Ezrin, radixin, and moesin are phosphorylated in response to 2-methoxyestradiol and modulate endothelial hyperpermeability

Natalia V. Bogatcheva, Marina A. Zemskova, Boris A Gorshkov, Kyung Mi Kim, Gregory A. Daglis, Christophe Poirier, Alexander Dmitriyevich Verin

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

We showed previously that microtubule disruptor 2-methoxyestradiol (2ME) induces hyperpermeability of the endothelial monolayer via mechanisms that include the activation of p38 and Rho kinase (ROCK) and rearrangement of the actin cytoskeleton. Using the protein kinase C (PKC) inhibitors Ro-31-7549 and Ro-32-0432, we show in vitro and in vivo that 2ME-induced barrier dysfunction is also PKC-dependent. The known PKC substrates ezrin, radixin, and moesin (ERM) were recently implicated in the regulation of endothelial permeability. This study tested the hypotheses thatERMproteins are phosphorylated in response to 2ME, and that this phosphorylation is involved in 2ME-induced barrier dysfunction. We show that the application of 2ME leads to a dramatic increase in the level of ERM phosphorylation. This increase is attenuated in cells pretreated with the microtubule stabilizer taxol. In human pulmonary artery endothelial cells (HPAECs), the phosphorylation of ERM occurs in a p38-dependent and PKC-dependent manner. The activation of p38 appears to occur upstream from the activation of PKC, in response to 2ME. Phosphorylated ERM are localized at the cell periphery during the early phase of response to 2ME (15 minutes), and colocalize with F-actin branching points during the later phase of response (60 minutes). Using the short interfering RNA approach, we also showed that individual ERM depletion significantly attenuates 2ME-induced hyperpermeability. HPAEC monolayers, depleted of ERM proteins and monolayers, overexpressing phosphorylationdeficientERMmutants, exhibit less attenuationof 2ME-induced barrier disruption in response to the PKC inhibitor Ro-31-7549. These results suggest a critical role of PKC activation in response to microtubuledisrupting agents, and implicate the phosphorylation of ERM in the barrier dysfunction induced by 2ME.

Original languageEnglish (US)
Pages (from-to)1185-1194
Number of pages10
JournalAmerican journal of respiratory cell and molecular biology
Volume45
Issue number6
DOIs
StatePublished - Dec 1 2011

Fingerprint

Protein Kinase C
Phosphorylation
Chemical activation
Monolayers
Protein C Inhibitor
Endothelial cells
Protein Kinase Inhibitors
Microtubules
Pulmonary Artery
Actins
Endothelial Cells
moesin
2-methoxyestradiol
radixin
ezrin
rho-Associated Kinases
p38 Mitogen-Activated Protein Kinases
Paclitaxel
Actin Cytoskeleton
Small Interfering RNA

Keywords

  • 2-methoxyestradiol
  • Barrier dysfunction
  • ERM
  • PKC
  • Phosphorylation

ASJC Scopus subject areas

  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

Cite this

Ezrin, radixin, and moesin are phosphorylated in response to 2-methoxyestradiol and modulate endothelial hyperpermeability. / Bogatcheva, Natalia V.; Zemskova, Marina A.; Gorshkov, Boris A; Kim, Kyung Mi; Daglis, Gregory A.; Poirier, Christophe; Verin, Alexander Dmitriyevich.

In: American journal of respiratory cell and molecular biology, Vol. 45, No. 6, 01.12.2011, p. 1185-1194.

Research output: Contribution to journalArticle

Bogatcheva, Natalia V. ; Zemskova, Marina A. ; Gorshkov, Boris A ; Kim, Kyung Mi ; Daglis, Gregory A. ; Poirier, Christophe ; Verin, Alexander Dmitriyevich. / Ezrin, radixin, and moesin are phosphorylated in response to 2-methoxyestradiol and modulate endothelial hyperpermeability. In: American journal of respiratory cell and molecular biology. 2011 ; Vol. 45, No. 6. pp. 1185-1194.
@article{d314100e12984bddbc904f3c4808fa41,
title = "Ezrin, radixin, and moesin are phosphorylated in response to 2-methoxyestradiol and modulate endothelial hyperpermeability",
abstract = "We showed previously that microtubule disruptor 2-methoxyestradiol (2ME) induces hyperpermeability of the endothelial monolayer via mechanisms that include the activation of p38 and Rho kinase (ROCK) and rearrangement of the actin cytoskeleton. Using the protein kinase C (PKC) inhibitors Ro-31-7549 and Ro-32-0432, we show in vitro and in vivo that 2ME-induced barrier dysfunction is also PKC-dependent. The known PKC substrates ezrin, radixin, and moesin (ERM) were recently implicated in the regulation of endothelial permeability. This study tested the hypotheses thatERMproteins are phosphorylated in response to 2ME, and that this phosphorylation is involved in 2ME-induced barrier dysfunction. We show that the application of 2ME leads to a dramatic increase in the level of ERM phosphorylation. This increase is attenuated in cells pretreated with the microtubule stabilizer taxol. In human pulmonary artery endothelial cells (HPAECs), the phosphorylation of ERM occurs in a p38-dependent and PKC-dependent manner. The activation of p38 appears to occur upstream from the activation of PKC, in response to 2ME. Phosphorylated ERM are localized at the cell periphery during the early phase of response to 2ME (15 minutes), and colocalize with F-actin branching points during the later phase of response (60 minutes). Using the short interfering RNA approach, we also showed that individual ERM depletion significantly attenuates 2ME-induced hyperpermeability. HPAEC monolayers, depleted of ERM proteins and monolayers, overexpressing phosphorylationdeficientERMmutants, exhibit less attenuationof 2ME-induced barrier disruption in response to the PKC inhibitor Ro-31-7549. These results suggest a critical role of PKC activation in response to microtubuledisrupting agents, and implicate the phosphorylation of ERM in the barrier dysfunction induced by 2ME.",
keywords = "2-methoxyestradiol, Barrier dysfunction, ERM, PKC, Phosphorylation",
author = "Bogatcheva, {Natalia V.} and Zemskova, {Marina A.} and Gorshkov, {Boris A} and Kim, {Kyung Mi} and Daglis, {Gregory A.} and Christophe Poirier and Verin, {Alexander Dmitriyevich}",
year = "2011",
month = "12",
day = "1",
doi = "10.1165/rcmb.2011-0092OC",
language = "English (US)",
volume = "45",
pages = "1185--1194",
journal = "American Journal of Respiratory Cell and Molecular Biology",
issn = "1044-1549",
publisher = "American Thoracic Society",
number = "6",

}

TY - JOUR

T1 - Ezrin, radixin, and moesin are phosphorylated in response to 2-methoxyestradiol and modulate endothelial hyperpermeability

AU - Bogatcheva, Natalia V.

AU - Zemskova, Marina A.

AU - Gorshkov, Boris A

AU - Kim, Kyung Mi

AU - Daglis, Gregory A.

AU - Poirier, Christophe

AU - Verin, Alexander Dmitriyevich

PY - 2011/12/1

Y1 - 2011/12/1

N2 - We showed previously that microtubule disruptor 2-methoxyestradiol (2ME) induces hyperpermeability of the endothelial monolayer via mechanisms that include the activation of p38 and Rho kinase (ROCK) and rearrangement of the actin cytoskeleton. Using the protein kinase C (PKC) inhibitors Ro-31-7549 and Ro-32-0432, we show in vitro and in vivo that 2ME-induced barrier dysfunction is also PKC-dependent. The known PKC substrates ezrin, radixin, and moesin (ERM) were recently implicated in the regulation of endothelial permeability. This study tested the hypotheses thatERMproteins are phosphorylated in response to 2ME, and that this phosphorylation is involved in 2ME-induced barrier dysfunction. We show that the application of 2ME leads to a dramatic increase in the level of ERM phosphorylation. This increase is attenuated in cells pretreated with the microtubule stabilizer taxol. In human pulmonary artery endothelial cells (HPAECs), the phosphorylation of ERM occurs in a p38-dependent and PKC-dependent manner. The activation of p38 appears to occur upstream from the activation of PKC, in response to 2ME. Phosphorylated ERM are localized at the cell periphery during the early phase of response to 2ME (15 minutes), and colocalize with F-actin branching points during the later phase of response (60 minutes). Using the short interfering RNA approach, we also showed that individual ERM depletion significantly attenuates 2ME-induced hyperpermeability. HPAEC monolayers, depleted of ERM proteins and monolayers, overexpressing phosphorylationdeficientERMmutants, exhibit less attenuationof 2ME-induced barrier disruption in response to the PKC inhibitor Ro-31-7549. These results suggest a critical role of PKC activation in response to microtubuledisrupting agents, and implicate the phosphorylation of ERM in the barrier dysfunction induced by 2ME.

AB - We showed previously that microtubule disruptor 2-methoxyestradiol (2ME) induces hyperpermeability of the endothelial monolayer via mechanisms that include the activation of p38 and Rho kinase (ROCK) and rearrangement of the actin cytoskeleton. Using the protein kinase C (PKC) inhibitors Ro-31-7549 and Ro-32-0432, we show in vitro and in vivo that 2ME-induced barrier dysfunction is also PKC-dependent. The known PKC substrates ezrin, radixin, and moesin (ERM) were recently implicated in the regulation of endothelial permeability. This study tested the hypotheses thatERMproteins are phosphorylated in response to 2ME, and that this phosphorylation is involved in 2ME-induced barrier dysfunction. We show that the application of 2ME leads to a dramatic increase in the level of ERM phosphorylation. This increase is attenuated in cells pretreated with the microtubule stabilizer taxol. In human pulmonary artery endothelial cells (HPAECs), the phosphorylation of ERM occurs in a p38-dependent and PKC-dependent manner. The activation of p38 appears to occur upstream from the activation of PKC, in response to 2ME. Phosphorylated ERM are localized at the cell periphery during the early phase of response to 2ME (15 minutes), and colocalize with F-actin branching points during the later phase of response (60 minutes). Using the short interfering RNA approach, we also showed that individual ERM depletion significantly attenuates 2ME-induced hyperpermeability. HPAEC monolayers, depleted of ERM proteins and monolayers, overexpressing phosphorylationdeficientERMmutants, exhibit less attenuationof 2ME-induced barrier disruption in response to the PKC inhibitor Ro-31-7549. These results suggest a critical role of PKC activation in response to microtubuledisrupting agents, and implicate the phosphorylation of ERM in the barrier dysfunction induced by 2ME.

KW - 2-methoxyestradiol

KW - Barrier dysfunction

KW - ERM

KW - PKC

KW - Phosphorylation

UR - http://www.scopus.com/inward/record.url?scp=82755165225&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=82755165225&partnerID=8YFLogxK

U2 - 10.1165/rcmb.2011-0092OC

DO - 10.1165/rcmb.2011-0092OC

M3 - Article

VL - 45

SP - 1185

EP - 1194

JO - American Journal of Respiratory Cell and Molecular Biology

JF - American Journal of Respiratory Cell and Molecular Biology

SN - 1044-1549

IS - 6

ER -