requires actin filaments.

Cystic fibrosis (CF), the most common lethal genetic disease in Caucasians, is characterized by chronic bacterial lung infection and excessive inflammation, which lead to progressive loss of lung function and premature death. Although ivacaftor (VX-770) alone and ivacaftor in combination with lumacaftor (VX-809) improve lung function in CF patients with the Gly551Asp and del508Phe mutations, respectively, the effects of these drugs on the function of human CF macrophages are unknown. Thus studies were conducted to examine the effects of lumacaftor alone and lumacaftor in combination with ivacaftor (i.e., ORKAMBI) on the ability of human CF ( del508Phe/ del508Phe) monocyte-derived macrophages (MDMs) to phagocytose and kill Pseudomonas aeruginosa. Lumacaftor alone restored the ability of CF MDMs to phagocytose and kill P. aeruginosa to levels observed in MDMs obtained from non-CF (WT-CFTR) donors. This effect contrasts with the partial (~15%) correction of del508Phe Cl secretion of airway epithelial cells by lumacaftor. Ivacaftor reduced the ability of lumacaftor to stimulate phagocytosis and killing of P. aeruginosa. Lumacaftor had no effect on P. aeruginosa-stimulated cytokine secretion by CF MDMs. Ivacaftor (5 µM) alone and ivacaftor in combination with lumacaftor reduced secretion of several proinflammatory cytokines. The clinical efficacy of ORKAMBI may be related in part to the ability of lumacaftor to stimulate phagocytosis and killing of P. aeruginosa by macrophages.