Transcription Factor Elf2 Signals Resolution of Lung Injury

转录因子 Elf2 发出肺损伤消退信号

• 批准号: 10586059
• 负责人: CHINNASWAMY TIRUPPATHI
• 金额: $ 51.97万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-15 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586059
• 关键词: Acute Lung Injury; Agonist; Azacitidine; Binding; Binding Proteins; Biochemical; Blood Vessels; Calcium; Calcium Signaling; Calmodulin; Cell Nucleus; Complex; CpG dinucleotide; DNA; DNA Methyltransferase Inhibitor; DNA Modification Methylases; DNMT3B gene; Data; Disease; Dissociation; Edema; Endothelial Cells; Endothelium; Epigenetic Process; G-Protein-Coupled Receptors; Gene Expression; Genes; Genetic Transcription; Goals; Homeostasis; Hypermethylation; Inflammatory; Investigation; Knock-out; Knockout Mice; Lung; Mediating; Methyl-CpG-Binding Protein 2; Methylation; Modeling; Molecular; Mus; Nucleic Acid Regulatory Sequences; Oranges; PAR-1 Receptor; Phosphorylation; Phosphotransferases; Predisposition; Promoter Regions; Receptor Protein-Tyrosine Kinases; Regulation; Repression; Resolution; Role; S-Adenosylhomocysteine; S-Adenosylmethionine; Seminal; Sepsis; Signal Induction; Signal Transduction; TIE-2 Receptor; TLR4 gene; Testing; Thrombin; Transferase; Vascular Endothelial Cell; Vascular Permeabilities; cadherin 5; demethylation; derepression; endothelial repair; gene repression; genome wide methylation; imaging approach; injury and repair; lung injury; lung repair; mortality; mouse model; novel; prevent; programs; promoter; receptor expression; repaired; response; restoration; sepsis induced acute lung injury; septic patients; therapeutic target; transcription factor; vascular endothelial cadherin-2; vascular endothelial dysfunction

Optimal expression of endothelial-enriched tunica interna endothelial cell kinase (Tie2) and vascular endothelial cadherin (VE-cad) in endothelial cells (ECs) is required to form restrictive endothelial barrier and to maintain vascular homeostasis. Acute lung injury (ALI) is a complex inflammatory disease associated with increased lung vascular permeability. Rapid reduction in the expression of Tie2 and VE-cad in ECs contributes to ALI. Studies proposed in this application will test the central hypothesis that the calcium/calmodulin (Ca2+/CaM)-dependent kinase CaMKKβ-mediated expression of the transcription factor Elf2 promotes the resolution of inflammatory lung injury through the expression of Tie2 and VE-cad in ECs. This project was inspired by our seminal observations that Camkkβ deficient (Camkkβ─/─) mice are unusually susceptible to LPS-induced lung injury and that expression of both CaMKKβ and Elf2 is downregulated in lung endothelia from septic patients. We discovered that CaMKKβ signaling downstream of TLR4 and PAR-1 (a GPCR) mediates EC expression of Elf2, which in turn induces EC-specific transcription of the receptor tyrosine kinase Tie2, and VE-cad. In Camkkβ─/─ mice, the DNA methyltransferase inhibitor 5-azacytidine or expression of wild type (WT) but not kinase-defective CaMKKβ, restored the expression of Tie2 and VE-cad. Genome-wide methylation analysis showed that the gene encoding the transcription factor Elf2 was hyper-methylated in ECs of Camkkβ─/─ mice. Further, methyl- CpG-binding protein 2 (MeCP2), which binds methylated-CpG and thereby represses transcription, was associated with regulatory regions of the Elf2 gene in Camkkβ─/─ mice. Consistent with these findings, Elf2 expression was markedly reduced in ECs of Camkkβ─/─ mice. Interestingly, EC-specific deletion of either DNA methyltransferase Dnmt3b (Dnmt3bEC─/─) or Mecp2 (Mecp2EC─/─) in mice, augmented Elf2 expression in ECs. Importantly, in EC-specific Elf2 knockout (Elf2EC─/─) mice, expression of Tie2 and VE-cad was dramatically reduced. Based on these novel findings, in Aim 1a, we will test the hypothesis that DNA methyl transferase DNMT3b mediates methylation of Elf2-gene promoter in quiescent ECs and in Aim 1b, we will test the hypothesis that the methyl CpG binding protein MeCP2, binds methylated-CpG in the promoter regions of the Elf2 gene and inactivates Elf2 transcription. In Aim 2, we will test the hypothesis that CaMKKβ activated downstream of TLR4 and/or PAR-1 mediates phosphorylation of MeCP2 residue S421, which in turn induces the expression of Elf2 in ECs. In Aim 3, we will test the hypothesis that Elf2 activation is required for the optimal expression of Tie2 and VE-cad in ECs and thus repair of the lung endothelial barrier. We will employ biochemical, molecular, and imaging approaches to define the underlying mechanisms. Importantly, we will use EC-restricted knockout mouse models (Dnmt3bEC─/─, Mecp2EC─/─, CamkkβEC─/─ and Elf2EC─/─) created by us to accomplish the goals. Our hope is that these studies will identify therapeutic targets to reverse sepsis-induced acute lung injury based on a deeper understanding of endogenous EC repair programs.

富含内皮的内膜内皮细胞激酶 (Tie2) 和血管内皮细胞的最佳表达 内皮细胞 (EC) 中的钙粘蛋白 (VE-cad) 需要形成限制性内皮屏障并维持 血管稳态。急性肺损伤 (ALI) 是一种与肺损伤相关的复杂炎症性疾病 血管通透性。 EC 中 Tie2 和 VE-cad 表达的快速减少导致 ALI。研究 本申请中提出的将测试钙/钙调蛋白（Ca2+/CaM）依赖性的中心假设 激酶 CaMKKβ 介导的转录因子 Elf2 的表达促进炎症消退 通过 EC 中 Tie2 和 VE-cad 的表达导致肺损伤。这个项目的灵感来自于我们的开创性成果 观察发现 Camkkβ 缺陷（Camkkβ─/─）小鼠异常容易受到 LPS 诱导的肺损伤，并且 脓毒症患者肺内皮细胞中 CaMKKβ 和 Elf2 的表达均下调。我们 发现 TLR4 和 PAR-1（GPCR）下游的 CaMKKβ 信号传导介导 Elf2 的 EC 表达， 进而诱导受体酪氨酸激酶 Tie2 和 VE-cad 的 EC 特异性转录。在Camkkβ──/── 小鼠，DNA 甲基转移酶抑制剂 5-氮杂胞苷或表达野生型 (WT)，但不存在激酶缺陷 CaMKKβ，恢复了 Tie2 和 VE-cad 的表达。全基因组甲基化分析表明 编码转录因子 Elf2 的基因在 Camkkβ─/─ 小鼠的 EC 中高度甲基化。此外，甲基- CpG 结合蛋白 2 (MeCP2) 与甲基化 CpG 结合，从而抑制转录， 与 Camkkβ─/─ 小鼠中 Elf2 基因的调控区域相关。与这些发现一致，Elf2 Camkkβ─/─ 小鼠 EC 中的表达显着降低。有趣的是，EC特异性删除任一DNA 小鼠体内的甲基转移酶 Dnmt3b (Dnmt3bEC─/─) 或 Mecp2 (Mecp2EC─/─) 增强了 EC 中的 Elf2 表达。 重要的是，在 EC 特异性 Elf2 敲除 (Elf2EC─/─) 小鼠中，Tie2 和 VE-cad 的表达显着增加。 减少。基于这些新发现，在目标 1a 中，我们将检验 DNA 甲基转移酶的假设 DNMT3b 介导静止 EC 中 Elf2 基因启动子的甲基化，在目标 1b 中，我们将测试 假设甲基 CpG 结合蛋白 MeCP2 与启动子区的甲基化 CpG 结合 Elf2 基因并使 Elf2 转录失活。在目标 2 中，我们将检验 CaMKKβ 激活的假设 TLR4 和/或 PAR-1 下游介导 MeCP2 残基 S421 的磷酸化，进而诱导 Elf2 在 EC 中的表达。在目标 3 中，我们将测试以下假设：Elf2 激活对于最优结果是必需的 Tie2 和 VE-cad 在 EC 中的表达，从而修复肺内皮屏障。我们将聘用 生物化学、分子和成像方法来定义潜在的机制。重要的是，我们将使用 我们创建的 EC 限制性基因敲除小鼠模型（Dnmt3bEC─/─、Mecp2EC─/─、CamkkβEC─/─ 和 Elf2EC─/─） 实现目标。我们希望这些研究能够确定逆转败血症引起的治疗靶点 急性肺损伤基于对内源性 EC 修复程序的更深入了解。