Calpain/talin/MLCP axis in pulmonary endothelial barrier regulation

钙蛋白酶/talin/MLCP轴在肺内皮屏障调节中的作用

基本信息

批准号：
10522290
负责人：
YUNCHAO SU
金额：
$ 72.2万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10522290
关键词：
Actins Actomyosin Acute Lung Injury Acute Respiratory Distress Syndrome Affect Antibiotics Attenuated Bacteria Bacterial Infections Bacterial Toxins Binding Calpain Caspase Catalytic Domain Cyclin-Dependent Kinase 5 Cytoskeletal Modeling Cytoskeleton Data Development Down-Regulation Ectopic Expression Endopeptidases Endothelial Cells Endothelium Escherichia coli Exposure to Family Feedback Focal Adhesions Gene Delivery Goals Head Human In Vitro Integrins Intensive Care Knock-in Mouse Knock-out Knockout Mice Lead Link Lipopolysaccharides Lung Mammalian Cell Mediating Membrane Molecular Mus Mutation Myosin Light Chains Pathologic Pathway interactions Patients Permeability Pharmacology Phosphorylation Plasma Protein Dephosphorylation Protein Family Proteins Proteolysis Pulmonary Edema Regulation Resistance Rod Role Sepsis Streptococcus pneumoniae Streptococcus pneumoniae plY protein Stress Fibers Tail Talin Testing Toxin Ubiquitination Vascular Endothelial Cell Vinculin Withdrawal ezrin in vivo interdisciplinary approach knock-down lung injury lung microvascular endothelial cells mimetics mortality mouse model mutant myosin phosphatase novel novel therapeutics overexpression prevent response rho septic tool

项目摘要

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.

项目概要急性肺损伤（ALI）的特点是肺血管内皮细胞（EC）屏障受损，导致肺水肿。而由大肠杆菌或肺炎链球菌（Spn）引起的细菌感染及其细菌毒素脂多糖（LPS）和肺炎球菌溶血素（PLY）分别是引起ALI的主要原因， LPS 或 PLY 诱导的 ALI 的分子机制尚不明确。科学前提来自我们新的初步数据表明，LPS 和 PLY 均可激活人肺中的肽链内切酶钙蛋白酶微血管内皮细胞 (HLMVEC) 以 ERK 依赖性方式进行，并且特定的钙蛋白酶抑制可防止 LPS- HLMVEC 中 PLY 诱导的 EC 屏障破坏以及 ALI 中 LPS 诱导的肺水肿。此外，我们发现小鼠中 ERK 介导的钙蛋白酶 Ser-50 磷酸化和钙蛋白酶活性要高得多从暴露于 LPS 的肺中分离出肺微血管 EC，表明 ERK-钙蛋白酶途径高度与阿里有关。我们发现 LPS/PLY 诱导踝蛋白裂解成头和杆结构域以及踝蛋白磷酸化在 HLMVEC 中，钙蛋白酶的过度表达会导致踝蛋白裂解，以及 Rho 介导的肌球蛋白抑制轻链磷酸酶（MLCP）。踝蛋白通过踝蛋白裂解或磷酸化被激活。塔林切割将头与杆结构域分开，从而消除自身抑制并刺激talin头与杆结构域的结合整合素，从而诱导粘着斑 (FA) 的激活，导致 RhoA 介导的 MLCP 抑制和肺EC通透性增加。我们发现，反对 ALI 中 EC 屏障妥协的 MLCP 正在下降—— MLCP 在 ARDS 患者肺部受到调节，凸显了 MLCP 抑制在人类 ALI 中的重要性。 MLCP 结合 talin 并诱导 talin 去磷酸化，这可能导致 talin 降解，随后发生 FA 分解。有趣的是，MLCP 使另一种钙蛋白酶底物埃兹蛋白 (ezrin) 去磷酸化，导致其失活和退出来自膜细胞骨架。 talin/ezrin 轴的激活参与 FA 的形成，我们的数据表明在 LPS 诱导的 EC 细胞骨架重塑中，talin 是 ezrin 的上游。结果表明，埃兹蛋白耗尽降低钙蛋白酶活性，表明 MLCP 抑制对 LPS/PLY 诱导的钙蛋白酶有正反馈作用通过埃兹蛋白磷酸化/膜附着激活和 EC 屏障损害。这些数据导致我们新假设：钙蛋白酶/MLCP 串扰协调踝蛋白激活，导致肺 EC 屏障破坏在阿里。为了验证这一假设，我们将采用独特的分子工具，例如鼠肺靶向基因传递体内EC特异性诱导钙蛋白酶和MLCP KO小鼠以及ERK不敏感敲入小鼠（Capn2S50A）。我们将：（1）评估ERK介导的钙蛋白酶激活是否参与肺微血管EC屏障破坏细菌毒素（LPS、PLY）、活细菌（大肠杆菌、Spn）诱导的 ALI 中的细胞骨架重组和脓毒症； (2)确定革兰氏阴性和革兰氏阳性细菌、其毒素和败血症是否诱导talin 激活（裂解/磷酸化）和 FA 强化导致肺微血管 EC 屏障破坏； (3)检测MLCP活性是否影响LPS/E.coli、PLY/Spn和脓毒症诱导的钙蛋白酶激活。