IADR Abstract Archives
Orai Channels Mediate Antimicrobial Peptide and Neuropeptide-induced Mast Cell Degranulation
Objectives: Rosacea is a common chronic inflammatory skin disease, prevalent among female adults with fair skin. Activation of human mast cells (MC) by the antimicrobial peptide LL-37 and the neuropeptide Substance P (SP) via Mas-related G protein coupled receptor X2 (MRGPRX2) contributes to the pathogenesis of rosacea. MRGPRX2-dependent MC activation is characterized by an influx of extracellular calcium, which is essential for degranulation. One study showed that transient receptor potential cation channel subfamily V member 4 (TRPV4) is involved in MRGPRX2-mediated MC degranulation. However, microRNA data show that it is not expressed in MCs; while, Orai channels are present in MCs. TRPV4 and Orai channels were studied to determine their roles on LL-37 and SP-induced calcium mobilization and degranulation in human MCs.
Methods: Calcium mobilization
LAD2 cells (human MC line) were loaded with 1µM Fura-2, stimulated with 3µM LL-37 or SP, then 1mM CaCl2 was added at 400 sec. Hitachi F-2700 Fluorescence Spectrophotometer was used to determine Ca2+ influx with excitation at 340nm and emission at 380nm.
Degranulation assay
LAD2 cells were seeded into 96-well plates and were divided into three groups – (1) control, (2) treated with 1μM TRPV4 inhibitors (TRAP4 and GSK-213874), and (3) treated with 10μM Orai inhibitors (Synta-66 and GSK-7975A). Cells were exposed to LL-37 (3µM) or SP (1µM) for 30 min, and β-hexosaminidase release was determined by measuring absorbance at 405 nm. In some studies, cells were cultured overnight with LL-37 (3µM), washed, and used to determine the effects of TRPV4 and Orai inhibitors on MC degranulation.
Results: LL-37 and SP induced robust calcium mobilization and degranulation in LAD2 cells. TRPV4 blockers did not inhibit LL-37 and SP-induced calcium mobilization nor degranulation in control LAD2 cells and in cells cultured overnight with LL-37. In contrast, Orai channel blockers caused substantial inhibition of LL-37 and SP-induced calcium mobilization and abolished MC degranulation in response to these MRGPRX2 agonists.
Conclusions: These findings suggest that Orai channels, but not TRPV4, participate in MRGPRX2-mediated calcium influx and MC degranulation. Thus, targeting Orai channels through inhibitors could serve as a novel therapeutic approach for the treatment of rosacea.
Methods: Calcium mobilization
LAD2 cells (human MC line) were loaded with 1µM Fura-2, stimulated with 3µM LL-37 or SP, then 1mM CaCl2 was added at 400 sec. Hitachi F-2700 Fluorescence Spectrophotometer was used to determine Ca2+ influx with excitation at 340nm and emission at 380nm.
Degranulation assay
LAD2 cells were seeded into 96-well plates and were divided into three groups – (1) control, (2) treated with 1μM TRPV4 inhibitors (TRAP4 and GSK-213874), and (3) treated with 10μM Orai inhibitors (Synta-66 and GSK-7975A). Cells were exposed to LL-37 (3µM) or SP (1µM) for 30 min, and β-hexosaminidase release was determined by measuring absorbance at 405 nm. In some studies, cells were cultured overnight with LL-37 (3µM), washed, and used to determine the effects of TRPV4 and Orai inhibitors on MC degranulation.
Results: LL-37 and SP induced robust calcium mobilization and degranulation in LAD2 cells. TRPV4 blockers did not inhibit LL-37 and SP-induced calcium mobilization nor degranulation in control LAD2 cells and in cells cultured overnight with LL-37. In contrast, Orai channel blockers caused substantial inhibition of LL-37 and SP-induced calcium mobilization and abolished MC degranulation in response to these MRGPRX2 agonists.
Conclusions: These findings suggest that Orai channels, but not TRPV4, participate in MRGPRX2-mediated calcium influx and MC degranulation. Thus, targeting Orai channels through inhibitors could serve as a novel therapeutic approach for the treatment of rosacea.
