a Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University , Chongqing , China.
b Department of Pharmacology and Toxicology , Georgia Regents University , Augusta , Georgia , USA.
Autophagy. 2018;14(10):1677-1692. doi: 10.1080/15548627.2018.1476811. Epub 2018 Jul 26.
Microvascular barrier dysfunction is the central pathophysiological feature of acute lung injury (ALI). RAB26 is a newly identified small GTPase involved in the regulation of endothelial cell (EC) permeability. However, the mechanism behind this protection has not been clearly elucidated. Here we found that RAB26 promoted the integrity of adherens junctions (AJs) in a macroautophagy/autophagy-dependent manner in ALI. RAB26 is frequently downregulated in mouse lungs after LPS treatment. Mice lacking Rab26 exhibited phosphorylated SRC expression and increased CDH5/VE-cadherin phosphorylation, leading to AJ destruction. rab26-null mice showed further aggravation of the effects of endotoxin insult on lung vascular permeability and water content. Depletion of RAB26 resulted in upregulation of phosphorylated SRC, enhancement of CDH5 phosphorylation, and aggravation of CDH5 internalization, thereby weakening AJ integrity and endothelial barrier function in human pulmonary microvascular endothelial cells (HPMECs). RAB26 overexpression caused active interaction between SRC and the autophagy marker LC3-II and promoted degradation of phosphorylated SRC. Furthermore, RAB26 was involved in a direct and activation-dependent manner in autophagy induction through interaction with ATG16L1 in its GTP-bound form. These findings demonstrate that RAB26 exerts a protective effect on endothelial cell (EC) permeability, which is in part dependent on autophagic targeting of active SRC, and the resultant CDH5 dephosphorylation maintains AJ stabilization. Thus, RAB26-mediated autophagic targeting of phosphorylated SRC can maintain barrier integrity when flux through the RAB26-SRC pathway is protected. These findings suggest that activation of RAB26-SRC signaling provides a new therapeutic opportunity to prevent vascular leakage in ALI.
AJs: adherens junctions; ALI: acute lung injury; ARDS: acute respiratory distress syndrome; ATG5: autophagy related 5; ATG12: autophagy related 12; ATG 16L1: autophagy related 16 like; 1 BALF: bronchoalveolar lavage fluidCQ: chloroquine; Ctrl: control; EC: endothelial cell; GFP: green fluorescent protein; HA-tagged; RAB26: HA-tagged wild-type; RAB26 HA-tagged; RAB26: HA-tagged; RAB26HA-tagged; RAB26: HA-tagged; RAB26HPMECs: human pulmonary microvascular endothelial cells; H&E: hematoxylin & eosin; IgG: immunoglobulin; GIF: immunofluorescence; IP: immunoprecipitationi;. p.: intraperitoneal; LPS: lipopolysaccharide; PBS: phosphate-buffered salinesi; RNA: small interfering;RNASQSTM1/p62, sequestosome; 1TBS: Tris-buffered saline; VEGF: vascular endothelial growth factor; WB: western blot; WT: wild-type.
微血管屏障功能障碍是急性肺损伤（ALI）的中心病理生理特征。RAB26 是一种新发现的小 GTPase，参与调节内皮细胞（EC）通透性。然而，这种保护的机制尚不清楚。在这里，我们发现 RAB26 以巨自噬/自噬依赖的方式促进 ALI 中粘着连接（AJs）的完整性。在 LPS 处理后，RAB26 在小鼠肺中频繁下调。缺乏 Rab26 的小鼠表现出 SRC 表达的磷酸化和增加的 CDH5/VE-钙粘蛋白磷酸化，导致 AJ 破坏。rab26 基因敲除小鼠显示出对内毒素损伤对肺血管通透性和含水量的影响的进一步加重。RAB26 的耗竭导致 SRC 磷酸化的上调、CDH5 磷酸化的增强和 CDH5 内化的加重，从而削弱了人肺微血管内皮细胞（HPMECs）中的 AJ 完整性和内皮屏障功能。RAB26 的过表达导致 SRC 与自噬标记物 LC3-II 之间的活性相互作用，并促进磷酸化 SRC 的降解。此外，RAB26 通过与其 GTP 结合形式的 ATG16L1 相互作用，以直接和激活依赖性方式参与自噬的诱导。这些发现表明，RAB26 对内皮细胞（EC）通透性具有保护作用，部分依赖于自噬靶向活性 SRC，由此产生的 CDH5 去磷酸化维持 AJ 稳定。因此，当 RAB26-SRC 通路的通量受到保护时，RAB26 介导的磷酸化 SRC 的自噬靶向可以维持屏障完整性。这些发现表明，激活 RAB26-SRC 信号提供了一种新的治疗机会，以防止急性呼吸窘迫综合征（ARDS）中的血管渗漏。
