Dexamethasone-induced apoptosis of thymocytes: role of glucocorticoid receptor-associated Src kinase and caspase-8 activation.
Glucocorticoid hormones (GCHs) regulate normal and neoplastic lymphocyte development by exerting antiproliferative and/or apoptotic effects. We have previously shown that dexamethasone (DEX)-activated thymocyte apoptosis requires a sequence of events including interaction with the glucocorticoid receptor (GR), phosphatidylinositol-specific phospholipase C (PI-PLC), and acidic sphingomyelinase (aSMase) activation. We analyzed the mechanisms of GCH-activated apoptosis by focusing on GR-associated Src kinase, cytochrome c release, and caspase-8, -9, and -3 activation. We show here that PI-PLC binds to GR-associated Src kinase, as indicated by coimmunoprecipitation experiments. Moreover, DEX treatment induces PI-PLC phosphorylation and activation. DEX-induced PI-PLC phosphorylation, activation, and apoptosis are inhibited by PP1, a Src kinase inhibitor, thus suggesting that Src-mediated PI-PLC activation is involved in DEX-induced apoptosis. Caspase-9, -8, and -3 activation and cytochrome c release can be detected 1 to 2 hours after DEX treatment. Caspase-9 inhibition does not counter cytochrome c release, caspase-8 and caspase-3 activation, and apoptosis. Caspase-8 inhibition counters cytochrome c release, caspase-9 and caspase-3 activation, and apoptosis, thus suggesting that caspase-8 inhibitor can directly inhibit caspase-9 and/or that DEX-induced caspase-8 activation is upstream to mitochondria and can regulate caspase-3 directly or through cytochrome c release and the consequent caspase-9/caspase-3 activation. DEX-induced caspase-8 activation, like ceramide-induced caspase-8 activation, correlates with the formation of Fas-associated death domain protein (FADD)/caspase-8 complex. Caspase-8 activation is countered by the inhibition of macromolecular synthesis and of Src kinase, PI-PLC, and aSMase activation, suggesting it is downstream in the DEX-activated apoptotic pathway of thymocytes.
Marchetti
Maria Cristina
MC
Department of Clinical and Experimental Medicine, Pharmacology Section, University of Perugia, University of L'Aquila, Italy.
Di Marco
Barbara
B
Cifone
Grazia
G
Migliorati
Graziella
G
Riccardi
Carlo
C
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D013961:Thyroid Gland / Q000166:cytology* / Q000187:drug effects; D010738:Type C Phospholipases / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism / Q000502:physiology*
D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D019061:src-Family Kinases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D043265:Phosphatidylinositol Diacylglycerol-Lyase