Transcription Factor Elf2 Signals Resolution of Lung Injury

内皮富集的图尼卡内膜内皮细胞激酶（Tie 2）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）是形成限制性内皮屏障和维持 血管稳态急性肺损伤（acute lung injury，ALI）是一种复杂的炎症性疾病， 血管通透性内皮细胞中Tie 2和VE-cad表达的快速减少有助于ALI的发生。研究 本申请中提出的方法将检验中心假设，即钙/钙调蛋白（Ca 2 +/CaM）依赖性 激酶CaMKKβ介导的转录因子Elf 2的表达促进炎症反应的消退 肺损伤与内皮细胞Tie 2和VE-cad表达有关。这个项目的灵感来自于我们的开创性的 观察到Camkkβ缺陷（Camkkβ─/─）小鼠对LPS诱导的肺损伤异常敏感， 脓毒症患者肺内皮细胞CaMKKβ和Elf 2表达下调。我们 发现TLR 4和PAR-1下游的CaMKKβ信号传导（a GPCR）介导Elf 2的EC表达， 其又诱导受体酪氨酸激酶Tie 2和VE-cad的EC特异性转录。在Camkkβ─/─ 小鼠中，DNA甲基转移酶抑制剂5-氮杂胞苷或表达野生型（WT）但没有激酶缺陷 CaMKKβ恢复Tie 2和VE-cad的表达。全基因组甲基化分析表明， 在Camkkβ─/─小鼠的内皮细胞中，编码转录因子Elf 2的基因被过度甲基化。此外，甲基- CpG结合蛋白2（MeCP 2），结合甲基化CpG，从而抑制转录， 与Camkkβ─/─小鼠Elf 2基因调控区相关。与这些发现一致，Elf 2 Camkkβ─/─小鼠EC表达明显降低。有趣的是，EC特异性缺失的DNA 甲基转移酶Dnmt 3b（Dnmt 3bEC ─/─）或Mecp 2（Mecp 2 EC ─/─）的作用均能增强内皮细胞Elf 2的表达。 重要的是，在EC特异性Elf 2基因敲除（Elf 2 EC ─/─）小鼠中，Tie 2和VE-cad的表达显著增加， 降低基于这些新的发现，在目标1a中，我们将检验DNA甲基转移酶 DNMT 3b介导的Elf 2基因启动子甲基化在静止EC和Aim 1b中，我们将测试DNMT 3b介导的Elf 2基因启动子甲基化在静止EC和Aim 1b中的表达。 假设甲基CpG结合蛋白MeCP 2结合甲基化CpG的启动子区， Elf 2基因并使Elf 2转录失活。在目标2中，我们将检验CaMKKβ激活 TLR 4和/或PAR-1的下游介导MeCP 2残基S421的磷酸化，这反过来又诱导 Elf 2在EC中的表达。在目标3中，我们将测试Elf 2激活是最佳选择所需的假设。 Tie 2和VE-cad在EC中的表达，从而修复肺内皮屏障。我们会委聘 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 我们建立了EC限制性基因敲除小鼠模型（Dnmt 3bEC <$/<$、Mecp 2 EC <$/<$、CamkkβEC <$/<$和Elf 2 EC <$/<$）， 实现目标。我们希望这些研究将确定治疗靶点，以逆转脓毒症诱导的 急性肺损伤的基础上更深入地了解内源性EC修复程序。

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.