Diacylglycerol kinase in airway smooth muscle functions

GQ偶联G蛋白偶联受体(GPCR)在气道平滑肌细胞(ASM)上的作用 哮喘引起的气道高反应性(AHR)和气道重塑的调节。GQ ASM中的信号涉及磷脂酶C的激活，该酶C转化为4，5-二磷酸(PIP2)。 二酰甘油(DAG)和1，4，5-三磷酸肌醇(IP3)。而IP3导致[Ca~(2+)]i升高， MLC20的磷酸化和ASM收缩，DAG直接激活PKC家族成员和RAS鸟苷 核苷酸释放蛋白。DAG在ASM中的信号和功能作用在很大程度上是未知的。达格是 已知被磷酸化，并被称为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，可以激活多种信号蛋白 因此，细胞内DAG和PA的水平受到严格的调控。建议进行研究的原动力是 我们的初步观察表明，缺乏亚型DGK的小鼠对卵蛋白诱导的AHR具有保护作用。 尽管有完全性的呼吸道炎症。在这项建议中，我们寻求建立一种机制，通过这种机制 DGK调节ASM的收缩和增殖，其核心假设是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)来阻断哮喘的气道反应 ASM细胞的功能。为了实现我们的研究目标，我们将使用各种最先进的工具，如 靶向脂质组学、PIP2/DAG的荧光传感器和DGK的遗传/药物抑制。我们 认为DGK的急性抑制会导致DAG的积累，而DAG起着负反馈调节的作用 并抑制ASM细胞的GQ-PLC信号转导。在目标1研究中，我们将发现DGK通过多种机制 异构体调节GQ偶联的GPCR介导的ASM收缩。额外的初步数据表明，PA是 ASM中的促有丝分裂信号分子和DGK的抑制导致ASM生长的抑制。因此， 目的2研究将建立DGK亚型调节ASM细胞的分子和细胞机制 扩散。最后，为了确定DGK抑制在体内的相关性，Aim 3研究将使用一所房子 尘螨(HDM)诱导的小鼠哮喘模型及DGK抑制变应原诱导的作用 AHR和ASM重塑。DGK抑制将通过使用平滑肌特异性条件性 DGK的缺失或用DGK的药理抑制剂治疗动物(R59022)。我们的成功在于 得益于一种独特的细胞类型特定的DGK亚型基因敲除小鼠的可用性，我们团队的能力 创造性地应用尖端成像方法，并使用多种互补方法来 辨别ASM细胞中脂质信号分子的复杂(Im)平衡。这些发现不仅会推动 ASM生物学的基础科学，但也确定DGK作为潜在的治疗靶点，其操作 可用于开发一种新的哮喘治疗方法，同时解决AHR和气道重塑问题。

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.