HDAC9 nuclear/cytoplasmic shuttling in pulmonary vascular endothelial barrier regulation

HDAC9核/细胞质穿梭在肺血管内皮屏障调节中的作用

• 批准号: 10597538
• 负责人: ALEXANDER D VERIN
• 金额: $ 38.5万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-02 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10597538
• 关键词: Acceleration; Acute Lung Injury; Acute Respiratory Distress Syndrome; Adherens Junction; Affect; Agonist; Antibiotics; Bacterial Toxins; Blood Vessels; Cell Nucleus; Clinical; Complex; Cytoplasm; Cytoplasmic Protein; Cytoskeletal Proteins; Cytosol; Data; Deacetylation; Development; Endothelial Cells; Endothelium; Escherichia coli; Functional disorder; GAB1 gene; Genetic Transcription; Goals; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; HDAC9 gene; HGF gene; Histone Deacetylase; Human; In Vitro; Inflammation; Inflammatory Response; Intensive Care; Investigation; Knockout Mice; Link; Lipopolysaccharides; Lung; Mediating; Modeling; Molecular; Mus; Nuclear; Nuclear Export; Nuclear Import; Pathologic; Patients; Permeability; Phosphorylation; Phosphotransferases; Property; Protein Dephosphorylation; Protein Inhibition; Protein phosphatase; Proteins; Pulmonary Circulation; Pulmonary Edema; Regulation; Respiratory distress; Role; Sepsis; Signal Transduction; Source; Syndrome; Testing; Transgenic Mice; Vascular Endothelial Cell; Vascular Endothelium; Vascular Permeabilities; analog; beta catenin; genetic corepressor; improved; in vivo; lung microvascular endothelial cells; mortality; novel; novel therapeutic intervention; overexpression; pharmacologic; plakoglobin; protective effect; protein activation; pulmonary function; receptor

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.

项目摘要 急性肺损伤（ALI）的特征是肺血管内皮（EC）屏障受损，导致 肺水肿革兰氏阴性（G-）细菌是细菌毒素如脂多糖的来源 (LPS)，这是ALI的强有力的触发器。尽管使用了强效抗生素和积极的重症监护 支持，LPS诱导的ALI的死亡率仍然很高，主要是因为复杂的分子机制， 欧盟屏障监管中涉及的问题定义不清。这个项目的科学假设来自我们的小说 初步数据表明，IIa类组蛋白脱乙酰酶HDAC 9在患者的肺中上调， 败血症和炎症反应因此，HDAC 9在转基因小鼠中的过表达是不稳定的。 小鼠增加基础肺血管通透性并加剧LPS诱导的血管屏障损害。 相反，HDAC 9基因的缺失改善了LPS诱导的小鼠ALI模型的肺功能。这些数据 提示HDAC 9表达在ALI中促水肿作用。HDAC 9是核质蛋白，其细胞内 定位受磷酸化调节。磷酸化导致HDAC 9核输出，从而抑制其表达。 核内的活动，并促进与核外目标的相互作用。我们证明LPS 增加HDAC 9磷酸化伴随其在EC中的核输出。相反， HDAC 9导致核输入，从而抑制其与细胞质蛋白的结合。我们发现HDAC 9 与蛋白磷酸酶2A（PP 2A）相互作用，PP 2A在EC中具有屏障保护作用。此外，我们的新数据 表明PP 2A被Gi介导的EC屏障保护激动剂HGF和稳定的ATP类似物激活， ATPγS，表明在EC屏障增强中Gi介导的信号传导和PP 2A之间的联系。而 Gi-mediated PP 2A激活的机制尚未被描述，我们的数据表明，它们可能包括 激活GAB 1/Shp 2介导的信号传导。此外，HDAC 9和PP 2A都与AJ蛋白相互作用， 斑珠蛋白提示功能复合体。然而，HDAC 9依赖性EC屏障调节是否 参与HDAC 9/PP 2A/斑珠蛋白相互作用，PP 2A的EC屏障保护作用是通过 HDAC 9磷酸化的降低尚未建立。因此，本提案的主要目标是 定义了HDAC 9/PP 2A串扰在AJ-mediated EC屏障调节中的新作用。我们假设 HDAC 9的磷酸化状态及其与PP 2A的相互作用改变细胞内信号传导以调节EC AJ组件上的阻挡通孔效应。SA是：1：评估HDAC 9磷酸化/核输出的作用 在LPS诱导的粘附连接介导的内皮屏障妥协在体外和体内。2：向 确定PP 2A介导的HDAC 9去磷酸化/核输入在AJs组装中的重要性 和内皮屏障增强。