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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

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