Serum- and glucocorticoid-inducible kinase SGK1 regulates reorganization of actin cytoskeleton in mast cells upon degranulation

Abstract

Aggregation of the high-affinity IgE receptor (FcεRI) on mast cells (MCs) causes MC degranulation, a process that involves cortical F-actin disassembly. Actin depolymerization may be triggered by increase of cytosolic Ca2+. Entry of Ca2+ through the Ca2+ release-activated Ca2+ (CRAC) channels is under powerful regulation by the serum- and glucocorticoid-inducible kinase SGK1. Moreover, FcεRI-dependent degranulation is decreased in SGK1-deficient ( sgk1 −/−) MCs. The present study addressed whether SGK1 is required for actin cytoskeleton rearrangement in MCs and whether modulation of actin architecture could underlie decreased degranulation of sgk1 −/− MCs. Confirming previous results, release of β-hexosaminidase reflecting FcεRI-dependent degranulation was impaired in sgk1 −/− MCs compared with sgk1 +/+ MCs. When CRAC channels were inhibited by 2-aminoethoxydiphenyl borate (2-APB; 50 μM), MC degranulation was strongly decreased in both sgk1 +/+ and sgk1 −/− MCs and the difference between genotypes was abolished. Moreover, degranulation was impaired by actin-stabilizing (phallacidin) and enhanced by actin-disrupting (cytochalasin B) agents to a similar extent in sgk1 +/+ MCs and sgk1 −/− MCs, implying a regulatory role of actin reorganization in this event. In line with this, measurements of monomeric (G) and filamentous (F) actin content by FACS analysis and Western blotting of detergent-soluble and -insoluble cell fractions indicated an increase of the G/F-actin ratio in sgk1 +/+ MCs but not in sgk1 −/− MCs upon FcεRI ligation, an observation reflecting actin depolymerization. In sgk1 +/+ MCs, FcεRI-induced actin depolymerization was abolished by 2-APB. The observed actin reorganization was confirmed by confocal laser microscopic analysis. Our observations uncover SGK1-dependent Ca2+ entry in mast cells as a novel mechanism regulating actin cytoskeleton.