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 antiproliferitive and/or apoptotic effects. We have previously shown that dexamethasone (DEX)-activated thymocyte apoptosis a requires a sequence of events including interaction with the glucocorticold receptor (GR), phosphatidlylinositol-specific phospholipase C (PI-PLC), and acidic sphingomye-linase (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, thug 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 caspage-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. (C) 2003 by The American Society of Hematology.