Arsenic promotes ubiquitinylation and lysosomal degradation of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in human airway epithelial cells

Abstract

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.

FIGURE 1.

The cytotoxic effects of arsenic were examined using CellTiter 96 AQ_ueous One Solution reagent (Promega). Data are expressed as the percentage of increase versus control. n = 3 for each dose and time. *, p < 0.0001 versus other doses at each time point. Control was vehicle (water)-treated.

FIGURE 2.

Arsenic reduces abundance and function of CFTR in airway epithelial cells. A, arsenic reduces the abundance of CFTR in the apical membrane and cell lysate. CFBE cells were treated with varying doses of arsenic (0.1–50 ppb) or vehicle (water) for 4 h, and cell surface biotinylation and Western blot analyses were performed. Representative blots are shown in the upper panels, and the summary of the data is shown in the lower panel. AP CFTR is apical membrane CFTR (black bars). WCL CFTR is whole cell lysate CFTR (striped bars). Data are normalized for ezrin in whole cell lysates and presented as the percentage of control (C). n = 4/group. *, p < 0.05 versus control. B, arsenic reduces the CFTR-mediated short circuit current. Polarized CFBE cells were treated with arsenic (10 ppb of arsenic for 4 h before Ussing chamber studies). Data are reported as the short circuit current inhibited by the CFTR channel blocker CFTR-172 in forskolin (10 μm)-treated cells (ΔIsc). n = 4/group. *, p < 0.05.

FIGURE 3.

Arsenic targets CFTR for lysosomal degradation. A, to determine whether arsenic promoted the lysosomal degradation of CFTR, CFBE cells were treated with vehicle (water) or arsenic (10 ppb) for 2 h in the presence of lysosomal or proteasomal inhibitors, and CFTR in cell lysates was detected by Western blot analysis. Arsenic reduced the amount of CFTR, confirming studies reported in Fig. 2A. Chloroquine (CHQ; 200 μm) and ammonium chloride (NH₄Cl; 50 mm), inhibitors of the lysosomal degradation of proteins, blocked the arsenic-induced decrease in CFTR abundance. Lactacystin (Lacta; 50 μm), an inhibitor of the proteasomal degradation of proteins, had no effect on the arsenic-induced decrease in CFTR abundance. n = 4/group.*, p < 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 = 3/group. *, p < 0.05.

FIGURE 4.

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 μm) 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 = 4/group. *, p < 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 = 3/group. Representative blots are shown above quantitation.

FIGURE 6.

Arsenic reduced CFTR abundance by phosphorylating c-Cbl and promoting interaction between c-Cbl and CFTR. A, siRNA knockdown of c-Cbl (sic-Cbl) reduces c-Cbl protein levels in CFBE cells, as assessed by Western blot analysis. A c-Cbl representative Western blot is shown for cell lysates from experiments in panel B, detecting apical membrane CFTR. siNeg, siNegative. B, to determine whether the E3 ubiquitin ligase c-Cbl mediates the effect of arsenic to promote CFTR degradation, c-Cbl expression was reduced in CFBE cells by siRNA-mediated knockdown, and the effect of arsenic on the amount of CFTR in the apical plasma membrane was determined by cell surface biotinylation and Western blot analysis. siRNA for c-Cbl completely inhibited the arsenic-induced decrease in CFTR. n = 4/group. *, p < 0.05 versus control. C, to determine whether arsenic altered the abundance of c-Cbl, CFBE cells were treated with varying doses of arsenic (0.1–10 ppb), and c-Cbl protein abundance was measured by Western blot analysis. Data are reported as the percentage of the control. n = 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 = 3/group. *, p < 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 = 3/group. *, p < 0.05 versus control.