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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：
10090626
负责人：
Deepak A Deshpande
金额：
$ 49.8万
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/10090626
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.

gq偶联G蛋白偶联受体（GPCR）在气道平滑肌（ASM）细胞中的作用至关重要