A novel tumor necrosis factor-mediated mechanism of direct epithelial sodium channel activation

Abstract

Rationale: Alveolar liquid clearance is regulated by Na(+) uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na(+)-K(+)-ATPase in type II alveolar epithelial cells. Dysfunction of these Na(+) transporters during pulmonary inflammation can contribute to pulmonary edema.

Objectives: In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na(+) uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY).

Methods: We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na(+) uptake stimulatory activity.

Measurements and main results: TIP peptide directly activates ENaC, but not the Na(+)-K(+)-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression.

Conclusions: These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.

Keywords: epithelial sodium channel; pneumonia; protein kinase C-α; pulmonary edema; tumor necrosis factor.

Figure 1.

(A) Whole-cell, voltage-clamped patch-clamp current measurements (pA) of H441 cells treated or not with TIP peptide (50 μg/ml) in the cell bath in the presence or absence of amiloride (10 μM, n = 3/group). Inset: Effect of N-glycosidase F pretreatment (100 U, 5 min) of H441 cells on TIP peptide–mediated increase in Na⁺ uptake. (B–D) Single-channel patch-clamp measurements of 2F3 cells treated for 10 minutes with TIP peptide (50 μg/ml, n = 5) (B) or with TIP peptide pretreated with N,N′-diacetylchitobiose (100 μg/ml, n = 6) (C) or cellobiose (100 μg/ml, n = 6) (D).

Figure 2.

(A) Representative pull-down experiment assessing binding of biotinylated human TNF (10 ng/ml) to the apical epithelial Na⁺ channel (ENaC)-α, -β, and -γ subunits in H441 cell lysates. (B) Measurement of binding of biotinylated TIP peptide to recombinant ENaC-α, -β, and -γ subunits (n = 5). (C) Upper panel: Representative pull-down experiment assessing binding of biotinylated TIP or mutant TIP peptide to endogenous or overexpressed ENaC-α in H441 cell lysates. Lower panel: Influence of preincubation of TIP peptide with either N,N′-diacetylchitobiose or cellobiose (both in 10-fold molar excess over TIP peptide) upon binding to ENaC-α. (D) Binding activity of biotinylated TIP or mutant TIP peptide to glutathione-S-transferase–coupled domains of ENaC-α. EL loop = extracellular loop.

Figure 3.

(A and B) Binding and uptake of rhodamine-labeled TIP peptide (red) in H441 cells pretreated or not with β-methylcyclodextrin (1 mM). (C) Quantification of uptake of rhodamine-labeled TIP peptide in H441 cells, pretreated or not with β-methylcyclodextrin (1 mM) and subsequently treated with trypsin to remove all extracellularly bound peptide (n = 20/group, mean [black bars] ± SD [gray bars]).

Figure 4.

(A) Effect of pneumolysin (PLY) (60 ng/ml, 24 h) treatment on apical epithelial Na⁺ channel (ENaC) expression in H441 cells, assessed in a representative Western blot (WB). (B) Representative WB used to demonstrate protein kinase C-α (PKC-α) activation, measured as the ratio of phosphorylated (phospho)Ser657/total PKC-α expression in the plasma membrane in H441 cells treated for 1 hour with PLY (60 ng/ml). Cells were pretreated for 30 minutes with TIP peptide (50 μg/ml), either in combination with amiloride (10 μM) or not. (C) Quantification of the phospho/total PKC-α ratio in H441 cells treated with PLY in the presence or absence of TIP peptide (50 μg/ml) and amiloride (10 μM) (mean ± SD data from three independent experiments in triplicates). (D) Representative WB used to assess activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in H441 cells treated for 1.5 hours with PLY (125 ng/ml) upon pretreatment or not with TIP peptide (50 μg/ml, 30 min). (E) Quantification of the phospho/total ERK1/2 ratio in H441 cells treated with PLY in the presence or absence of TIP peptide (means ± SD of three independent experiments in triplicates). (F) Effect of preincubation with the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609 (30 μM) or with TIP peptide (50 μg/ml) upon PC-PLC activation in basal or PLY-treated H441 cells. (G) Ro32-0432 (10 nM) preserves ENaC-α expression in the presence of PLY (125 ng/ml) in H441 cells. (H) Influence of the PKC-α inhibitor Ro32-0432 (10 nM, 30 min) upon PKC-δ activation in H441 cells in the presence or absence of PLY (125 ng/ml). (I) Effect of TIP peptide pretreatment on PKC-δ and ERK1/2 activation in control and PLY-treated H441 cells.

Figure 5.

(A) Representative immunoprecipitation Western blot showing that the association between apical epithelial Na⁺ channel (ENaC)and myristoylated alanine-rich C kinase substrate (MARCKS) is attenuated after treating Xenopus 2F3 cells with pneumolysin (PLY) (60 ng/ml) for 30 minutes, in comparison to vehicle-treated cells. The reduced association between ENaC and MARCKS was less pronounced in cells treated with TIP (50 μg/ml) 5 minutes before PLY treatment. WCL = whole-cell lysate. (B) Densitometry of immunoreactive bands corresponding to the ENaC-α subunit from three independent experiments. All values were normalized to the background signal.

Figure 6.

(A) Protein concentration in bronchoalveolar lavage fluid (BALF) in wild-type (WT) or triple-mutant TNF knock-in (TKI) mice (n = 6, mean [black bars] ± SD [gray bars]) treated for 24 hours with pneumolysin (PLY) (1.5 μg/kg). (B) TNF and IL-6 concentrations (in pg/ml; MCYTOMAG-70K assay; EMD Millipore, Billerica, MA) in BALF from WT or TKI mice treated or not with PLY (1.5 μg/kg, n = 4, mean [black bars] ± SD [gray bars]). (C) Capillary leak, measured as Evans blue dye incorporation (n = 6, mean [black bars] ± SD [gray bars]). (D) Lung wet-to-dry ratio in WT or TKI mice treated for 24 hours with PLY (1.5 μg/kg, n = 6, mean [black bars] ± SD [gray bars]). (E) Quantification of ENaC-α expression upon PLY instillation normalized to basal level in either WT or TKI mice (1.5 μg/kg PLY, n = 6, mean [black bars] ± SD [gray bars]). (F) Representative Western blot of apical epithelial Na⁺ channel (ENaC)-α, -β, and -γ subunit expression in lung homogenates from WT or TKI mice treated or not with PLY.