Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados, Sede Sur, Calzada de los Tenorios 235, Col Granjas Coapa, CP 14330 Mexico City, Mexico.
Biochem Biophys Res Commun. 2010 Jan 22;391(4):1714-20. doi: 10.1016/j.bbrc.2009.12.139. Epub 2009 Dec 30.
IgE-antigen-dependent crosslinking of the high affinity IgE receptor (FcepsilonRI) on mast cells leads to degranulation, leukotriene synthesis and cytokine production. Calcium (Ca(2+)) mobilization is a sine qua non requisite for degranulation, allowing the rapid secretion of stored pro-inflammatory mediators responsible for allergy symptoms. Fyn is a Src-family kinase that positively controls FcepsilonRI-induced mast cell degranulation. However, our understanding of the mechanism connecting Fyn activation to secretion of pre-synthesized mediators is very limited. We analyzed FcepsilonRI-dependent Ca(2+) mobilization in bone marrow-derived mast cells (BMMCs) differentiated from WT and Fyn -/- knock out mice. Fyn -/- BMMCs showed a marked defect in extracellular Ca(2+) influx after FcepsilonRI crosslinking but not after thapsigargin addition. High concentrations of Gadolinium (Gd(3+)) partially blocked FcepsilonRI-induced Ca(2+) influx in WT cells but, in contrast, completely inhibited Ca(2+) mobilization in Fyn -/- cells. Low concentrations of an inhibitor of the canonical transient receptor potential (TRPC) Ca(2+) channels (2-aminoethoxyphenyl-borane, 2-APB) blocked FcepsilonRI-induced maximal Ca(2+) rise in WT but not in Fyn -/- cells. Ca(2+) entry through Fyn-controlled, 2-APB sensitive channels was found to be important for full degranulation and IL-2 mRNA accumulation in WT cells. Immunoprecipitation assays showed that Fyn kinase interacts with TRPC 3/6/7 channels after IgE-antigen stimulation, but its association is not related to protein tyrosine phosphorylation. Results indicate Fyn kinase mediates the receptor-dependent activation of TRPC channels that contribute to degranulation in FcepsilonRI-stimulated mast cells.
IgE 抗原依赖性交联高亲和力 IgE 受体 (FcepsilonRI) 在肥大细胞上导致脱颗粒、白三烯合成和细胞因子产生。钙 (Ca(2+)) 动员是脱颗粒的必要条件，允许快速分泌储存的促炎介质，导致过敏症状。Fyn 是一种Src 家族激酶，可正向控制 FcepsilonRI 诱导的肥大细胞脱颗粒。然而，我们对连接 Fyn 激活与预先合成介质分泌的机制的理解非常有限。我们分析了从 WT 和 Fyn -/- 敲除小鼠分化而来的骨髓来源的肥大细胞 (BMMC) 中 FcepsilonRI 依赖性 Ca(2+) 动员。Fyn -/- BMMC 在 FcepsilonRI 交联后表现出明显的细胞外 Ca(2+) 内流缺陷，但在添加 thapsigargin 后则没有。高浓度的钆 (Gd(3+)) 部分阻断了 WT 细胞中 FcepsilonRI 诱导的 Ca(2+) 内流，但相反，完全抑制了 Fyn -/- 细胞中的 Ca(2+) 动员。经典瞬时受体电位 (TRPC) Ca(2+) 通道的低浓度抑制剂 (2-氨基乙氧基苯硼酸，2-APB) 阻断了 WT 细胞中 FcepsilonRI 诱导的最大 Ca(2+) 上升，但在 Fyn -/- 细胞中则没有。发现 Fyn 控制的、2-APB 敏感的通道中的 Ca(2+) 内流对于 WT 细胞的完全脱颗粒和 IL-2 mRNA 积累非常重要。免疫沉淀测定表明，Fyn 激酶在 IgE 抗原刺激后与 TRPC3/6/7 通道相互作用，但它的关联与蛋白酪氨酸磷酸化无关。结果表明，Fyn 激酶介导了受体依赖性的 TRPC 通道的激活，该激活有助于 FcepsilonRI 刺激的肥大细胞中的脱颗粒。
