TIMAP is a positive regulator of pulmonary endothelial barrier function
TIMAP is a positive regulator of pulmonary endothelial barrier function
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DOI：
10.1152/ajplung.00325.2007
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发表时间：
2008-09-01
影响因子：
4.9
通讯作者：
Verin, Alexander D.
中科院分区：
文献类型：
--
作者：
Csortos, Csilla;Czikora, Istvan;Verin, Alexander D.
转化生长因子-β抑制的膜相关蛋白TIMAP在内皮细胞中高水平表达。它被认为是蛋白磷酸酶1(PP1)调节亚基中MYPT(肌球蛋白磷酸酶靶标亚基)家族的成员，但其在EC中的功能尚不清楚。在我们的下拉实验中，重组TIMAP优先结合来自肺动脉EC的PP1催化亚基的β-亚基(PP1cβ)。由于PP1cβ而不是PP1cα与MYPT1结合形成功能复合体，这些结果表明TIMAP是EC中肌球蛋白磷酸酶的一个新的调节亚基。用小干扰RNA(SiRNA)技术耗尽TIMAP可减弱EC屏障保护剂(1-鞘氨醇-1-磷酸，ATP)引起的跨内皮细胞电阻的增加，并增强屏障损害剂(凝血酶、诺可达唑)的作用，表明TIMAP在EC中具有屏障保护作用。免疫荧光染色显示TIMAP与膜/细胞骨架蛋白moesin共存。此外，TIMAP与moesin免疫共沉淀提示TIMAP/moesin相互作用参与TIMAP介导的EC屏障增强。Forskolin激活cAMP/PKA级联反应，对凝血酶诱导的EC通透性具有屏障保护作用，减弱凝血酶诱导的Moesin在对照siRNA处理的EC细胞外围的磷酸化。相反，在TIMAP缺失的EC中，forskolin不能影响细胞边缘Moesin的磷酸化水平。这些结果提示TIMAP参与了PKA介导的moesin去磷酸化，以及这种去磷酸化在TIMAP介导的EC屏障保护中的重要性。
TGF-beta-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 beta-isoform of the catalytic subunit of PP1 (PP1c beta) from pulmonary artery EC. As PP1c beta, but not PP1c alpha, 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.