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

Abstract

The recent discovery of MRGPRX2 explains mast cell (MC)-dependent symptoms independently of FcεRI-activation. Because of its novelty, signaling cascades triggered by MRGPRX2 are rudimentarily understood, especially in cutaneous MCs, by which MRGPRX2 is chiefly expressed. Here, MCs purified from human skin were used following preculture or ex vivo and stimulated by FcεRI-aggregation or MRGPRX2 agonists (compound 48/80, Substance P) in the presence/absence of inhibitors. Degranulation was assessed by β-hexosaminidase or histamine release. Phosphorylation events were studied by immunoblotting. As a G protein-coupled receptor, MRGPRX2 signals by activating G proteins; however, their nature has remained controversial. In skin MCs, G_αi and G_αq were required for degranulation, but G_αi was clearly more relevant. Ca++ channels were likewise crucial. Downstream, PI3K was essential for granule discharge initiated by MRGPRX2 or FcεRI. ERK1/2 and JNK were additional participants, especially in the allergic route. Addressing possible points of intersection between early and later events, pERK1/2 and pAKT were found to depend on G_αi, further highlighting its significance. G_αq and Ca++ channels made some contributions to the phosphorylation of ERK. Ca++ differentially affected PI3K activation in FcεRI- vis-à-vis MRGPRX2-signaling, as channel inhibition increased pAKT only when triggered via FcεRI. Collectively, our study significantly extends our understanding of the molecular framework behind granule secretion from skin MCs.

Keywords: ERK1/2; FcεRI; G proteins; MAP kinases; MRGPRX2; PI3K/AKT; degranulation; mast cells; signal transduction; skin.

Figure 1

MC degranulation via MRGPRX2 relies on G_αi, G_αq and calcium mobilization. (A–C) Cultured or (D–F) ex vivo skin-derived MCs were pretreated with 200 ng/mL PTX, 10 µM YM-254890, 100 µM 2-APB or 1 µM La3+. Then cells were triggered by 10 µg/mL c48/80, 30 µM SP or IgER-CL (cross-linking) (0.1 µg/mL anti-FcεRI-antibody AER-37), β-hexosaminidase release was determined as described in methods. Data are from 5–11 independent experiments, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, skMCs: skin mast cells.

Figure 2

Both ERK and PI3K contribute to the degranulation of skin MCs stimulated by MRGPRX2 or FcεRI. (A–C) Cultured and (D–F) ex vivo skin MCs were pretreated with inhibitors targeting ERK1 and ERK2 indiscriminately (SCH772984, 10 µM), ERK2 only (Vx-11e, 2 µM), JNK (SP600125, 5 µM), or PI3K (Pictilisib, 5 µM). β-hexosaminidase release stimulated by c48/80 (10 µg/mL), SP (30 µM) or IgER-cross-linking (CL) (by AER-37 antibody for cultured MCs at 0.1 µg/mL; by anti-FcεRIα-Ab 29C6 for ex vivo MCs at 0.5 µg/mL) was determined. The data are from 7–11 independent experiments, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. skMCs: skin mast cells.

Figure 3

C48/80 activated pERK depends on G_αi, G_αq, and Ca++, while pAKT exclusively requires the action of G_αi only. Cultured skin-derived MCs were pretreated with PTX (200 ng/mL), YM-254890 (10 µM), 2-APB (100 µM), or no inhibitor (w/o Inh) then stimulated with c48/80 (10 µg/mL) for 1 min. Cells receiving no inhibitor or stimulus were the negative control (w/o stim). (A–C) Phosphorylation signals detected consecutively on the same membranes for ERK1/2, p38 and AKT, quantified and normalized to cyclophilin B as described in methods. Mean ± SEM of 10–15 independent experiments (individual cultures). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (D–F) Representative blots of A–C.

Figure 4

Interconnections between early and later signals induced by SP are comparable to those elicited by c48/80. MCs were pretreated with inhibitors, as shown in Figure 3, then stimulated with SP (30 µM) for 1 min. (A–C) Signals of pERK1/2, pp38 and pAKT were quantified and normalized to cyclophilin B as described in methods. (D–F) Representative blots of A–C. Data shown are Mean ± SEM of 11–13 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

Ca++ channel inhibition boosts AKT activation downstream of IgER-CL. MCs were pretreated with inhibitors, as described in Figure 3, then stimulated for 30 min with AER-37 (0.1 µg/mL) for FcεRI-aggregation. (A–C) Signals of pERK1/2, pp38 and pAKT were quantified and normalized to cyclophilin B as described in methods. (D–F) Representative blots of A–C. n = 10–13, Mean ± SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001.