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

• 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
• 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

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）是至关重要的 哮喘时发生的气道高反应性（AHR）和气道重塑的调节剂。GQ ASM中的信号传导涉及磷脂酶C的活化，磷脂酶C将磷酸肌醇4，5-二磷酸（PIP 2） 转化为二酰基甘油（DAG）和肌醇1，4，5-三磷酸（IP 3）。虽然IP 3导致[Ca 2 +]i增加， DAG通过MLC 20磷酸化和ASM收缩，直接激活PKC家族成员和Ras鸟苷酸， 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是未知的。DAG是 已知被磷酸化并进一步被称为DAG的酶转化为磷脂酸（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，DAG和PA的细胞内水平受到严格调节。拟议研究的动力是 我们初步观察到，缺乏DGK亚型的小鼠， 尽管气道炎症得到充分补充。在这项建议中，我们寻求建立机制， DGK调节ASM的收缩和增殖，其中心假设是DGK在以下方面起关键作用： 调节ASM收缩（通过PIP 2-DAG/IP 3轴）和增殖（通过PIP 2-DAG/PA轴），以及抑制 DGK通过影响收缩性（AHR）和增殖性（重塑）来阻断哮喘气道反应 ASM细胞的功能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、PIP 2/DAG的荧光传感器和DGK的遗传/药理学抑制。我们 认为DGK的急性抑制导致DAG的积累，其作为负反馈调节剂起作用 并抑制ASM细胞中的Gq-PLC信号传导。在目标1研究中，我们将识别DGK的多种机制， 同种型调节Gq偶联的GPCR介导的ASM收缩。其他初步数据表明，PA ASM和DGK抑制中的促有丝分裂信号分子导致ASM生长减弱。因此，我们建议， 目的2研究DGK亚型调控ASM细胞的分子和细胞机制 增殖最后，为了确定DGK抑制的体内相关性，Aim 3研究将使用一个房子， 粉尘螨（HDM）诱导的小鼠哮喘模型，并测试DGK抑制变应原诱导的哮喘的效果。 AHR和ASM重塑。DGK抑制将通过使用平滑肌特异性条件刺激来实现。 缺失DGK β或通过用DGK的药理学抑制剂（R59022）治疗动物。我们的成功是 由于获得了独特的细胞类型特异性DGK同种型敲除小鼠，我们的团队能够 创造性地应用最先进的成像方法，并使用多种互补的方法， 识别ASM细胞中脂质信号分子的复杂（不）平衡。这些发现不仅会推动 ASM生物学的基础科学，而且还将DGK确定为潜在的治疗靶点，其操作 可以用于开发一种新的哮喘治疗，解决AHR和气道重塑。