Division of Pulmonary, Allergy, and Critical Care Medicine, Center for Translational Medicine, Jane and Leonard Korman Respiratory Institute, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania.
Rutgers Institute for Translational Medicine and Science, Rutgers, The State University of New Jersey, New Brunswick, New Jersey; and.
Am J Respir Cell Mol Biol. 2021 Dec;65(6):658-671. doi: 10.1165/rcmb.2021-0106OC.
Exaggerated airway smooth muscle (ASM) contraction regulated by the Gq family of G protein-coupled receptors causes airway hyperresponsiveness in asthma. Activation of Gq-coupled G protein-coupled receptors leads to phospholipase C (PLC)-mediated generation of inositol triphosphate (IP) and diacylglycerol (DAG). DAG signaling is terminated by the action of DAG kinase (DGK) that converts DAG into phosphatidic acid (PA). Our previous study demonstrated that DGKζ and α isoform knockout mice are protected from the development of allergen-induced airway hyperresponsiveness. Here we aimed to determine the mechanism by which DGK regulates ASM contraction. Activity of DGK isoforms was inhibited in human ASM cells by siRNA-mediated knockdown of DGKα and ζ, whereas pharmacological inhibition was achieved by pan DGK inhibitor I (R59022). Effects of DGK inhibition on contractile agonist-induced activation of PLC and myosin light chain (MLC) kinase, elevation of IP and calcium levels were assessed. Furthermore, we used precision-cut human lung slices and assessed the role of DGK in agonist-induced bronchoconstriction. DGK inhibitor I attenuated histamine- and methacholine-induced bronchoconstriction. DGKα and ζ knockdown or pretreatment with DGK inhibitor I resulted in attenuated agonist-induced phosphorylation of MLC and MLC phosphatase in ASM cells. Furthermore, DGK inhibition decreased Gq agonist-induced calcium elevation and generation of IP and increased histamine-induced production of PA. Finally, DGK inhibition or treatment with DAG analog resulted in attenuation of activation of PLC in human ASM cells. Our findings suggest that DGK inhibition perturbed the DAG:PA ratio, resulting in inhibition of Gq-PLC activation in a negative feedback manner, resulting in protection against ASM contraction.
Gq 家族 G 蛋白偶联受体调节的气道平滑肌（ASM）过度收缩导致哮喘中的气道高反应性。Gq 偶联 G 蛋白偶联受体的激活导致磷脂酶 C（PLC）介导的三磷酸肌醇（IP）和二酰基甘油（DAG）的产生。DAG 信号通过 DAG 激酶（DGK）的作用终止，该酶将 DAG 转化为磷酸脂酸（PA）。我们之前的研究表明，DGKζ 和α同工型敲除小鼠免受变应原诱导的气道高反应性的发展。在这里，我们旨在确定 DGK 调节 ASM 收缩的机制。通过 siRNA 介导的 DGKα 和 ζ 的敲低抑制人 ASM 细胞中 DGK 同工型的活性，而通过泛 DGK 抑制剂 I（R59022）实现药理学抑制。评估 DGK 抑制对收缩激动剂诱导的 PLC 和肌球蛋白轻链（MLC）激酶激活、IP 和钙水平升高的影响。此外，我们使用精密切割的人肺切片评估 DGK 在激动剂诱导的支气管收缩中的作用。DGK 抑制剂 I 减弱了组胺和乙酰甲胆碱诱导的支气管收缩。DGKα 和 ζ 敲低或用 DGK 抑制剂 I 预处理导致激动剂诱导的 ASM 细胞中 MLC 和 MLC 磷酸酶磷酸化减弱。此外，DGK 抑制降低了 Gq 激动剂诱导的钙升高、IP 的产生和组胺诱导的 PA 产生。最后，DGK 抑制或 DAG 类似物处理导致人 ASM 细胞中 PLC 的激活减弱。我们的研究结果表明，DGK 抑制扰乱了 DAG：PA 比率，以负反馈方式抑制 Gq-PLC 激活，从而防止 ASM 收缩。
