Calpain/talin/MLCP axis in pulmonary endothelial barrier regulation

项目摘要 急性肺损伤（ALI）的特征是肺血管内皮细胞（EC）屏障受损， 肺水肿而由大肠杆菌或肺炎链球菌（Spn）诱导的细菌感染及其 细菌毒素脂多糖（LPS）和肺炎球菌溶血素（Pneumolysin，Pneumolysin）是引起ALI的主要原因， 涉及LPS或PLY诱导的ALI的分子机制尚不清楚。科学原理来自于 我们新的初步数据表明，LPS和LPS都能激活人肺内的内肽酶钙蛋白酶， 微血管内皮细胞（HLMVECs）的ERK依赖性的方式和特异性钙蛋白酶抑制剂防止LPS- PLY诱导的HLMVECs EC屏障破坏和LPS诱导的ALI肺水肿。我们还 发现ERK介导的钙蛋白酶Ser-50磷酸化和钙蛋白酶活性在小鼠中高得多， 从暴露于LPS的肺中分离的肺微血管EC，表明ERK-钙蛋白酶通路高度依赖于肺微血管内皮细胞。 与阿里有关我们发现，LPS/LPS诱导talin裂解成头和杆结构域和talin磷酸化 在HLMVECs中，钙蛋白酶过表达引起塔林蛋白裂解，Rho介导的肌球蛋白抑制 轻链磷酸酶（MLCP）。塔林蛋白通过塔林蛋白裂解或磷酸化被激活。Talin 切割将头部与杆状结构域分开，从而消除了自身抑制并刺激塔林素头部结合到 整合素，从而诱导粘着斑（FA）活化，导致RhoA介导的MLCP抑制， 增加肺EC渗透性。我们发现，MLCP，反对EC屏障妥协在ALI，是下降- 在ARDS患者的肺中调节MLCP，突出了MLCP抑制在人ALI中的重要性。MLCP结合 talin和诱导talin去磷酸化，这可能导致talin降解，随后FAs分解。 有趣的是，MLCP使另一种钙蛋白酶底物ezrin去磷酸化，导致其失活和撤回 从细胞膜骨架中分离出来talin/ezrin轴的激活参与FA的形成，我们的数据表明， talin在LPS诱导的EC细胞骨架重塑中位于ezrin的上游。结果表明，埃兹林耗竭 降低钙蛋白酶活性，表明MLCP抑制对LPS/PLY诱导的钙蛋白酶具有正反馈效应 激活和EC屏障通过埃兹蛋白磷酸化/膜附着妥协。这些数据导致我们 钙蛋白酶/MLCP串扰协调talin激活导致肺EC屏障破坏的新假设 在阿里。为了验证这一假设，我们将采用独特的分子工具，如小鼠肺靶向基因传递 在体内，EC特异性可诱导钙蛋白酶和MLCP KO小鼠和ERK不敏感敲入小鼠（Capn 2S 50 A）。我们 将：（1）评价ERK介导的钙蛋白酶激活是否参与肺微血管EC屏障破坏 在细菌毒素（LPS、LPS）、活菌（E.coli、Spn）诱导的ALI中， 脓毒症条件下;（2）确定是否革兰氏和革兰氏+细菌，其毒素和脓毒症诱导塔林 激活（裂解/磷酸化）和FA强化导致肺微血管EC屏障破坏; (3)检查MLCP活性是否影响由LPS/大肠杆菌、LPS/Spn和脓毒症诱导的钙蛋白酶活化。

PROJECT SUMMARY Acute lung injury (ALI) is characterized by lung vascular endothelial cell (EC) barrier compromise resulting in pulmonary edema. While bacterial infections induced by E.coli or Streptococcus pneumoniae (Spn), and their respective bacteria toxin lipopolysaccharides (LPS) and pneumolysin (PLY) are the major causes for ALI, the molecular mechanisms involved in LPS- or PLY-induced ALI are ill-defined. The Scientific Premise comes from our novel preliminary data, which show that both LPS and PLY activate endopeptidase calpain in human lung microvascular ECs (HLMVECs) in ERK-dependent manner and that specific calpain inhibition prevents LPS- and PLY-induced disruption of EC barrier in HLMVECs and LPS-induced pulmonary edema in ALI. Further, we found that ERK-mediated calpain phosphorylation at Ser-50 and calpain activity were much higher in murine lung microvascular ECs isolated from lungs exposed to LPS, indicating that ERK-calpain pathway is highly related to ALI. We show that LPS/PLY induces talin cleavage into head and rod domain and talin phosphorylation in HLMVECs and that overexpression of calpain causes talin cleavage, and Rho-mediated inhibition of myosin light chain phosphatase (MLCP). Talin is activated through either talin cleavage or phosphorylation. Talin cleavage separates head from rod domain thus removing auto-inhibition and stimulating talin head binding to integrin and thus induces activation of focal adhesions (FAs), leading to RhoA-mediated MLCP inhibition and increased lung EC permeability. We found that MLCP, which opposes EC barrier compromise in ALI, is down- regulated in lungs of ARDS patients, highlighting the importance of MLCP inhibition in human ALI. MLCP binds talin and induced talin dephosphorylation, which may lead to talin degradation followed by FAs disassembly. Interestingly, MLCP dephosphorylates another calpain substrate, ezrin, leading to its deactivation and withdrawal from membrane cytoskeleton. Activation of talin/ezrin axis is involved in FA formation and our data suggest that talin is upstream of ezrin in LPS-induced EC cytoskeletal remodeling. It was shown that ezrin depletion decreases calpain activity suggesting positive feedback effect of MLCP inhibition on LPS/PLY-induced calpain activation and EC barrier compromise via ezrin phosphorylation/membrane attachment. These data led to our novel hypothesis that calpain/MLCP crosstalk coordinates talin activation leading to lung EC barrier disruption in ALI. To test this hypothesis we will employ unique molecular tools such as murine lung-targeting gene delivery in vivo, EC-specific inducible calpain and MLCP KO mice and ERK insensitive knockin mouse (Capn2S50A). We will: (1) evaluate whether ERK-mediated calpain activation is involved in lung microvascular EC barrier disruption and cytoskeletal reorganization in ALI induced by bacterial toxins (LPS, PLY), live bacteria (E.coli, Spn) and in sepsic conditions; (2) determine whether Gram- and Gram+ bacteria, their toxins and sepsis induce talin activation (cleavage/phosphorylation) and FA strengthening leading to lung microvascular EC barrier disruption; (3) examine whether MLCP activity affects calpain activation induced by LPS/E.coli, PLY/Spn and sepsis.