The protective role of MLCP-mediated ERM dephosphorylation in endotoxin-induced lung injury in vitro and in vivo

Anita Kovacs-Kasa, Boris A Gorshkov, Kyung Mi Kim, Sanjiv Kumar, Stephen Matthew Black, David J Fulton, Christiana Dimitropoulou, John D. Catravas, Alexander Dmitriyevich Verin

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Abstract

The goal of this study was to investigate the role of MLC phosphatase (MLCP) in a LPS model of acute lung injury (ALI). We demonstrate that ectopic expression of a constitutively-active (C/A) MLCP regulatory subunit (MYPT1) attenuates the ability of LPS to increase endothelial (EC) permeability. Down-regulation of MYPT1 exacerbates LPS-induced expression of ICAM1 suggesting an anti-inflammatory role of MLCP. To determine whether MLCP contributes to LPS-induced ALI in vivo, we utilized a nanoparticle DNA delivery method to specifically target lung EC. Expression of a C/A MYPT1 reduced LPS-induced lung inflammation and vascular permeability. Further, increased expression of the CS1β (MLCP catalytic subunit) also reduced LPS-induced lung inflammation, whereas the inactive CS1β mutant increased vascular leak. We next examined the role of the cytoskeletal targets of MLCP, the ERM proteins (Ezrin/Radixin/Moesin), in mediating barrier dysfunction. LPS-induced increase in EC permeability was accompanied by PKC-mediated increase in ERM phosphorylation, which was more prominent in CS1β-depleted cells. Depletion of Moesin and Ezrin, but not Radixin attenuated LPS-induced increases in permeability. Further, delivery of a Moesin phospho-null mutant into murine lung endothelium attenuated LPS-induced lung inflammation and vascular leak suggesting that MLCP opposes LPS-induced ALI by mediating the dephosphorylation of Moesin and Ezrin.

Original languageEnglish (US)
Article number39018
JournalScientific Reports
Volume6
DOIs
StatePublished - Dec 15 2016

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Lung Injury
Phosphoric Monoester Hydrolases
Endotoxins
Acute Lung Injury
Permeability
Pneumonia
Blood Vessels
Lung
Phosphoprotein Phosphatases
Capillary Permeability
Nanoparticles
Endothelium
In Vitro Techniques
Catalytic Domain
Anti-Inflammatory Agents
Down-Regulation
Phosphorylation
moesin
DNA
ezrin

ASJC Scopus subject areas

  • General

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The protective role of MLCP-mediated ERM dephosphorylation in endotoxin-induced lung injury in vitro and in vivo. / Kovacs-Kasa, Anita; Gorshkov, Boris A; Kim, Kyung Mi; Kumar, Sanjiv; Black, Stephen Matthew; Fulton, David J; Dimitropoulou, Christiana; Catravas, John D.; Verin, Alexander Dmitriyevich.

In: Scientific Reports, Vol. 6, 39018, 15.12.2016.

Research output: Contribution to journalArticle

Kovacs-Kasa, Anita ; Gorshkov, Boris A ; Kim, Kyung Mi ; Kumar, Sanjiv ; Black, Stephen Matthew ; Fulton, David J ; Dimitropoulou, Christiana ; Catravas, John D. ; Verin, Alexander Dmitriyevich. / The protective role of MLCP-mediated ERM dephosphorylation in endotoxin-induced lung injury in vitro and in vivo. In: Scientific Reports. 2016 ; Vol. 6.
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abstract = "The goal of this study was to investigate the role of MLC phosphatase (MLCP) in a LPS model of acute lung injury (ALI). We demonstrate that ectopic expression of a constitutively-active (C/A) MLCP regulatory subunit (MYPT1) attenuates the ability of LPS to increase endothelial (EC) permeability. Down-regulation of MYPT1 exacerbates LPS-induced expression of ICAM1 suggesting an anti-inflammatory role of MLCP. To determine whether MLCP contributes to LPS-induced ALI in vivo, we utilized a nanoparticle DNA delivery method to specifically target lung EC. Expression of a C/A MYPT1 reduced LPS-induced lung inflammation and vascular permeability. Further, increased expression of the CS1β (MLCP catalytic subunit) also reduced LPS-induced lung inflammation, whereas the inactive CS1β mutant increased vascular leak. We next examined the role of the cytoskeletal targets of MLCP, the ERM proteins (Ezrin/Radixin/Moesin), in mediating barrier dysfunction. LPS-induced increase in EC permeability was accompanied by PKC-mediated increase in ERM phosphorylation, which was more prominent in CS1β-depleted cells. Depletion of Moesin and Ezrin, but not Radixin attenuated LPS-induced increases in permeability. Further, delivery of a Moesin phospho-null mutant into murine lung endothelium attenuated LPS-induced lung inflammation and vascular leak suggesting that MLCP opposes LPS-induced ALI by mediating the dephosphorylation of Moesin and Ezrin.",
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