RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response.
RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response.
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DOI：
10.1186/1475-2840-11-7
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发表时间：
2012-01-17
影响因子：
9.3
通讯作者：
Huang Q
中科院分区：
文献类型：
--
作者：
Wang J;Liu H;Chen B;Li Q;Huang X;Wang L;Guo X;Huang Q
晚期糖基化终末产物（AGEs）在糖尿病尤其是糖尿病并发症的发生发展中的作用已被许多报道所强调。AGEs在血管系统中的积累引发了内皮细胞（EC）的一系列形态和功能变化，并诱导内皮通透性增加。本研究旨在探讨RhoA/ROCK依赖的膜突蛋白磷酸化在AGEs诱导的内皮细胞异常中的作用。以人真皮微血管内皮细胞（human dermal microvascular endothelial cells，HMVECs）为材料，通过测定细胞单层通透性和F-肌动蛋白染色，观察人血清白蛋白修饰的AGEs（human serum albumin modified-AGEs，AGE-HSA）对HMVECs内皮细胞的影响。通过基于发光的测定和免疫印迹法测定RhoA和ROCK的激活。转染RhoA显性阴性的重组腺病毒（RhoA N19）以下调RhoA表达，而转染具有组成型激活的RhoA的腺病毒（RhoA L 63）以引起HMVECs中RhoA的过表达。H-1152用于特异性阻断ROCK的活化。免疫共沉淀法进一步证实了ROCK与其下游靶膜突蛋白的相互作用。为了鉴定AGE/ROCK诱导的膜突蛋白磷酸化位点，将两种突变体pcDNA 3/HA-moesinT 558 A和pcDNA 3/HA-moesinT 558 D分别转染内皮细胞。结果表明，AGE-HSA增加了HMVEC单层的通透性，并触发了F-actin阳性应力纤维的形成。AGE-HSA以时间和剂量依赖性方式增强RhoA活性以及ROCK磷酸化。用RhoA N19转染下调RhoA表达可消除这些AGE诱导的变化，而RhoA L 63转染可再现AGE诱导的变化。H-1152减弱AGE诱导的单层通透性和细胞骨架的改变。结果也证实了AGE诱导ROCK和膜突蛋白的直接相互作用。Thr 558被进一步鉴定为膜突蛋白在AGE诱导的内皮反应中的磷酸化位点。这些结果证实了RhoA/ROCK通路和随后的膜突蛋白Thr 558磷酸化参与AGE介导的内皮功能障碍。
The role of advanced glycation end products (AGEs) in the development of diabetes, especially diabetic complications, has been emphasized in many reports. Accumulation of AGEs in the vasculature triggers a series of morphological and functional changes in endothelial cells (ECs) and induces an increase of endothelial permeability. This study was to investigate the involvement of RhoA/ROCK-dependent moesin phosphorylation in endothelial abnormalities induced by AGEs. Using human dermal microvascular endothelial cells (HMVECs), the effects of human serum albumin modified-AGEs (AGE-HSA) on the endothelium were assessed by measuring monolayer permeability and staining of F-actin in HMVECs. Activations of RhoA and ROCK were determined by a luminescence-based assay and immunoblotting. Transfection of recombinant adenovirus that was dominant negative for RhoA (RhoA N19) was done to down-regulate RhoA expression, while adenovirus with constitutively activated RhoA (RhoA L63) was transfected to cause overexpression of RhoA in HMVECs. H-1152 was employed to specifically block activation of ROCK. Co-immunoprecipitation was used to further confirm the interaction of ROCK and its downstream target moesin. To identify AGE/ROCK-induced phosphorylation site in moesin, two mutants pcDNA3/HA-moesinT558A and pcDNA3/HA-moesinT558D were applied in endothelial cells. The results showed that AGE-HSA increased the permeability of HMVEC monolayer and triggered the formation of F-actin-positive stress fibers. AGE-HSA enhanced RhoA activity as well as phosphorylation of ROCK in a time- and dose-dependent manner. Down-regulation of RhoA expression with RhoA N19 transfection abolished these AGE-induced changes, while transfection of RhoA L63 reproduced the AGE-evoked changes. H-1152 attenuated the AGE-induced alteration in monolayer permeability and cytoskeleton. The results also confirmed the AGE-induced direct interaction of ROCK and moesin. Thr558 was further identified as the phosphorylating site of moesin in AGE-evoked endothelial responses. These results confirm the involvement of RhoA/ROCK pathway and subsequent moesin Thr558 phosphorylation in AGE-mediated endothelial dysfunction.
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影响因子：
4.8
作者：
Essler, M;Retzer, M;Siess, W
通讯作者：
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4.8
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