Arsenic Promotes Ubiquitinylation and Lysosomal Degradation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Channels in Human Airway Epithelial Cells*

Background: Arsenic increases respiratory bacterial infections by an unknown mechanism. Results: Arsenic increased the c-Cbl-mediated ubiquitinylation and degradation of CFTR in human lung cells and reduced CFTR chloride secretion. Conclusion: The reduction in chloride secretion is proposed to decrease mucociliary clearance of respiratory pathogens. Significance: Low levels of arsenic commonly found in food and water suppress the innate immune function of the lung. Arsenic exposure significantly increases respiratory bacterial infections and reduces the ability of the innate immune system to eliminate bacterial infections. Recently, we observed in the gill of killifish, an environmental model organism, that arsenic exposure induced the ubiquitinylation and degradation of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is essential for the mucociliary clearance of respiratory pathogens in humans. Accordingly, in this study, we tested the hypothesis that low dose arsenic exposure reduces the abundance and function of CFTR in human airway epithelial cells. Arsenic induced a time- and dose-dependent increase in multiubiquitinylated CFTR, which led to its lysosomal degradation, and a decrease in CFTR-mediated chloride secretion. Although arsenic had no effect on the abundance or activity of USP10, a deubiquitinylating enzyme, siRNA-mediated knockdown of c-Cbl, an E3 ubiquitin ligase, abolished the arsenic-stimulated degradation of CFTR. Arsenic enhanced the degradation of CFTR by increasing phosphorylated c-Cbl, which increased its interaction with CFTR, and subsequent ubiquitinylation of CFTR. Because epidemiological studies have shown that arsenic increases the incidence of respiratory infections, this study suggests that one potential mechanism of this effect involves arsenic-induced ubiquitinylation and degradation of CFTR, which decreases chloride secretion and airway surface liquid volume, effects that would be proposed to reduce mucociliary clearance of respiratory pathogens.

The World Health Organization has identified arsenic as the number one environmental chemical of concern with regard to human health. In the United States, arsenic is listed as the number one chemical of concern in the Agency for Toxic Substances and Disease Registry list of priority pollutants in the environment (1). Arsenic exposure has been associated with increased rates of cancer, cardiovascular disease, diabetes, and reproductive and developmental problems (2)(3)(4)(5)(6)(7)(8)(9)(10). Exposure to arsenic in drinking water in utero or during early childhood has pronounced pulmonary effects in humans, greatly increasing subsequent mortality from both malignant and nonmalignant lung disease, including chronic bacterial infections and bronchiectasis, which is characterized by chronic bacterial infections (11)(12)(13)(14)(15)(16).
In studies on experimental animals, environmentally relevant levels of arsenic inhibit the ability of the innate immune system to eliminate bacterial and viral infections. For example, as little as 2 ppb of arsenic in the swim water of zebrafish dramatically reduces their ability to clear both viral and bacterial infections (17). In addition, 100 ppb of arsenic in the drinking water of mice significantly increases mortality in response to infection by the H1N1 influenza virus (18). Although gene array studies in mice reveal that arsenic down-regulates the expression of innate immune genes in the lungs (19), notably the expression of cytokines that enhance the migration into the lungs of phagocytic neutrophils, an essential component of the innate immune response, very little is known about the molecular mechanisms whereby low levels of arsenic inhibit the innate immune response of the lungs to bacterial infection.
Another vital component of the innate immune response to respiratory bacterial infection is mucociliary clearance (20). The cystic fibrosis transmembrane conductance regulator (CFTR), 2 a cyclic AMP-regulated chloride channel in the apical membrane of airway epithelial cells, plays an essential role in mucociliary clearance by secreting chloride into the periciliary space, which drives the secretion of sodium across the paracellular pathway (21)(22)(23)(24)(25). Sodium chloride secretion establishes an osmotic gradient across the airway epithelium that promotes fluid secretion. Thus, CFTR regulates the volume of airway surface liquid, which is an important component of the mucociliary escalator (20,26). Individuals with defective CFTR function, for example patients with cystic fibrosis, have an inability to clear respiratory pathogens, which results in chronic respiratory infections, the primary cause of morbidity and mortality in cystic fibrosis (21)(22)(23)(24)(25).
In recent studies on CFTR in the gill of killifish, an environmental model organism, we observed that arsenic induced the ubiquitinylation and subsequent degradation of CFTR (27,28). Although several other studies have shown that arsenic increases protein ubiquitinylation, the cellular mechanism whereby arsenic increases the ubiquitinylation of CFTR is unknown, as is the relevance of this observation to the function of the human lungs (29,30). Thus, the goal of this study was two-fold. First, we tested the hypothesis that environmentally relevant levels of arsenic enhance the ubiquitinylation and degradation of CFTR in human airway epithelial cells. Second, we began to elucidate the cellular mechanism by which arsenic promotes respiratory infections. Our results demonstrate that arsenic promotes the activation of the E3 ubiquitin ligase, c-Cbl, through enhanced tyrosine phosphorylation, resulting in an increase in the ubiquitinylation and lysosomal degradation of CFTR. Because epidemiological studies have shown that arsenic increases the incidence of respiratory infections, this study suggests that one mechanism of this effect involves arsenic-induced ubiquitinylation and degradation of CFTR, which decreases sodium and chloride secretion and is predicted to decrease airway surface liquid volume, effects that would be proposed to reduce mucociliary clearance of respiratory pathogens.

EXPERIMENTAL PROCEDURES
Cell Culture-The role of arsenic in regulating CFTR function was studied in human airway epithelial cells (CFBE41o Ϫ cells, homozygous for the ⌬F508 mutation) stably expressing WT-CFTR. Details on the stable transfection, characterization of CFBE41o Ϫ cells expressing WT-CFTR (hereafter called CFBE cells), and cell culture conditions have been described in detail by several laboratories (31)(32)(33). CFBE cells between passages 18 and 27 were grown in an air-liquid interface culture at 37°C for 7-10 days, as described previously (33). Sodium arsenite (NaAsO 2 ; 0.1-50 ppb) was added to the cell culture medium for a variety of times (2 h to 4 days). The concentrations of arsenic examined were selected based on measurements of arsenic in human plasma obtained from individuals exposed to arsenic in drinking water (34).
Ubiquitinylation Assay-To measure the amount of ubiquitinylated CFTR in CFBE cells, a protocol was adapted from Urbé et al. (35) as described previously in detail by our laboratory (36 -38). Briefly, CFTR was immunoprecipitated, and the immunoprecipitated CFTR was examined by SDS-PAGE and Western blot analysis and probed with anti-ubiquitin antibodies (FK1 and FK2). The quantitation of ubiquitinylated CFTR was calculated as the signal obtained with the FK antibodies normalized for immunoprecipitated CFTR detected with a CFTR antibody (36 -38).
Immunoprecipitation-To determine whether c-Cbl, an E3 ligase, interacts with CFTR, c-Cbl was immunoprecipitated from CFBE cell lysates by methods described previously in detail (32). Briefly, CFBE cells grown as polarized monolayers on 75-mm Transwell filters were washed with ice-cold PBS, scraped, and spun at 200 ϫ g at 4°C. The cell pellet was lysed in immunoprecipitation buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 5 mM MgCl 2 , 5 mM EDTA, 1 mM EGTA, 1% Triton X-100 (Bio-Rad), Complete protease inhibitor tablet without EDTA (Roche Applied Science), and Phos-STOP tablet (Roche Applied Science) for 30 min at 4°C and then centrifuged at 14,000 ϫ g for 10 min at 4°C. The supernatant was collected in a new tube and rotated at 4°C for 2 h. After centrifuging at 14,000 ϫ g for 15 min, the lysates were added to protein A-Sepharose bead antibody complexes. c-Cbl was immunoprecipitated with rabbit c-Cbl C-15 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Rabbit IgG (Bethyl Laboratories, Montgomery, TX) was used as a nonimmune control. After overnight incubation, the beads were washed, and proteins were eluted by incubation at 37°C for 30 min. Immunoprecipitated c-Cbl was analyzed by Western blots, and CFTR immunoprecipitated with c-Cbl was detected with Western blot analysis using the CFTR antibody described above.
Identification of Active Deubiquitinylating Enzymes (DUBs)-To test the hypothesis that arsenic enhances the ubiquitinylation and degradation of CFTR by inhibiting active DUBs, we used a biochemical activity assay described by Dr. Hidde Ploegh (39 -41) and described previously by our laboratory in detail (36 -38).
Biochemical Determination of Apical Membrane CFTR-The biochemical determination of apical membrane CFTR was performed by domain-selective cell surface biotinylation using EZ-Link TM Sulfo-NHS-LC-Biotin (Pierce), as described previously in detail by our laboratory (42)(43)(44).
Cytotoxicity Assay-To determine whether arsenic treatment was cytotoxic to airway cells, CFBE cells were incubated with sodium arsenite in serum-free medium for 2 h to 4 days, at concentrations between 0.1 and 50 ppb. Cytotoxicity was measured using the CellTiter 96 AQ ueous One solution reagent (Promega, Madison, WI), according to the manufacturer's protocol as published previously by our laboratory (45).
Intracellular Vesicle Isolation-To assess the effect of arsenic on the endosomal trafficking of CFTR, differential centrifugation combined with immunoprecipitation was used to isolate early endosomes, late endosomes, and recycling endosomes from airway epithelial cells, as described previously by our laboratory (38,44,48) and originally described by Barile et al. (49). Briefly, CFBE cells were incubated in the presence or absence of 2 ppb of arsenic for 4 h at 37°C, washed with PBS (pH 7.4), and scraped from filters into isolation buffer (10 mM triethanolamine, 250 mM sucrose, pH to 7.6, 8 mg/liter PMSF, and 0.08 mg/liter leupeptin). Cells were homogenized with a plastic tube homogenizer and centrifuged at 4000 ϫ g for 10 min at 4°C. Supernatant was saved, and homogenization was repeated on the pellet. Pooled supernatants were centrifuged at 17,000 ϫ g for 20 min at 4°C, and then supernatant was centrifuged at 200,000 ϫ g for 1 h at 4°C. The pellet was resuspended via homogenization and centrifuged a second time for 1 h at 200,000 ϫ g at 4°C. The pellet was resuspended in 500 l of PBS containing protease inhibitors, and CFTR was immunoprecipitated from this fraction (intracellular vesicle fraction). Rab 5 (early endosomes)-, Rab 7 (late endosomes)-, and Rab 11 (recycling endosomes)-specific antibodies were used to identify CFTR localization in the corresponding endosomal population.
Antibodies and Reagents-The antibodies used were: mouse anti-human CFTR C terminus antibody (clone 24-1; R&D systems, Minneapolis, MN); mouse anti-CFTR antibody (clone 596; University of North Carolina Cystic Fibrosis Center, Chapel Hill, NC); mouse anti-ezrin antibody, mouse anti-HA antibody, rabbit anti-c-Cbl, and mouse anti-phospho-tyrosine antibody (Santa Cruz Biotechnology); mouse anti-ubiquitin antibodies (clones FK2 and FK1) (BioMol, Plymouth Meeting, PA); rabbit anti-USP10 antibody and rabbit anti-IgG antibody (Bethyl Laboratories); and horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (Bio-Rad). All antibodies and reagents were used at the concentrations recommended by the manufacturers.
Data Analysis and Statistics-Statistical analysis of the data was performed using GraphPad Prism version 5.0 for Macintosh (GraphPad Software, San Diego, CA). Means were compared using a t test or analysis of variance followed by Tukey's test, as appropriate. p Ͻ 0.05 was considered significant. Data are expressed as the mean Ϯ S.E.

Environmentally Relevant Levels of Arsenic Are Not
Cytotoxic-To determine whether arsenic is toxic to human airway epithelial cells, CFBE cells were exposed to arsenic (0.1-50 ppb) for a variety of times (2 h to 4 days), and cytotoxicity was determined by the CellTiter 96 AQ ueous One cytotoxicity assay (45). The concentrations of arsenic tested were selected based on measurements of arsenic in human plasma obtained from individuals exposed to arsenic in drinking water (34). Arsenic was not cytotoxic at 0.1, 2.0, or 10 ppb for all times studied (Fig. 1). The highest concentration of arsenic examined (50 ppb) was not cytotoxic for up to 8 h of exposure. However, longer exposure to 50 ppb of arsenic was cytotoxic to CFBE cells. Because our goal was to study the noncytotoxic effects of arsenic, all subsequent studies were conducted on human airway epithelial cells exposed to arsenic for 4 h or less.
Arsenic Reduces CFTR Abundance and Function in Human Airway Epithelial Cells-The effect of arsenic on CFTR abundance is shown in Fig. 2A. Arsenic exposure for 4 h induced a dose-dependent decrease in CFTR abundance in both the apical membrane of cells, as determined by cell surface biotinylation, and the total cell lysates ( Fig. 2A). As little as 2 ppb of arsenic significantly decreased CFTR abundance in total cell lysate, and 10 ppb significantly decreased CFTR apical membrane abundance. To determine whether the arsenic-induced decrease in plasma membrane CFTR reduced chloride secretion into the airway surface liquid, monolayers of CFBE cells were mounted in Ussing chambers, and the forskolin (i.e. cAMP)-stimulated, CFTR-mediated short circuit current was measured. Arsenic exposure for 4 h (10 ppb) significantly reduced forskolin-stimulated CFTR-mediated chloride secretion by ϳ50% from 14.4 Ϯ 1.4 A/cm 2 in control to 7.3 Ϯ 0.5 A/cm 2 in the presence of arsenic (Fig. 2B). Thus, arsenic reduced both plasma membrane CFTR abundance and CFTRmediated chloride secretion by ϳ50%.
Arsenic Promotes Ubiquitin-mediated Lysosomal Degradation of CFTR-To begin to explore the mechanism whereby arsenic reduced CFTR abundance, we conducted studies to determine whether arsenic enhanced the lysosomal or proteasomal degradation of CFTR using pharmacological inhibitors. To this end, we treated cells with vehicle or arsenic (10 ppb for 2 h) and examined the effect of pharmacological inhibitors of lysosomal (ammonium chloride or chloroquine) or proteasomal (lactacystin) degradation of proteins on CFTR abundance. Lysosomal inhibitors, but not a proteasomal inhibitor, blocked the ability of arsenic to decrease CFTR abundance (Fig.  3A). This observation demonstrates that arsenic promotes the lysosomal degradation of CFTR.
To provide additional support for the observation that arsenic increases the lysosomal degradation of CFTR, studies were conducted to examine the effect of arsenic on the subcellular distribution of CFTR in endosomes of airway cells. In previous studies, we and others demonstrated that CFTR is rapidly endocytosed from the apical membrane of human airway epithelial cells and avidly recycled back to the plasma membrane  (33, 38, 43, 50 -52). We hypothesized that arsenic altered the endosomal trafficking of CFTR, redirecting CFTR from the endosomal recycling pathway to the lysosomal pathway for degradation. To this end, we purified endosomes from CFBE cells, treated with or without arsenic, using differential centrifugation and density gradient fractionation, as described under "Experimental Procedures." Arsenic had no effect on the amount of CFTR in recycling endosomes, but increased the amount of CFTR in both early endosomes and late endosomes by over 2-fold (Fig. 3B). Taken together, these observations suggest that arsenic reduced plasma membrane CFTR by increasing both CFTR endocytosis and the amount of CFTR directed to late endosomes and subsequently to lysosomes for degradation.
To determine whether arsenic reduced CFTR abundance by increasing the ubiquitinylation of CFTR, CFBE cells were exposed to vehicle (water) or arsenic (10 ppb) for 30 min, 1 h, 2 h, 3 h, or 8 h, and then CFTR was immunoprecipitated, and the immunoprecipitated CFTR was separated by SDS-PAGE followed by Western blot analysis using an antibody that detects mono-, multi-, and polyubiquitinylated proteins (FK2) or an antibody that detects only polyubiquitinylated proteins (FK1). In these experiments, cells were also treated with chloroquine to inhibit the lysosomal degradation of CFTR. The addition of chloroquine is required to detect ubiquitinylated CFTR because ubiquitinylated proteins are rapidly degraded in lysosomes (36 -38). Arsenic produced a time-dependent increase in the amount of ubiquitinylated CFTR (Fig. 4A, FK2  antibody). However, the same experiment repeated with pro-  , an inhibitor of the proteasomal degradation of proteins, had no effect on the arsenic-induced decrease in CFTR abundance. n ϭ 4/group.*, p Ͻ 0.05 versus control. B, to determine whether arsenic redirects CFTR from the recycling pathway to the lysosomal pathway, CFBE cells were treated with 10 ppb of arsenic for 4 h, and intracellular vesicle isolation and co-immunoprecipitation studies were conducted to determine the subcellular location of CFTR in cells treated with or without arsenic. Intracellular vesicles were purified with density gradient centrifugation, CFTR-containing vesicles were immunoprecipitated using a monoclonal CFTR antibody (clone M3A7, Upstate Biotech Millipore), and endosomes were identified by SDS-PAGE and Western blot analysis. Early endosomes were identified with Rab5a, recycling endosomes were identified with Rab11a, and late endosomes were identified with Rab7a. Data are presented as the amount of CFTR in each endosomal compartment normalized for the total amount of CFTR immunoprecipitated. n ϭ 3/group. *, p Ͻ 0.05. teasomal inhibition and an antibody specific for polyubiquitinylated proteins (FK1) did not detect ubiquitinylated CFTR in these experiments (data not shown). Taken together, the FK2 and FK1 antibody studies suggest that arsenic increased the amount of mono-and/or multiubiquitinylated CFTR. Because most of the ubiquitinylated CFTR had a molecular mass greater than 200 kDa (Fig. 4A), and CFTR detected by Western blot using a CFTR antibody was ϳ180 kDa ( Figs. 2A and 4B), and a single ubiquitin has a molecular mass of 8 kDa, we infer that most of the ubiquitinylated CFTR was multiubiquitinylated, a signal that targets proteins for lysosomal degradation (53)(54)(55)(56). Studies were also conducted to examine the effect of arsenic on CFTR abundance in CFBE cells exposed to arsenic (10 ppb) for 30 min, 1 h, 2 h, 3 h, or 8 h in the absence of chloroquine. SDS-PAGE followed by Western blot analysis revealed that 10 ppb of arsenic elicited a time-dependent decrease in CFTR abundance (Fig. 4B) that was inversely related to the amount of ubiquitinylated CFTR at each time point (compare Fig. 4, A and  B). These observations are consistent with the idea that arsenic promotes the ubiquitin-mediated degradation of CFTR in the lysosome.
Arsenic Activates the E3 Ubiquitin Ligase c-Cbl to Promote Degradation of CFTR-In previous studies, we demonstrated that the ubiquitinylated status of CFTR depends on the relative activities of the E3 ubiquitin ligase c-Cbl, which adds ubiquitin to CFTR, and USP10, a deubiquitinylating enzyme that removes ubiquitin from CFTR (37,38). To determine whether arsenic inhibits the activity of USP10, and thereby increases the amount of ubiquitinylated CFTR, we used a deubiquitinylating enzyme activity assay described previously (39 -41). CFBE cells were treated for 4 h with vehicle or arsenic (2 ppb), cells were lysed, and an HA-tagged ubiquitin-vinyl methyl ester probe (HA-UbVME) was added to the lysates. The HA-UbVME probe forms an irreversible, covalent bond with active DUBs. Subsequently, HA-UbVME-DUB complex(s) were immunoprecipitated with an anti-HA antibody, and immunoprecipitated complex(s) were analyzed by SDS-PAGE followed by Western blotting using an anti-USP10 antibody. As illustrated in Fig. 5, arsenic had no effect on USP10 activity. Using this same assay and silver-stained gels of cell lysates, we saw no changes in the activity of any other active DUBS in CFBE cells exposed to arsenic (2 ppb for 4 h, data not shown) as compared with control. Thus, arsenic did not increase ubiquitinylated CFTR by inhibiting USP10.
To determine whether arsenic increased the amount of ubiquitinylated CFTR by a mechanism involving the E3 ligase, c-Cbl, we used siRNA to knock down c-Cbl abundance. siRNA . Arsenic (10 ppb) increases multiubiquitinylation and degradation of CFTR in a time-dependent manner. A, cells were treated with arsenic for the times indicated, CFTR was immunoprecipitated using a monoclonal antibody (clone M3A7, Upstate Biotech Millipore), and ubiquitinylated CFTR was detected via Western blot analysis using an anti-ubiquitin antibody (FK2, BioMol). Cells were treated with chloroquine (200 M) to allow the accumulation of ubiquitinylated CFTR (Ub-CFTR) in quantities that could be detected by Western blot analysis. Similar amounts of CFTR were immunoprecipitated in all cases (data not shown); thus, differences in the amount of ubiquitinylated CFTR detected were not due to differences in the efficiency of the immunoprecipitation step for CFTR. Representative blots are shown. B, cells were treated with arsenic for the times indicated in the absence of chloroquine, and Western blot analysis was performed to examine the time-dependent effect of arsenic on CFTR abundance. Data were normalized for ezrin abundance. n ϭ 4/group. *, p Ͻ 0.05 versus control. FIGURE 5. Arsenic does not alter USP10 DUB activity. Cells were treated with arsenic (2 ppb) or vehicle (water) for 4 h, cells were lysed, and the lysates were incubated with the HA-UbVME probe to identify active DUBs. The HA-UbVME-DUB complex was immunoprecipitated with an anti-HA antibody, and the immunoprecipitated complex was analyzed by Western blot analysis using an anti-USP10 antibody. The nonimmune IgG did not immunoprecipitate USP10 and thus served as a negative control (data not shown). n ϭ 3/group. Representative blots are shown above quantitation.
for c-Cbl reduced c-Cbl protein abundance by 59 Ϯ 7.3% (Fig.  6A) as determined by SDS-PAGE and Western blot analysis. A reduction in c-Cbl protein abundance completely eliminated the arsenic (10 ppb for 4 h)-induced decrease in plasma mem-brane CFTR (Fig. 6B). Thus, arsenic increased the amount of ubiquitinylated CFTR by a mechanism that requires c-Cbl.
To elucidate the mechanism whereby arsenic increased the c-Cbl-mediated ubiquitinylation of CFTR, studies were con-  1-10 ppb), and c-Cbl protein abundance was measured by Western blot analysis. Data are reported as the percentage of the control. n ϭ 4/group. D, to determine whether arsenic promotes CFTR interaction with c-Cbl, CFBE cells were treated with arsenic (10 ppb) for 4 h, and cells were lysed in the presence of phosphatase inhibitors. c-Cbl was immunoprecipitated, and immunoprecipitated c-Cbl was analyzed by Western blot with a CFTR antibody (clone M3A7, Upstate Biotech Millipore). Results are normalized for c-Cbl immunoprecipitation efficiency and expressed as the percentage of the control. n ϭ 3/group. *, p Ͻ 0.05 versus control. E, to determine whether arsenic increases the phosphorylation of c-Cbl, cells were treated with arsenic (10 ppb) or vehicle (water) for 4 h and lysed in the presence of phosphatase inhibitors, and c-Cbl was immunoprecipitated and analyzed by Western blot using a phospho-tyrosine (pTyr)-specific antibody. Results are normalized for c-Cbl immunoprecipitation efficiency and expressed as the percentage of the control. n ϭ 3/group. *, p Ͻ 0.05 versus control. ducted to determine whether arsenic increased c-Cbl abundance, the interaction between c-Cbl and CFTR, and/or the tyrosine phosphorylation of c-Cbl, which increases c-Cbl activity (57,58). The ability of E3 ligases to ubiquitinylate target proteins is regulated by phosphorylation and by E3 ligase interaction with target proteins (59). Arsenic had no effect on c-Cbl protein abundance (Fig. 6C), but significantly increased the amount of c-Cbl-CFTR interaction by 48% Ϯ 13% as determined by co-immunoprecipitation studies (Fig. 6D). To determine whether arsenic promoted the tyrosine phosphorylation of c-Cbl, we immunoprecipitated c-Cbl from CFBE cells that had been exposed to vehicle or arsenic (10 ppb) for 4 h and probed the immunoprecipitated c-Cbl with a phospho-tyrosine-specific antibody. Fig. 6E demonstrates that arsenic increased tyrosine phosphorylation of c-Cbl by 46 Ϯ 17% (Fig.  6E). Taken together, these results suggest that arsenic promotes the multiubiquitinylation of CFTR, and its subsequent degradation in lysosomes, by enhancing the tyrosine phosphorylation of c-Cbl, and thus, interaction of c-Cbl with CFTR.

DISCUSSION
The results of the current study demonstrate that arsenic promotes the time-and dose-dependent reduction in CFTR abundance and chloride secretion in polarized human airway epithelial cells. We present evidence that this occurs through an arsenic-mediated increase in the phosphorylation and activation of the E3 ubiquitin ligase, c-Cbl, which promotes increased interaction of c-Cbl with CFTR and multiubiquitinylation of CFTR, targeting ubiquitinylated CFTR for lysosomal degradation. Decreased CFTR-mediated chloride secretion has been shown to reduce airway surface liquid volume and ciliary beat frequency, effects that decrease mucociliary clearance of respiratory pathogens (20,26). Because environmental exposure to arsenic increases the incidence of respiratory infections (11)(12)(13)(14)(15)(16), this study suggests that one mechanism of this effect involves arsenic-induced ubiquitinylation and degradation of CFTR.
The effects of arsenic on CFTR were observed at levels in the cell culture medium as low as 2 ppb, a concentration that is well within the range of values measured in the blood of individuals exposed to arsenic in the drinking water. For example, individuals in Bangladesh with drinking water with a mean arsenic concentration of 90 ppb, levels not uncommon in the United States, have levels of inorganic arsenic in their blood ranging from 1.4 to 11.0 ppb, with a mean value of 5.5 ppb (34). Thus, the levels of arsenic used in the present study are environmentally relevant. Accordingly, the results of the present study provide a novel insight into the cellular mechanism whereby environmentally relevant levels of arsenic are associated with increased incidence of respiratory infections.
We and others have demonstrated that the plasma membrane expression of CFTR in human airway epithelial cells depends on its rate of synthesis and delivery to the plasma membrane, as well as its rate of internalization from the plasma membrane (i.e. endocytosis), the rate of recycling of endocytosed CFTR back to the plasma membrane, and its ubiquitinylation and degradation in the lysosome (43, 60 -66). Recently, we demonstrated that c-Cbl ubiquitinylates endocytosed CFTR, thereby targeting CFTR for degradation in the lysosome (66). In the early endosome, USP10 deubiquitinylates CFTR, which directs CFTR back to the plasma membrane (37,38). Thus, the balance between c-Cbl-mediated ubiquitinylation and USP10mediated deubiquitinylation of CFTR modulates the plasma membrane abundance of CFTR, which in turn determines the rate of chloride secretion (37,38,66). Although arsenic had no effect on the activity or abundance of USP10, we observed that arsenic increased the tyrosine phosphorylation of c-Cbl, which enhanced the interaction between c-Cbl and CFTR, an effect that increased the amount of CFTR in early and late endosomes as well as the amount of ubiquitinylated CFTR, which is directed to the lysosome for degradation. Previous studies have shown that tyrosine phosphorylation of c-Cbl stimulates its E3 ubiquitin ligase activity (57,58), observations consistent with the results in the present study. It has been suggested that because arsenic has similar chemistry to phosphate, it mimics a phosphorylated residue on a protein (67). The present results are consistent with, but do not prove, the view that arsenic binds to and activates c-Cbl. It is also possible that arsenic may increase the phosphorylation of c-Cbl by another, unknown mechanism.
Previous studies have shown that arsenic regulates the expression and activity of genes involved in protein ubiquitinylation and degradation. Arsenic increases the amount of ubiquitinylated proteins in human uroepithelial cells and HEK cells (68,69) and induces the expression of genes involved in protein ubiquitinylation and degradation (29,30). Interestingly, arsenic trioxide up-regulates the expression of Cbl-b and directly binds to the RING finger domain of c-Cbl and inhibits its self-ubiquitinylation and degradation (70 -72). The present work confirms and extends these studies by demonstrating that arsenic activates c-Cbl by increasing its tyrosine phosphorylation and increasing the ubiquitinylation and degradation of CFTR, a novel target of c-Cbl.
Several studies have shown that arsenic inhibits the innate immune system in the lung by down-regulating the expression of interleukin 1b, Toll-like receptors, and several cytokines and cytokine receptor genes (18,19). Furthermore arsenic has immunosuppressive effects on human T lymphocytes and impairs macrophage function (73,74). Thus, arsenic suppresses the innate immune system in the lungs by several mechanisms.
In conclusion, we have demonstrated that arsenic decreases CFTR-mediated chloride secretion by human airway epithelial cells, an effect that will decrease airway surface liquid volume, and thereby, mucociliary clearance of respiratory pathogens. This is the first study to demonstrate that environmentally relevant levels of arsenic inhibit the innate immune function of human airway epithelial cells by increasing the ubiquitinylation and degradation of the CFTR chloride channel, and it also provides novel insight into the mechanism of this action of arsenic. Our findings are particularly relevant because the World Health Organization has determined that the global disease burden of lung infections exceeds that of HIV/AIDS, cancer, and heart disease and has since 1990 and has estimated that 500 million people are exposed to arsenic in their drinking water (75).