Calpain/talin/MLCP axis in pulmonary endothelial barrier regulation

项目总结 急性肺损伤(ALI)的特征是肺血管内皮细胞(EC)屏障受损，导致 肺水肿。而由大肠埃希菌或肺炎链球菌(Spn)引起的细菌感染及其 细菌毒素脂多糖(LPS)和肺溶素(PLY)是引起ALI的主要原因 内毒素或PLY诱导的ALI的分子机制尚不明确。科学前提来自于 我们新的初步数据显示，内毒素和PLY都激活了人肺内的内肽酶Calain 微血管内皮细胞(HLMVECs)以ERK依赖的方式被激活，而特异性的钙蛋白酶抑制可阻止内毒素- PLY诱导HLMVECs内皮细胞屏障的破坏和内毒素诱导的ALI肺水肿。此外，我们 发现ERK介导的小鼠钙蛋白酶Ser-50的磷酸化和活性显著高于正常小鼠 肺微血管内皮细胞的分离表明ERK-calain通路高度激活 与阿里有关。我们发现，脂多糖/PLY可诱导talin裂解成头部和杆状结构域，并使talin磷酸化 在HLMVECs中，钙蛋白酶过表达导致Talin切割和Rho介导的肌球蛋白抑制 轻链磷酸酶(MLCP)。Talin通过Talin裂解或磷酸化被激活。塔林 切割使头部与杆状结构域分离，从而消除自身抑制并刺激Talin头部与 整合素，从而诱导焦点粘连(FA)的激活，导致RhoA介导的MLCP抑制和 肺内皮细胞通透性增加。我们发现，在ALI中反对EC壁垒妥协的MLCP出现了下降- 在ARDS患者的肺中进行调节，强调了MLCP抑制在人ALI中的重要性。MLCP绑定 Talin和诱导talin去磷酸化，这可能导致talin降解，继而FAs解体。 有趣的是，MLCP使另一种钙蛋白底物Ezrin去磷酸化，导致其失活和退出 从膜细胞骨架。Talin/Ezrin轴的激活参与了FA的形成，我们的数据提示 Talin在脂多糖诱导的EC细胞骨架重塑中位于Ezrin的上游。结果表明，Ezrin枯竭 降低钙蛋白酶活性提示MLCP抑制对内毒素/PLY诱导的钙蛋白酶有正反馈作用 通过Ezrin磷酸化/膜附着的激活和EC屏障妥协。这些数据导致我们的 Calain/MLCP串扰协调talin激活导致肺内皮细胞屏障破坏的新假说 在阿里。为了验证这一假设，我们将使用独特的分子工具，如小鼠肺靶向基因传递 在体内，EC特异性诱导的Calain和MLCP KO小鼠和ERK不敏感的敲打小鼠(Capn2S50A)。我们 Will：(1)评估ERK介导的calain激活是否参与肺微血管内皮细胞屏障的破坏 细菌毒素(内毒素，PLY)、活细菌(E.Coli，Spn)和白蛋白诱导的ALI细胞骨架重组 败血症条件；(2)确定革兰氏和革兰氏+细菌、它们的毒素和败血症是否会导致Talin 激活(切割/磷酸化)和FA增强导致肺微血管内皮细胞屏障破坏； (3)检测MLCP活性是否影响内毒素/大肠杆菌、PLY/Spn和脓毒症诱导的钙蛋白激活。

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.