GEF-H1 Transduces FcεRI Signaling in Mast Cells to Activate RhoA and Focal Adhesion Formation during Exocytosis

by Selleckchem | Jun 13 2023

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.

Comments:

The passage describes a research study that investigated the role of GEF-H1, a Rho guanine nucleotide exchange factor (GEF), in mast cell activation and the subsequent release of inflammatory mediators. Mast cells are immune cells involved in allergic reactions and immune responses.

The study found that when mast cells are stimulated by an antigen, GEF-H1 acts as a signal transducer, transmitting the antigen stimulation signal to activate RhoA, a small GTPase protein. This activation of RhoA promotes mast cell spreading through the formation of focal adhesions (FA), which are important structures for cell attachment and movement.

The researchers used RNA interference to deplete GEF-H1 in mast cells and observed that this depletion resulted in reduced cell spreading, granule movement, and exocytosis (the release of preformed inflammatory mediators from cytoplasmic granules). Additionally, RhoA activation was decreased in GEF-H1-depleted cells, leading to reduced formation of stress fibers (bundles of actin filaments) without affecting lamellipodia formation (sheet-like structures at the leading edge of migrating cells).

To further confirm the role of RhoA in the observed effects, the researchers introduced a constitutively active mutant form of RhoA into GEF-H1-depleted cells. This ectopic expression of active RhoA restored normal cell morphology in GEF-H1-depleted cells, suggesting that RhoA activation is crucial for mast cell spreading.

The formation of focal adhesions during antigen stimulation was found to depend on GEF-H1, indicating that GEF-H1 acts as a downstream target of the GEF-H1-RhoA signaling axis. GEF-H1 itself was activated through phosphorylation in response to antigen stimulation.

Furthermore, the study explored the involvement of Syk kinase, which is linked to the FcεRI signaling pathway—a pathway involved in mast cell activation. The researchers used a Syk inhibitor called GS-9973 and found that it blocked GEF-H1 activation and also suppressed cell spreading, granule movement, and exocytosis.

In summary, the study demonstrated that GEF-H1 serves as a mediator of antigen stimulation in mast cells, relaying the signal to activate RhoA and promote cell spreading through the formation of focal adhesions. This mechanism facilitates the exocytosis of inflammatory mediators, which is important for immune responses and allergic reactions. The activation of GEF-H1 is dependent on Syk kinase, further implicating its involvement in the FcεRI signaling pathway.