Epithelial sodium channel-α mediates the protective effect of the TNF-derived TIP peptide in pneumolysin-induced endothelial barrier dysfunction

Istvan Czikora, Abdel A. Alli, Supriya Sridhar, Michael A. Matthay, Helena Pillich, Martina Hudel, Besim Berisha, Boris A Gorshkov, Maritza Josefina Romero Lucas, Joyce N Gonzales, Guangyu Wu, Yuqing Huo, Yunchao Su, Alexander Dmitriyevich Verin, David J Fulton, Trinad Chakraborty, Douglas C. Eaton, Rudolf Lucas

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Background: Streptococcus pneumoniae is a major etiologic agent of bacterial pneumonia. Autolysis and antibiotic-mediated lysis of pneumococci induce release of the pore-forming toxin, pneumolysin (PLY), their major virulence factor, which is a prominent cause of acute lung injury. PLY inhibits alveolar liquid clearance and severely compromises alveolar-capillary barrier function, leading to permeability edema associated with pneumonia. As a consequence, alveolar flooding occurs, which can precipitate lethal hypoxemia by impairing gas exchange. The a subunit of the epithelial sodium channel (ENaC) is crucial for promoting Na+ reabsorption across Na+-transporting epithelia. However, it is not known if human lung microvascular endothelial cells (HL-MVEC) also express ENaC-α and whether this subunit is involved in the regulation of their barrier function. Methods: The presence of α, β, and γ subunits of ENaC and protein phosphorylation status in HL-MVEC were assessed in western blotting. The role of ENaC-α in monolayer resistance of HL-MVEC was examined by depletion of this subunit by specific siRNA and by employing the TNF-derived TIP peptide, a specific activator that directly binds to ENaC-α. Results: HL-MVEC express all three subunits of ENaC, as well as acid-sensing ion channel 1a (ASIC1a), which has the capacity to form hybrid non-selective cation channels with ENaC-α. Both TIP peptide, which specifically binds to ENaC-α, and the specific ASIC1a activator MitTx significantly strengthened barrier function in PLY-treated HL-MVEC. ENaC-α depletion significantly increased sensitivity to PLY-induced hyperpermeability and in addition, blunted the protective effect of both the TIP peptide and MitTx, indicating an important role for ENaC-α and for hybrid NSC channels in barrier function of HL-MVEC. TIP peptide blunted PLY-induced phosphorylation of both calmodulin-dependent kinase II (CaMKII) and of its substrate, the actin-binding protein filamin A (FLN-A), requiring the expression of both ENaC-α and ASIC1a. Since non-phosphorylated FLN-A promotes ENaC channel open probability and blunts stress fiber formation, modulation of this activity represents an attractive target for the protective actions of ENaC-α in both barrier function and liquid clearance. Conclusion: Our results in cultured endothelial cells demonstrate a previously unrecognized role for ENaC-α in strengthening capillary barrier function that may apply to the human lung. Strategies aiming to activate endothelial NSC channels that contain ENaC-α should be further investigated as a novel approach to improve barrier function in the capillary endothelium during pneumonia.

Original languageEnglish (US)
Article number842
JournalFrontiers in immunology
Volume8
Issue numberJUL
DOIs
StatePublished - Jul 21 2017

Fingerprint

Epithelial Sodium Channels
Endothelial Cells
Acid Sensing Ion Channels
Lung
Peptides
Filamins
Streptococcus pneumoniae
Pneumonia
Phosphorylation
Bacterial Pneumonia
Autolysis
Microfilament Proteins
Stress Fibers
Calcium-Calmodulin-Dependent Protein Kinases
Acute Lung Injury
Vascular Endothelium
Protein Subunits
Virulence Factors
Streptococcus pneumoniae plY protein
Small Interfering RNA

Keywords

  • Endothelial barrier function
  • Epithelial sodium channel
  • Non-selective cation channel
  • Pneumolysin
  • Pneumonia
  • TNF

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

Epithelial sodium channel-α mediates the protective effect of the TNF-derived TIP peptide in pneumolysin-induced endothelial barrier dysfunction. / Czikora, Istvan; Alli, Abdel A.; Sridhar, Supriya; Matthay, Michael A.; Pillich, Helena; Hudel, Martina; Berisha, Besim; Gorshkov, Boris A; Romero Lucas, Maritza Josefina; Gonzales, Joyce N; Wu, Guangyu; Huo, Yuqing; Su, Yunchao; Verin, Alexander Dmitriyevich; Fulton, David J; Chakraborty, Trinad; Eaton, Douglas C.; Lucas, Rudolf.

In: Frontiers in immunology, Vol. 8, No. JUL, 842, 21.07.2017.

Research output: Contribution to journalArticle

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abstract = "Background: Streptococcus pneumoniae is a major etiologic agent of bacterial pneumonia. Autolysis and antibiotic-mediated lysis of pneumococci induce release of the pore-forming toxin, pneumolysin (PLY), their major virulence factor, which is a prominent cause of acute lung injury. PLY inhibits alveolar liquid clearance and severely compromises alveolar-capillary barrier function, leading to permeability edema associated with pneumonia. As a consequence, alveolar flooding occurs, which can precipitate lethal hypoxemia by impairing gas exchange. The a subunit of the epithelial sodium channel (ENaC) is crucial for promoting Na+ reabsorption across Na+-transporting epithelia. However, it is not known if human lung microvascular endothelial cells (HL-MVEC) also express ENaC-α and whether this subunit is involved in the regulation of their barrier function. Methods: The presence of α, β, and γ subunits of ENaC and protein phosphorylation status in HL-MVEC were assessed in western blotting. The role of ENaC-α in monolayer resistance of HL-MVEC was examined by depletion of this subunit by specific siRNA and by employing the TNF-derived TIP peptide, a specific activator that directly binds to ENaC-α. Results: HL-MVEC express all three subunits of ENaC, as well as acid-sensing ion channel 1a (ASIC1a), which has the capacity to form hybrid non-selective cation channels with ENaC-α. Both TIP peptide, which specifically binds to ENaC-α, and the specific ASIC1a activator MitTx significantly strengthened barrier function in PLY-treated HL-MVEC. ENaC-α depletion significantly increased sensitivity to PLY-induced hyperpermeability and in addition, blunted the protective effect of both the TIP peptide and MitTx, indicating an important role for ENaC-α and for hybrid NSC channels in barrier function of HL-MVEC. TIP peptide blunted PLY-induced phosphorylation of both calmodulin-dependent kinase II (CaMKII) and of its substrate, the actin-binding protein filamin A (FLN-A), requiring the expression of both ENaC-α and ASIC1a. Since non-phosphorylated FLN-A promotes ENaC channel open probability and blunts stress fiber formation, modulation of this activity represents an attractive target for the protective actions of ENaC-α in both barrier function and liquid clearance. Conclusion: Our results in cultured endothelial cells demonstrate a previously unrecognized role for ENaC-α in strengthening capillary barrier function that may apply to the human lung. Strategies aiming to activate endothelial NSC channels that contain ENaC-α should be further investigated as a novel approach to improve barrier function in the capillary endothelium during pneumonia.",
keywords = "Endothelial barrier function, Epithelial sodium channel, Non-selective cation channel, Pneumolysin, Pneumonia, TNF",
author = "Istvan Czikora and Alli, {Abdel A.} and Supriya Sridhar and Matthay, {Michael A.} and Helena Pillich and Martina Hudel and Besim Berisha and Gorshkov, {Boris A} and {Romero Lucas}, {Maritza Josefina} and Gonzales, {Joyce N} and Guangyu Wu and Yuqing Huo and Yunchao Su and Verin, {Alexander Dmitriyevich} and Fulton, {David J} and Trinad Chakraborty and Eaton, {Douglas C.} and Rudolf Lucas",
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language = "English (US)",
volume = "8",
journal = "Frontiers in Immunology",
issn = "1664-3224",
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TY - JOUR

T1 - Epithelial sodium channel-α mediates the protective effect of the TNF-derived TIP peptide in pneumolysin-induced endothelial barrier dysfunction

AU - Czikora, Istvan

AU - Alli, Abdel A.

AU - Sridhar, Supriya

AU - Matthay, Michael A.

AU - Pillich, Helena

AU - Hudel, Martina

AU - Berisha, Besim

AU - Gorshkov, Boris A

AU - Romero Lucas, Maritza Josefina

AU - Gonzales, Joyce N

AU - Wu, Guangyu

AU - Huo, Yuqing

AU - Su, Yunchao

AU - Verin, Alexander Dmitriyevich

AU - Fulton, David J

AU - Chakraborty, Trinad

AU - Eaton, Douglas C.

AU - Lucas, Rudolf

PY - 2017/7/21

Y1 - 2017/7/21

N2 - Background: Streptococcus pneumoniae is a major etiologic agent of bacterial pneumonia. Autolysis and antibiotic-mediated lysis of pneumococci induce release of the pore-forming toxin, pneumolysin (PLY), their major virulence factor, which is a prominent cause of acute lung injury. PLY inhibits alveolar liquid clearance and severely compromises alveolar-capillary barrier function, leading to permeability edema associated with pneumonia. As a consequence, alveolar flooding occurs, which can precipitate lethal hypoxemia by impairing gas exchange. The a subunit of the epithelial sodium channel (ENaC) is crucial for promoting Na+ reabsorption across Na+-transporting epithelia. However, it is not known if human lung microvascular endothelial cells (HL-MVEC) also express ENaC-α and whether this subunit is involved in the regulation of their barrier function. Methods: The presence of α, β, and γ subunits of ENaC and protein phosphorylation status in HL-MVEC were assessed in western blotting. The role of ENaC-α in monolayer resistance of HL-MVEC was examined by depletion of this subunit by specific siRNA and by employing the TNF-derived TIP peptide, a specific activator that directly binds to ENaC-α. Results: HL-MVEC express all three subunits of ENaC, as well as acid-sensing ion channel 1a (ASIC1a), which has the capacity to form hybrid non-selective cation channels with ENaC-α. Both TIP peptide, which specifically binds to ENaC-α, and the specific ASIC1a activator MitTx significantly strengthened barrier function in PLY-treated HL-MVEC. ENaC-α depletion significantly increased sensitivity to PLY-induced hyperpermeability and in addition, blunted the protective effect of both the TIP peptide and MitTx, indicating an important role for ENaC-α and for hybrid NSC channels in barrier function of HL-MVEC. TIP peptide blunted PLY-induced phosphorylation of both calmodulin-dependent kinase II (CaMKII) and of its substrate, the actin-binding protein filamin A (FLN-A), requiring the expression of both ENaC-α and ASIC1a. Since non-phosphorylated FLN-A promotes ENaC channel open probability and blunts stress fiber formation, modulation of this activity represents an attractive target for the protective actions of ENaC-α in both barrier function and liquid clearance. Conclusion: Our results in cultured endothelial cells demonstrate a previously unrecognized role for ENaC-α in strengthening capillary barrier function that may apply to the human lung. Strategies aiming to activate endothelial NSC channels that contain ENaC-α should be further investigated as a novel approach to improve barrier function in the capillary endothelium during pneumonia.

AB - Background: Streptococcus pneumoniae is a major etiologic agent of bacterial pneumonia. Autolysis and antibiotic-mediated lysis of pneumococci induce release of the pore-forming toxin, pneumolysin (PLY), their major virulence factor, which is a prominent cause of acute lung injury. PLY inhibits alveolar liquid clearance and severely compromises alveolar-capillary barrier function, leading to permeability edema associated with pneumonia. As a consequence, alveolar flooding occurs, which can precipitate lethal hypoxemia by impairing gas exchange. The a subunit of the epithelial sodium channel (ENaC) is crucial for promoting Na+ reabsorption across Na+-transporting epithelia. However, it is not known if human lung microvascular endothelial cells (HL-MVEC) also express ENaC-α and whether this subunit is involved in the regulation of their barrier function. Methods: The presence of α, β, and γ subunits of ENaC and protein phosphorylation status in HL-MVEC were assessed in western blotting. The role of ENaC-α in monolayer resistance of HL-MVEC was examined by depletion of this subunit by specific siRNA and by employing the TNF-derived TIP peptide, a specific activator that directly binds to ENaC-α. Results: HL-MVEC express all three subunits of ENaC, as well as acid-sensing ion channel 1a (ASIC1a), which has the capacity to form hybrid non-selective cation channels with ENaC-α. Both TIP peptide, which specifically binds to ENaC-α, and the specific ASIC1a activator MitTx significantly strengthened barrier function in PLY-treated HL-MVEC. ENaC-α depletion significantly increased sensitivity to PLY-induced hyperpermeability and in addition, blunted the protective effect of both the TIP peptide and MitTx, indicating an important role for ENaC-α and for hybrid NSC channels in barrier function of HL-MVEC. TIP peptide blunted PLY-induced phosphorylation of both calmodulin-dependent kinase II (CaMKII) and of its substrate, the actin-binding protein filamin A (FLN-A), requiring the expression of both ENaC-α and ASIC1a. Since non-phosphorylated FLN-A promotes ENaC channel open probability and blunts stress fiber formation, modulation of this activity represents an attractive target for the protective actions of ENaC-α in both barrier function and liquid clearance. Conclusion: Our results in cultured endothelial cells demonstrate a previously unrecognized role for ENaC-α in strengthening capillary barrier function that may apply to the human lung. Strategies aiming to activate endothelial NSC channels that contain ENaC-α should be further investigated as a novel approach to improve barrier function in the capillary endothelium during pneumonia.

KW - Endothelial barrier function

KW - Epithelial sodium channel

KW - Non-selective cation channel

KW - Pneumolysin

KW - Pneumonia

KW - TNF

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