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：
10349442
负责人：
Deepak A Deshpande
金额：
$ 49.63万
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 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10349442
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 Persons 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 antagonist 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 kinase inhibitor lipid mediator lipidomics methacholine microscopic imaging mouse model new therapeutic target novel obstructive airway disease 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耦合G蛋白偶联受体（GPCR）至关重要哮喘发生的气道高反应性（AHR）和气道重塑的调节器。 GQ ASM中的信号传导涉及转化磷脂酶C的激活，该磷脂酶C转化4,5-双磷酸盐（PIP2）进入二酰基甘油（DAG）和肌醇1,4,5-三磷酸（IP3）。而IP3导致[Ca2+] i的增加， MLC20和ASM收缩的磷酸化，DAG直接激活PKC家族成员和RAS Guanyl 核苷酸释放蛋白。 DAG在ASM中的信号传导和功能作用在很大程度上未知。 DAG是已知被称为DAG的酶进一步转化为磷脂酸（PA）激酶（DGK）。 PA和DAG都是重要的脂质介质，可以激活许多信号蛋白因此，细胞内DAG和PA的水平受到严格调节。拟议研究的动力是我们的初步观察到缺乏DGK的小鼠受到卵巢蛋白诱导的AHR的保护气道炎症完全完成。在此提案中，我们试图建立一种机制 DGK通过中心假设调节ASM收缩和增殖，即DGK在调节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）来阻止哮喘气道响应 ASM细胞的功能。为了实现我们的研究目标，我们将采用潜水员，最先进的工具，例如靶向脂质组学，PIP2/DAG的荧光传感器以及DGK的遗传/药理抑制。我们争辩说，急性抑制DGK会导致DAG积累，而DAG充当负面反馈调节器并抑制ASM细胞中的GQ-PLC信号传导。在AIM 1研究中，我们将辨别DGK的多种机制同工型调节GQ耦合的GPCR介导的ASM收缩。其他初步数据表明PA是 ASM和DGK抑制中的促触点信号分子会导致ASM生长的衰减。所以， AIM 2研究将建立DGK同工型调节ASM细胞的分子和细胞机制增殖。最后，为了建立DGK抑制的体内相关性，AIM 3研究将雇用房屋尘螨（HDM）诱导的哮喘小鼠模型，并测试DGK抑制对过敏原诱导的影响 AHR和ASM重塑。 DGK抑制将通过使用平滑肌特异性条件来实现 DGK的缺失或用DGK的药物抑制剂（R59022）治疗动物。我们的成功是由于提供独特的细胞类型的特定DGK同工敲除小鼠而受到青睐，我们的团队的能力创造性地采用尖端成像方法，并使用多种完善方法辨别ASM细胞中脂质信号分子的复合物平衡。这些发现不仅会进步 ASM生物学的基础科学，但也将DGK识别为一种潜在的治疗靶点可以探索用于开发一种针对AHR和气道重塑的新型哮喘治疗。