Dept. of Physiology, University of Tübingen, Gmelinstr. 5, D-72072 Tübingen, Germany.
Am J Physiol Cell Physiol. 2013 Jan 1;304(1):C49-55. doi: 10.1152/ajpcell.00179.2012. Epub 2012 Sep 26.
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 Ca(2+). Entry of Ca(2+) through the Ca(2+) release-activated Ca(2+) (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 Ca(2+) entry in mast cells as a novel mechanism regulating actin cytoskeleton.
高亲和力 IgE 受体 (FcεRI) 在肥大细胞 (MC) 上的聚集导致 MC 脱颗粒，这一过程涉及皮质 F-肌动蛋白解聚。肌动蛋白解聚可能是由细胞质 Ca(2+)增加触发的。通过 Ca(2+)释放激活的 Ca(2+) (CRAC) 通道进入 Ca(2+)受到血清和糖皮质激素诱导激酶 SGK1 的有力调节。此外，SGK1 缺陷 (sgk1(-/-)) MC 中 FcεRI 依赖性脱颗粒减少。本研究旨在探讨 SGK1 是否是 MC 中肌动蛋白细胞骨架重排所必需的，以及肌动蛋白结构的调节是否是 sgk1(-/-) MC 脱颗粒减少的基础。证实了先前的结果，反映 FcεRI 依赖性脱颗粒的 β-己糖胺酶释放在 sgk1(-/-) MC 中比 sgk1(+/+) MC 受损。当 CRAC 通道被 2-氨基乙氧基二苯硼酸盐 (2-APB; 50 μM) 抑制时，sgk1(+/+) 和 sgk1(-/-) MC 中的 MC 脱颗粒均明显减少，并且基因型之间的差异被消除。此外，肌动蛋白稳定剂 (鬼笔环肽) 和肌动蛋白破坏剂 (细胞松弛素 B) 对 sgk1(+/+) MC 和 sgk1(-/-) MC 的脱颗粒作用程度相似，这表明肌动蛋白重排在此事件中具有调节作用。与此一致，通过流式细胞术分析和去污剂可溶性和不溶性细胞部分的 Western blot 测定，单体 (G) 和丝状 (F) 肌动蛋白含量的测量表明，sgk1(+/+) MC 中 FcεRI 交联后 G/F-肌动蛋白比值增加，但 sgk1(-/-) MC 中没有增加，这一观察结果反映了肌动蛋白解聚。在 sgk1(+/+) MC 中，2-APB 消除了 FcεRI 诱导的肌动蛋白解聚。通过共聚焦激光显微镜分析证实了观察到的肌动蛋白重排。我们的观察结果揭示了 SGK1 依赖性 Ca(2+) 进入肥大细胞作为调节肌动蛋白细胞骨架的新机制。
