Department of Medicine, University of Alberta, Edmonton, AB T6G 2H7, Canada.
Department of Cell Biology, University of Alberta, Edmonton, AB T6G 2H7, Canada.
Cells. 2023 Feb 7;12(4):537. doi: 10.3390/cells12040537.
When antigen-stimulated, mast cells release preformed inflammatory mediators stored in cytoplasmic granules. This occurs via a robust exocytosis mechanism termed degranulation. Our previous studies revealed that RhoA and Rac1 are activated during mast cell antigen stimulation and are required for mediator release. Here, we show that the RhoGEF, GEF-H1, acts as a signal transducer of antigen stimulation to activate RhoA and promote mast cell spreading via focal adhesion (FA) formation. Cell spreading, granule movement, and exocytosis were all reduced in antigen-stimulated mast cells when GEF-H1 was depleted by RNA interference. GEF-H1-depleted cells also showed a significant reduction in RhoA activation, resulting in reduced stress fiber formation without altering lamellipodia formation. Ectopic expression of a constitutively active RhoA mutant restored normal morphology in GEF-H1-depleted cells. FA formation during antigen stimulation required GEF-H1, suggesting it is a downstream target of the GEF-H1-RhoA signaling axis. GEF-H1 was activated by phosphorylation in conjunction with antigen stimulation. Syk kinase is linked to the FcεRI signaling pathway and the Syk inhibitor, GS-9973, blocked GEF-H1 activation and also suppressed cell spreading, granule movement, and exocytosis. We concluded that during FcεRI receptor stimulation, GEF-H1 transmits signals to RhoA activation and FA formation to facilitate the exocytosis mechanism.
当受到抗原刺激时，肥大细胞会释放储存在细胞质颗粒中的预先形成的炎症介质。这是通过一种称为脱粒的强大胞吐作用机制发生的。我们之前的研究表明，RhoA 和 Rac1 在肥大细胞抗原刺激期间被激活，并且是介质释放所必需的。在这里，我们表明 RhoGEF，GEF-H1，作为抗原刺激的信号转导物起作用，通过形成焦点粘附（FA）来激活 RhoA 并促进肥大细胞扩展。在用 RNA 干扰耗尽 GEF-H1 时，抗原刺激的肥大细胞中的细胞扩展、颗粒运动和胞吐作用均减少。GEF-H1 耗尽的细胞还显示出 RhoA 激活的显着减少，导致应力纤维形成减少，而不会改变片状伪足形成。组成性激活的 RhoA 突变体的异位表达恢复了 GEF-H1 耗尽细胞的正常形态。在抗原刺激过程中 FA 的形成需要 GEF-H1，这表明它是 GEF-H1-RhoA 信号轴的下游靶标。GEF-H1 通过与抗原刺激结合的磷酸化而被激活。Syk 激酶与 FcεRI 信号通路相关联，并且 Syk 抑制剂 GS-9973 阻断了 GEF-H1 的激活，并且还抑制了细胞扩展、颗粒运动和胞吐作用。我们得出的结论是，在 FcεRI 受体刺激期间，GEF-H1 将信号传递给 RhoA 激活和 FA 形成，以促进胞吐作用机制。
