PKC-dependent phosphorylation of eNOS at T495 regulates eNOS coupling and endothelial barrier function in response to G+ -toxins

Feng Chen, Sanjiv Kumar, Yanfang Yu, Saurabh Aggarwal, Christine Gross, Yusi Wang, Trinad Chakraborty, Alexander D. Verin, John D. Catravas, Rudolf Lucas, Stephen M. Black, David J.R. Fulton

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Gram positive (G+) infections make up ∼50% of all acute lung injury cases which are characterized by extensive permeability edema secondary to disruption of endothelial cell (EC) barrier integrity. A primary cause of increased permeability are cholesterol-dependent cytolysins (CDCs) of G +-bacteria, such as pneumolysin (PLY) and listeriolysin-O (LLO) which create plasma membrane pores, promoting Ca2+-influx and activation of PKCα. In human lung microvascular endothelial cells (HLMVEC), pretreatment with the nitric oxide synthase (NOS) inhibitor, ETU reduced the ability of LLO to increase microvascular cell permeability suggesting an endothelial nitric oxide synthase (eNOS)-dependent mechanism. LLO stimulated superoxide production from HLMVEC and this was prevented by silencing PKCα or NOS inhibition suggesting a link between these pathways. Both LLO and PLY stimulated eNOS T495 phosphorylation in a PKC-dependent manner. Expression of a phosphomimetic T495D eNOS (human isoform) resulted in increased superoxide and diminished nitric oxide (NO) production. Transduction of HLMVEC with an active form of PKCα resulted in the robust phosphorylation of T495 and increased peroxynitrite production, indicative of eNOS uncoupling. To determine the mechanisms underlying eNOS uncoupling, HLMVEC were stimulated with LLO and the amount of hsp90 and caveolin-1 bound to eNOS determined. LLO stimulated the dissociation of hsp90, and in particular, caveolin-1 from eNOS. Both hsp90 and caveolin-1 have been shown to influence eNOS uncoupling and a peptide mimicking the scaffolding domain of caveolin-1 blocked the ability of PKCα to stimulate eNOS-derived superoxide. Collectively, these results suggest that the G+ pore-forming toxins promote increased EC permeability via activation of PKCα, phosphorylation of eNOS-T495, loss of hsp90 and caveolin-1 binding which collectively promote eNOS uncoupling and the production of barrier disruptive superoxide.

Original languageEnglish (US)
Article numbere99823
JournalPloS one
Volume9
Issue number7
DOIs
StatePublished - Jul 14 2014

Fingerprint

Phosphorylation
Nitric Oxide Synthase Type III
phosphorylation
toxins
Endothelial cells
Caveolin 1
endothelial cells
Endothelial Cells
Superoxides
superoxide anion
lungs
permeability
Permeability
Lung
nitric oxide synthase
Nitric Oxide Synthase
endothelial nitric oxide synthase
Chemical activation
Peroxynitrous Acid
Acute Lung Injury

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

PKC-dependent phosphorylation of eNOS at T495 regulates eNOS coupling and endothelial barrier function in response to G+ -toxins. / Chen, Feng; Kumar, Sanjiv; Yu, Yanfang; Aggarwal, Saurabh; Gross, Christine; Wang, Yusi; Chakraborty, Trinad; Verin, Alexander D.; Catravas, John D.; Lucas, Rudolf; Black, Stephen M.; Fulton, David J.R.

In: PloS one, Vol. 9, No. 7, e99823, 14.07.2014.

Research output: Contribution to journalArticle

Chen, Feng ; Kumar, Sanjiv ; Yu, Yanfang ; Aggarwal, Saurabh ; Gross, Christine ; Wang, Yusi ; Chakraborty, Trinad ; Verin, Alexander D. ; Catravas, John D. ; Lucas, Rudolf ; Black, Stephen M. ; Fulton, David J.R. / PKC-dependent phosphorylation of eNOS at T495 regulates eNOS coupling and endothelial barrier function in response to G+ -toxins. In: PloS one. 2014 ; Vol. 9, No. 7.
@article{be9495e7cb7148f0a114a8299d5475ab,
title = "PKC-dependent phosphorylation of eNOS at T495 regulates eNOS coupling and endothelial barrier function in response to G+ -toxins",
abstract = "Gram positive (G+) infections make up ∼50{\%} of all acute lung injury cases which are characterized by extensive permeability edema secondary to disruption of endothelial cell (EC) barrier integrity. A primary cause of increased permeability are cholesterol-dependent cytolysins (CDCs) of G +-bacteria, such as pneumolysin (PLY) and listeriolysin-O (LLO) which create plasma membrane pores, promoting Ca2+-influx and activation of PKCα. In human lung microvascular endothelial cells (HLMVEC), pretreatment with the nitric oxide synthase (NOS) inhibitor, ETU reduced the ability of LLO to increase microvascular cell permeability suggesting an endothelial nitric oxide synthase (eNOS)-dependent mechanism. LLO stimulated superoxide production from HLMVEC and this was prevented by silencing PKCα or NOS inhibition suggesting a link between these pathways. Both LLO and PLY stimulated eNOS T495 phosphorylation in a PKC-dependent manner. Expression of a phosphomimetic T495D eNOS (human isoform) resulted in increased superoxide and diminished nitric oxide (NO) production. Transduction of HLMVEC with an active form of PKCα resulted in the robust phosphorylation of T495 and increased peroxynitrite production, indicative of eNOS uncoupling. To determine the mechanisms underlying eNOS uncoupling, HLMVEC were stimulated with LLO and the amount of hsp90 and caveolin-1 bound to eNOS determined. LLO stimulated the dissociation of hsp90, and in particular, caveolin-1 from eNOS. Both hsp90 and caveolin-1 have been shown to influence eNOS uncoupling and a peptide mimicking the scaffolding domain of caveolin-1 blocked the ability of PKCα to stimulate eNOS-derived superoxide. Collectively, these results suggest that the G+ pore-forming toxins promote increased EC permeability via activation of PKCα, phosphorylation of eNOS-T495, loss of hsp90 and caveolin-1 binding which collectively promote eNOS uncoupling and the production of barrier disruptive superoxide.",
author = "Feng Chen and Sanjiv Kumar and Yanfang Yu and Saurabh Aggarwal and Christine Gross and Yusi Wang and Trinad Chakraborty and Verin, {Alexander D.} and Catravas, {John D.} and Rudolf Lucas and Black, {Stephen M.} and Fulton, {David J.R.}",
year = "2014",
month = "7",
day = "14",
doi = "10.1371/journal.pone.0099823",
language = "English (US)",
volume = "9",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "7",

}

TY - JOUR

T1 - PKC-dependent phosphorylation of eNOS at T495 regulates eNOS coupling and endothelial barrier function in response to G+ -toxins

AU - Chen, Feng

AU - Kumar, Sanjiv

AU - Yu, Yanfang

AU - Aggarwal, Saurabh

AU - Gross, Christine

AU - Wang, Yusi

AU - Chakraborty, Trinad

AU - Verin, Alexander D.

AU - Catravas, John D.

AU - Lucas, Rudolf

AU - Black, Stephen M.

AU - Fulton, David J.R.

PY - 2014/7/14

Y1 - 2014/7/14

N2 - Gram positive (G+) infections make up ∼50% of all acute lung injury cases which are characterized by extensive permeability edema secondary to disruption of endothelial cell (EC) barrier integrity. A primary cause of increased permeability are cholesterol-dependent cytolysins (CDCs) of G +-bacteria, such as pneumolysin (PLY) and listeriolysin-O (LLO) which create plasma membrane pores, promoting Ca2+-influx and activation of PKCα. In human lung microvascular endothelial cells (HLMVEC), pretreatment with the nitric oxide synthase (NOS) inhibitor, ETU reduced the ability of LLO to increase microvascular cell permeability suggesting an endothelial nitric oxide synthase (eNOS)-dependent mechanism. LLO stimulated superoxide production from HLMVEC and this was prevented by silencing PKCα or NOS inhibition suggesting a link between these pathways. Both LLO and PLY stimulated eNOS T495 phosphorylation in a PKC-dependent manner. Expression of a phosphomimetic T495D eNOS (human isoform) resulted in increased superoxide and diminished nitric oxide (NO) production. Transduction of HLMVEC with an active form of PKCα resulted in the robust phosphorylation of T495 and increased peroxynitrite production, indicative of eNOS uncoupling. To determine the mechanisms underlying eNOS uncoupling, HLMVEC were stimulated with LLO and the amount of hsp90 and caveolin-1 bound to eNOS determined. LLO stimulated the dissociation of hsp90, and in particular, caveolin-1 from eNOS. Both hsp90 and caveolin-1 have been shown to influence eNOS uncoupling and a peptide mimicking the scaffolding domain of caveolin-1 blocked the ability of PKCα to stimulate eNOS-derived superoxide. Collectively, these results suggest that the G+ pore-forming toxins promote increased EC permeability via activation of PKCα, phosphorylation of eNOS-T495, loss of hsp90 and caveolin-1 binding which collectively promote eNOS uncoupling and the production of barrier disruptive superoxide.

AB - Gram positive (G+) infections make up ∼50% of all acute lung injury cases which are characterized by extensive permeability edema secondary to disruption of endothelial cell (EC) barrier integrity. A primary cause of increased permeability are cholesterol-dependent cytolysins (CDCs) of G +-bacteria, such as pneumolysin (PLY) and listeriolysin-O (LLO) which create plasma membrane pores, promoting Ca2+-influx and activation of PKCα. In human lung microvascular endothelial cells (HLMVEC), pretreatment with the nitric oxide synthase (NOS) inhibitor, ETU reduced the ability of LLO to increase microvascular cell permeability suggesting an endothelial nitric oxide synthase (eNOS)-dependent mechanism. LLO stimulated superoxide production from HLMVEC and this was prevented by silencing PKCα or NOS inhibition suggesting a link between these pathways. Both LLO and PLY stimulated eNOS T495 phosphorylation in a PKC-dependent manner. Expression of a phosphomimetic T495D eNOS (human isoform) resulted in increased superoxide and diminished nitric oxide (NO) production. Transduction of HLMVEC with an active form of PKCα resulted in the robust phosphorylation of T495 and increased peroxynitrite production, indicative of eNOS uncoupling. To determine the mechanisms underlying eNOS uncoupling, HLMVEC were stimulated with LLO and the amount of hsp90 and caveolin-1 bound to eNOS determined. LLO stimulated the dissociation of hsp90, and in particular, caveolin-1 from eNOS. Both hsp90 and caveolin-1 have been shown to influence eNOS uncoupling and a peptide mimicking the scaffolding domain of caveolin-1 blocked the ability of PKCα to stimulate eNOS-derived superoxide. Collectively, these results suggest that the G+ pore-forming toxins promote increased EC permeability via activation of PKCα, phosphorylation of eNOS-T495, loss of hsp90 and caveolin-1 binding which collectively promote eNOS uncoupling and the production of barrier disruptive superoxide.

UR - http://www.scopus.com/inward/record.url?scp=84904266553&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84904266553&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0099823

DO - 10.1371/journal.pone.0099823

M3 - Article

C2 - 25020117

AN - SCOPUS:84904266553

VL - 9

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 7

M1 - e99823

ER -