HDAC9 nuclear/cytoplasmic shuttling in pulmonary vascular 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。革兰氏阴性(G-)细菌是内毒素等细菌毒素的来源 (LP)，这是ALI的有力触发因素。尽管使用了强效抗生素和积极的重症监护 支持，内毒素诱导的ALI的死亡率仍然很高，主要是因为复杂的分子机制 参与欧共体壁垒监管的人定义不清。这个项目的科学前提来自于我们的小说 初步数据显示，IIa类组蛋白去乙酰基酶hdac9在患者的肺部表达上调。 脓毒症与炎症反应相一致。因此，HDAC9在转基因系统中的过表达 小鼠基础肺血管通透性增加，加重脂多糖诱导的血管屏障破坏。 反之，HDAC9基因缺失可改善脂多糖诱导的小鼠ALI模型的肺功能。这些数据 提示HDAC9在ALI中的表达具有促水肿性作用。HDAC9是一种核质蛋白，其细胞 定位受磷酸化的调节。磷酸化导致HDAC9核输出，从而抑制其 核内的活动，并促进与核外靶子的相互作用。我们证明了内毒素 在欧共体增加HDAC9的磷酸化，同时增加其核出口。相反，去磷酸化 HDAC9导致核进口，从而抑制其与细胞质蛋白的结合。我们展示了HDAC9 与蛋白磷酸酶2A(PP2A)相互作用，在EC中起到屏障保护作用。此外，我们的新数据 提示PP2A被Gi介导的EC屏障保护性激动剂HGF和稳定的ATP类似物激活， γS，提示GI介导的信号转导和PP2A在EC屏障增强中的联系。而当 GI介导的PP2A激活机制尚未描述，我们的数据表明，它们可能包括 GAB1/Shp2介导的信号通路的激活。此外，HDAC9和PP2A都与AJ蛋白相互作用 白蛋白提示有功能复合体。然而，依赖HDAC9的欧盟壁垒监管是否 参与HDAC9/PP2A/PLAKHb的相互作用和PP2A的EC屏障保护作用是通过 HDAC9的磷酸化水平降低还没有确定。因此，这项提案的主要目标将是 确定HDAC9/PP2A串扰在AJ介导的EC屏障调节中的新作用。我们假设 HDAC9的磷酸化状态及其与PP2A的相互作用改变细胞内信号调节EC 屏障通孔对AJ组装的影响。SA是：1：评估HDAC9磷酸化/核输出的作用 在脂多糖诱导的黏附连接介导的内皮屏障破坏的体内外。2：至 确定PP2A介导的HDAC9去磷酸化/核导入在AJs组装中的重要性 体内、体外内皮屏障增强。

PROJECT SUMMARY Acute lung injury (ALI) is characterized by lung vascular endothelial (EC) barrier compromise resulting in pulmonary edema. Gram negative (G-) bacteria are the source of bacterial toxins such as lipopolysaccharides (LPS), which are potent triggers of ALI. Despite the use of potent antibiotics and aggressive intensive care support, the mortality of LPS-induced ALI remains high, primarily because the complex molecular mechanisms involved in EC barrier regulation are ill-defined. The Scientific Premise of this Project derives from our novel preliminary data indicating that the class IIa histone deacetylase, HDAC9, is upregulated in the lungs of patients with sepsis in concert with the inflammatory responses. Accordingly, the overexpression of HDAC9 in transgenic mice increases basal lung vascular permeability and exacerbates vascular barrier compromise induced by LPS. Conversely, deletion of HDAC9 gene improves lung function in LPS-induced murine ALI model. These data suggest pro-edemagenic role of HDAC9 expression in ALI. HDAC9 is nuclear-cytoplasmic protein and its cellular localization is regulated by phosphorylation. Phosphorylation leads to HDAC9 nuclear export thus inhibiting its activity in the nucleus and promoting interactions with extra-nuclear targets. We demonstrated that LPS increases HDAC9 phosphorylation accompanied by its nuclear export in EC. In contrary, de-phosphorylation of HDAC9 leads to nuclear import thus inhibiting its association with cytoplasmic proteins. We showed that HDAC9 interacts with protein phosphatase 2A (PP2A), which has a barrier-protective role in EC. Further, our novel data indicated that PP2A is activated by Gi-mediated EC barrier-protective agonists, HGF and the stable ATP analog, ATPγS, suggesting the link between Gi-mediated signaling and PP2A in EC barrier enhancement. While the mechanisms of Gi-mediated PP2A activation have not been described, our data suggest that they may include the activation of GAB1/Shp2-mediated signaling. Further, both HDAC9 and PP2A interact with AJ protein plakoglobin suggesting functional complex. However, whether the HDAC9-dependent EC barrier regulation involved HDAC9/PP2A/plakoglobin interaction and EC barrier-protective effect of PP2A is mediated through decreased HDAC9 phosphorylation is not yet established. Therefore, a primary goal of this Proposal will be to define a novel role for HDAC9/PP2A crosstalk in AJ-mediated EC barrier regulation. We hypothesize that the phosphorylation status of HDAC9 and its interaction with PP2A alter intracellular signaling to regulate the EC barrier via effect on AJ assembly. The SAs are: 1: To evaluate the role of HDAC9 phosphorylation/nuclear export in the LPS-induced adherens junctions-mediated endothelial barrier compromise in vitro and in vivo. 2: To determine the importance of PP2A-mediated HDAC9 dephosphorylation/nuclear import in the assembly of AJs and endothelial barrier strengthening in vitro and in vivo.