Little is known about the signals downstream of PI3K which regulate mast cell homeostasis and function following FcεRI aggregation and Kit ligation. In this study, we investigated the role of the mammalian target of rapamycin complex 1 (mTORC1) pathway in these responses. In human and mouse mast cells, stimulation via FcεRI or Kit resulted in a marked PI3K-dependent activation of the mTORC1 pathway, as revealed by the wortmannin-sensitive sequential phosphorylation of tuberin, mTOR, p70S6 kinase (p70S6K), and 4E-BP1. In contrast, in human tumor mast cells, the mTORC1 pathway was constitutively activated and this was associated with markedly elevated levels of mTORC1 pathway components. Rapamycin, a specific inhibitor of mTORC1, selectively and completely blocked the FcεRI- and Kit-induced mTORC1-dependent p70S6K phosphorylation and partially blocked the 4E-BP1 phosphorylation. In parallel, although rapamycin had no effect on FcεRI-mediated degranulation or Kit-mediated cell adhesion, it inhibited cytokine production, and kit-mediated chemotaxis and cell survival. Furthermore, Rapamycin also blocked the constitutive activation of the mTORC1 pathway and inhibited cell survival of tumor mast cells. These data provide evidence that mTORC1 is a point of divergency for the PI3K-regulated downstream events of FcεRI and Kit for the selective regulation of mast cell functions. Specifically, the mTORC1 pathway may play a critical role in normal and dysregulated control of mast cell homeostasis.

Mast cells play a central role in the initiation of inflammatory responses associated with asthma and other allergic disorders. Receptor-mediated mast cell growth, differentiation, homing to their target tissues, survival, and activation, are all controlled, to varying degrees, by phosphoinositide-3-kinase (PI3K)-driven pathways. It is not fully understood how such diverse responses can be differentially regulated by PI3K. However, recent studies have provided greater insight into the mechanisms which control, and those which are controlled by, different PI3K subunit isoforms in mast cells. In this review, we discuss how PI3K influences the mast cell processes described above. Furthermore, we describe how different mast cell receptors utilize alternative isoforms of PI3K for these functions, and discuss potential downstream targets of these isoforms.

Antigen/IgE-mediated mast cell activation via FcεRI can be markedly enhanced by the activation of other receptors expressed on mast cells and these receptors may thus contribute to the allergic response in vivo. One such receptor family is the G protein-coupled receptors (GPCRs). Although the signaling cascade linking FcεRI aggregation to mast cell activation has been extensively investigated, the mechanisms by which GPCRs amplify this response are relatively unknown. To investigate this, we utilized prostaglandin (PG)E 2 based on initial studies demonstrating its greater ability to augment antigen-mediated degranulation in mouse mast cells than other GPCR agonists examined. This enhancement, and the ability of PGE 2 to amplify antigen-induced calcium mobilization, was independent of phosphoinositide 3-kinase but was linked to a pertussis toxinsensitive synergistic translocation to the membrane of phospholipase (PL)Cγ and PLCβ and to an enhancement of PLCγ phosphorylation. This "trans-synergistic" activation of PLCβ and γ, in turn, enhanced production of inositol 1,4,5-trisphosphate, store operated calcium entry, and activation of protein kinase C (PKC) (α and β). These responses were critical for the promotion of degranulation. This is the first report of synergistic activation between PLCγ and PLCβ that permits reinforcement of signals for degranulation in mast cells.

Activated mast cells are a major source of the eicosanoids PGD2 and leukotriene C4 (LTC4), which contribute to allergic responses. These eicosanoids are produced following the ERK1/2-dependent activation of cytosolic phospholipase A2, thus liberating arachidonic acid, which is subsequently metabolized by the actions of 5-lipoxygenase and cyclooxygenase to form LTC4 and PGD2, respectively. These pathways also generate reactive oxygen species (ROS), which have been proposed to contribute to FcεRI-mediated signaling in mast cells. In this study, we demonstrate that, in addition to ERK1/2-dependent pathways, ERK1/2-independent pathways also regulate FcεRI-mediated eicosanoid and ROS production in mast cells. A role for the Tec kinase Btk in the ERK1/2-independent regulatory pathway was revealed by the significantly attenuated FcεRI-dependent PGD2, LTC4, and ROS production in bone marrow-derived mast cells of Btk−/− mice. The FcεRI-dependent activation of Btk and eicosanoid and ROS generation in bone marrow-derived mast cells and human mast cells were similarly blocked by the PI3K inhibitors, Wortmannin and LY294002, indicating that Btk-regulated eicosanoid and ROS production occurs downstream of PI3K. In contrast to ERK1/2, the PI3K/Btk pathway does not regulate cytosolic phospholipase A2 phosphorylation but rather appears to regulate the generation of ROS, LTC4, and PGD2 by contributing to the necessary Ca2+ signal for the production of these molecules. These data demonstrate that strategies to decrease mast cell production of ROS and eicosanoids would have to target both ERK1/2- and PI3K/Btk-dependent pathways.