Distinct Phosphoinositide 3-Kinases Mediate Mast Cell Degranulation in Response to G-protein-coupled VersusFcεRI Receptors

Abstract

Chem. 276, 17213-17220). We now examine the role of Class IA PI 3-kinases isoforms in degranulation itself, using a single-cell degranulation assay that measures the binding of fluorescently tagged annexin V to phosphatidylserine in the outer leaflet of the plasma membrane of degranulated mast cells. Consistent with previous data, antibodies against p110␦ and p110␤ blocked Fc⑀R1-mediated degranulation in response to Fc⑀RI ligation. However, antigen-stimulated degranulation was also inhibited by antibodies against p110␣, despite the fact that these antibodies have no effect on antigeninduced calcium flux. These data suggest that p110␣ mediates a calcium-independent signal during degranulation. In contrast, only p110␤ was required for enhancement of antigen-stimulated degranulation by adenosine, which augments mast cell-mediated airway inflammation in asthma. Finally, we examined carbachol-stimulated degranulation in RBL2H3 cells stably expressing the M1 muscarinic receptor (RBL-2H3-M1 cells). Surprisingly, carbachol-stimulated degranulation was blocked by antibody-mediated inhibition of the Class III PI 3-kinase hVPS34 or by titration of its product with FYVE domains. Antibodies against Class IA PI 3-kinases had no effect. These data demonstrate: (a) a calcium-independent role for p110␣ in antigen-stimulated degranulation; (b) a requirement for p110␤ in adenosine receptor signaling; and (c) a requirement for hVPS34 during M1 muscarinic receptor signaling. Elucidation of the intersections between these distinct pathways will lead to new insights into mast cell degranulation.Mast cells are important cellular mediators of allergic responses in humans (1). Moreover, increased levels of mast cells and mast cell-derived inflammatory mediators are found in brochoalveolar lavage fluid from asthmatics, suggesting a role for mast cells in the etiology of clinical asthma (2-4). Crosslinking of cell surface Fc⑀RI receptors leads to the release of pre-formed mediators present in mast cell granules, as well as the induction of cytokines and bioactive lipids (5). Release of these inflammatory molecules in the lung is likely to contribute to inflammation and vasoconstriction during asthma. Antigenmediated degranulation is enhanced by co-stimulation of mast cells with adenosine, which is an important contributor to airway inflammation in asthma (6).The initial signaling events during antigen-stimulated degranulation have been well studied. Fc⑀RI cross-linking leads to recruitment and activation of lyn and syk tyrosine kinases, with subsequent phosphorylation of tyrosine residues in the Fc⑀RI ␥-chain (5, 7). This leads to the recruitment, phosphorylation and activation of phospholipase C␥, and generation of inositol trisphosphate and diacylglycerol from the hydrolysis of plasma membrane phosphatidylinositol (4,5)-bisphosphate. Inositol trisphosphate-mediated release of intracellular calcium stores and activation of classical and novel isoforms of protein kinase C (8) are required for the opening of plasma membrane calcium channels...

Phospholipase Cγ negatively regulates Rac/Cdc42 activation in antigen‐stimulated mast cells

“…This could explain the presence of a calciumdependent early Rac and Cdc42 activation phase, even in the presence of the PLCc inhibitor. Work from our laboratory and others showed a requirement for PI3K in mast cell degranulation and calcium influx [8,10,12], and PLCc-independent phosphatidylinositol 3,4,5-triphosphate-sensitive calcium channels at the plasma membrane have been characterized [9]. However, PI3K is not required for activation of Rac and Cdc42 in mast cells [10,13,24], where it may in fact be downstream of Rac [24,52].…”

“…Previously, we have shown that PI3K is essential for degranulation in antigen-responsive mast cells [11,12]. However, it has been reported that Rac activation in response to antigen stimulation in mast cells is PI3K-independent [24].…”

“…These two second messengers lead to increases in cytoplasmic calcium and activation of calcium/diacylglycerol-regulated isoforms of protein kinase C (PKC) which culminate in granule margination and exocytosis [7]. PI3K plays an important regulatory role as an upstream activator of PLCc1, as well as a regulator of calcium flux during mast cell degranulation [8][9][10][11][12][13].…”

“…These two second messengers lead to increases in cytoplasmic calcium and activation of calcium/diacylglycerol-regulated isoforms of protein kinase C (PKC) which culminate in granule margination and exocytosis [7]. PI3K plays an important regulatory role as an upstream activator of PLCc1, as well as a regulator of calcium flux during mast cell degranulation [8][9][10][11][12][13].Members of the Rho family of small GTPases also regulate signaling events in mast cells [14][15][16]. In the rat basophilic leukemia (RBL-2H3) cells, Rac and Cdc42 have been implicated in the control of the FceRImediated phagocytosis and the regulation of migration, In conjunction with the secretory response, antigenstimulated mast cells undergo dramatic cytoskeletal responses and morphological changes [25].…”

Regulation of Leukocyte Degranulation by cGMP-Dependent Protein Kinase and Phosphoinositide 3-Kinase: Potential Roles in Phosphorylation of Target Membrane SNARE Complex Proteins in Rat Mast Cells

“…Vesicular fusion to plasma membrane was visualized with FITC-annexin V, as described previously (32). Briefly, RBL-FPR cells were grown on a glass coverslip for 48 h, washed once with HBSS, and incubated with 10 M cytochalasin B and a 1/20 dilution of FITC-annexin V in HBSS-HB for 15 min on ice and then 15 min at 37°C.…”