title: Thapsigargin-induced degranulation of mast cells is dependent on transient activation of phosphatidylinositol-3 kinase.
abstract: Thapsigargin, which elevates cytosolic calcium levels by inhibiting the sarcoplasmic/endoplasmic reticulum calcium-dependent ATPase, was tested for its ability to degranulate bone marrow-derived mast cells (BMMCs) from src homology 2-containing inositol phosphatase +/+ (SHIP+/+) and SHIP-/- mice. As was found previously with steel factor, thapsigargin stimulated far more degranulation in SHIP-/- than in SHIP+/+ BMMCs, and this was blocked with the phosphatidylinositol-3 (PI-3) kinase inhibitors, LY294002 and wortmannin. In contrast to steel factor, however, this heightened degranulation of SHIP-/- BMMCs was not due to a greater calcium influx into these cells, nor was the thapsigargin-induced calcium influx inhibited by LY294002, suggesting that the heightened thapsigargin-induced degranulation of SHIP-/- BMMCs was due to a PI-3 kinase-regulated step distinct from that regulating calcium entry. An investigation of thapsigargin-stimulated pathways in both cell types revealed that MAPK was heavily but equally phosphorylated. Interestingly, the protein kinase C inhibitor, bisindolylmaleimide (compound 3), totally blocked thapsigargin-induced degranulation in both SHIP+/+ and SHIP-/- BMMCs. As well, thapsigargin stimulated a PI-3 kinase-dependent, transient activation of protein kinase B, and this activation was far greater in SHIP-/- than in SHIP+/+ BMMCs. Consistent with this, thapsigargin was found to be a potent survival factor, following cytokine withdrawal, for both cell types and was more potent with SHIP-/- cells. These studies have both identified an additional PI-3 kinase-dependent step within the mast cell degranulation process, possibly involving 3-phosphoinositide-dependent protein kinase-1 and a diacylglycerol-independent protein kinase C isoform, and shown that the tumor-promoting activity of thapsigargin may be due to its activation of protein kinase B and subsequent promotion of cell survival.
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Huber
authors.forename: M R
authors.initials: MR
authors.lastname: Hughes
authors.forename: G
authors.initials: G
authors.lastname: Krystal
mesh_terms: D000276:Adjuvants, Immunologic / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects / Q000201:enzymology / Q000276:immunology; D015550:Cell Degranulation / Q000187:drug effects* / Q000276:immunology; D002470:Cell Survival / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects / Q000276:immunology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008407:Mast Cells / Q000187:drug effects* / Q000201:enzymology* / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism* / Q000502:physiology; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010744:Phosphoric Monoester Hydrolases / Q000172:deficiency / Q000235:genetics; D011493:Protein Kinase C / Q000502:physiology; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D019284:Thapsigargin / Q000494:pharmacology*; D018909:src Homology Domains / Q000276:immunology
chemical_list: D000276:Adjuvants, Immunologic; D004791:Enzyme Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010744:Phosphoric Monoester Hydrolases; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019284:Thapsigargin; C098706:INPPL1 protein, human