Critical Signaling Events in the Mechanoactivation of Human Mast Cells through p.C492Y-ADGRE2

Abstract

A role for the adhesion G-protein coupled receptor ADGRE2 or EMR2 in mechanosensing was revealed by the finding of a missense substitution (p.C492Y) associated with familial vibratory urticaria. In these patients, friction of the skin induces mast cell hyper-degranulation through p.C492Y-ADGRE2, causing localized hives, flushing, and hypotension. We have now characterized the responses and intracellular signals elicited by mechanical activation in human mast cells expressing p.C492Y-ADGRE2 and attached to dermatan sulfate, a ligand for ADGRE2. The presence of p.C492Y-ADGRE2 reduced the threshold to activation and increased the extent of degranulation along with the percentage of mast cells responding. Vibration caused phospholipase C activation, transient increases in cytosolic calcium, and downstream activation of phosphoinositide 3-kinase and extracellular signal-regulated kinases 1 and 2 by Gβγ, Gα_q/11, and Gα_i/o-independent mechanisms. Degranulation induced by vibration was dependent on phospholipase C pathways, including calcium, protein kinase C, and phosphoinositide 3-kinase but not extracellular signal-regulated kinases 1/2 pathways, along with pertussis toxin-sensitive signals. In addition, mechanoactivation of mast cells stimulated the synthesis and release of prostaglandin D₂, to our knowledge a previously unreported mediator in vibratory urticaria, and extracellular signal-regulated kinases 1/2 activation was required for this response together with calcium, protein kinase C, and to some extent, phosphoinositide 3-kinase. Our studies thus identified critical molecular events initiated by mechanical forces and potential therapeutic targets for patients with vibratory urticaria.

Figure 1.. Characterization of vibration-induced degranulation.

(a) Depiction of ADGRE2. The NTF and CTF, translated as a single polypeptide and self-cleaved in the GAIN domain in the ER, remain non-covalently linked (Kwakkenbos et al., 2002). Arrows indicate the location of p.C492Y and binding sites for DS and the 2A1 antibody. (b) β-hexosaminidase release induced by vibration (20 min) in nonmutated (NM)- or p.C492Y-ADGRE2 cells. Data are mean±SD. Student t-tests were used for the point by point comparisons and a two-way ANOVA for comparison between the curves (side bracket). (c) Fold changes in degranulation (from data in b). (d) Images of CD63 surface expression 5 min after vibration (750 rpm). Scale bar=10 μm. (e) Anti-CD63-APC fluorescent intensity 10 min after vibration; (n≥77). (f) Anti-CD63-APC intensity after vibration in responsive cells; (n=10). (g) Percentage of responsive cells. Data are mean±SEM in e–g. *p<0.05; ** p<0.005; **** p<0.0001.

Figure 2.. Vibration causes transient calcium mobilization.

(a) Changes in intracellular calcium measured by Fura-2 in NM- or p.C492Y-ADGRE2 cells. Data are mean±SD of 2 experiments performed in triplicate. (b) Images of changes in intracellular calcium after vibration (Vb) using Fluo-8. Scale bar=10 μm (c, d) Average Fluo-8 fluorescence intensity of all cells 5 min after vibration (c) or in responding cells (with signals above baseline) overtime (d), with or without extracellular calcium, and in cells pretreated with inhibitors, as indicated. Data are mean±SEM (n≥19). (e) Percentage of responsive cells. Data are mean±SEM (n≥77 cells). (f–g) Pearson correlation between calcium changes (Fluo-8 intensity) and degranulation (anti-CD63-APC intensity) from n=4 experiments normalized to average intensities. A.U., arbitrary units. ****p<0.0001. Two-way ANOVA was used in a and d.

Figure 3.. Activation of PI3K and ERK1/2 signaling pathways and their potential crosstalk by mechanoactivation in mast cells with p.C492Y-ADGRE2.

DS-bound cells expressing nonmutated (NM)- or p.C492Y-ADGRE2 were lysed after vibration (+Vb) for 5 min at 750 rpm. (a, b) Phosphorylation of AKT (a), ERK1/2, SAPK/JNK and p38 (b). (c, d) Effects of inhibition of PKC, PI3K, and MEK1/2 on AKT (c) and ERK1/2 (d) phosphorylation. Inhibitors were added 20 min before vibration. The histograms in a–d show the quantification of band intensities (normalized by total AKT/ERK1/2 protein or β-actin) from n≥3 experiments and expressed as fold change compared to non-vibrated, NM-ADGRE2- expressing cells. Data are mean±SEM. * p<0.05; *** p≤0.0001.

Figure 4.. PLC activation and calcium mobilization are critical for PI3K and ERK1/2 activation independently of βγ, α_q/11/14 and α_i/o G-protein subunits.

Effects of inhibitors for PLC, IP3 and SOCE channels (a and b), or inhibitors of G-protein subunits βγ, α_i/o and α_q/11 (c and d), as indicated, on vibration-induced AKT (a, c) and ERK1/2 phosphorylation (b, d). DS-attached LAD2 cells expressing nonmutated (NM)-ADGRE2 or p.C492Y-ADGRE2 were treated with inhibitors for 20 min and vibrated 5 min at 750 rpm. Histograms represent band intensities (normalized by total AKT/ERK1/2 protein or β-actin) from n≥3 experiments and calculated as fold change compared to non-vibrated, NM-ADGRE2 expressing cells. Data are mean±SEM. * p<0.05; ** p<0.01; *** p<0.001.

Figure 5.. PLC/calcium and PKC axis and PTX-sensitive pathways are critical for vibration-induced degranulation, while MEK/ERK1/2 is dispensable.

LAD2 cells expressing NM-ADGRE2 or p.C492Y-ADGRE2 plated on DS-coated dishes were treated with the indicated inhibitors for 20 min before vibration. (a) Representative confocal images of anti-CD63-APC on the membrane after vibration for 5 min. Scale bar=20 μm. (b) Quantification of degranulation from data shown in a. Fluorescent intensity of anti-CD63-APC in individual cells (n> 264 cells) was determined, and expressed as % decrement (Δ) of the maximal response (i.e. fluorescent intensity of cells expressing p.C492Y-ADGRE2), being the minimal response fluorescent intensity of anti-CD63-APC in cells with NM-ADGRE2. (c) β-hexosaminidase release after 20 min vibration. Data are mean±SEM, n≥12 *p<0.05; **p<0.005; ***p<0.001; ****p<0.0001.

Figure 6.. Vibration-induced PGD₂ release is dependent on PLC/calcium, PKC and MEK/ERK1/2; and vortex challenge in patients with VU increases serum PGD₂.

(a, b) PGD₂ release induced by vibration in mast cells expressing NM or p.C492Y-ADGRE2 in the presence or absence of the indicated inhibitors. Supernatants of vibrated (750 rpm) and non-vibrated samples after 10 min. Data are mean±SEM; n≥9. *p<0.05; **p<0.005;****p<0.0001. (c) PGD₂ in the serum of 2 patients with VU and 2 control subjects after a vibratory challenge. (d) Representation of the proposed signaling pathways involved in mechanical activation of mast cells. Doted lines are potential ways of crosstalk based on our data or the literature (Cullen and Lockyer, 2002, Danciu et al., 2003, Mendoza et al., 2011, Yano et al., 1998).