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This Article Full Text Full Text (PDF) All Versions of this Article: 295/3/L440    most recent 00325.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Csortos, C. Articles by Verin, A. D. PubMed PubMed Citation Articles by Csortos, C. Articles by Verin, A. D.

TIMAP is a positive regulator of pulmonary endothelial barrier function

¹Department of Medical Chemistry, Research Center for Molecular Medicine, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary; ²Department of Medicine, Division of Biological Sciences, University of Chicago, Chicago, Illinois; and ³Medical College of Georgia, Vascular Biology Center, Augusta, Georgia

Submitted 14 August 2007 ; accepted in final form 25 June 2008

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 PP1c, 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.

transendothelial electrical resistance; small interfering RNA; moesin interaction with protein phosphatase 1