Thapsigargin-induced degranulation of mast cells is dependent on transientactivation of phosphatidylinositol-3 kinase

Thapsigargin, which elevates cytosolic calcium levels bg inhibiting the sar coplasmic/endoplasmic reticulum calcium-dependent ATPase, was tested for it s ability to degranulate hone marrow-derived mast cells (BMMCs) from src ho mology 2-containing inositol phosphatase +/+ (SHIP+/+) and SHIP-/- mice. As was found previously with steel factor, thapsigargin stimulated far more d egranulation in SHIP-/- than in SHIP+/+ BMMCs, and this was blocked with th e phosphatidylinositol-3 (PI-3) kinase inhibitors, LY294002 and wortmannin. In contrast to steel factor, however, this heightened degranulation of SHI P-/- BMMCs was not due to a greater calcium influx into these cells, nor wa s the thapsigargin-induced calcium influx inhibited by LY294002, suggesting that the heightened thapsigargin-induced degranulation of SHIP-/- BMMCs wa s due to a PI-3 kinase-regulated step distinct from that regulating calcium entry. An investigation of thapsigargin-stimulated pathways in both cell t ypes revealed that MAPK was heavily but equally phosphorylated, Interesting ly, the protein kinase C inhibitor, bisindolylmaleimide (compound 3), total ly blocked thapsigargin-induced degranulation in both SHIP+/+ and SHIP-/- B MMCs. As well, thapsipargin stimulated a PI-3 kinase-dependent, transient a ctivation of protein kinase B, and this activation was far greater in SHIP- /- than in SHIP+/+ BMMCs. Consistent with this, thapsigargin was found to b e a potent survival factor, following cytokine withdrawal, for both cell ty pes and was more potent with SHIP-/- cells. These studies have both identif ied an additional PI-3 kinase-dependent step within the mast cell degranula tion process, possibly involving 3-phosphoinositide-dependent protein kinas e-1 and a diacylglycerol-independent protein kinase C isoform, and shown th at the tumor-promoting activity of thapsigargin may be due to its activatio n of protein kinase B and subsequent promotion of cell survival.