Constitutive, Basal, and β-Alanine-Mediated Activation of the Human Mas-Related G Protein-Coupled Receptor D Induces Release of the Inflammatory Cytokine IL-6 and Is Dependent on NF-κB Signaling

Abstract

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.

MRGPRX2-Mediated Degranulation of Human Skin Mast Cells Requires the Operation of Gαi, Gαq, Ca++ Channels, ERK1/2 and PI3K—Interconnection between Early and Late Signaling

“…The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11060953/s1, Figure S1: The inhibitors employed in main Figure 1 exert no cytotoxicity on cultured skin MC; Figure S2: MC degranulation via MRGPRX2 relies on Gαi, Gαq and calcium mobilization as evidenced by histamine release; Figure S3: The survival of ex vivo skin MC is not perceptibly compromised by the inhibitors employed in main Figure 1; Figure S4: Differences between c48/80 and SP from main Figure 1; Figure S5: Comparison between cultured and ex vivo skin MCs from main Figure 1; Figure S6: The inhibitors employed in main Figure 2 exert no cytotoxicity on cultured skin MC; Figure S7: The survival of ex vivo skin MCs is not perceptibly compromised by the inhibitors employed in main Figure 2; Figure S8: Differences between c48/80 and SP from main Figure 2; S1: Inhibitors employed and their primary targets. References [15,30,31,33,36,38,39,44,49,51,55,[60][61][62][86][87][88][89][90][91][92][93][94][95][96] are cited in the Supplementary materials.…”

Role of signal transduction pathways in IL‐1β‐induced apoptosis: Pathological and therapeutic aspects

“… 33 IL‐1β is one of these pro‐inflammatory factors to activate NF‐κB. The activation of NF‐κB upregulates the release of IL‐1β, IL‐6, 42 and tumor necrosis factor‐α (TNF‐α), 43 which fuels apoptosis 44 , 45 and inflammation. 46 , 47 ROS activated by IL‐1β is another pro‐inflammatory factors.…”

β-Aminoisobutyric acid (L-BAIBA) is a novel regulator of mitochondrial biogenesis and respiratory function in human podocytes

“…Our results showed that human podocytes express MRGPRD, the expression of which increased upon extracellular L-BAIBA supplementation. MRGPRD belongs to Mas-related G protein-coupled receptors and has been mostly associated with neuronal excitability and the sensation of pain and itch 20 . MRGPRD was first discovered in sensory neurons of the mouse dorsal root ganglion.…”

“…Its expression was subsequently found in other organs and tissues, including arteries, testes, the urinary bladder, adipose tissue, and the gastrointestinal tract 21 . Although β-alanine is the best known MRGPRD ligand, it can also be stimulated by other molecules, such as alamandine 22 , angiotensin II 20 , and L-BAIBA 15 . The downstream effects of MRGPRD activation are multidirectional.…”