TIMAP is a positive regulator of pulmonary endothelial barrier function

Csilla Csortos, Istvan Czikora, Natalia V. Bogatcheva, Djanybek M. Adyshev, Christophe Poirier, Gabor Olah, Alexander D. Verin

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

TGF-β-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the β-isoform of the catalytic subunit of PP1 (PP1cβ) from pulmonary artery EC. As PP1cβ, but not PP1cα, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume295
Issue number3
DOIs
StatePublished - Sep 2008
Externally publishedYes

Fingerprint

Endothelial Cells
Lung
Myosin-Light-Chain Phosphatase
Thrombin
Protein Phosphatase 1
Colforsin
Small Interfering RNA
Membrane Proteins
Phosphorylation
Nocodazole
Protective Agents
Cytoskeletal Proteins
Cytoprotection
Electric Impedance
Pulmonary Artery
moesin
Permeability
Protein Isoforms
Adenosine Triphosphate
Staining and Labeling

Keywords

  • Moesin interaction with protein phosphatase 1
  • Small interfering RNA
  • Transendothelial electrical resistance

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology
  • Physiology

Cite this

TIMAP is a positive regulator of pulmonary endothelial barrier function. / Csortos, Csilla; Czikora, Istvan; Bogatcheva, Natalia V.; Adyshev, Djanybek M.; Poirier, Christophe; Olah, Gabor; Verin, Alexander D.

In: American Journal of Physiology - Lung Cellular and Molecular Physiology, Vol. 295, No. 3, 09.2008.

Research output: Contribution to journalArticle

Csortos, Csilla ; Czikora, Istvan ; Bogatcheva, Natalia V. ; Adyshev, Djanybek M. ; Poirier, Christophe ; Olah, Gabor ; Verin, Alexander D. / TIMAP is a positive regulator of pulmonary endothelial barrier function. In: American Journal of Physiology - Lung Cellular and Molecular Physiology. 2008 ; Vol. 295, No. 3.
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AU - Csortos, Csilla

AU - Czikora, Istvan

AU - Bogatcheva, Natalia V.

AU - Adyshev, Djanybek M.

AU - Poirier, Christophe

AU - Olah, Gabor

AU - Verin, Alexander D.

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AB - TGF-β-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the β-isoform of the catalytic subunit of PP1 (PP1cβ) from pulmonary artery EC. As PP1cβ, but not PP1cα, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.

KW - Moesin interaction with protein phosphatase 1

KW - Small interfering RNA

KW - Transendothelial electrical resistance

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