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. 2021 Dec 9;22(24):13254.
doi: 10.3390/ijms222413254.

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

Rohit Arora  1   2 , Kenny M Van Theemsche  2 , Samuel Van Remoortel  1 , Dirk J Snyders  2 , Alain J Labro  2   3 , Jean-Pierre Timmermans  1
Affiliations

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

Rohit Arora et al. Int J Mol Sci. .

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.

Keywords: GPCR; Gq inhibitor; MRGPRD; NF-kB; constitutive receptor; interleukin 6; β-alanine.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in (a) the design of the study, (b) in the collection, analyses, or interpretation of data, (c) in the writing of the manuscript, or (d) in the decision to publish the results.

Figures

Figure 1
Figure 1
Activation of MRGPRD mediates IL-6 release. HeLa cells transiently expressing empty vector (pCMV-GS) or MRGPRD-HA were stimulated with either vehicle control or β-alanine (100 µM; final concentration/well). The normalized IL-6 release is represented in ng/mg. β-alanine- or vehicle-treated cells expressing MRGPRD released high and intermittent levels of IL-6, respectively. Whereas vehicle- or β-alanine-treated cells expressing the empty vector did not release significant amounts of IL-6. On the right, a representative Western blot of whole cell lysates from HeLa cells transiently transfected with either empty vector (pCMV-GS) or MRGPRD-HA plasmids is displayed. Expression of the MRGPRD-HA receptor (~37 kDa) was observed in both vehicle and β-alanine (100 µM)-treated MRGPRD-transfected cells, whereas cells transfected with the empty vector showed no expression (i.e., absence of a protein band of ~37 kDa). The graph represents the mean ± s.e.m values from three independent experiments. Statistical significance was determined using one-way analysis of variance (ANOVA), and Sidak’s post hoc test was applied for multiple comparisons. * p ≤ 0.05 was considered significant and ‘ns’ is non-significant.
Figure 2
Figure 2
Basal activity of MRGPRD. HeLa cells expressing increasing concentrations of MRGPRD-HA and PAR2-HA receptors (transfected with 0.03, 0.1, 0.3, 1, and 2 µg of cDNAs per well). When treated with vehicle control, the MRGPRD-transfected cells did show an MRGPRD expression-dependent release of IL-6 (A), while the PAR2-transfected cells did not show PAR2 expression-dependent release of IL-6 (B). Furthermore, when cells expressing increasing concentrations of MRGPRD-HA or PAR2-HA were treated with their respective agonists, β-alanine (100 µM) or the PAR2-agonist SLIGKV-NH2 (100 µM), a receptor concentration-dependent gradual IL-6 release was noticed (A,B). The release of IL-6 from vehicle-treated MRGPRD-HA cells points to basal activity of the receptor. Below the graphs, representative Western blot analysis of whole cell lysates from stimulated and non-stimulated HeLa cells transiently expressing increasing concentrations of MRGPRD-HA and PAR2-HA are shown. Each graph represents the mean ± s.e.m values from three independent experiments. The comparison between the two groups was analyzed by one-way ANOVA with Sidak’s post hoc test. **** p ≤ 0.0001 was considered significant and ‘ns’ is non-significant.
Figure 3
Figure 3
IL-6 assay optimization. To achieve a high dynamic window for the IL-6 detection, the experiment timeline was optimized. (A) At 24 h post-transfection, medium was replaced by FBS-free medium, and cells were treated with vehicle or β-alanine (100 µM). At 24 h post-treatment, i.e., at 48 h post-transfection, samples were collected for IL-6 detection. IL-6 levels from β-alanine-treated MRGPRD-expressing cells were 2-fold higher when compared to those from vehicle-treated MRGPRD-expressing cells. (B) Similarly, at 24 h post-transfection, medium was replaced by FBS-free medium for another 24 h, and at 48 h, the medium was once again replaced by FBS-free medium, and cells were treated with vehicle or β-alanine (100 µM). Samples were collected for IL-6 detection after 24 h of treatment, i.e., at 72 h post-transfection. IL-6 levels from β-alanine-treated MRGPRD-expressing cells were ~5-fold higher when compared to those from only vehicle-treated MRGPRD-expressing cells, which is substantially higher than for the protocol used in (A). The cells expressing empty vector (pCMV-GS), treated with β-alanine or vehicle, did not show IL-6 release above the basal level (A,B). Below the graphs are representative Western blots of cell lysates from HeLa cells transiently transfected with empty vector (pCMV-GS) or MRGPRD-HA plasmid, showing receptor expression in vehicle or β-alanine (100 µM)-treated MRGPRD-expressing cells subjected to the experiment timeline protocols illustrated in (A,B), respectively. Each graph represents the mean ± s.e.m values from three independent experiments. Statistical significance was determined using one-way ANOVA, and Sidak’s post hoc test was applied for multiple comparisons. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 were considered significant and ‘ns’ is non-significant.
Figure 4
Figure 4
Agonistic effect of FBS and constitutive activity of hMRGPRD. HeLa cells expressing MRGPRD-HA or empty vector (pCMV-GS) were challenged with medium containing 10%, 3 %, 1% FBS and FBS-free (0%) medium. Significantly increased IL-6 levels were observed with increasing concentration of FBS from MRGPRD-expressing cells as compared to control cells (empty vector). β-alanine (100 µM)-treated MRGPRD-expressing cells served as positive control for this experiment. β-alanine- or vehicle-treated cells expressing the empty vector (pCMV-GS) did not show IL-6 release above the basal level. On the right, representative western blot of whole cell lysates from HeLa cells transiently transfected with empty vector or MRGPRD-HA plasmids, treated with vehicle or β-alanine (100 µM) under FBS (10%, 3%, and 1%) or FBS-free (0%) conditions. The graph represents the mean ± s.e.m values from three independent experiments. The comparison between the two groups was analyzed by one-way ANOVA with Sidak’s post hoc test. * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001 were considered significant and ‘ns’ is non-significant.
Figure 5
Figure 5
Concentration–effect curves of β-alanine obtained in the presence or absence of FBS. HeLa cells expressing the empty vector (pCMV-GS; square symbol) and MRGPRD-HA (triangle symbol) were treated with increasing concentrations of β-alanine in the absence or presence of FBS. Cells expressing MRGPRD induced the release of IL-6 in a concentration-dependent manner when treated with β-alanine with FBS (A) or without FBS (B). A higher basal activity of MRGPRD was observed in cells treated under FBS conditions as compared to non-FBS conditions. No significant IL-6 release was observed from cells expressing the empty vector (pCMV-GS). The dynamic range obtained when cells were treated in FBS (A) conditions was 2.85, whereas it increased to 4.62 when cells were treated in FBS-free conditions (B). Each graph represents the mean ± s.e.m values from at least three independent experiments.
Figure 6
Figure 6
β-alanine-stimulated MRGPRD-induced IL-6 release is dependent on NF-kB activation. (A) HeLa cells stably expressing MRGPRD-NLuc were pre-treated with either vehicle control or the Gαq inhibitor (YM-254890; 10 µM), the pan-PLC inhibitor (U-73122; 10 µM) or the IKK inhibitor (IKK-16; 5 µM) for 1 h before being stimulated with β-alanine (1mM). β-alanine-stimulated MRGPRD-expressing HeLa cells induced NF-κB phosphorylation, whereas no induction was observed in cells pre-treated with either of the three applied inhibitors. (B) Similarly, HeLa cells stably expressing MRGPRD-NLuc were pre-treated with either vehicle control or Gαq (YM-254890; 10 µM) or pan-PLC (U-73122; 10 µM) or IKK (IKK-16; 5 µM) inhibitors for 1 h before being subjected to β-alanine (100 µM). MRGPRD-expressing cells upon stimulation with β-alanine showed a strongly reduced IL-6 release after being pretreated with either YM-254890 or IKK-16 and a less prominent but still significantly reduced IL-6 release compared to β-alanine stimulated MRGPRD expressing cells that were not pre-exposed with the inhibitor. Each graph represents the mean ± s.e.m values from three independent experiments. The comparison between the two groups was analyzed by one-way ANOVA with Sidak’s post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 were considered significant and ‘ns’ is non-significant.
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