Protein kinase Cdelta functions downstream of Ca2+ mobilization in FcepsilonRI signaling to degranulation in mast cells.

BACKGROUND: Mobilization of Ca 2+ plays an important role in the degranulation of mast cells. Although events upstream of Ca 2+ mobilization in the regulation of degranulation are relatively well characterized, the downstream mediators of Ca 2+ remain largely unknown. OBJECTIVE: We sought to characterize the downstream signaling mechanism by which Ca 2+ mobilization mediates degranulation in antigen-stimulated mast cells. METHODS: The effect of various inhibitors was examined in the antigen-induced or Ca 2+ ionophore A23187-induced degranulation process, and the effect of inhibitors on histamine release was tested in the mouse model of asthma. RESULTS: The delta isoform of protein kinase C (PKC) functions downstream of Ca 2+ in the signaling pathway from FcepsilonRI to degranulation in RBL-2H3 mast cells. Stimulation of cells with either antigen or the Ca 2+ ionophore A23187 induced a rapid translocation of PKC-delta from the cytosol to the cellular membranes, and either treatment with the PKC-delta-specific inhibitor rottlerin or infection with an adenovirus encoding a dominant negative mutant of PKC-delta markedly inhibited degranulation induced with antigen or A23187. Furthermore, both the translocation of PKC-delta and degranulation induced by A23187 were inhibited by prevention of the accumulation of reactive oxygen species normally elicited by the ionophore. Finally, intraperitoneal injection of rottlerin prevented the increase in the concentration of histamine in bronchoalveolar lavage fluid induced by means of antigen challenge in a mouse model of allergic asthma. conclusion: PKC-delta plays an essential downstream mediatory role in the degranulation elicited by Ca 2+ mobilization, and reactive oxygen species mediate the activation of PKC-delta by Ca 2+ in the regulation of degranulation.

Eotaxin induces migration of RBL-2H3 mast cells via a Rac-ERK-dependent pathway.

Eotaxin is a potent chemokine that acts via CC chemokine receptor 3 (CCR3) to induce chemotaxis, mainly on eosinophils. Here we show that eotaxin also induces chemotactic migration in rat basophilic leukemia (RBL-2H3) mast cells. This effect was dose-dependently inhibited by compound X, a selective CCR3 antagonist, indicating that, as in eosinophils, the effect was mediated by CCR3. Eotaxin-induced cell migration was completely blocked in RBL-RacN17 cells expressing a dominant negative Rac1 mutant, suggesting a crucial role for Rac1 in eotaxin signaling to chemotactic migration. ERK activation also proved essential for eotaxin signaling and it too was absent in RBL-RacN17 cells. Finally, we found that activation of Rac and ERK was correlated with eotaxin-induced actin reorganization known to be necessary for cell motility. It thus appears that Rac1 acts upstream of ERK to signal chemotaxis in these cells, and that a Rac-ERK-dependent cascade mediates the eotaxin-induced chemotactic motility of RBL-2H3 mast cells.