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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.

Mast cells are tissue-resident immune cells that have numerous cytoplasmic granules which contain preformed pro-inflammatory mediators. Upon antigen stimulation, sensitized mast cells undergo profound changes to their morphology and rapidly release granule mediators by regulated exocytosis, also known as degranulation. We have previously shown that Rho GTPases regulate exocytosis, which suggests that cytoskeleton remodeling is involved in granule transport. Here, we used live-cell imaging to analyze cytoskeleton remodeling and granule transport in real-time as mast cells were antigen stimulated. We found that granule transport to the cell periphery was coordinated by de novo microtubule formation and not F-actin. Kinesore, a drug that activates the microtubule motor kinesin-1 in the absence of cargo, inhibited microtubule-granule association and significantly reduced exocytosis. Likewise, shRNA knock-down of Kif5b, the kinesin-1 heavy chain, also reduced exocytosis. Imaging showed granules accumulated in the perinuclear region after kinesore treatment or Kif5b knock-down. Complete microtubule depolymerization with nocodazole or colchicine resulted in the same effect. A biochemically enriched granule fraction showed kinesin-1 levels increase in antigen-stimulated cells, but are reduced by pre-treatment with kinesore. Kinesore had no effect on the levels of Slp3, a mast cell granule cargo adaptor, in the granule-enriched fraction which suggests that cargo adaptor recruitment to granules is independent of motor association. Taken together, these results show that granules associate with microtubules and are driven by kinesin-1 to facilitate exocytosis., Competing Interests: The authors have declared that no competing interests exist.

Peptide based therapeutics are desirable owing to their high biological specificity. However, a number of these fail in clinical testing due to an adverse inflammatory response. Mast cells play a key role in directing the host response to drugs and related products. Although the role of FcεRI receptor is well known, Mas-related G-protein coupled receptor X2 (MRGPRX2) binding of endogenous peptides, and drugs will activate mast cells independent of FcεRI. Identifying peptides that activate mast cells through MRGPRX2, and their respective activation potency, can be used to reduce the failure rate of peptide therapeutics at clinical trial. Moreover, it will allow for peptide design where mast cell activation is actually desired. It was found that FRKKW and WNKWAL are two motifs that activate human LAD2 cells similar to PAMP-12 controls. Peptide activators of MRGPRX2 could be reduced to X a -(Y) (n ≥ 3) -X b where: X a is an aromatic residue; X b is a hydrophobic residue; and Y is a minimum 3 residue long sequence, containing a minimum of one positively charged residue with the remainder being uncharged residues. Artificial peptides WKKKW and FKKKF were constructed to test this structural functionality and were similar to PAMP-12 controls. Peptides with different activation potentials were found where FRKKW = WKKKW = FKKKF > PAMP-12 = WNKWAL > YKKKY > FRKKANKWALSR = FRKKWNKAALSR > KWKWK > FRKK = WNKWA > KYKYK > NKWALSR = YKKY = WNK. These sequences should be considered when designing peptide-based therapeutics. STATEMENT OF SIGNIFICANCE: Mast cells release immune regulating molecules upon activation that direct host's immune response. MRGPRX2 receptor provides an alternate pathway for mast cell activation that is independent of FcεRI receptor. It is thought that mast cell activation through MRGPRX2 plays a critical role in high failure rates of drugs in clinical trials. Identifying peptide sequences that activate mast cells through MRGPRX2 can serve two important purposes, namely, sequences to avoid when designing peptide therapeutics, and artificial peptides with different activation potentials for mast cells. Herein, we have identified a general amino acid sequence that induces mast cell activation through MRGPRX2. Furthermore, by modulating the identified sequence, artificial peptides have been designed which activate mast cells by varying degrees for therapeutic applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Mast cells are tissue-resident immune cells that produce potent proinflammatory mediators, which are stored in cytoplasmic granules. Stimulation triggers degranulation, a process that mobilizes granules to dock and fuse to the plasma membrane, releasing mediators. Mast cell degranulation has an important role in immunity but can also intensify inflammation and contribute to allergic disorders. Hence, it is important to understand signaling pathways that regulate mast cell degranulation. Here, we examined the role of Rho proteins in regulating mast cell activation leading to degranulation. RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) were stimulated through aggregation of FcεRI receptors. Stimulated cells showed a large increase in the levels of activated Rac and, to a lesser extent, RhoA. Drugs were used to acutely inhibit the function of specific Rho proteins. The Rac inhibitor EHT-1864 and the RhoA inhibitor rhosin inhibited degranulation. Microscopic characterization showed that, upon stimulation, RBL-2H3 cells formed surface ridges that grew into large protrusions reminiscent of circular dorsal ruffles, which flattened into large lamellipodia. LysoTracker-labeled cells showed granules stream into peripheral protrusions. EHT-1864 reduced granule motility, whereas rhosin increased motility; both drugs affected the formation of peripheral protrusions. These results showed that, in response to stimuli, Rho proteins control discrete cytoskeletal remodeling processes that are needed for granule exocytosis. Rac is required to stimulate the remodeling of mast cells, triggering actin-mediated flattening of the cell periphery to create an active degranulation zone, whereas RhoA controls the streaming of highly motile granules into the active zone., (© Society for Leukocyte Biology.)

The release of preformed mediators from immune cells is through a process described as exocytosis. In mast cells, exocytosis is regulated by several coordinated intracellular signaling pathways. Here, we investigated the role of the hematopoietic-specific Rho GTPase, Rac2, and the ubiquitously expressed Rac1, in controlling mast cell exocytosis. These two isoforms showed equivalent levels of expression in mouse BMMCs. Although Rac1 and Rac2 share 92% sequence identity, they were not functionally redundant, as Rac2 -/- BMMCs were defective in exocytosis, even though Rac1 levels were unaffected. Antigen-stimulated WT mast cells underwent a series of morphological transitions: initial flattening, followed by actin-mediated peripheral membrane ruffling and calcium influx, which preceded exocytosis. Whereas membrane ruffling was unaffected in Rac2 -/- BMMCs, calcium influx was decreased significantly. Calcium influx was studied further by examining SOCE. In Rac2 -/- BMMCs, the activation of PLCγ1 and calcium release from intracellular stores occurred normally; however, activation of plasma membrane calcium channels was defective, shown by the lack of extracellular calcium influx and a reduction of YFP-STIM1 puncta at the plasma membrane. Additionally, we used the small molecule Rac inhibitor, EHT 1864, to target Rac signaling acutely in WT BMMCs. EHT 1864 blocked exocytosis and membrane ruffling completely in conjunction with exocytosis. Our findings suggest that antigen-stimulated membrane ruffling in mast cells is a Rac1-mediated process, as this persisted in the absence of Rac2. Therefore, we define distinct modes of Rac-regulated mast cell exocytosis: Rac2-mediated calcium influx and Rac1-mediated membrane ruffling., (© 2014 Society for Leukocyte Biology.)