Regulation of the Epithelial Sodium Channel by Serine Proteases in Human Airways

Donaldson, Scott H.; Hirsh, Andrew J.; Li, Dong Chen; Holloway, Ginger; Chao, Julie; Boucher, Richard C.; Gabriel, Sherif E.

doi:10.1074/jbc.m105044200

Cited by 232 publications

(241 citation statements)

References 26 publications

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“…The onset of inhibition (30-60 min; Fig. 1 C and D) is comparable with aprotinin-inhibition rates in human bronchial epithelial cultures (8,9), 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.…”

Section: Resultssupporting

confidence: 71%

“…X. laevis oocytes were prepared and injected as described (9). Oocytes were studied 24 h after RNA injection using the 2 electrode voltage clamp technique as previously described (9).…”

Section: Methodsmentioning

confidence: 99%

“…ENaC can also be cleaved and activated by exogenous serine proteases such as trypsin, an action that is attenuated by the protease inhibitor aprotinin (6). 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 (8,9). 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 (10,11).…”

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confidence: 99%

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

Garcı́a-Caballero

Rasmussen

Gaillard

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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140

182

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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.

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Section: Resultssupporting

confidence: 71%

“…X. laevis oocytes were prepared and injected as described (9). Oocytes were studied 24 h after RNA injection using the 2 electrode voltage clamp technique as previously described (9).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Garcı́a-Caballero

Rasmussen

Gaillard

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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140

182

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“…However, our results suggest that these trypsin-sensitive sites are not targeted by proteases when channels are expressed in CHO or MDCK cells. Whether CAPs (39) or other proteases participate in the post-translational processing of ENaC subunits remains to be determined. There is evidence for proteolytic processing of other channels.…”

Section: N-glycans Are Processed On All Three Subunits Of Enac-mentioning

confidence: 99%

Maturation of the Epithelial Na+ Channel Involves Proteolytic Processing of the α- and γ-Subunits

Hughey¹,

Mueller²,

Bruns³

et al. 2003

Journal of Biological Chemistry

248

264

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The epithelial Na ؉ channel (ENaC) is a tetramer of two ␣-, one ␤-, and one ␥-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone ␣-(95 kDa), ␤-(96 kDa), and ␥-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of ␣␤␥ENaC subunits revealed a second band for each subunit (65 kDa for ␣, 110 kDa for ␤, and 75 kDa for ␥) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [ 3 H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed ␣-and ␥-subunits is also consistent with proteolytic cleavage. By using ␣-and ␥-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the ␣-and ␥-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for ␣ and 18 kDa for ␥) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.The amiloride-sensitive epithelial Na ϩ channel (ENaC) 1 is composed of three structurally related subunits, termed ␣-, ␤-, and ␥-ENaC. The three subunits exhibit limited amino acid sequence identity (30 -40%) but are structurally similar with two membrane-spanning domains and cytosolic amino and carboxyl termini. We and others have shown that ENaC expressed in Xenopus oocytes has a subunit stoichiometry of two ␣-, one ␤-, and one ␥-subunit (1, 2

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“…Prostasin, the human ortholog of CAP1, is a glycosylphosphatidylinositol (GPI)-anchored serine protease that activates ENaC (4,8,13,44). Donaldson et al (13) demonstrated that prostasin is expressed in airway epithelium and increases the Na ϩ current by ϳ75% when coexpressed with ENaC in Xenopus oocytes.…”

mentioning

confidence: 99%

Prostasin expression is regulated by airway surface liquid volume and is increased in cystic fibrosis

Myerburg

McKenna²,

Luke³

et al. 2008

American Journal of Physiology-Lung Cellular and Molecular Phys

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Airway surface liquid (ASL) absorption is initiated by Na+ entry via epithelial Na+ channels (ENaC), which establishes an osmotic gradient that drives fluid from the luminal to serosal airway surface. We and others have recently reported that a protease/anti-protease balance regulates ENaC in human airway epithelial cells (HAEC) and provides a mechanism for autoregulation of ASL volume. In cystic fibrosis (CF), this balance is disturbed, leading to constitutive proteolytic activation of ENaC and the pathological Na+ hyperabsorption characteristic of this airway disease. Prostasin is a glycosylphosphatidylinositol-anchored serine protease that activates ENaC and is expressed on the surface epithelium lining the airway. In this report we present evidence that prostasin expression is regulated by the ASL volume, allowing for increased proteolytic activation of ENaC when the ASL volume is high. Prostasin activity is further regulated by the cognate serpin protease nexin-1 (PN-1), which is expressed in HAEC and inhibits Na+ absorption by forming an inactive complex with prostasin and preventing the proteolytic processing of prostasin. Whereas these mechanisms regulate prostasin expression in response to ASL volume in non-CF epithelia, HAEC cultured from CF patients express >50% more prostasin on the epithelial surface. These findings suggest that a proteolytic cascade involving prostasin, an upstream prostasin-activating protease, and PN-1 regulate Na+ absorption in the airway and that abnormal prostasin expression contributes to excessive proteolytic activation of ENaC in CF patients.

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Regulation of the Epithelial Sodium Channel by Serine Proteases in Human Airways

Cited by 232 publications

References 26 publications

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

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

Maturation of the Epithelial Na+ Channel Involves Proteolytic Processing of the α- and γ-Subunits

Prostasin expression is regulated by airway surface liquid volume and is increased in cystic fibrosis

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