Diacylglycerol kinase in airway smooth muscle functions

二酰甘油激酶在气道平滑肌功能中的作用

基本信息

批准号：
10204427
负责人：
Deepak A Deshpande
金额：
$ 5.11万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10204427
关键词：
Ablation Acetylcholine Acute Address Affect Airway Disease Allergens Animals Asthma Attenuated Basic Science Biochemical Biological Assay Biology Breathing Bronchoconstriction Calcium Cell Proliferation Cell membrane Cell physiology Cell surface Cells Clinical effectiveness Complement Complex Confocal Microscopy Coupled Cytoskeletal Modeling Data Diacylglycerol Kinase Diglycerides Disease Drug Targeting Enzymes Equilibrium Extracellular Signal Regulated Kinases Family Family member Feedback Fluorescence Future G Protein-Coupled Receptor Signaling G alpha q Protein G-Protein-Coupled Receptors Generations Genetic Goals Growth Health Histamine Human Individual Inositol Knockout Mice Leukotrienes Lipids Lung Mediating Mediator of activation protein Mitogens Molecular Mus Muscarinics Muscle Contraction Muscle function Myosin Light Chains Neurons Nucleotides Obstructive Lung Diseases Ovalbumin Pathogenesis Pathologic Pathway interactions Pharmacology Phosphatidic Acid Phosphatidylinositol 4,5-Diphosphate Phospholipase C Phosphorylation Play Process Production Prostaglandins Protein Isoforms Protein Kinase C Proteins Proto-Oncogene Proteins c-akt Pyroglyphidae Regulation Regulatory Pathway Research Role Signal Pathway Signal Transduction Signaling Molecule Signaling Protein Slice Small Interfering RNA Smooth Muscle Smooth Muscle Myocytes Spatial Distribution Testing airway hyperresponsiveness airway inflammation airway remodeling asthma model asthmatic asthmatic airway attenuation cell type chronic inflammatory disease cysteinyl-leukotriene drug development drug discovery experimental study imaging approach in vivo inflammatory milieu inhibitor/antagonist kinase inhibitor lipid mediator lipidomics methacholine microscopic imaging mouse model new therapeutic target novel prevent protein activation public health relevance receptor respiratory smooth muscle response sensor success therapeutic target tool tripolyphosphate

项目摘要

Gq-coupled G protein coupled receptors (GPCR) on airway smooth muscle (ASM) cells are critical regulators of the airway hyperresponsiveness (AHR) and airway remodeling that occurs with asthma. Gq signaling in ASM involves activation of phospholipase C that converts phosphoinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). While IP3 leads to increases in [Ca2+]i, phosphorylation of MLC20 and ASM contraction, DAG directly activates PKC family members and Ras guanyl nucleotide-releasing protein. The signaling and functional role of DAG in ASM is largely unknown. DAG is known to be phosphorylated and further converted into phosphatidic acid (PA) by enzymes known as DAG kinases (DGK). Both PA and DAG are important lipid mediators that can activate numerous signaling proteins and therefore, intracellular levels of DAG and PA are tightly regulated. The impetus for the proposed studies is our preliminary observation that mice lacking  isoform of DGK are protected from ovalbumin-induced AHR in spite of full complement of airway inflammation. In this proposal we seek to establish the mechanisms by which DGK regulates ASM contraction and proliferation with the central hypothesis that DGKs play a key role in regulating ASM contraction (via PIP2-DAG/IP3 axis) and proliferation (via PIP2-DAG/PA axis), and inhibition of DGK blocks the asthmatic airway response by affecting the contractile (AHR) and proliferative (remodeling) function of ASM cells. To achieve our research goals, we will employ diverse, state-of-the-art tools such as targeted lipidomics, fluorescence sensors of PIP2/DAG and genetic/pharmacological inhibition of DGK. We contend that acute inhibition of DGK results in accumulation of DAG that acts as a negative feedback regulator and inhibit Gq-PLC signaling in ASM cells. In Aim 1 studies we will discern multiple mechanisms by which DGK isoforms regulate Gq-coupled GPCR-mediated ASM contraction. Additional preliminary data suggest that PA is a pro-mitogenic signaling molecule in ASM and DGK inhibition leads to attenuation of ASM growth. Therefore, Aim 2 studies will establish the molecular and cellular mechanisms by which DGK isoforms regulate ASM cell proliferation. Finally, to establish the in vivo relevance of DGK inhibition, Aim 3 studies will employ a house dust mite (HDM)-induced mouse model of asthma and test the effect of DGK inhibition on allergen-induced AHR and ASM remodeling. DGK inhibition will be achieved either by using smooth muscle specific conditional deletion of DGK or by treating animals with a pharmacological inhibitor of DGK (R59022). Our success is favored by the availability of a unique cell-type specific DGK isoform knockout mice, our team’s ability to creatively apply cutting edge imaging approaches, and the use of multiple complementary approaches to discern the complex (im)balance of lipid signaling molecules in ASM cells. The findings will not only advance the basic science of ASM biology, but also identify DGK as a potential therapeutic target whose manipulation can be exploited for developing a novel asthma therapy that addresses both AHR and airway remodeling.

气道平滑肌 (ASM) 细胞上的 Gq 偶联 G 蛋白偶联受体 (GPCR) 至关重要哮喘气道高反应性 (AHR) 和气道重塑的调节因子 Gq。 ASM 中的信号传导涉及磷脂酶 C 的激活，该酶将磷酸肌醇 4,5-二磷酸 (PIP2) 转化为磷酸肌醇转化为二酰基甘油 (DAG) 和肌醇 1,4,5-三磷酸 (IP3) 而 IP3 会导致 [Ca2+]i 增加， MLC20磷酸化和ASM收缩，DAG直接激活PKC家族成员和Ras鸟苷 DAG 在 ASM 中的信号传导和功能作用目前尚不清楚。已知会被称为 DAG 的酶磷酸化并进一步转化为磷脂酸 (PA) PA 和 DAG 都是重要的脂质介质，可以激活多种信号蛋白。因此，DAG 和 PA 的细胞内水平受到严格调控。我们的初步观察结果表明，缺乏 DGK 亚型的小鼠可以免受卵清蛋白诱导的 AHR 的影响。尽管气道炎症得到充分补充，但在本提案中，我们寻求建立机制。 DGK 调节 ASM 收缩和增殖，其中心假设是 DGK 在 ASM 收缩和增殖中发挥关键作用调节 ASM 收缩（通过 PIP2-DAG/IP3 轴）和增殖（通过 PIP2-DAG/PA 轴），并抑制 DGK 通过影响收缩 (AHR) 和增殖（重塑）来阻断哮喘气道反应为了实现我们的研究目标，我们将采用多种最先进的工具，例如靶向脂质组学、PIP2/DAG 荧光传感器和 DGK We 的遗传/药理学抑制。认为 DGK 的急性抑制会导致 DAG 的积累，DAG 充当负反馈调节剂并抑制 ASM 细胞中的 Gq-PLC 信号传导。在 Aim 1 研究中，我们将了解 DGK 的多种机制。亚型调节 Gq 偶联的 GPCR 介导的 ASM 收缩。 ASM 和 DGK 抑制中的促有丝分裂信号分子会导致 ASM 生长减弱。目标 2 研究将建立 DGK 亚型调节 ASM 细胞的分子和细胞机制最后，为了确定 DGK 抑制的体内相关性，Aim 3 研究将使用一个房屋。尘螨 (HDM) 诱发的小鼠哮喘模型并测试 DGK 抑制对过敏原诱发的影响 AHR 和 ASM 重塑可通过使用平滑肌特异性条件来实现。删除 DGK 或用 DGK 药理学抑制剂治疗动物（R59022）。得益于独特的细胞类型特异性 DGK 同种型敲除小鼠的可用性，我们的团队能够创造性地应用尖端成像方法，并使用多种互补方法辨别 ASM 细胞中脂质信号分子的复杂（不）平衡，这些发现不仅会取得进展。 ASM 生物学的基础科学，同时也将 DGK 确定为潜在的治疗靶点，其操纵可用于开发一种同时解决 AHR 和气道重塑问题的新型哮喘疗法。