Agnieszka Swiatecka-Urban

PDZ Domain Interaction Controls the Endocytic Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator

The C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-ΔTRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-ΔTRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from ∼24 to ∼13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-ΔTRL did not differ. However, endocytic recycling of CFTR-ΔTRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells.

The Short Apical Membrane Half-life of Rescued ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Results from Accelerated Endocytosis of ΔF508-CFTR in Polarized Human Airway Epithelial Cells

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, ΔF508, causes retention of ΔF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl- channels in the apical plasma membrane. Rescue of ΔF508-CFTR by reduced temperature or chemical means reveals that the ΔF508 mutation reduces the half-life of ΔF508-CFTR in the apical plasma membrane. Because ΔF508-CFTR retains some Cl- channel activity, increased expression of ΔF508-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 ΔF508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued ΔF508-CFTR that lead to the decreased apical membrane half-life of ΔF508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o-) the ΔF508 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 ΔF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing DeltaF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and DeltaF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on DeltaF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing DeltaF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The DeltaF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

The Pseudomonas aeruginosa secreted protein PA2934 decreases apical membrane expression of the cystic fibrosis transmembrane conductance regulator.

ABSTRACT We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl− ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in α/β hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.

Myosin Vb Is Required for Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator in Rab11a-specific Apical Recycling Endosomes in Polarized Human Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl- channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and ΔF508-CFTR in the apical membrane and decreased CFTR-mediated Cl- secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.

Myosin VI Regulates Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator

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.

c-Cbl Facilitates Endocytosis and Lysosomal Degradation of Cystic Fibrosis Transmembrane Conductance Regulator in Human Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl− channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl− currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.

Anti-Interleukin-2 Receptor Antibodies for the Prevention of Rejection in Pediatric Renal Transplant Patients Current Status

The anti-interleukin-2 receptor (anti-IL-2R) antibody therapy is an exciting approach to the prevention of acute rejection after renal allograft transplantation whereby immunosuppression is exerted by a selective and competitive inhibition of IL-2-induced T cell proliferation, a critical pathway of allorecognition. The anti-IL-2R antibodies specifically block the α-subunit of the IL-2R on activated T cells, and prevent T cell proliferation and activation of the effector arms of the immune system. The anti-IL-2R antibodies are used as induction therapy, immediately after renal transplantation, for prevention of acute cellular rejection in children and adults. During acute rejection, the IL-2Rα chain is no longer expressed on T cells; thus, the antibodies cannot be used to treat an existing acute rejection.Two anti-IL-2R monoclonal antibodies are currently in clinical use: daclizumab and basiliximab. In placebo-controlled phase III clinical trials in adults, daclizumab and basiliximab in combination with calcineurin inhibitor-based immunosuppression, significantly reduced the incidence of acute rejection and corticosteroid-resistant acute rejection without increasing the risk of infectious or malignant complications, and neither antibody was associated with the cytokine-release syndrome.Children who receive calcineurin inhibitors and corticosteroids for maintenance immunosuppression, as well as children who receive augmented immunosuppression to treat acute rejection, are at increased risk of growth impairment, hypertension, hyperlipidemia, lymphoproliferative disorders, diabetes mellitus, and cosmetic changes. In older children, the cosmetic adverse effects frequently reduce compliance with the treatment, and subsequently increase the risk of allograft loss. Being effective and well tolerated in children, the anti-IL-2R antibodies reduce the need for calcineurin inhibitors while maintaining the overall efficacy of the regimen; thus, the anti-IL-2R antibodies increase the safety margin (less toxicity, fewer adverse effects) of the baseline immunosuppression. Secondly, the anti-IL-2R antibodies decrease the need for corticosteroids and muromonab CD3 (OKT3) in children as a result of decreased incidence of acute rejection.The recommended pediatric dose of daclizumab is 1 mg/kg intravenously every 14 days for five doses, with the first dose administered within 24 hours pre-transplantation. This administration regimen maintains daclizumab levels necessary to completely saturate the IL-2Rα (5–10 µg/mL) in children for at least 12 weeks.The recommended pediatric dose of basiliximab for recipients <35kg is 10mg, and 20mg for recipients ≥35kg, intravenously on days 0 and 4 post-transplantation. This administration regimen maintains basiliximab levels necessary to completely saturate the IL-2Rα (>0.2 µg/mL) in children for at least 3 weeks.

Disabled-2 Protein Facilitates Assembly Polypeptide-2-independent Recruitment of Cystic Fibrosis Transmembrane Conductance Regulator to Endocytic Vesicles in Polarized Human Airway Epithelial Cells

Background: The proteins that control CFTR endocytosis in epithelial cells have only been partially explored. Results: In human airway epithelial cells Disabled-2 mediates recruitment of CFTR to clathrin-coated vesicles (CCVs) independent of the assembly polypeptide-2 (AP-2). Conclusion: AP-2 is not essential for CFTR recruitment to CCVs in these cells. Significance: Regulating CFTR interaction with Dab2 may stabilize CFTR in the plasma membrane. Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl− channel expressed in the apical plasma membrane of fluid-transporting epithelia, where the plasma membrane abundance of CFTR is in part controlled by clathrin-mediated endocytosis. The protein networks that control CFTR endocytosis in epithelial cells have only been partially explored. The assembly polypeptide-2 complex (AP-2) is the prototypical endocytic adaptor critical for optimal clathrin coat formation. AP-2 is essential for recruitment of cargo proteins bearing the YXXΦ motif. Although AP-2 interacts directly with CFTR in vitro and facilitates CFTR endocytosis in some cell types, it remains unknown whether it is critical for CFTR uptake into clathrin-coated vesicles (CCVs). Disabled-2 (Dab2) is a clathrin-associated sorting protein (CLASP) that contributes to clathrin recruitment, vesicle formation, and cargo selection. In intestinal epithelial cells Dab2 was not found to play a direct role in CFTR endocytosis. By contrast, AP-2 and Dab2 were shown to facilitate CFTR endocytosis in human airway epithelial cells, although the specific mechanism remains unknown. Our data demonstrate that Dab2 mediates AP-2 independent recruitment of CFTR to CCVs in polarized human airway epithelial cells. As a result, it facilitates CFTR endocytosis and reduces CFTR abundance and stability in the plasma membrane. These effects are mediated by the DAB homology domain. Moreover, we show that in human airway epithelial cells AP-2 is not essential for CFTR recruitment to CCVs.

Tissue-specific control of CFTR endocytosis by Dab2 Cargo recruitment as a therapeutic target

Clathrin-mediated endocytosis dynamically regulates cell membrane abundance of CFTR and plays an essential role in CFTR-dependent Cl- conductance in fluid-transporting epithelia. It requires two closely related, but distinct processes: assembly of the clathrin coat and recruitment of cargo proteins for endocytosis. The assembly polypeptide-2 complex (AP-2) is the prototypical endocytic adaptor responsible for optimal clathrin coat formation. Disabled-2 (Dab2) is a clathrin associated sorting protein (CLASP) that also mediates clathrin assembly and cargo selection. Both of these complexes have clearly been shown to play roles in CFTR endocytosis in cells that endogenously express the channel. However, their precise functions exhibit cell-specific differences. While Dab2 appears to play a central role in CFTR recruitment to the clathrin coat in airway epithelial cells, it does not play a direct role in CFTR endocytosis in intestinal epithelial cells. Here, we review our current understanding of the role of Dab2 in CFTR endocytosis in different tissues. Next, we present new data demonstrating the role of Dab2 in endocytosis of the most commonly mutated CFTR gene product, ∆F508-CFTR, in human airwy epithelial cells. Finally we discuss the potential therapeutic implications of targeting the functional interaction between ∆F508-CFTR and Dab2.