Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, 2610 Wilrijk, Belgium.
Laboratory for Molecular, Cellular and Network Excitability, Department of Biomedical Sciences, University of Antwerp, 2610 Wilrijk, Belgium.
Int J Mol Sci. 2021 Dec 9;22(24):13254. doi: 10.3390/ijms222413254.
G protein-coupled receptors (GPCRs) have emerged as key players in regulating (patho)physiological processes, including inflammation. Members of the Mas-related G protein coupled receptors (MRGPRs), a subfamily of GPCRs, are largely expressed by sensory neurons and known to modulate itch and pain. Several members of MRGPRs are also expressed in mast cells, macrophages, and in cardiovascular tissue, linking them to pseudo-allergic drug reactions and suggesting a pivotal role in the cardiovascular system. However, involvement of the human Mas-related G-protein coupled receptor D (MRGPRD) in the regulation of the inflammatory mediator interleukin 6 (IL-6) has not been demonstrated to date. By stimulating human MRGPRD-expressing HeLa cells with the agonist β-alanine, we observed a release of IL-6. β-alanine-induced signaling through MRGPRD was investigated further by probing downstream signaling effectors along the Gαq/Phospholipase C (PLC) pathway, which results in an IkB kinases (IKK)-mediated canonical activation of nuclear factor kappa-B (NF-κB) and stimulation of IL-6 release. This IL-6 release could be blocked by a Gαq inhibitor (YM-254890), an IKK complex inhibitor (IKK-16), and partly by a PLC inhibitor (U-73122). Additionally, we investigated the constitutive (ligand-independent) and basal activity of MRGPRD and concluded that the observed basal activity of MRGPRD is dependent on the presence of fetal bovine serum (FBS) in the culture medium. Consequently, the dynamic range for IL-6 detection as an assay for β-alanine-mediated activation of MRGPRD is substantially increased by culturing the cells in FBS free medium before treatment. Overall, the observation that MRGPRD mediates the release of IL-6 in an in vitro system, hints at a role as an inflammatory mediator and supports the notion that IL-6 can be used as a marker for MRGPRD activation in an in vitro drug screening assay.
G 蛋白偶联受体（GPCRs）已成为调节（病理）生理过程的关键因素，包括炎症。Mas 相关 G 蛋白偶联受体（MRGPRs）的成员是 GPCRs 的一个亚家族，主要由感觉神经元表达，已知可调节瘙痒和疼痛。MRGPRs 的几个成员也在肥大细胞、巨噬细胞和心血管组织中表达，将它们与假性过敏药物反应联系起来，并表明它们在心血管系统中具有关键作用。然而，到目前为止，人类 Mas 相关 G 蛋白偶联受体 D（MRGPRD）在调节炎症介质白细胞介素 6（IL-6）中的作用尚未得到证实。通过用激动剂β-丙氨酸刺激表达人 MRGPRD 的 HeLa 细胞，我们观察到 IL-6 的释放。通过探测沿着 Gαq/磷脂酶 C（PLC）途径的下游信号效应物，进一步研究了β-丙氨酸通过 MRGPRD 诱导的信号，这导致 IkB 激酶（IKK）介导的核因子 kappa-B（NF-κB）的经典激活和 IL-6 释放的刺激。这种 IL-6 释放可以被 Gαq 抑制剂（YM-254890）、IKK 复合物抑制剂（IKK-16）和部分 PLC 抑制剂（U-73122）阻断。此外，我们研究了 MRGPRD 的组成型（配体非依赖性）和基础活性，并得出结论，观察到的 MRGPRD 的基础活性依赖于培养基中胎牛血清（FBS）的存在。因此，通过在治疗前在无 FBS 的培养基中培养细胞，可大大增加 IL-6 检测作为测定β-丙氨酸介导的 MRGPRD 激活的动态范围。总的来说，MRGPRD 在体外系统中介导 IL-6 的释放这一观察结果表明其作为炎症介质的作用，并支持这样一种观点，即 IL-6 可作为体外药物筛选测定中 MRGPRD 激活的标志物。
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