Transcription Factor Elf2 Signals Resolution of Lung Injury

内皮富集膜内皮细胞激酶Tie2和血管内皮细胞的优化表达 内皮细胞(ECs)中的钙粘附素(VE-cad)是形成限制性内皮屏障和维持内皮功能的必需物质。 血管动态平衡。急性肺损伤(ALI)是一种复杂的炎症性疾病，与肺组织增厚有关。 血管通透性。内皮细胞中Tie2和VE-cad表达的快速降低参与了ALI的发生。研究 将检验钙/钙调蛋白(钙/钙调素)依赖的中心假设 KCAMKKβ介导的转录因子elF2的表达促进炎症消退 内皮细胞表达Tie2和VE-cad引起的肺损伤。这个项目的灵感来自于我们的开创性 观察到Camkkβ缺陷(Camkkβ─/─)小鼠对脂多糖诱导的肺损伤和 脓毒症患者肺内皮细胞中CaMKKβ和elF2的表达均下调。我们 发现CaMKKβ信号在TLR4和PAR-1下游(一种GPCR)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-氮胞苷或表达野生型(WT)但不存在激酶缺陷 CaMKKβ，恢复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─/─小鼠的内皮细胞中发生了超甲基化。此外，甲基- CpG结合蛋白2(MeCP2)与甲基化的CpG结合，从而抑制转录。 与β─/─小鼠elF2基因调控区相关。与这些发现一致，elF2 KAMKKβ─/─小鼠内皮细胞表达明显降低。有趣的是，EC特有的DNA缺失 甲基转移酶DNMT3b(DNMT3bEC─/─)或MeCP2(Mecp2EC─/─)可增强ECs中ELF2的表达。 重要的是，在EC特异性elF2基因敲除(Elf2EC─/─)小鼠中，Tie2和VE-cad的表达显著增加 减少了。基于这些新发现，在目标1a中，我们将检验DNA甲基转移酶 Dnmt3b在静止的内皮细胞中介导elF2基因启动子的甲基化，在Aim 1b中，我们将测试 假设甲基CpG结合蛋白MeCP2与甲基化的CpG结合在 ElF2基因，失活elF2转录。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 内皮细胞中elF2的表达。在目标3中，我们将测试以下假设：最佳状态需要elF2激活 Tie2和VE-cad在内皮细胞中的表达及其对肺内皮细胞屏障的修复我们将聘用 生物化学、分子和成像方法，以确定潜在的机制。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─/─) 完成目标。我们希望这些研究将确定逆转败血症诱导的治疗靶点。 急性肺损伤基于对内源性EC修复计划的更深入了解。

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.