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Cathepsin B contributes to Na+ hyperabsorption in cystic fibrosis airway epithelial cultures Chong Da Tan1 , Carey Hobbs1 , Mansoureh Sameni2 , Bonnie F. Sloane2 , M. Jackson Stutts1 and Robert Tarran1 1 2

Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC, USA Department of Pharmacology, Wayne State University, School of Medicine, Detroit, MI, USA

Key points

The Journal of Physiology

r The epithelial Na+ channel (ENaC) is hyperactive in cystic fibrosis (CF) airway epithelia, and r r r r

contributes to excessive Na+ absorption and dehydration of the airway surface liquid (ASL) that lines the lungs – a major cause of CF lung disease. The CF gene product (CFTR) is a cAMP-regulated anion channel and how CFTR dysfunction contributes to ENaC hyperactivity is poorly understood. Of note, ENaC must be proteolytically cleaved to be active. CF ASL is moderately acidic, due to the absence of HCO3 − secretion through CFTR. The acid protease cathepsin B is present in the apical membrane of airway epithelia and is secreted into the ASL, suggesting that this protease may be active in the acidic CF environment. We have found that cathepsin B induces activation of ENaC in both Xenopus laevis oocytes and in airway epithelia. Cathepsin B is also highly expressed in airway epithelia derived from patients with CF. Inhibition of cathepsin B prevents Na+ /ASL hyperabsorption in CF airway cultures, suggesting a hitherto unrecognized role for this protease in CF pathogenesis. Treatments directed at normalizing CF ASL pH or inhibiting cathepsin B may be useful in the treatment of CF lung disease.

Abstract In cystic fibrosis (CF) lung disease, the absence of functional CF transmembrane conductance regulator results in Cl− /HCO3 − hyposecretion and triggers Na+ hyperabsorption through the epithelial Na+ channel (ENaC), which contribute to reduced airway surface liquid (ASL) pH and volume. Prostasin, a membrane-anchored serine protease with trypsin-like substrate specificity has previously been shown to activate ENaC in CF airways. However, prostasin is typically inactive below pH 7.0, suggesting that it may be less relevant in acidic CF airways. Cathepsin B (CTSB) is present in both normal and CF epithelia and is secreted into ASL, but little is known about its function in the airways. We hypothesized that the acidic ASL seen in CF airways may stimulate CTSB to activate ENaC, contributing to Na+ hyperabsorption and depletion of CF ASL volume. In Xenopus laevis oocytes, CTSB triggered α- and γENaC cleavage and induced an increase in ENaC activity. In bronchial epithelia from both normal and CF donor lungs, CTSB localized to the apical membrane. In normal and CF human bronchial epithelial cultures, CTSB was detected at the apical plasma membrane and in the ASL. CTSB activity was significantly elevated in acidic ASL, which correlated with increased abundance of ENaC in the plasma membrane and a reduction in ASL volume. This acid/CTSB-dependent activation of ENaC was ameliorated with the cell impermeable, CTSB-selective inhibitor CA074, suggesting that CTSB inhibition may have therapeutic relevance. Taken together, our data suggest that CTSB is a pathophysiologically relevant protease that activates ENaC in CF airways.

 C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

DOI: 10.1113/jphysiol.2013.267286

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(Received 23 October 2013; accepted after revision 15 September 2014; first published online 25 September 2014) Corresponding author R. Tarran: Cystic Fibrosis/Pulmonary Research & Treatment Center, 7125 Thurston Bowles Bldg., University of North Carolina, Chapel Hill, NC 27599-7248, USA. Email: [email protected] Abbreviations IAmil , amiloride-sensitive current; ASL, airway surface liquid; CAPs, channel-activating proteases; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; CTSB, cathepsin B; ENaC, epithelial Na+ channel; FBS, fetal bovine serum; HBECs, human bronchial epithelial cultures; MBS-Ca2+ , modified Barth’s saline with calcium; PBS, phosphate-buffered saline; sulpho-NHS-biotin, N-hydroxysulphosuccinimide-biotin.

Introduction −

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Human airway epithelia secrete Cl /HCO3 through the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) and associated proteins such as SLC26 (Poulsen et al. 1994; El Khouri & Tour´e, 2014). In CF, mutations in the CFTR gene result in this cAMP-regulated Cl− /HCO3 − channel being dysfunctional (Kerem et al. 1989; Riordan et al. 1989). Reduced Cl− secretion through CFTR leads to a decreased airway surface liquid (ASL) volume, which compromises mucus clearance (Boucher, 2007). The loss of CFTR-mediated HCO3 − secretion also contributes to reduced ASL pH, which has been proposed to be causal for decreased bacterial killing, altered mucus rheology and abnormal activation of an epithelial Na+ channel (ENaC) (Coakley et al. 2003; Song et al. 2003; Pezzulo et al. 2012; Garland et al. 2013). In the case of abnormal ENaC regulation, the underlying mechanism is not fully clear, but it may include abnormal stimulation of ENaC by intracellular cAMP (Stutts et al. 1997), excessive proteolytic activation and/or failure to be regulated by natural inhibitors such as short palate lung and nasal epithelial clone 1 (SPLUNC1) (Garcia-Caballero et al. 2009; Garland et al. 2013). Increased ENaC activity leads to ASL dehydration and decreased mucus clearance (Gaillard et al. 2010). This inability to clear mucus results in persistent bacterial infection and inflammation in patients with CF (Boucher, 2007; Hartl et al. 2012). ENaC belongs to the degenerins/ENaC family of proteins and is characterized by sensitivity to amiloride (Benos & Stanton, 1999). It forms the apical conduit for Na+ absorption across epithelia and plays an important role in fluid homeostasis (Garty & Palmer, 1997). Although the stoichiometry of ENaC remains debatable, recent crystallization of acid sensing ion channel 1 indicated that channels in the degenerins/ENaC family consist of a trimer of α, β and γ subunits (Jasti et al. 2007). In the lung, the cephalad movement of ASL leads to an excess volume of liquid when two airways converge. Thus, Na+ absorption via ENaC is required to remove excess Na+ and water, which maintains a constant ASL height and preserves efficient mucocilliary clearance (Boucher, 2007; Gaillard et al. 2010). The first evidence that ENaC was regulated by proteolytic cleavage came from studies in toad bladder epithelium where aprotinin, a Kunitz-type serine protease inhibitor, decreased the amiloride-sensitive short circuit

currents (Orce et al. 1980). Subsequently, it was found that secreted and membrane-anchored serine proteases can also cleave and activate ENaC in the plasma membrane, and furin-type convertases can cleave α- and γENaC intracellularly (Hughey et al. 2004; Rossier & Stutts, 2008). A class of membrane-anchored, serine proteases known as the channel-activating proteases (CAPs), which includes prostasin and matriptase, have been shown to activate ENaC in CF epithelia (Tong et al. 2004; Planes et al. 2005). However, such experiments were performed with the mucosal solution clamped at pH 7.4. In contrast, we and others have shown that CF ASL is slightly acidic (6.5) (Coakley et al. 2003; Pezzulo et al. 2012; Garland et al. 2013) and prostasin is inactive below pH 7 (Yu et al. 1994), suggesting that prostasin and other CAPs may be less relevant under the pathophysiological conditions in CF airways. As CAPs are less active in the acidic pH of CF ASL, we searched for protease(s) that remain active or become active in acidic pH. Using mass spectrometry, Kesimer et al. (2009) recently detected cathepsin B (CTSB) in apical secretions from normal, well-differentiated, primary human bronchial epithelial cultures (HBECs) grown at an air–liquid interface, suggesting that airway epithelium can also be a source of CTSB. However, the role of CTSB in the airways remains to be investigated. CTSB has previously been shown to cleave ENaC in Xenopus 2F3 cells (Alli et al. 2012). As CF airways are acidic and CTSB is present in CF ASL, we tested the hypothesis that CTSB, and not CAPs, was the pathophysiologically relevant protease that triggered Na+ hyperabsorption and CF ASL volume depletion.

Methods Two-electrode voltage clamp

Approval was obtained from the University of North Carolina’s Institutional Animal Care and Use Committee for all Xenopus laevis oocyte studies. Mature female X. laevis were anaesthetized in 0.1% MS222 (Sigma, St. Louis, MO, USA) and the ovarian lobes surgically removed. The frogs were then killed by surgical transection of the spinal cord at the level of the brainstem (pithing). Ovarian lobes were harvested and enzymatically digested to obtain defolliculated stage V–VI oocytes.  C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

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Cathepsin B and ENaC

X. laevis oocytes were injected with complementary RNAs of rat αβγENaC subunits (0.3 ng each) with or without human CTSB (1 ng each) and studied 24 h postinjection using the two-electrode voltage clamp technique as described previously (Garcia-Caballero et al. 2009). Oocytes were bathed in modified Barth’s saline with calcium (MBS-Ca2+ ) [in mM: 88 NaCl, 1 KCl, 2.4 NaHCO3 , 0.3 Ca(NO3 )2 , 0.41 CaCl2 , 0.82 MgSO4 and 15 Hepes]. During recordings, oocytes were clamped at a holding potential of −60 mV. The amiloride-sensitive current (IAmil ) was determined as the difference between whole cell currents measured in the presence and absence of amiloride (10 μM; Sigma). Recordings were performed in frog Ringer’s solution (containing, in mM, 120 NaCl, 2.5 KCl, 1.8 CaCl2 , 10 Hepes, pH 7.2). Where noted, CTSB (100 μg ml−1 ; Sigma), trypsin (10 μg ml−1 ; Sigma) and chymotrypsin (10 μg ml−1 ; Sigma) were added for  5 min before amiloride. Surface biotinylation in Xenopus laevis oocytes

Batches of 40 X. laevis oocytes were washed three times with ice-cold MBS-Ca2+ and the surface membrane of the oocytes was biotinylated with 0.5 mg ml−1 N-hydroxysulphosuccinimide-biotin (sulpho-NHS-biotin) (Pierce, Rockford, IL, USA) diluted in biotinylation buffer (in mM: 10 triethanolamine, 150 NaCl, 2 CaCl2 , pH 9.5) for 20 min on ice. Unbound sulpho-NHS-biotin was quenched with MBS-Ca2+ supplemented with 100 mM glycine. Oocytes were washed three times with ice-cold MBS-Ca2+ and lysed by passing them through a 27.5 gauge needle in lysis buffer containing 1% Triton X-100, 500 mM NaCl, 5 mM EDTA, 50 mM Tris-Cl and protease inhibitor (5 μg ml−1 ; Roche, Indianapolis, IN, USA). The lysates were vortexed for 30 s and centrifuged at 10,000 g for 10 min. Supernatants were incubated with 50 μl of neutravidin-agarose beads (Pierce) overnight at 4°C with agitation. Beads were washed three times with lysis buffer and eluted in Laemmli buffer supplemented with 10% (v/v) β-mercaptoethanol by first incubating at room temperature for 10 min, followed by heating at 95°C for another 10 min. Human lung procurement and human bronchial epithelial cell culture

Human donor lungs were obtained by the UNC CF Center Tissue Core (Chapel Hill, NC, USA) under protocols approved by the UNC Institutional Committee for the Protection of the Rights of Human Subjects. Bronchial epithelia were isolated as described, plated on 12 mm transwell clear culture inserts (Corning, Manassas, VA, USA) and studied after 3–5 weeks of culture (Tarran et al. 2013).  C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

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Human embryonic kidney 293T cell culture

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium alpha medium with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. Cells were maintained in a humidified atmosphere at 37°C with 5% CO2 . HEK293T cells were transfected with 0.75 μg human αβγENaC cDNA (0.25 μg per subunit) ± 0.5 μg human CTSB or control vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s instructions. Protease activity assays

The mucosal pH was clamped at 6.0, 6.5, 7.0 or 7.5 as previously described (Garland et al. 2013). Mucosal pH was adjusted by adding 20 μl of modified Ringer’s solution [in mM: 86 NaCl, 5 KCl, 1.2 CaCl2 , 1.2 MgCl2 and either 100 Hepes (pH 7.5 and 7.0) or 100 MES (pH 6.5 and 6)]. To detect CTSB activity, a fluorogenic substrate (Z-Arg-Arg-MCA; excitation wavelength 340 nm, emission wavelength 440 nm; 50 μM) (Peptide International, Louisville, KY, USA) was added with Ringer’s solution to ASL or to mucosally washed HBECs. CTSB activity was determined by the fluorescence signal generated from the cleavage of the substrate and was read using an Infinte 1000 plate reader (Tecan, San Jose, CA, USA) and substrate specificity was confirmed using the highly selective and cell impermeant CTSB antagonist CA074 (10 μM; Sigma). For the CAP activity assay, Boc-Gln-Ala-Arg-MCA (Peptide International) (50 μM) was used. Immunolocalization of cathepsin B

HBECs and airways from human donor lungs were embedded in OCT (Sakura Finetek, Torrance, CA, USA) and snap frozen on dry ice. Eight micron thick frozen sections were then prepared, fixed with 4% (w/v) paraformaldehyde and incubated in phosphate-buffered saline (PBS) supplemented with 5% (v/v) normal goat serum and 1% (w/v) bovine serum albumin to prevent non-specific binding. CTSB was immunostained using an antibody generated in house at Wayne State University, USA and visualized with Dylight 488-labelled secondary antiserum (Pierce). Actin was stained with Alexa fluor 649-labelled phalloidin (Invitrogen). HBEC sections were imaged using a Leica SP5 confocal microscope (Wetzlar, Germany) and a 63× glycerol lens objective. Apical membrane biotinylation in human bronchial epithelial cultures

Apical membrane proteins were biotinylated as previously described (Tarran et al. 2013). Polarized HBECs were

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washed three times with PBS supplemented with 1 mM MgCl2 and 1 mM CaCl2 (PBS2+ ). Sulpho-NHS-biotin (0.5 mg ml−1 ) in borate buffer (in mM: 85 NaCl, 4 KCl, 15 Na2 B4 O7 , pH 9) was applied apically and incubated for 30 min with gentle agitation. PBS2+ supplemented with 10% (v/v) FBS was added to the basolateral bath to prevent biotinylation of basolateral proteins. Unbound sulpho-NHS-biotin was quenched with PBS2+ supplemented with 10% (v/v) FBS. Cells were lysed in lysis buffer (0.4% sodium deoxycholate, 1% NP-40, 50 mM EGTA, 10 mM Tris-Cl, pH 7.4 and protease inhibitor (Roche) and protein concentration was determined by bicinchoninic acid assay. Three hundred micrograms of total protein per sample was incubated overnight with 100 μl of neutravidin-agarose beads at 4 °C with agitation. Biotinylated proteins bound to beads were washed three times with lysis buffer and eluted in 30 μl of Laemmli buffer supplemented with 10% (v/v) β-mercaptoethanol by first incubating at room temperature for 10 min, followed by heating at 95 °C for another 10 min. Western blotting

After biotinylation, protein samples were fractionated on 4–12% Bis–Tris gel (Invitrogen) alongside prestained protein standards (Invitrogen) and transferred on to polyvinylidene fluoride membranes. Total protein lysate samples were also run/shown in data presented. Membranes were incubated in PBS-Tween containing 5% (w/v) non-fat milk powder for 1 h at room temperature before incubation with primary antibodies; mouse anti-αENaC, rabbit anti-γENaC (UNC) (diluted 1:2000), mouse anti-CTSB (1:5000), mouse anti-V5 (Invitrogen) (1:5000), mouse anti-actin (Abcam, Cambridge, MA, USA) (1:5000), mouse anti-C9 (Pierce), rabbit anti-mCherry (Abcam) or mouse anti-GAPDH (Abcam) (1:5000), followed by incubation with species-specific horseradish peroxidase-conjugated secondary antisera (GE Healthcare, Westborough, MA, USA). The anti-αENaC antibody was generated using a polypeptide corresponding to the 54th–81st amino acids on the N-terminus of human αENaC. The characterization of this antibody has previously been described (Gentzsch et al. 2010). The polyclonal anti-γENaC antibody against cleaved γENaC was generated by Gilead Sciences (Foster City, CA, USA). The immunostained proteins were visualized with ECL chemiluminescent substrate (GE Healthcare, Westborough, MA, USA). Densitometric quantification was performed using ImageJ (NIH Freeware, Bethesda, MD, USA). Airway surface liquid height measurements

To measure the height of the ASL, PBS (20 μl) containing 2 mg ml−1 rhodamine-dextran (10 kDa; Invitrogen) was

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added to cultures at the start of the experiment. In some cases, after addition of PBS, all available fluid was aspirated with a Pasteur pipette to bring ASL volume down to minimal levels (Tarran et al. 2006). Five predetermined points (one central, four 2 mm from the edge of the culture) were XZ scanned using a confocal microscope (Leica SP5; glycerol 63× immersion lens) as described (Tarran et al. 2006). Cultures were returned to the incubator between time points. For all studies, perfluorocarbon (FC-77; 3M, St. Paul, MN, USA) was added mucosally during imaging to prevent evaporation of the ASL.

Statistical analyses

All data were presented as means ± S.E.M. Values of n refer to the number of cultures or oocytes used in each group as appropriate. All data were inspected for normal distribution. For normally distributed data, paired or unpaired Student’s t tests were used as appropriate. If data were not normally distributed, then the Mann–Whitney U test or Wilcoxon matched pairs test were used as appropriate. For comparisons of multiple groups, ANOVA tests were used. For experiments using HBECs, a minimum of four different donors supplied cultures for each experiment. For experiments utilizing oocytes, all experiments were performed at least three times.

Results Cathepsin B cleaves α and γ epithelial Na+ channel and stimulates epithelial Na+ channel activity in Xenopus laevis oocytes

To determine if CTSB was active extracellularly, the bathing media of X. laevis oocytes, co-injected with αβγENaC cRNA ± CTSB cRNA, were harvested and incubated with the fluorogenic substrate (Z-Arg-Arg-MCA). The fluorescence signal emitted as a result of Z-Arg-Arg-MCA cleavage by CTSB was then measured over time and the peak signal was averaged. CTSB activity was detected in the bathing media harvested from these oocytes (P < 0.001, n = 3) and activity was inhibited by the CTSB-selective inhibitor CA074 (Fig. 1A; P < 0.01; n = 3). To investigate the effect of CTSB on ENaC activity, we measured amiloride-sensitive Na+ currents (IAmil ) in X. laevis oocytes injected with αβγENaC cRNA ± human CTSB cRNA using the two-electrode voltage clamp system. IAmil was significantly greater in the presence of CTSB (5112 ± 153.7 nA) than for ENaC alone (2082 ± 393.4 nA; Fig. 1B; P < 0.01; n = 3). CA074 did not significantly affect basal ENaC activity but attenuated CTSB-induced activation of ENaC (Fig. 1B). To determine  C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

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oocytes co-injected with αβγENaC ± CTSB by Western blot. In all cases, we utilized α- and γENaC constructs with C-terminal V5 tags. A change in the banding pattern for γENaC was observed around 64 kDa (Fig. 1D; n = 3). To corroborate this observation, we performed surface biotinylation followed by Western blot analysis for α- and γENaC in the presence and absence of CTSB. CTSB was detected in the lysate, suggesting that CTSB was expressed (Fig. 1E and G). Our data indicated that αENaC-V5 was

further if CTSB acted extracellularly, we measured IAmil in X. laevis oocytes injected with αβγENaC cRNA ± exposure to purified CTSB. IAmil was significantly greater in X. laevis oocytes preincubated with purified CTSB (2915 ± 488.3 nA) than in oocytes incubated in bathing media alone (1173 ± 161.9 nA) (Fig. 1C; P < 0.01; n = 10). To determine whether CTSB stimulated ENaC through cleavage of ENaC subunits, we performed a preliminary examination on crude membrane preparations of X. laevis

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Figure 1. CTSB increased ENaC activity in X. laevis oocytes A, activity of CTSB secreted from X. laevis was assayed by measuring the cleavage of a fluoreogenic peptide (Z-Arg-Arg-MCA; 10 μM; n = 3). B, wild-type α-, β- and γ ENaC ± CTSB cRNA were injected into oocytes. 24 h post-injection, two-electrode voltage clamp assays were performed to measure ENaC currents (n = 3). C, two-electrode voltage clamp assays were performed on oocytes injected with wild-type α-, β- and γ ENaC cRNA ± exposure to purified CTSB (1 unit ml−1 ) for 30 min to measure ENaC currents (n = 10). D, representative Western blot obtained from crude membrane fractions from oocytes co-injected with αβγ v5ENaC cRNA ± CTSB cRNA and probed for γ ENaC. E and G, representative Western blots of surface biotinylated oocytes co-injected with αv5βγ ENaC (D) or αβγ v5ENaC (F) cRNA ± CTSB cRNA (all n = 3–4). F and H, mean densitometry analysis of α- and γ ENaC from Western blots as shown in (E) and (G), respectively. Batches of oocytes were extracted from three to five different frogs. Results are expressed as the means ± S.E.M. ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001 different from ENaC alone. † P < 0.01 different from ENaC + CTSB. CTSB, cathepsin B; ENaC, epithelial Na+ channel.

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represented by 91, 74 and 63 kDa bands in the plasma membrane of X. laevis oocytes co-injected with αβγENaC (Fig. 1E and F; n = 4). In oocytes co-injected with αβγENaC and human CTSB, there was a significant reduction in abundance of the 91 kDa αENaC band and a significant increase in the abundance of the 74 kDa band of αENaC (Fig. 1E and F; P < 0.05; both n = 4). However, there was no significant change in the 63 kDa band (Fig. 1E and F; n = 4). As the 63 kDa band is thought to constitute the active moiety, the increased abundance of 74 kDa αENaC may not contribute to the observed increase in ENaC activity. In X. laevis oocytes where V5-tagged γENaC was co-injected with untagged α- and βENaC, both 85 kDa and 64 kDa bands were detected by Western blot (Fig. 1G and H). When oocytes were co-injected with ENaC and CTSB, no significant change in band intensity occurred. However, a significant shift in the mobility of the smaller band, which corresponded to a change in size of 2.4 ± 0.3 kDa, was observed (Fig. 1G; P < 0.05; n = 3), suggesting that γENaC has been cleaved by CTSB. Cathepsin B cleaves the γ epithelial Na+ channel subunit in HEK293T cells

To determine if CTSB also cleaves γENaC in a mammalian expression system, we performed surface biotinylation followed by Western blot analysis on HEK293T cells co-transfected with αV5 βγENaC, αβV5 γENaC or αβγ V5 ENaC in the presence and absence of mCherry-tagged CTSB. Using an antibody against mCherry, CTSB was detected in the plasma membrane, suggesting that CTSB was expressed (Fig. 2A, C and E). αENaC-V5 was represented by 100 and 85 kDa bands (Fig. 2A and B; n = 3) while βENaC-V5 was seen as a 91 kDa band (Fig. 2C and D; n = 3) and γENaC was seen as a 69 kDa band (Fig. 2E and F; n = 5). Co-transfecting αβγENaC with human CTSB did not affect the membrane abundance of either αENaC (Fig. 2B; n = 3) or βENaC (Fig. 2D; n = 3). However, CTSB co-transfection resulted in a significant shift in the mobility of γENaC, which corresponded to a change in size of 7.0 ± 0.7 kDa (P < 0.001; n = 5). The membrane abundance of cleaved γENaC was also significantly reduced in the presence of CTSB (Fig. 2F; P < 0.05; n = 3). Cathepsin B may activate epithelial Na+ channel in a similar fashion as chymotrypsin

In X. laevis oocytes co-injected with αβγENaC cRNA, when either purified trypsin or chymotrypsin were added to the bath solution, increased IAmil from 1505.2 ± 175.9 to 5148 ± 195.8 and 681.7 ± 61.4 to 3377 ± 69.2 nA respectively (Fig. 3A and B; P < 0.0001; n = 6). After

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activation of ENaC with CTSB, trypsin further increased IAmil from 3348 ± 273.3 to 5557 ± 247.1 nA (Fig. 3A; P < 0.0001; n = 6). On the other hand, chymotrypsin did not result in a significant increase in IAmil in the presence of CTSB (Fig. 3B). Chymotrypsin also had no additional effect when added after purified CTSB (Fig. 3C). To probe ENaC activation by CTSB further, we co-injected X. laevis oocytes with wild-type αβγENaC cRNA or a protease-resistant ENaC where known cleavage sites were mutated to non-cleavable residues (αENaC, deficient in furin cleavage sites, wild-type βENaC and γENaC deficient in one of its two polybasic sequences; Fig. 3D) ± CTSB cRNA and performed two-electrode voltage clamp. Compared to oocytes expressing wild-type αβγENaC, expression of α202Q,205A,228Q,231A βγ 135QQQQ138 ENaC reduced IAmil from 1608 ± 231.5 nA to 195.6 ± 21.6 nA (Fig. 3E; P < 0.0001; n = 7). Co-injection of α202Q,205A,228Q,231A βγ 135QQQQ138 ENaC with CTSB increased IAmil from 195.6 ± 21.6 nA to 881.7 ± 234.7 nA (Fig. 3E; P < 0.05; n = 15). Trypsin was still able to activate α202Q,205A,228Q,231A βγ 135QQQQ138 ENaC after CTSB exposure, but failed to attain the level of activation seen for wild-type ENaC (Fig. 3F; n = 15). To understand if (i) the reduced IAmil in oocytes expressing mutant ENaC was a result of reduced membrane expression, and (ii) CTSB cleaves mutant γ 135QQQQ138 ENaC, we performed surface biotinylation followed by Western blot analysis on HEK293T cells co-transfected with αβENaC together with wild-type γENaC or γ 135QQQQ138 ENaC in the presence and absence of mCherry-tagged CTSB. α202Q,205A,228Q,231A ENaC (see Fig. 3D) was excluded from this experiment because CTSB exposure did not fully cleave αENaC (see Figs 1F and 2A). Expression of wild-type γENaC in the plasma membrane of HEK293T cells was significantly greater than γ 135QQQQ138 ENaC (Fig. 3G and H; P < 0.05; n = 3). The presence of γ 135QQQQ138 ENaC prevented CTSB-induced cleavage (Fig. 3G; n = 3). However, the membrane abundance of γ 135QQQQ138 ENaC in HEK293T cells co-transfected αβENaC and CTSB was significantly increased (Fig. 3G and H; P < 0.05; n = 3).

Immunostaining reveals that cathepsin B is present in airway epithelial apical membranes and in cystic fibrosis mucus plugs

CTSB has previously been detected in plasma membrane/endosomal fractions harvested from metastatic tumours (Sloane et al. 1986, 1987; Rozhin et al. 1987). To investigate the expression and distribution of CTSB in human airways, we utilized a mouse monoclonal antibody that recognizes several forms of CTSB, including procathepsin B (43 kDa) as well as single  C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

Cathepsin B and ENaC

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did not occur in human airway sections (Fig. 4B). Next, frozen sections of normal and CF human donor bronchi were probed for CTSB expression. In keeping with CTSB’s established role in lysosomal degradation, we observed colocalization of CTSB and lysosome-associated membrane protein 1 intracellularly in both normal and

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chain (31 kDa) and double chain (25 kDa) forms of the mature enzyme by Western blot (Moin et al. 1992; Sloane et al. 1994; Cavallo-Medved et al. 2005). This antibody detected CTSB that was transiently expressed in HEK293T cells (Fig. 4A). As an additional control, we ensured that non-specific binding of secondary antibodies

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Figure 2. CTSB cleaves γENaC in HEK293T cells A, C and E, representative Western blots of surface biotinylated HEK293T cells co-transfected with αv5βγ ENaC, αβv5γ ENaC or αβγ v5ENaC cDNA ± CTSB-mCherry cDNA respectively (n = 3–5). B, D and F, mean densitometry taken from (A), (C) and (E) respectively with (closed bars) or without (open bars) CTSB cDNA, respectively. Results are expressed as the means ± S.E.M. ∗ P < 0.05 different from ENaC alone. CTSB, cathepsin B; ENaC, epithelial Na+ channel.  C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

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CF bronchial epithelia (Fig. 4C). This staining pattern was maintained in culture and colocalization between CTSB and lysosome-associated membrane protein 1 was detected in normal HBECs (Fig. 4D). CTSB was also detected in the apical membrane of both airway epithelia and HBECs (Fig. 4C and D respectively). Consistent with previous reports that CTSB was detected in CF sputum (Martin et al. 2010; Naudin et al. 2011; Moffitt et al. 2014), we observed CTSB staining in mucus plugs that were adherent to CF luminal surfaces (Fig. 4C). Taken together, these data suggest that airway epithelia may be a significant source of CTSB in the plasma membrane and in airway secretions.

present in the apical membrane of normal and CF HBECs (Fig. 5C and E respectively). We performed densitometry on the Western blot of apical membrane biotinylation, which was then normalized to the optical intensity of the same gel after Coomassie staining, which served as a loading control. Exposure to pH 6.0 Ringer’s solution did not affect the abundance of apical membrane bound proand mature CTSB in normal HBECs. However, exposure to pH 6.0 Ringer’s solution resulted in a significant increase in the abundance of 30 kDa mature CTSB in CF HBECs (Fig. 5F). There was also a corresponding decrease in the 46 kDa pro-CTSB in the apical membrane of CF HBECs (Fig. 5F).

Normal and cystic fibrosis human bronchial epithelial cultures have extracellular, apical membrane-associated cathepsin B activity

Acidic airway surface liquid pH increases the activity of secreted cathepsin B independent of changes in protein abundance

The volume of ASL over HBECs grown on a 12 mm culture insert has previously been determined to be 1–2 μl (Harvey et al. 2011). By diluting the ASL with 20 μl of modified Ringer’s solution, ASL pH can be clamped at 6.0, 6.5, 7.0 or 7.5 for >1 h in normal and CF HBECs (Garland et al. 2013). We extensively washed HBEC mucosal surfaces to remove secreted CTSB. We then performed a fluorogenic assay to determine if pH could affect CTSB activity and then performed surface biotinylation to measure apical plasma membrane CTSB protein density under pH-clamped conditions. CTSB activity was significantly greater in cultures exposed to 20 μl of pH 6.0 Ringer’s solution compared to those exposed to pH 7.5, 7.0 and 6.5 Ringer’s solution (Fig. 5A and B; P < 0.01; n = 9). Previously, researchers have demonstrated that CTSB translocates from the perinuclear region to the plasma membrane in responses to extracellular acidic stimuli (Rozhin et al. 1994). Therefore, we next asked if acid-induced activation of CTSB in HBECs was associated with increased trafficking of CTSB to the apical membrane in HBECs. Western blot analysis of apical membrane biotinylation suggested that pro-CTSB (46 kDa) and mature CTSB (26 and 30 kDa) were

To investigate secreted CTSB, ASL was harvested and the fluorescence that resulted from the cleavage of Z-Arg-Arg-MCA was measured over time. CTSB activity was significantly greater in both normal and CF ASL set to pH 6.0 than at pH 7.5, 7.0 and 6.5 (Fig. 6A and B; P < 0.01; n = 9). We next asked whether acid-induced CTSB activation was associated with increased CTSB secretion into the ASL. Western blot analysis revealed that pro-CTSB (>46 kDa) and mature CTSB (26 and 30 kDa) were present in normal and CF ASLs (Fig. 6C and E respectively). Densitometry analysis indicated that exposure to pH 6.0 Ringer’s solution did not increase the secretion of CTSB into normal and CF ASL (Fig. 6D and F respectively).

Channel-activating protease activity is diminished in acidic airway surface liquid

Our data thus far demonstrate that: (i) CTSB can cleave and activate ENaC; (ii) active CTSB is present in the apical membrane of airway epithelia; and (iii) CTSB is more active at acidic pH. However, we and others have

WT α-, β- and γ ENaC ± CTSB cRNA were co-injected into oocytes and studied 24 h later. Trypsin-induced (A) and chymotryspin-induced (B), amiloride-sensitive whole cell currents were compared to the basal amiloride-sensitive currents in oocytes co-injected with αβγ ENaC ± CTSB cRNA (n = 6). C, chymotrypsin-induced amiloride-sensitive whole cell currents in oocytes co-injected with αβγ ENaC cRNA ± extracellular purified CTSB (n = 10). D, schematic of α (α 202Q,205A,228Q,231A ) and γ ENaC mutants (γ 135QQQQ138 ) deficient in Furin cleavage sites and one of its two polybasic sequences, respectively. E, cRNA from WT ENaC or mutants shown in (D) were injected into oocytes (n = 7–15). F, trypsin-induced whole cell amiloride-sensitive currents in oocytes co-injected with αβγ ENaC cRNA or α 202Q,205A,228Q,231A βγ 135QQQQ138 ENaC cRNA ± CTSB cRNA. Basal current (open bars), CTSB (closed bars) and CTSB followed by trypsin (grey bars). (n = 15). G, representative Western blots of surface biotinylated HEK293T cells co-transfected with αβγ ENaC or αβγ 135QQQQ138 ENaC cDNA ± CTSB cDNA (n = 3). H, mean densitometry analysis of γ ENaC from Western blots shown in (G). Batches of oocytes were extracted from three to five different frogs. Results are expressed as the means ± S.E.M. ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001 different from WT ENaC alone. † P < 0.01 different from WT ENaC with CTSB or mutant ENaC alone. Chymo, chymotrypsin; CTSB, cathepsin B; ENaC, epithelial Na+ channel; WT, wild-type.  C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

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previously shown that CAPs are expressed in airway epithelia, and have implicated them in abnormal activation in CF airways (Donaldson et al. 2002; Tong et al. 2004; Adebamiro et al. 2005; Tarran et al. 2006). As CF ASL pH is lower than normal ASL pH (Coakley et al. 2003; Pezzulo et al. 2012; Garland et al. 2013), we tested the ability of endogenous CAPs to cleave a fluorogenic substrate (BOC-Gly-Gln-Ala-MCA) at pH 6.0–8.5. Consistent with previous reports (Yu et al. 1994), we observed an approximately three-fold reduction in CAP activity at pH 7.0 and below, suggesting that this class of protease may be less relevant at the acidic pH seen in CF airways (Fig. 7A). We have previously shown that aprotinin, an inhibitor of CAPs, inhibits ENaC activity and ASL hyperabsorption in CF HBECs under thin film conditions (Tarran et al. 2006). However, aprotinin may not be specific for CAPs. Indeed, our

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CA074 prevents airway surface liquid volume depletion in cystic fibrosis but not normal human bronchial epithelial cultures

To investigate whether CTSB affected ENaC abundance at the apical plasma membrane, surface biotinylation was performed on HBECs exposed to purified CTSB or vehicle. To remove exogenous ASL, all cultures were

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Figure 4. CTSB is detected in the apical membrane of airway epithelia and in the mucus plugs of patients with cystic fibrosis A, HEK293T cells ± CTSB cDNA immunostained for CTSB. B, confocal images of human airway sections incubated with Dylight 488- and Alexa fluor 568-labelled secondary antisera only. Sections of airways and human bronchial epithelial cultures were fixed and immunostained for CTSB and LAMP1. Actin was stained with phalloidin. CTSB and LAMP1 were visualized with Dylight 488and Alexa fluor 568-labelled secondary antisera. Confocal images of normal (C) and (D) cystic fibrosis human airways. Scale bars are 50 μm. E, confocal images of polarized normal human bronchial epithelial cultures. Scale bar is 5 μm. Ab, antibody; CTSB, cathepsin B; LAMP1, lysosome-associated membrane protein 1.

 C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

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exposed to 20 μl Ringer solution mucosally for 1 h before experimentation. Exposure to CTSB resulted in a significant time-dependent increase in α- and γENaC (Fig. 8A and B; P < 0.05; n = 3). We next tested whether pH-induced cleavage of ENaC was affected by inhibition of endogenous CTSB. To this end, we apically exposed HBECs to pH 7.5 or pH 6.0 Ringer’s solution ± CA074. This manoeuvre reduced acid-induced γENaC cleavage in both normal and CF HBECs (Fig. 8C and D; P < 0.05; n = 3). Following a 20 μl mucosal volume challenge, normal HBECs absorb excess ASL through an ENaC-led process and then inactivate ENaC to maintain ASL height at 7 μm. Conversely, CF HBECs fail to autoregulate after a volume challenge leading to ASL volume depletion (Tarran et al. 2006). As predicted by the Western blot data (Fig. 8C), CA074 had little effect on normal ASL height (Fig. 8E and F). However, this inhibitor significantly prevented CF ASL volume depletion (Fig. 8E and F). As a control, we also exposed normal and CF HBECs to the cathepsin D

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inhibitor pepstatin A (10 μM). This compound failed to have any significant effect on either normal or CF ASL height at either pH. Discussion As CTSB has previously been shown to cleave ENaC in an X. laevis-derived cell line (Alli et al. 2012), we first tested whether CTSB could cleave and activate ENaC expressed in X. laevis oocytes. Expression of CTSB significantly enhanced αβγENaC currents in oocytes, suggesting that CTSB does indeed activate ENaC (Fig. 1). The cell impermeable CTSB inhibitor CA074 attenuated CTSB-induced activation of ENaC, suggesting that CTSB acted extracellularly (Fig. 1A and B). Consistent with this observation, purified CTSB added to the oocyte bath also increased ENaC activity (Fig. 1C). ENaC has been reported to be cleaved by several proteases, including CAP1 and CAP3 (Rossier & Stutts,

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Figure 5. Extracellular, plasma membrane-associated CTSB is more active at acidic pH in normal and CF HBECs Normal (A) and CF (B) HBECs were washed mucosally three times and incubated with Ringer’s solutions of different pH (6.0, 6.5, 7.0 and 7.5) and assayed for CTSB activity by measuring fluorescence of Z-Arg-Arg-MCA over time as an indicator of peptide cleavage (50 μM; n = 9). C, representative Western blot of apical membrane biotinylation from normal HBECs exposed to pH 6.0 (open bars) or pH 7.5 (grey bars) modified Ringer’s solution for 1 h (n = 4). D, densitometry analysis of Western blot taken from (C) were normalized to Coomassie blue G-250-based loading control staining and expressed as a fold of pH 6.0 (open bars). E, representative Western blot of apical membrane biotinylation from CF HBECs exposed to pH 6.0 (open bars) or pH 7.5 (grey bars) modified Ringer’s solution for 1 h (n = 3). F, densitometry analysis of Western blot taken from (E). Results are shown as means ± S.E.M. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 different from pH 7.5. #P < 0.01 and † P < 0.001 different from pH 6.0. ASL, airway surface liquid; CF, cystic fibrosis; CTSB, cathepsin B; HBECs, human bronchial epithelial cultures.

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2008) and from the banding pattern on the Western blot (Fig. 1D, E and G), we hypothesized that CTSB cleaved ENaC on its extracellular domains. Both full length (95 kDa) and cleaved (74 and 63 kDa) αENaC were detected in the plasma membrane (Fig. 1E and F). However, CTSB mostly increased the abundance of the 74 kDa αENaC band and reduced the 95 kDa αENaC without affecting the 63 kDa band (Fig. 1E and F). As the 74 kDa band probably represents partially cleaved/less active ENaC while 63 kDa band is thought to represent the twice-cleaved/active αENaC moiety, we conclude that while CTSB cleaves αENaC, this cleavage may not contribute to CTSB-induced activation of ENaC. In contrast, CTSB resulted in a 2.4 kDa shift in the molecular weight/banding pattern of the γENaC band that is detected around 63 kDa in oocytes, and a

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7 kDa shift in this band in HEK293T cells (Fig. 2E). This shift in molecular weight is equivalent to 21–40 amino acid residues being removed from γENaC. A 43 residue inhibitory tract cleaved from γENaC by furin and prostasin has previously been described (Bruns et al. 2007). This 43 amino acid sequence was further refined to a minimal 11 amino acid peptide that was capable of inhibiting ENaC (Passero et al. 2010). We speculate that the 2.4 kDa shift in γENaC seen with CTSB corresponds to the removal of a similar inhibitory domain and leads to activation of the channel. The discrepancy in the CTSB-induced molecular weight shift could be the result of differences between rat and human ENaC or between the heterologous expression systems. However, further studies will be required to confirm its existence and to determine its identity.

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Figure 6. Secreted CTSB is more active at acidic pH in normal and CF HBECs Normal (A) and CF (B) HBECs were harvested using Ringer’s solutions of different pHs (6.0, 6.5, 7.0 and 7.5) and assayed for CTSB activity by measuring fluorescence of Z-Arg-Arg-MCA over time as an indicator of peptide cleavage (50 μM; n = 9). C, representative Western blot of ASL lavages harvested from normal HBECs exposed to pH 6.0 (open bars) or pH 7.5 (grey bars) modified Ringer’s solution for 1 h (n = 5). D, densitometry analysis of Western blot taken from (C) normalized to Coomassie blue G-250 based loading control staining and expressed as a fold of pH 6.0 (open bars). E, representative Western blot of ASL lavages harvested from CF HBECs exposed to pH 6.0 (open bars) or pH 7.5 (grey bars) modified Ringer’s solution for 1 h (n = 3). F, densitometry analysis of Western blot taken from (E). Results are shown as means ± S.E.M. ∗∗ P < 0.01 different from pH 7.5. ASL, airway surface liquid; CF, cystic fibrosis; CTSB, cathepsin B; HBECs, human bronchial epithelial cultures.

 C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

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CTSB is a highly promiscuous protease and it is not possible to predict a cleavage sequence for it (http:// merops.sanger.ac.uk/cgi-bin/substrates?id=C01.060). After CTSB exposure, addition of trypsin further increased the current, suggesting that CTSB had not fully activated the channel. In contrast, chymotrypsin had no further effect when added after CTSB (Fig. 3B and C). Thus, while the precise location of CTSB cleavage remains to be identified, it is possible that CTSB and chymotrypsin share similar cleavage sites. However, we next expressed mutants of α (α202Q,205A,228Q,231A ) and γENaC (γ 135QQQQ138 ) that were deficient of αENaC furin cleavage sites and one of the γENaC polybasic sequences (Fig. 3D). In oocytes, expressing α202Q,205A,228Q,231A βγ 135QQQQ138 ENaC greatly reduced IAmil (Fig. 3E). This is consistent with previous

observations that cleavage by furin and a second protease in the polybasic sequence (e.g. 135 RKRR138 ) is required to activate ENaC fully (Hughey et al. 2003, 2004; Kleyman et al. 2009). Interestingly, CTSB was unable to cleave the γ 135QQQQ138 ENaC in HEK293T cells (Fig. 3G), suggesting that the polybasic sequence is required for CTSB-induced cleavage of γENaC. Despite preventing the cleavage of γENaC by CTSB, when the γ 135QQQQ138 ENaC was expressed in oocytes, the amiloride-sensitive whole cell current in oocytes co-injected with ENaC and CTSB was still greater than oocytes injected with ENaC alone (Fig. 3E). This is probably due to the increased γENaC membrane abundance of the αβγ 135QQQQ138 ENaC, suggesting that under some circumstances, CTSB may also regulate ENaC through other mechanism(s) such as

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Figure 7. Comparison of CAP vs. CTSB activity in HBEC ASL A, CAP activity on normal (open bars) and CF (closed bars) HBECs was determined using fluorogenic peptide Boc-Gly-Gln-Ala-MCA (50 μM) at various pH (6.0–8.5). B, effect of aprotinin (2 units ml−1 ) on CTSB activity in normal HBECs was determined using the fluorogenic peptide Z-Arg-Arg-MCA (50 μM) (n = 3). C and D, effect of cell impermeable CTSB inhibitor CA074 (10 μM) on CTSB activity in normal and CF HBECs respectively (n = 3). E and F, effect of CA074 (10 μM) on the ability of CAPs or purified trypsin to cleave Boc-Gly-Gln-Ala-MCA in normal HBECs (all n = 3). Results are shown as means ± S.E.M. ∗ P < 0.05 and ∗∗∗ P < 0.001, different from pH 6.0 or vehicle control respectively. ASL, airway surface liquid; CAP, channel-activating protease; CF, cystic fibrosis; CTSB, cathepsin B; HBECs, human bronchial epithelial cultures.  C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

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speculate that the reduced trypsin-stimulated amiloridesensitive whole cell current in α202Q,205A,228Q,231A βγ 135QQQQ138 ENaC is probably the result of reduced membrane expression of γENaC. Our results also suggested that the two-electrode voltage clamp experiments performed on the cleavage resistant mutant must be interpreted with care due to the potential difference in membrane abundance of wild-type vs. mutant ENaC. CTSB is expressed in lysosomes (Barrett & Kirschke, 1981). However, CTSB is also known to be secreted, particularly from tumour cells, in a manner that is not fully

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trafficking (Fig. 3G and H). However, why CTSB affected the surface density of the mutant but not wild-type ENaC remains to be determined. The absolute value of IAmil in oocytes expressing α202Q,205A,228Q,231A βγ 135QQQQ138 ENaC with or without CTSB was reduced compared to oocytes expressing wild-type γENaC (Fig. 3F). Although we have not performed the surface biotinylation experiment to determine the ability of trypsin to cleave the γ 135QQQQ138 ENaC ± CTSB, we observed a reduction in plasma membrane γ 135QQQQ138 ENaC compared to wild-type γENaC in HEK293T cells (Fig. 3H). We

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Figure 8. The CTSB inhibitor CA074 prevents acid-induced ASL hyperabsorption A, representative Western blots of apically biotinylated protein from normal HBECs mucosally exposed to purified CTSB (1 unit ml−1 ) or Ringer’s solution for the indicated amount of time and probed with antibodies against αand γ ENaC (n = 3). B, densitometry analysis of protein abundance of αENaC (open bars) and γ ENaC (closed bars) from Western blots taken from (A). C, representative Western blots against γ ENaC of apically biotinylated protein from normal and CF HBECs exposed for 1 h to pH 7.5 Ringer’s solution or pH 6.0 Ringer’s solution ± CA074 (10 μM) (all n = 3). D, densitometry analysis of protein abundance of γ ENaC from normal (open bars) and CF (closed bars) HBECs shown (C). E, representative XZ confocal images of ASL from normal and CF HBECs exposed mucosally to pH 6.0 Ringer’s solution in the presence of vehicle (dimethyl sulphoxide) or CA074 (10 μM). F, mean 2 h ASL height following exposure to vehicle, CA074 (10 μM) and pepstatin A (10 μM) (all n = 6–11). Normal (open bars) and CF (closed bars). Results are shown as means ± S.E.M. ∗ P < 0.05 different to t = 0 or pH 7.5 as appropriate. † P < 0.05 different to pH 6.0. ‡ P < 0.05 different to vehicle. ASL, airway surface liquid; CF, cystic fibrosis; CTSB, cathepsin B; ENaC, epithelial Na+ channel; HBECs, human bronchial epithelial cultures; NL, normal; Pep, pepstatin A; Veh, vehicle.  C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

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understood (Roshy et al. 2003). Although CTSB activity in the sputum has been reported to correlate positively with the inflammation status in CF lungs (Martin et al. 2010), it is not known if CF sputum, in the absence of exacerbation, contains higher CTSB protein levels. However, the ability to define the activity of CTSB in CF airways, in the absence of exacerbation, may also be limited by the assay condition, since pH is usually clamped at neutral or alkaline pH that favours CTSB inactivation. It was previously postulated that the increase in CTSB activity during inflammation was primarily due to damaged and/or immune cells (Reddy et al. 1995). However, data mining revealed that CTSB is present in the ASL of our HBEC culture system, suggesting that CTSB secretion from epithelia may have contributed to CTSB detected in the sputum (Kesimer et al. 2009). Three forms of CTSB, with molecular weights of 45, 30 and 25 kDa, were detected in the apical membrane and ASL of normal and CF HBECs (Figs 5 and 6). This is consistent with the molecular weight reported for the inactive (46 kDa) CTSB proenzyme and the two active forms (31 kDa and 25 kDa) (Moin et al. 1992). While we did not detect an increase in CTSB activity in CF HBECs, the effects of inflammation on CTSB secretion remain to be tested. We have previously shown that CAP1 (prostasin), CAP2 (TMPRSS4), CAP3 (matriptase) and human airway trypsin-like protease are expressed in normal and CF HBECs, and that CAP1 is upregulated in CF HBECs (Tarran et al. 2006). Several groups have shown that CAPs, including CAP1 activate ENaC in normal and CF airway epithelia (Donaldson et al. 2002; Tong et al. 2004; Adebamiro et al. 2005; Coote et al. 2009). However, these experiments were all performed with the epithelia exposed to Ringer’s solution where the pH was adjusted to 7.4, i.e. almost 1 pH unit greater than the pH seen on CF epithelia. As many CAPs are more active at alkaline pH and may even be inactive at acidic pH (Yu et al. 1994), we question whether CAPs are relevant in the acidic CF lumen. Indeed, our data suggest that proteases such as CTSB may be more pertinent in CF airways. CTSB can be activated by other proteases such as urokinase-type plasminogen activator, elastase and cathepsin G as part of a proteolytic cascade (Reiser et al. 2010). Therefore it will also be important to understand whether CTSB acts directly on ENaC, or whether it is part of a proteolytic cascade in the airways. Other than CTSB, neutrophil-derived elastase and cathepsin S also cleave and activate ENaC (Caldwell et al. 2005; Haerteis et al. 2012). Neutrophil elastase remains active under acidic pH (unpublished data). This suggests that other, non-epithelial proteases may further exacerbate Na+ hyperabsorption beyond the effects exerted by CTSB during times of chronic inflammation in CF airways. Both α- and γENaC surface densities were significantly increased by CTSB exposure (Fig. 8A and B). However, we did not see a significant change in the migration  C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

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rates of α- and γENaC on the Western blots, suggesting that CTSB may modify ENaC trafficking rather than cleavage in HBECs. It has previously been suggested that altered proteolytic activity induces Brefeldin A-sensitive trafficking of ENaC to the plasma membrane (Myerburg et al. 2010; Tan et al. 2011) and clearly, further investigation will be required to fully understand this phenomenon. Of note, while we detected the 63 kDa (i.e. active) γENaC band, we only detected the partially cleaved 75 kDa αENaC in the HBEC plasma membrane, suggesting that ENaC activity may be predominantly set by γENaC in HBECs grown and assayed under thin film conditions. CFTR conducts both Cl− and HCO3 − (Poulsen et al. 1994), which is required to keep normal ASL pH at neutral or slightly alkaline levels (7.0–7.4) (Coakley et al. 2003; Garland et al. 2013). As such, CF ASL is thought to be moderately acidic due to the lack of HCO3 − secretion through CFTR (Coakley et al. 2003) and ASL pH in the range 6.0–6.9 have been reported (Garland et al. 2013). The normal vs. CF pH difference is probably limited to only 0.5 units because the paracellular pathway is permeable to HCO3 − , leading to a backflux of HCO3 − into the ASL that offsets the lack of HCO3 − secreted by CFTR. None the less, this pH difference has been shown adversely to affect bacterial killing, mucus rheology and SPLUNC1’s ability to regulate ENaC (Quinton, 2010; Pezzulo et al. 2012; Garland et al. 2013). In the latter case, SPLUNC1 is secreted into the ASL and acts as a natural inhibitor of ENaC (Garcia-Caballero et al. 2009). SPLUNC1 is highly pH-sensitive and despite its presence in CF ASL, CF SPLUNC1 is non-functional and unable to restrain ENaC activity (Garland et al. 2013). Importantly, in the context of ENaC, SPLUNC1’s inability to regulate ENaC at acidic pH means that ENaC is present on the cell surface and can be proteolytically cleaved leading to CF ASL volume depletion (Garland et al. 2013). In agreement with our previous study (Garland et al. 2013), changing ASL pH for 1 h strongly influenced the cleavage state of γENaC in both normal and CF HBECs (Fig. 8C and D). pH-induced cleavage was abrogated by CA074 in both normal and CF HBECs, suggesting that CTSB plays a role in the abnormal ENaC cleavage at low pH but not at pH 7.5. This sensitivity to CA074 was extended to ASL height measurements in both normal and CF HBECs at acidic pH (Fig. 8E and F). That is, ASL absorption under acidic conditions was significantly retarded by CA074, again suggesting that CTSB may be a pathophysiologically relevant protease that is active in CF airways. CTSB is a non-specific protease. Thus, in addition to activating ENaC, it may exert other detrimental effects in CF lungs. For example, CTSB degrades extracellular matrixes, which in cancer is associated with tumour invasion and metastasis (Sloane et al. 2005). Mucosally secreted CTSB should not come into contact with, or affect the extracellular matrix. However, in damaged CF lungs where the epithelia have been partially

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denuded, CTSB would then have access to the extracellular matrix and may exacerbate this damage and increase cellular detachment. Despite the availability of specific CTSB inhibitors, to date, none of these antagonists have been used clinically. Reasons for this include poor bioavailability, off-target side effects and cytotoxicity. Recently, 5-nitro-8-hydroxyquinoline (nitroxoline) was identified to be a potent and reversible inhibitor of CTSB that has also been used as an antimicrobial agent in the treatment of urinary tract infections caused by Escherichia coli (Pelletier et al. 1995; Mirkovic et al. 2011). However, it is not known if proteases such as CTSB have a role in the antimicrobial action of nitroxoline. While the search to identify new small molecule inhibitors that selectively inhibit CTSB continues, our data further emphasize the possibility that inhaled CTSB inhibitors may be beneficial for the treatment of CF lung disease. In conclusion, CTSB is present in the apical membrane of both normal and CF human airways epithelia and is secreted into their ASL. However, CTSB is more active at the slightly acidic pH seen in CF ASL and appears to be a physiologically relevant protease that drives Na+ hyperabsorption in CF HBECs. Inhibition of extracellular CTSB is predicted to normalize ENaC activity in CF lungs and may help limit or reverse the CF mucus dehydration/stasis.

References Adebamiro A, Cheng Y, Johnson JP & Bridges RJ (2005). Endogenous protease activation of ENaC: effect of serine protease inhibition on ENaC single channel properties. J Gen Physiol 126, 339–352. Alli AA, Song JZ, Al-Khalili O, Bao HF, Ma HP & Eaton DC (2012). Cathepsin B is secreted apically from Xenopus 2F3 cells and cleaves the epithelial sodium channel (ENaC) to increase its activity. J Biol Chem 287, 30073–30083. Barrett AJ & Kirschke H (1981). Cathepsin B, cathepsin H, and cathepsin L. Methods Enzymol 80 (Pt C), 535–561. Benos DJ & Stanton BA (1999). Functional domains within the degenerin/epithelial sodium channel (Deg/ENaC) superfamily of ion channels. J Physiol 520(Pt 3), 631–644. Boucher RC (2007). Evidence for airway surface dehydration as the initiating event in CF airway disease. J Intern Med 261, 5–16. Bruns JB, Carattino MD, Sheng S, Maarouf AB, Weisz OA, Pilewski JM, Hughey RP & Kleyman TR (2007). Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the gamma-subunit. J Biol Chem 282, 6153–6160. Caldwell RA, Boucher RC & Stutts MJ (2005). Neutrophil elastase activates near-silent epithelial Na+ channels and increases airway epithelial Na+ transport. Am J Physiol Lung Cell Mol Physiol 288, L813–L819.

J Physiol 592.23

Cavallo-Medved D, Mai J, Dosescu J, Sameni M & Sloane BF (2005). Caveolin-1 mediates the expression and localization of cathepsin B, pro-urokinase plasminogen activator and their cell-surface receptors in human colorectal carcinoma cells. J Cell Sci 118, 1493–1503. Coakley RD, Grubb BR, Paradiso AM, Gatzy JT, Johnson LG, Kreda SM, O’Neal WK & Boucher RC (2003). Abnormal surface liquid pH regulation by cultured cystic fibrosis bronchial epithelium. Proc Natl Acad Sci USA 100, 16083–16088. Coote K, Atherton-Watson HC, Sugar R, Young A, MacKenzie-Beevor A, Gosling M, Bhalay G, Bloomfield G, Dunstan A, Bridges RJ, Sabater JR, Abraham WM, Tully D, Pacoma R, Schumacher A, Harris J & Danahay H (2009). Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease. J Pharmacol Exp Ther 329, 764–774. Donaldson SH, Hirsh A, Li DC, Holloway G, Chao J, Boucher RC & Gabriel SE (2002). Regulation of the epithelial sodium channel by serine proteases in human airways. J Biol Chem 277, 8338–8345. El Khouri E & Tour´e A (2014). Functional interaction of the cystic fibrosis transmembrane conductance regulator with members of the SLC26 family of anion transporters (SLC26A8 and SLC26A9): physiological and pathophysiological relevance. Int J Biochem Cell Biol 52, 58–67. Gaillard EA, Kota P, Gentzsch M, Dokholyan NV, Stutts MJ & Tarran R (2010). Regulation of the epithelial Na+ channel and airway surface liquid volume by serine proteases. Pflugers Arch 460, 1–17. Garcia-Caballero A, Rasmussen JE, Gaillard E, Watson MJ, Olsen JC, Donaldson SH, Stutts MJ & Tarran R (2009). SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage. Proc Natl Acad Sci USA 106, 11412–11417. Garland AL, Walton WG, Coakley RD, Tan CD, Gilmore RC, Hobbs CA, Tripathy A, Clunes LA, Bencharit S, Stutts MJ, Betts L, Redinbo MR & Tarran R (2013). Molecular basis for pH-dependent mucosal dehydration in cystic fibrosis airways. Proc Natl Acad Sci USA 110 (40), 15973–15978. Garty H & Palmer LG (1997). Epithelial sodium channels: function, structure, and regulation. Physiol Rev 77, 359–396. Gentzsch M, Dang H, Dang Y, Garcia-Caballero A, Suchindran H, Boucher RC & Stutts MJ (2010). The cystic fibrosis transmembrane conductance regulator impedes proteolytic stimulation of the epithelial Na+ channel. J Biol Chem 285 (42), 32227–32232. Haerteis S, Krappitz M, Bertog M, Krappitz A, Baraznenok V, Henderson I, Lindstrom E, Murphy JE, Bunnett NW & Korbmacher C (2012). Proteolytic activation of the epithelial sodium channel (ENaC) by the cysteine protease cathepsin-S. Pflugers Arch 464, 353–365. Hartl D, Gaggar A, Bruscia E, Hector A, Marcos V, Jung A, Greene C, McElvaney G, Mall M & Doring G (2012). Innate immunity in cystic fibrosis lung disease. J Cyst Fibros 11, 363–382.

 C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

J Physiol 592.23

Cathepsin B and ENaC

Harvey PR, Tarran R, Garoff S & Myerburg MM (2011). Measurement of the airway surface liquid volume with simple light refraction microscopy. Am J Respir Cell Mol Biol 45, 592–599. Hughey RP, Mueller GM, Bruns JB, Kinlough CL, Poland PA, Harkleroad KL, Carattino MD & Kleyman TR (2003). Maturation of the epithelial Na+ channel involves proteolytic processing of the α- and γ-subunits. J Biol Chem 278, 37073–37082. Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD & Kleyman TR (2004). Epithelial sodium channels are activated by furin-dependent proteolysis. J Biol Chem 279, 18111–18114. Jasti J, Furukawa H, Gonzales EB & Gouaux E (2007). Structure of acid-sensing ion channel 1 at 1.9 A˚ resolution and low pH. Nature 449, 316–323. Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M & Tsui LC (1989). Identification of the cystic fibrosis gene: genetic analysis. Science 245, 1073–1080. Kesimer M, Kirkham S, Pickles RJ, Henderson AG, Alexis NE, Demaria G, Knight D, Thornton DJ & Sheehan JK (2009). Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways? Am J Physiol Lung Cell Mol Physiol 296, L92–L100. Kleyman TR, Carattino MD & Hughey RP (2009). ENaC at the cutting edge: regulation of epithelial sodium channels by proteases. J Biol Chem 284, 20447–20451. Martin SL, Moffitt KL, McDowell A, Greenan C, Bright-Thomas RJ, Jones AM, Webb AK & Elborn JS (2010). Association of airway cathepsin B and S with inflammation in cystic fibrosis. Pediatr Pulmonol 45, 860–868. Mirkovic B, Renko M, Turk S, Sosic I, Jevnikar Z, Obermajer N, Turk D, Gobec S & Kos J (2011). Novel mechanism of cathepsin B inhibition by antibiotic nitroxoline and related compounds. ChemMedChem 6, 1351–1356. Moffitt KL, Martin SL, Jones AM, Webb AK, Cardwell C, Tunney MM & Elborn JS (2014). Inflammatory and immunological biomarkers are not related to survival in adults with Cystic Fibrosis. J Cyst Fibros 13, 63–68. Moin K, Day NA, Sameni M, Hasnain S, Hirama T & Sloane BF (1992). Human tumour cathepsin B. Comparison with normal liver cathepsin B. Biochem J 285(Pt 2), 427–434. Myerburg MM, Harvey PR, Heidrich EM, Pilewski JM & Butterworth MB (2010). Acute regulation of the epithelial sodium channel in airway epithelia by proteases and trafficking. Am J Respir Cell Mol Biol 43, 712–719. Naudin C, Joulin-Giet A, Couetdic G, Plesiat P, Szymanska A, Gorna E, Gauthier F, Kasprzykowski F, Lecaille F & Lalmanach G (2011). Human cysteine cathepsins are not reliable markers of infection by Pseudomonas aeruginosa in cystic fibrosis. PLoS One 6, e25577. Orce GG, Castillo GA & Margolius HS (1980). Inhibition of short-circuit current in toad urinary bladder by inhibitors of glandular kallikrein. Am J PhysiolRenal Physiol 239, F459–F465.

 C 2014 The Authors. The Journal of Physiology  C 2014 The Physiological Society

5267

Passero CJ, Carattino MD, Kashlan OB, Myerburg MM, Hughey RP & Kleyman TR (2010). Defining an inhibitory domain in the gamma subunit of the epithelial sodium channel. Am J Physiol Renal Physiol 299, F854–F861. Pelletier C, Prognon P & Bourlioux P (1995). Roles of divalent cations and pH in mechanism of action of nitroxoline against Escherichia coli strains. Antimicrob Agents Chemother 39, 707–713. Pezzulo AA, Tang XX, Hoegger MJ, Alaiwa MH, Ramachandran S, Moninger TO, Karp PH, Wohlford-Lenane CL, Haagsman HP, van Eijk M, Banfi B, Horswill AR, Stoltz DA, McCray PB, Jr., Welsh MJ & Zabner J (2012). Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung. Nature 487, 109–113. Planes C, Leyvraz C, Uchida T, Angelova MA, Vuagniaux G, Hummler E, Matthay M, Clerici C & Rossier B (2005). In vitro and in vivo regulation of transepithelial lung alveolar sodium transport by serine proteases. Am J Physiol Lung Cell Mol Physiol 288, L1099–L1109. Poulsen JH, Fischer H, Illek B & Machen TE (1994). Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci USA 91, 5340–5344. Quinton PM (2010). Role of epithelial HCO3 transport in mucin secretion: lessons from cystic fibrosis. Am J Physiol Cell Physiol 299, C1222–C1233. Reddy VY, Zhang QY & Weiss SJ (1995). Pericellular mobilization of the tissue-destructive cysteine proteinases, cathepsins B, L, and S, by human monocyte-derived macrophages. Proc Natl Acad Sci USA 92, 3849–3853. Reiser J, Adair B & Reinheckel T (2010). Specialized roles for cysteine cathepsins in health and disease. J Clin Invest 120, 3421–3431. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, et al. (1989). Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066–1073. Roshy S, Sloane BF & Moin K (2003). Pericellular cathepsin B and malignant progression. Cancer Metastasis Rev 22, 271–286. Rossier BC & Stutts MJ (2008). Activation of the epithelial sodium channel (ENaC) by serine proteases. Annu Rev Physiol 283 (12), 7455–7463. Rozhin J, Robinson D, Stevens MA, Lah TT, Honn KV, Ryan RE & Sloane BF (1987). Properties of a plasma membrane-associated cathepsin B-like cysteine proteinase in metastatic B16 melanoma variants. Cancer Res 47, 6620–6628. Rozhin J, Sameni M, Ziegler G & Sloane BF (1994). Pericellular pH affects distribution and secretion of cathepsin B in malignant cells. Cancer Res 54, 6517–6525. Sloane BF, Rozhin J, Johnson K, Taylor H, Crissman JD & Honn KV (1986). Cathepsin B: association with plasma membrane in metastatic tumors. Proc Natl Acad Sci USA 83, 2483–2487. Sloane BF, Rozhin J, Hatfield JS, Crissman JD & Honn KV (1987). Plasma membrane-associated cysteine proteinases in human and animal tumors. Exp Cell Biol 55, 209–224.

5268

C. Da Tan and others

Sloane BF, Moin K, Sameni M, Tait LR, Rozhin J & Ziegler G (1994). Membrane association of cathepsin B can be induced by transfection of human breast epithelial cells with c-Ha-ras oncogene. J Cell Sci 107(Pt 2), 373–384. Sloane BF, Yan S, Podgorski I, Linebaugh BE, Cher ML, Mai J, Cavallo-Medved D, Sameni M, Dosescu J & Moin K (2005). Cathepsin B and tumor proteolysis: contribution of the tumor microenvironment. Semin Cancer Biol 15, 149–157. Song Y, Thiagarajah J & Verkman AS (2003). Sodium and chloride concentrations, pH, and depth of airway surface liquid in distal airways. J Gen Physiol 122, 511–519. Stutts MJ, Rossier BC & Boucher RC (1997). Cystic fibrosis transmembrane conductance regulator inverts protein kinase A-mediated regulation of epithelial sodium channel single channel kinetics. J Biol Chem 272, 14037–14040. Tan CD, Selvanathar IA & Baines DL (2011). Cleavage of endogenous γENaC and elevated abundance of αENaC are associated with increased Na+ transport in response to apical fluid volume expansion in human H441 airway epithelial cells. Pflugers Arch 462, 431–441. Tarran R, Trout L, Donaldson SH & Boucher RC (2006). Soluble mediators, not cilia, determine airway surface liquid volume in normal and cystic fibrosis superficial airway epithelia. J Gen Physiol 127, 591–604. Tarran R, Sabater JR, Clarke TC, Tan CD, Davies CM, Liu J, Yeung A, Garland AL, Stutts MJ, Abraham WM, Phillips G, Baker WR, Wright CD & Wilbert S (2013). Nonantibiotic macrolides prevent human neutrophil elastase-induced mucus stasis and airway surface liquid volume depletion. Am J Physiol Lung Cell Mol Physiol 304, L746–L756. Tong Z, Illek B, Bhagwandin VJ, Verghese GM & Caughey GH (2004). Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line. Am J Physiol Lung Cell Mol Physiol 287, L928–L935. Yu JX, Chao L & Chao J (1994). Prostasin is a novel human serine proteinase from seminal fluid. Purification, tissue distribution, and localization in prostate gland. J Biol Chem 269, 18843–18848.

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Additional information Competing interests None declared.

Author contributions C.D.T., C.A.H., M.S., B.F.S., M.J.S. and R.T. carried out the conception and design of research. C.D.T., C.A.H., M.S. and R.T. performed the experiments. C.D.T., C.A.H., M.S., M.J.S., B.F.S. and R.T. analysed data and interpreted results of the experiments. C.D.T., C.A.H., M.S., M.J.S. and R.T. prepared the figures. C.D.T. and R.T. drafted the manuscript. C.D.T., C.A.H., M.S., B.F.S., M.J.S. and R.T. edited and revised the manuscript. C.D.T., C.A.H., M.S., B.F.S., M.J.S. and R.T. approved the final version of manuscript. All protease assays, oocyte experiments and ASL height measurements were performed at the UNC-Chapel Hill. Western blot analysis was performed at both the UNC-Chapel Hill and Wayne State University.

Funding Funded by NIH R01HL108927, PPG P01HL034322, British American Tobacco and CFF RDP R026.

Acknowledgements We gratefully acknowledge the technical assistance of Hong He, Kimberly Burns and Yan Dang, and we thank the UNC CF Center Molecular, Tissue and Histology Cores for their help with this project.

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