SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage

Abstract

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.

Fig. 1.

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 added to both groups; 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.

Fig. 2.

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 (gray bars; n = 13) or coinjected with SPLUNC1 cRNA (1 ng; closed bars; 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 (gray bars; 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 alone; †, P < 0.05 different from SPLUNC1.

Fig. 3.

SPLUNC1 prevents proteolysis of ENaC. (A and B) Western blottings of lystate from oocytes coinjected with αβγ ENaC and CAP2 ± SPLUNC1. UI, uninjected control oocytes; <, α and γ ENaC cleavage fragments; 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 (α_R205,231K,β,γ_R138K 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 alone; †, P < 0.05 different ± SPLUNC1.

Fig. 4.

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. K_d was 55 ng/mL.

Fig. 5.

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-luciferase; ●, 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 different to control; †, P < 0.05 different ± trypsin.