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SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage
Agustin Garcia-Caballero, Julia E. Rasmussen, Erol Gaillard, Michael J. Watson, John C. Olsen, Scott H. Donaldson, M. Jackson Stutts, and Robert Tarran
PNAS 106 (27) ;
Agustin Garcia-Caballero
Julia E. Rasmussen
Erol Gaillard
Michael J. Watson
John C. Olsen
Scott H. Donaldson
M. Jackson Stutts
Robert Tarran
Edited by Peter C. Agre, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, and approved May 1, 2009 (received for review April 6, 2009)
This article has a correction. Please see:
Many epithelia, including the superficial epithelia of the airways, are thought to secrete “volume sensors,” which regulate the volume of the mucosal lining fluid. The epithelial Na+ channel (ENaC) is often the rate limiting factor in fluid absorption, and must be cleaved by extracellular and/or intracellular proteases before it can conduct Na+ and absorb excess mucosal liquid, a process that can be blocked by proteases inhibitors. In the airways, airway surface liquid dilution or removal activates ENaC. Therefore, we hypothesized that endogenous proteases are membrane-anchored, whereas endogenous proteolysis inhibitors are soluble and can function as airway surface liquid volume sensors to inhibit ENaC activity. Using a proteomic approach, we identified short palate, lung, and nasal epithelial clone (SPLUNC)1 as a candidate volume sensor. Recombinant SPLUNC1 inhibited ENaC activity in both human bronchial epithelial cultures and Xenopus oocytes. Knockdown of SPLUNC1 by shRNA resulted in a failure of bronchial epithelial cultures to regulate ENaC activity and airway surface liquid volume, which was restored by adding recombinant SPLUNC1 to the airway surface liquid. Despite being able to inhibit ENaC, recombinant SPLUNC1 had little effect on extracellular serine protease activity. However, SPLUNC1 specifically bound to ENaC, preventing its cleavage and activation by serine proteases. SPLUNC1 is highly expressed in the airways, as well as in colon and kidney. Thus, we propose that SPLUNC1 is secreted onto mucosal surfaces as a soluble volume sensor whose concentration and dilution can regulate ENaC activity and mucosal volumes, including that of airway surface liquid.Epithelial mucosal surfaces are lined with fluids whose volume and composition are precisely controlled. In the airways, a thin film of airway surface liquid helps protect mammalian airways from infection by acting as a lubricant for efficient mucus clearance (, ). This layer moves cephalad during mucus clearance, and excess liquid that accumulates as 2 airways converge is eliminated by Na+-led airway surface liquid absorption with Na+ passing through the epithelial Na+ channel (ENaC) (). How ENaC activity is sensed and controlled by the airways is poorly understood. However, there is evidence that reporter molecules in the airway surface liquid can serve as volume sensing signals whose dilution or concentration can alter specific cell surface receptors, which control ion transport rates to either absorb or secrete airway surface liquid as needed (). ENaC must be cleaved by intracellular furin-type proteases and/or extracellular channel activating proteases (CAPs) such as prostasin to be active and to conduct Na+ (–). ENaC can also be cleaved and activated by exogenous serine proteases such as trypsin, an action that is attenuated by the protease inhibitor aprotinin (). When human bronchial epithelial cultures are mounted in Ussing chambers where native airway surface liquid is washed away, ENaC is predominantly active, suggesting that cell attached proteases are predominant (, ). In contrast, under thin film conditions, where native airway surface liquid is present, ENaC activity is reduced, suggesting that airway surface liquid contains soluble proteases inhibitors (, ). Thus, based on the observation that diluting airway surface liquid with Ringer resulted in an increase in ENaC activity (, ), we speculated that unidentified protease inhibitors resided in the airway surface liquid. Accordingly, we incubated airway surface liquid with trypsin-coated beads, and used MS to identify bound proteins. Short palate, lung, and nasal epithelial clone (SPLUNC)1 was identified as a candidate protease inhibitor, because its binding to trypsin beads was reduced by the protease-inhibitor aprotinin.The PLUNC family are secreted proteins that are subdivided into short (SPLUNCs) and long (LPLUNCs) members, which contain either 1 or 2 domains respectively (, ). The original PLUNC gene, which is now called SPLUNC1, comprises up to 10% of total protein in the airway surface liquid, and can readily be detected in both nasal lavage and tracheal secretions (–). SPLUNC1 is expressed in both submucosal glands, the superficial epithelia and in neutrophils, and in theory, is present in the correct regions of the lung to be a volume sensing molecule, because it can be secreted onto the mucosal surface of the superficial epithelial where ENaC is expressed (, ). Thus, because SPLUNC1 bound to trypsin, we initiated studies to better characterize SPLUNC1 function and to determine whether it was a soluble regulator of ENaC.
Results and Discussion
Based on the ability of normal human bronchial epithelial cultures to regulate airway surface liquid height to 7 μm, which was paralleled by a decrease in trypsin-sensitive ENaC activity, we speculated that a soluble protease inhibitor is present in the airway surface liquid during normal airway surface liquid volume homeostasis. We searched for potential protease inhibitors/ENaC regulators by incubating trypsin-coated beads with airway surface liquid and performing a proteomic analysis. We collected airway surface liquid from well-differentiated normal human bronchial epithelial cultures, and incubated this lavage overnight at 4 °C with trypsin-agarose beads ± aprotinin. Proteins were eluted from the beads, separated by SDS/PAGE, and visualized with a silver stain (). The MS analysis allowed us to identify SPLUNC1 as one of the major proteins that bound to the trypsin-beads (A; ). The presence of SPLUNC1 was confirmed in airway surface liquid by Western blotting (B).
SPLUNC1 regulates trypsin-sensitive airway ion transport. (A) Example of an MS/MS spectrum used to specifically identify SPLUNC1. The corresponding amino acids are labeled on the spectrum. (B) Western blotting showing absence of SPLUNC1 on mucosal surfaces of HBECs washed with Ringer (lane 1) or the presence of SPLUNC1 in ASL that was left to accumulate for 24 h (lane 2). (C) Transepithelial voltage (PD) with time in human bronchial epithelial cultures prewashed to remove SPLUNC1 followed by addition of either 50 ng/mL recombinant SPLUNC1 (●) or vehicle (■). After 60 min, 1 unit/mL trypsin was
both n = 7. (D) Transepithelial voltage with time in cultures exposed to either 50 ng/mL rSPLUNC1 (●) or aprotinin (3 units/mL; ■); both n = 6. (E) Mean 30 min transepithelial voltage in washed human bronchial epithelial cultures with vehicle or aprotinin (open bars), with SPLUNC1 (black bars) or human bronchial epithelial cultures where airway surface liquid was allowed to accumulate undisturbed for 24 h ± rSPLUNC1 (gray bars); n = 6–7. *, P & 0.05 different from t = 0; +, P & 0.05 different to vehicle.
To better study SPLUNC1, we stably transfected V5/6His-tagged SPLUNC1 into HEK293 cells, and purified secreted V5/6His-SPLUNC1 from HEK293 media over a nickel column. Recombinant (r)SPLUNC1 could be detected using the anti-V5 antibody (), and a brief (30 min) incubation with trypsin resulted in the appearance of cleavage products of C-terminally V5-tagged rSPLUNC1, indicating that SPLUNC1 is a substrate for serine proteases (). To test whether SPLUNC1 was capable of altering airway ion transport, we then measured the transepithelial voltage under thin film conditions in human bronchial epithelial cultures ± rSPLUNC1 with time. Washing the mucosal surface of human bronchial epithelial cultures with PBS has previously been shown to maximally activate ENaC (), and removes endogenous SPLUNC1 (B). Under these conditions, 20 μL of Ringer containing 50 ng/mL of rSPLUNC1 significantly reduced the transepithelial voltage (C). In contrast, a purified SPLUNC1-free fraction of HEK293 media was without effect (C). To confirm that this inhibition was due to altered ENaC regulation, we exposed human bronchial epithelial cultures to trypsin for 30 min after the 1-h rSPLUNC1 exposure. Trypsin was without effect in the control human bronchial epithelial cultures, suggesting that ENaC remained fully active. However, mucosal trypsin exposure significantly raised the transepithelial voltage in the SPLUNC1-exposed group, suggesting that ENaC had been inhibited by rSPLUNC1 (C). Inhibition of the transepithelial voltage occurred at identical rates after both rSPLUNC1 and aprotinin addition, and the effects of these compounds were not additive. However, in both cases, these effects were reversed by trypsin-exposure ( D and E). Together, these data suggest that (i) both molecules operated through a common pathway, and (ii) this result was an ENaC-specific effect ( D and E). Also, when airway surface liquid was left to accumulate on human bronchial epithelial culture surfaces for 24 h (i.e., the cultures were not prewashed with Ringer), the transepithelial voltage was significantly lower than in washed cultures and rSPLUNC1 addition was without further effect, suggesting that the spontaneous accumulation of an endogenous inhibitor in the airway surface liquid reduces the transepithelial voltage, and that maximum inhibition is reached at steady state (Fig, 1E).
To test whether passive fluxes were affected by SPLUNC1 exposure, we measured the effects of amiloride on transepithelial voltage vs. transepithelial electrical resistance ± rSPLUC1. Amiloride reduced the transepithelial voltage by 54% (n = 12), and rSPLUNC1 addition to the same amiloride-treated cultures was without further effect (n = 12). In parallel, both amiloride and rSPLUNC1 increased the transepithelial electrical resistance by ≈33%, and again the effects were not additive, suggesting that amiloride and SPLUNC1 both act on ENaC in the apical membrane ().
To further investigate how SPLUNC1 regulated ion transport and ENaC in particular, we expressed αβγENaC in Xenopus laevis oocytes, and either exposed oocytes to rSPLUNC1 or coinjected SPLUNC1 cRNA into the oocytes with αβγENaC. ENaC currents were reduced by ≈70% when oocytes were incubated with rSPLUNC1 before recording (A). Due to the larger volumes required for oocyte incubations, SPLUNC1 was added at a 10× lower concentration than in the human bronchial epithelial cultures (5 ng/mL). Similarly, coexpression of αβγENaC and SPLUNC1 also resulted in ENaC inhibition by ≈70% (A). SPLUNC1 could not be detected in media from oocytes injected with αβγENaC (B). However, SPLUNC1 was readily detected in the media after coinjection of SPLUNC1 and αβγENaC cRNAs (B). Because SPLUNC1 could be detected in the oocyte media (), it is likely that coexpressed SPLUNC1 was secreted by the oocytes and inhibited ENaC externally in a similar fashion to rSPLUNC1.
SPLUNC1 inhibits ENaC, but not CFTR currents in Xenopus oocytes. (A) Bar graphs of mean, normalized amiloride-sensitive (ENaC) currents in oocytes coinjected with 0.3 ng αβγENaC subunits and incubated for 1 h with 5 ng/mL recombinant SPLUNC1 ( n = 13) or coinjected with SPLUNC1 cRNA (1 n = 19). (B) Western blotting using anti-V5 antibody to detect SPLUNC1 in media from oocytes injected with (i) αβγENaC alone, and (ii) αβγENaC with SPLUNC1. (C) Mean, normalized isoproterenol-activated CFTR currents in oocytes coinjected with the β2 adrenergic receptor (β2A-R), respectively, ± 5 ng/mL rSPLUNC1 ( n = 11), or coexpressed with 1 ng SPLUNC1 (n = 12). Mean basal ENaC and CFTR currents were 2,300 ± 377 and 868 ± 200 nA, respectively. *, P & 0.05 different from ENaC or CFTR +, P & 0.05 different from SPLUNC1.
To test whether SPLUNC1 specifically inhibited ENaC, we either exposed cystic fibrosis transmembrane conductance regulator (CFTR)-expressing oocytes to 5 ng/mL rSPLUNC1 or coexpressed SPLUNC1 and CFTR. In both cases, we coexpressed CFTR with the β2 adrenergic receptor (β2AR), which can be stimulated with isoproterenol to raise cAMP and stimulate CFTR (). After 10 μM isoproterenol exposure, CFTR was robustly activated, and unlike with ENaC, rSPLUNC1 exposure or injection of SPLUNC1 cRNA had no inhibitory effect on CFTR activity, suggesting that the inhibitory effects of SPLUNC1 are specific for ENaC (C).
Because SPLUNC1 bound to trypsin-agarose beads () and affected the trypsin sensitivity of ENaC (C), we next tested whether SPLUNC1 could alter serine protease activity. Despite rSPLUNC1 being capable of inhibiting ENaC by ≈70% in both human bronchial epithelia and oocytes ( and ), 50 ng/mL rSPLUNC1 had only a modest affect (≈10%) on the ability of either 1.0 or 0.3 units/mL trypsin to cleave a fluorogenic substrate (Di-tert-butyl dicarbonate-Gln-Ala-Arg-7-methoxycoumarin-4-yl)acetyl (BGAR-MCA), unlike 2 units/mL aprotinin, which inhibited trypsin activity by ≈100% (). Airway epithelia express serine proteases (, ), and mucosal addition of Ringer solution containing BGAR-MCA to human bronchial epithelial cultures resulted in spontaneous BGAR-MCA cleavage with time that was inhibited by aprotinin addition, confirming that serine proteases are indeed active on the mucosal surface of airway epithelia (). Recombinant SPLUNC1 had no significant affect on BGAR-MCA cleavage in human bronchial epithelial mucosal surfaces, suggesting that SPLUNC1 does not inhibit ENaC by inhibiting serine protease activity ().
Proteolytic cleavage of α and γ subunits is required for ENaC activation (, ), and because CAP2 is highly expressed in human bronchial epithelial cultures (), we tested whether SPLUNC1 could alter α and γ ENaC cleavage by CAP2. Due to the relative scarcity of purified rSPLUNC1, we elected to coexpress SPLUNC1 and ENaC, rather than use rSPLUNC1 for subsequent oocyte studies, because SPLUNC1 is secreted at sufficient quantities from oocytes to inhibit ENaC (). When αβγENaC and CAP2 were coexpressed in oocytes, both full-length ENaC subunits and cleaved α and γ fragments were detected with a V5 antibody ( A and B). However, in the presence of SPLUNC1, only full-length α and γ ENaC subunits could be observed, suggesting that SPLUNC1 protects ENaC from proteolytic cleavage despite SPLUNC1 having no observable intrinsic anti-protease activity ( A and B). Because we probed with a V5 antibody, SPLUNC, which is also V5-tagged, was visible as a 26-kDa band. However, SPLUNC1 could be differentiated from ENaC cleavage fragments based on its size and position on the gel ( A and B). To confirm that SPLUNC1 prevented functional activation of ENaC by CAPs, we coexpressed ENaC ± SPLUNC1 with prostasin (CAP1) and CAP2 both of which are present in the airways. As previously described, both CAPs significantly increased basal ENaC currents (). However, the ability of both proteases to activate ENaC was significantly reduced by SPLUNC1 (C). Similarly, trypsin-exposure also increased ENaC activity, and this stimulation was attenuated by SPLUNC1 (D). The furin-insensitive αR205,231K,β,γR138K ENaC mutant was also inhibited by SPLUNC1, suggesting that this effect is not mediated by convertases such as furin (E) ().
SPLUNC1 prevents proteolysis of ENaC. (A and B) Western blottings of lystate from oocytes coinjected with αβγ ENaC and CAP2 ± SPLUNC1. UI, uninje &, α and γ ENaC and ←, SPLUNC1. (C and D) Relative ENaC currents in oocytes coinjected with 0.3 ng αβγENaC ± SPLUNC1 (1 ng) and either coinjected with either prostasin (1 ng), CAP2 (1 ng) or exposed to trypsin. (E) Relative ENaC currents in oocytes coinjected with 0.3 ng furin-insensitive ENaC mutant (αR205,231K,β,γR138K ENaC) ± SPLUNC1 (1 ng). Currents were normalized to currents obtained from oocytes injected with ENaC alone. All n = 6. *, P & 0.05 different to ENaC +, P & 0.05 different ± SPLUNC1.
Because SPLUNC1 prevented cleavage and activation of ENaC, but did not appear to be a serine protease inhibitor in the same fashion as aprotinin, we hypothesized that SPLUNC1 could specifically bind to ENaC to protect it from proteolysis. To test this hypothesis, we used an airway cell line (JME cells) that had been extensively passaged and did not express ENaC, which we used to measure nonspecific binding after infection with a lentivirus containing an empty vector. To measure specific binding, we then infected these cells with a lentivirus containing yfp-tagged αENaC, because the αENaC subunit alone has previously been shown to form functional ENaCs, although with a significantly smaller conductance (, ). Stable αENaC expression was confirmed by Western blotting (). To qualitatively test the relationship between yfp-αENaC expression and SPLUNC1 binding, we plated JME cells on glass coverslips, and incubated these cells with varying concentrations of Texas red-labeled rSPLUNC1. As can be seen in A, Texas red-rSPLUNC1 and yfp-αENaC clearly colocalize. whereas rSPLUNC1 binding to JME cells infected with the lentivial vector alone was reduced and more diffuse (A). To quantitatively address this issue, we then polarized these cells on filters for 7 days. We then incubated these polarized cells with varying concentrations of Texas red-rSPLUNC1 for 30 min followed by a 5× wash with PBS. The subsequent binding isotherm shows a clear difference between nonspecific (empty vector-transfected) binding that did not saturate, and specific (yfp-αENaC) binding that was significantly greater and saturable (B). Using this graph, we calculated that the Kd was 55 ng/mL (B). Thus, if SPLUNC1 is indeed a volume sensor in the airways, these data suggests that it will be able to change ENaC activity over a narrow range of concentrations.
SPLUNC1 specifically binds to αENaC in polarized airway epithelia. JME airway epithelial cultures lacking ENaC were stably infected with a lentivirus containing yfp-tagged αENaC or a lentivirus/empty vector. (A) Representative images of yfpαENaC or control JME cells (vector) exposed to varying concentrations of rSPLUNC1 labeled with Texas Red for 30 min followed by a 5× PBS wash. (B) SPLUNC1-αENaC binding isotherm obtained from total rSPLUNC1 binding in αENaC expressing JME cells minus nonspecific rSPLUNC1 binding from control rSPLUNC1-expressing cells. All n = 6. Kd was 55 ng/mL.
Xenopus oocytes are autofluorescent, making this type of fluorescent binding assay difficult, and to see whether SPLUNC1 also bound to ENaC expressed in oocytes, we coexpressed α, β, and γ ENaC subunits in and SPLUNC1 in oocytes and immunoprecipitated ENaC. We then probed for SPLUNC1, and found that SPLUNC1 bound to all 3 ENaC subunits (). Thus, rather than being a protease inhibitor, we propose that SPLUNC1 protects α and γ ENaC from being cleaved by serine proteases, perhaps being cleaved itself in the process.
We have previously shown that human bronchial epithelial cultures rapidly absorb excess airway surface liquid, then absorption slows, and a steady state airway surface liquid height of ≈7 μm is maintained (). To ask whether endogenous SPLUNC1 was required as part of this homeostatic mechanism, we knocked down SPLUNC1 using 2 different anti-SPLUNC1 shRNA sequences, which were incorporated into retroviruses that were used to infect human bronchial epithelial cultures. Successful knockdown was confirmed by qPCR and Western blotting ( A and B), and because no difference in knockdown was detected between each sequence, the subsequent results were pooled. Human bronchial epithelial cultures infected with a control shRNA (antiluciferase), rapidly absorbed a test solution of 20 μL Ringer until an airway surface liquid height of 7 μm was reached, after which time absorption slowed, and airway surface liquid height was maintained at 7 μm as has previously been described for noninfected human bronchial epithelial cultures ( C and D) (, ). This regulation was paralleled by a decline in the transepithelial voltage that could be restored by mucosal exposure to trypsin (E). Importantly, cultures lacking SPLUNC1 failed to regulate airway surface liquid height with time, and exhibited increased airway surface liquid absorption during the initial phase followed by a failure to maintain steady-state airway surface liquid height at 7 μm ( C and D). Also, the transepithelial voltage failed to decline in human bronchial epithelial cultures lacking SPLUNC1 and remained both elevated and trypsin-insensitive, suggesting that ENaC remained fully activated. Regulation of both airway surface liquid height and the transepithelial voltage was restored by the addition of 50 ng/mL rSPLUNC1 to the airway surface liquid ( C–E), suggesting that SPLUNC1 indeed acts as a reporter molecule in the airway surface liquid that regulates ENaC activity to maintain appropriate airway surface liquid volume control.
SPLUNC1 is required for airway surface liquid volume homeostasis. (A) Real time (q)PCR of SPLUNC1 expression relative to GAPDH in cells expressing anti-luciferase shRNA (open bars) and 2 different anti-SPLUNC1 shRNAs (black and gray bars), all n = 6. (B) Western blotting showing the presence and absence of SPLUNC1 in airway surface liquid. SPLUNC1 in naive human bronchial epithelial cultures (lane 1), those infected with adenoviruses containing 2 different anti-SPLUNC1 shRNAs (lanes 2 and 3), anti-SPLUNC1 shRNA with 50 ng/mL rSPLUNC1 (lane 4), anti-luciferase shRNA (lane 5). (C) Confocal images of airway surface liquid 24 h after mucosal Ringer addition, or Ringer containing aprotinin or 50 ng/mL rSPLUNC1 to human bronchial epithelial cultures infected with retrovirus containing either antiluciferase or anti-SPLUNC1 shRNA. (D) Mean airway surface liquid height taken from C. ■, anti- ●, anti-SPLUNC1; ?, anti-SPLUNC1 with rSPLUNC1; ?, anti-SPLUNC1 with aprotinin. All n = 6–8. (E) A 24 h transepithelial voltage (PD) in human bronchial epithelial cultuers infected with antiluciferase or anti-SPLUNC1 shRNAs (open bars) and after 30-min exposure to trypsin (closed bars). All n = 6. *, P & 0.05
+, P & 0.05 different ± trypsin.
Due to the structural similarity between SPLUNC1 and antibacterial proteins such as lipopolysaccharide binding protein (LPB), it has been suggested that SPLUNC1 has an important role in airways innate host defense (). However, this hypothesis is controversial, and some investigators have found that SPLUNC1 appears to have little antimicrobial activity against Escherichia coli, Listeria monocytogenes, or Pseudomonas aeruginosa (), and does not appear to bind lipopolysaccharide (). However, SPLUNC1 has recently been shown to inhibit Mycoplasma pneumoniae in airway cultures (), and bind to lipopolysaccharide and disrupt Epstein–Barr virus proliferation (). Although there is no current explanation for these discrepancies, we do not know whether SPLUNC1 is posttranslationally modified and/or how labile SPLUNC1 is once purified, and it is possible that the purification and storing of SPLUNC1 may alter its biological activity, leading to differing results. Also, it will be interesting to determine whether the ability of SPLUNC1 to interfere with proteolysis may have a role in its putative antimicrobial actions.
In contrast to its proposed antibacterial actions, we propose that SPLUNC1 binds specifically to an extracellular domain of ENaC, preventing the channel from being cleaved and activated by serine proteases. It has been proposed that the extracellular loops of ENaC have a role in channel gating, and the α and γ subunits of ENaC have been reported to contain short inhibitory segments that are removed during proteolytic cleavage to activate the channel (, ). We speculate that ENaC subunits that have already been cleaved by extracellular serine proteases are likely to be SPLUNC1-insensitive. However, as new ENaCs are inserted in the plasma membrane, SPLUNC1 binds to them, preventing their cleavage and resulting in a decline in ENaC-mediated currents. The onset of inhibition (30–60
C and D) is comparable with aprotinin-inhibition rates in human bronchial epithelial cultures (, ), and is consistent with this model. However, we cannot as yet formally exclude the possibility that SPLUNC1 can bind to ENaC and induce a conformational change in the extracellular loops and/or directly block the channel pore itself. Also, in this study, we did not differentiate between possible effects of SPLUNC1 on the number of ENaC channels vs. their open probability. Because both basal and protease-activated ENaC currents were reduced in the presence of SPLUNC1, the relative increase from basal to protease-activated currents is similar ± SPLUNC1 (). Thus, we cannot exclude the possibility that SPLUNC1 decreases the number of ENaC channels in the plasma membrane. If this was the case, then the pool of surface ENaCs available to be cleaved would be reduced, which could explain both the reduction in ENaC cleavage and protease-activated currents in the presence of SPLUNC.
In addition to being expressed in the airways, ENaC is also expressed in aldosterone-sensitive epithelial cells in the colon and kidney where it has an important role in the control of sodium balance, blood volume, and blood pressure (, ). In the colon, the primary flux is in the absorptive direction. However, ion transport can switch from being absorptive to being secretory to help regulate salt balance (, ). In the kidney, ENaC is the rate-limiting step for salt reabsorption in the collecting duct (), and aldosterone induces a shift in the molecular mass of γ ENaC from 85 to ≈75 kDa, consistent with physiological proteolytic clipping of the extracellular loop (). Although SPLUNC1 expression has previously only been reported in the airways, oral and nasal cavities (), we find that it is also expressed in the colon and kidney (). Thus, SPLUNC1 expression in these tissues could potentially add an additional layer of regulation to further modulate ENaC activity and salt absorption.
SPLUNC1 is up-regulated by inflammation (), which may decrease ENaC activity and shift the fluid balance toward secretion in inflamed regions of the airways, colon and/or kidneys. In times of infection, this action may help to “flush out” the afflicted organ. For example, in the airways, this inhibition of ENaC would increase the driving force for Cl- and apical surface liquid (ASL) secretion, which would be predicted to increase mucus clearance (), and a similar effect would also be predicted in the kidneys and gastrointestinal tract.
SPLUNC1 expression is increased in CF lungs, especially in the surface epithelium of the proximal and distal airways () and this up-regulation may be due to the increased inflammation in seen CF lungs (). However, CF lungs are typified by Na+ hyperabsorption and mucus dehydration (), so it is unlikely that SPLUNC1 exerts any significant inhibitory effect on ENaC under these conditions. Also, we have previously demonstrated that CF bronchial epithelial cultures do not decrease ENaC activity with time (, ). CFTR expression is not required for SPLUNC1 to inhibit ENaC, as demonstrated in our oocytes studies, suggesting that this inability of SPLUNC1 to regulate ENaC is not an innate property of CF airways (A). However, the serine proteases that activate ENaC are up-regulated in CF airway epithelia (, ) and neutrophil elastase, which also activates ENaC, is increased in CF airways (–). Thus, it is possible that the excessive protease up-regulation seen in CF airways (, ) interferes with the normal regulation of ENaC by SPLUNC1 and other potential ENaC regulators, and thus, shifts the balance from anti-proteases and less ENaC activity to a protease-replete state with more ENaC activity, overwhelming the ability of SPLUNC1 to inactivate ENaC and contributing to CF airway surface liquid volume depletion.
In summary, we have identified SPLUNC1 as a previously undescribed extracellular protein inhibitor of ENaC that is present in the airway surface liquid. In normal airways, SPLUNC1 is highly expressed in submucosal glands with moderate expression in surface epithelium of the proximal airways with little expression in the distal airways (). Thus, we propose that SPLUNC1 is secreted from glands and surface epithelium where it serves as a reporter molecule whose dilution or concentration can adjust ENaC activity to regulate airways hydration and mucus clearance. Because SPLUNC1 is secreted by proximal airways, we propose that this regulation primarily occurs in the proximal airways, with little effect in the distal airways. However, if SPLUNC1 secretion is indeed up-regulated by inflammation (), this secretion may serve to decrease ENaC activity and increase mucus clearance in the distal airways during these times. In conclusion, ENaC is a soluble regulator of ENaC that is expressed in multiple organs. It will be interesting to understand how SPLUNC1 affects the protease-protease inhibitor balance in these ENaC-expressing tissues, and whether SPLUNC1 affects ENaC activity in response to inflammation.
Materials and Methods
This section is a summary of the most important techniques used. A detailed description of all methods can be found in .
Tissue Procurement and Cell Culture.
Cells were harvested by enzymatic digestion from human bronchial tissue as previously described under a protocol approved by the University of North Carolina (UNC) School of Medicine Institutional Review Board (). All preparations were maintained at an air-liquid interface and used 2–5 weeks after seeding on 12 mm Transwell permeable supports (Corning Costar).
Identification of SPLUNC1.
Airway surface liquid was collected from human bronchial epithelial cultures, and was incubated overnight with trypsin-agarose beads. The proteins were eluted and separated by SDS/PAGE per the UNC–Duke Michael Hooker Proteomics Center procedures (). Visible bands were excised and prepared for MALDI-MS/MS analysis as described previously ().
Microelectrode Studies.
A single-barreled potential difference-sensing electrode was placed in the airway surface liquid by micromanipulator and used in conjunction with a macroelectrode in the serosal solution to measure the transepithelial voltage as described previously ().
Oocyte Studies.
X. laevis oocytes were prepared and injected as described (). Oocytes were studied 24 h after RNA injection using the 2 electrode voltage clamp technique as previously described (). Oocytes were clamped at a holding potential of -60 mV. The change in amiloride-sensitive whole cell current (ΔIamil) as an indicator of ENaC activity was determined by subtracting the corresponding current value measured in the presence of 10 μM amiloride from that measured before the application of amiloride.
Western Blotting.
Airway surface liquid was lavaged from human bronchial epithelial cultures, and SPLUNC1 was resolved using SDS/PAGE and transferred to PVDF. The membrane was then probed using a αSPLUNC1 or αV5 antibodies (R&D).
Binding Assay.
JME nasal epithelial cells that did not express ENaC were stably infected with a lentivirus containing yfp-αENaC or empty vector (control). JME cells were incubated with varying concentrations of Texas red-labeled SPLUNC1 for 30 min followed by a 5× wash with PBS. After this time, images were acquired with a Nikon Ti-S inverted microscope, and were quantified to obtain specific and nonspecific binding using Image J. Data were then fitted with a Hill Plot to obtain the Kd.
Short Hairpin RNA-Induced Knockdown of SPLUNC1.
Viruses encoding anti-SPLUNC1 shRNA were used to infect airway cells to knockdown SPLUNC1. At the time of the functional assays, we measured airway surface liquid SPLUNC1 protein levels by Western blotting to verify stable knockdown. An anti-luciferase shRNA-expressing adenovirus was infected separately as a control.
Confocal Microscopy.
Cultures incubated with fluorescent dextrans were placed in a chamber on the stage of a Leica 5 points per culture were scanned and an average airway surface liquid height determined. For detailed methods, see ref. .
Acknowledgments
We thank Yan Dang, Hong He, Brett Rollins, and Hamsa Succindran for
Drs. Martina Gentzsch and Mehmet Kesimer for critical readin and the University of North Carolina at Chapel Hill Cystic Fibrosis Center Tissue and Molecular Cores. The yfp-αENaC was a kind gift from Dr. Bakhrom Berdiev (Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL). This work was supported by the National Institutes of Health Grants HL034322, HL074158, HL084934, and CFF TARRAN07G0.
Footnotes1To whom correspondence should be addressed. E-mail: robert_tarran{at}med.unc.edu
Author contributions: A.G.-C., M.J.W., S.H.D., M.J.S., and R.T. A.G.-C., J.E.R., E.G., M.J.W., J.C.O., S.H.D., and R.T. J.C.O. contributed new reagents/ A.G.-C., J.E.R., and R.T. and M.J.S. and R.T. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at .References
PB () State of the art: Why do the lungs of patients with cystic fibrosis become infected and why can't they clear the infection? Respir Res 4:8.
MR, Boucher
RC () Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 109:571–577.
LA, Rollins
BM, Tarran
R () Liquid movement across the surface epithelium of large airways. Respir Physiol Neurobiol 159:256–270.
C, Caughey
GH () Regulation of the epithelial Na+ channel by peptidases. Curr Top Dev Biol 78:23–46.
BC () The epithelial sodium channel: Activation by membrane-bound serine proteases. Proc Am Thorac Soc 1:4–9.
V, et al. () An epithelial serine protease activates the amiloride-sensitive sodium channel. Nature 389:607–610.
A, et al. () Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes. J Gen Physiol 111:127–138.
RJ, et al. () Na+ transport in normal and CF human bronchial epithelial cells is inhibited by BAY 39–9437. Am J Physiol Lung Cell Mol Physiol 281:L16–L23.
SH, et al. () Regulation of the epithelial sodium channel by serine proteases in human airways. J Biol Chem 277:8338–8345.
MM, et al. () Airway surface liquid volume regulates ENaC by altering the serine protease-protease inhibitor balance: A mechanism for sodium hypersabsorption in cystic fibrosis. J Biol Chem 281:27942–27949.
L, Donaldson
SH, Boucher
RC () Soluble mediators, not cilia, determine airway surface liquid volume in normal and cystic fibrosis superficial airway epithelia. J Gen Physiol 127:591–604.
CD, Bingle
L () Characterisation of the human plunc gene, a gene product with an upper airways and nasopharyngeal restricted expression pattern. Biochim Biophys Acta 1493:363–367.
WM, et al. () Differential display identification of plunc, a novel gene expressed in embryonic palate, nasal epithelium, and adult lung. J Biol Chem 274:13698–13703.
CD, Craven
CJ () PLUNC: A novel family of candidate host defence proteins expressed in the upper airways and nasopharynx. Hum Mol Genet 11:937–943.
MA, et al. () Purification and characterization of PLUNC from human tracheobronchial secretions. Am J Respir cell Mol Biol 30:184–192.
M, Stahlbom
B, Tagesson
C () Identification of a new potential airway irritation marker, palate lung nasal epithelial clone protein, in human nasal lavage fluid with two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight. Electrophoresis 22:1795–1800.
JA, et al. () PLUNC is a secreted product of neutrophil granules. J Leukoc Biol 83:1201–1206.
L, et al. () SPLUNC1 (PLUNC) is expressed in glandular tissues of the respiratory tract and in lung tumours with a glandular phenotype. J Pathol 205:491–497.
Y, et al. () Receptors that couple to 2 classes of G proteins increase cAMP and activate CFTR expressed in Xenopus oocytes. Receptors Channels 1:233–241.
BC, Stutts
MJ () Activation of the Epithelial Sodium Channel (ENaC) by Serine Proteases. Annu Rev Physiol 71:361–379.
GM, et al. () Epithelial sodium channel exit from the endoplasmic reticulum is regulated by a signal within the carboxyl cytoplasmic domain of the alpha subunit. J Biol Chem 282:33475–33483.
A, et al. () A segment of gamma ENaC mediates elastase activation of Na+ transport. J Gen Physiol 130:611–629.
G, et al. () Synergistic activation of ENaC by three membrane-bound channel-activating serine proteases (mCAP1, mCAP2, and mCAP3) and serum- and glucocorticoid-regulated kinase (Sgk1) in Xenopus Oocytes. J Gen Physiol 120:191–201.
RP, et al. () Epithelial sodium channels are activated by furin-dependent proteolysis. J Biol Chem 279:18111–18114.
XL, Hruska
K () Reconstitution of stretch-activated cation channels by expression of the alpha-subunit of the epithelial sodium channel cloned from osteoblasts. Proc Natl Acad Sci USA 94:1013–1018.
MP, Snyder
MJ () Cloning and expression of the beta- and gamma-subunits of the human epithelial sodium channel. Am J Physiol 268:C1157–C1163.
R, et al. () Normal and cystic fibrosis airway surface liquid homeostasis. The effects of phasic shear stress and viral infections. J Biol Chem 280:35751–35759.
HW, et al. () Function and regulation of SPLUNC1 protein in Mycoplasma infection and allergic inflammation. J Immunol 179:3995–4002.
HD, et al. () Intracellular co-localization of SPLUNC1 protein with nanobacteria in nasopharyngeal carcinoma epithelia HNE1 cells depended on the bactericidal permeability increasing protein domain. Mol Immunol 43:1864–1871.
MD, Hughey
RP, Kleyman
TR () Proteolytic processing of the epithelial sodium channel gamma subunit has a dominant role in channel activation. J Biol Chem 283:25290–25295.
MD, et al. () Defining an inhibitory domain in the alpha-subunit of the epithelial sodium channel. Am J Physiol Renal Physiol 294:F47–F52.
Kunzelmann
M () Electrolyte transport in the mammalian colon: Mechanisms and implications for disease. Physiol Rev 82:245–289.
BC, Pradervand
L, Hummler
E () Epithelial sodium channel and the control of sodium balance: Interaction between genetic and environmental factors. Annu Rev Physiol 64:877–897.
AN, Feldman
GM () Systemic acid-base disorders and intestinal electrolyte transport. Am J Physiol 247:G1–G12.
LG () Epithelial sodium channels: Function, structure, and regulation. Physiol Rev 77:359–396.
Masilamani
S, et al. () Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney. J Clin Invest 104:R19–R23.
RC () Human airway ion transport. Part one. Am J Respir Crit Care Med 150:271–281.
L, et al. () Differential epithelial expression of the putative innate immune molecule SPLUNC1 in cystic fibrosis. Respir Res 8:79.
RC () Regulation of airway surface liquid volume by human airway epithelia. Pflugers Arch 445:495–498.
MM, et al. () Prostasin expression is regulated by airway surface liquid volume and is increased in cystic fibrosis. Am J Physiol Lung Cell Mol Physiol 294:L932–L941.
P, et al. () Protease-antiprotease imbalance in the lungs of children with cystic fibrosis. Am J Respir Crit Care Med 150:207–213.
RA, Boucher
RC, Stutts
MJ () Neutrophil elastase activates near-silent epithelial Na+ channels and increases airway epithelial Na+ transport. Am J Physiol Lung Cell Mol Physiol 288:L813–L819.
MW, Hilliard
KA, Norvell
TM, Berger
M () Bronchoalveolar lavage findings in cystic fibrosis patients with stable, clinically mild lung disease suggest ongoing infection and inflammation. Am J Respir Crit Care Med 150:448–454.
DR, et al. () Improved protein identification through the use of unstained gels. J Proteome Res 4:992–997.
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Agustin Garcia-Caballero, Julia E. Rasmussen, Erol Gaillard, Michael J. Watson, John C. Olsen, Scott H. Donaldson, M. Jackson Stutts, Robert Tarran
Proceedings of the National Academy of Sciences 106 (27) ; DOI: 10.1073/pnas.
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SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage
Agustin Garcia-Caballero, Julia E. Rasmussen, Erol Gaillard, Michael J. Watson, John C. Olsen, Scott H. Donaldson, M. Jackson Stutts, Robert Tarran
Proceedings of the National Academy of Sciences 106 (27) ; DOI: 10.1073/pnas.
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