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.

[1]  A. Rosenzweig,et al.  Importance of FADD Signaling in Serum Deprivation- and Hypoxia-induced Cardiomyocyte Apoptosis* , 2002, Journal of Biological Chemistry.

[2]  S. Ghosh,et al.  CD8+ T cell apoptosis induced by Escherichia coli heat‐labile enterotoxin B subunit occurs via a novel pathway involving NF‐κB‐dependent caspase activation , 2002, European journal of immunology.

[3]  I. Boulven,et al.  Differential involvement of Src family kinases in pervanadate-mediated responses in rat myometrial cells. , 2002, Cellular signalling.

[4]  Steven Sun,et al.  Apoptosis of airway epithelial cells induced by corticosteroids. , 2001, American journal of respiratory and critical care medicine.

[5]  T. Ohmori,et al.  Wheat germ agglutinin-induced platelet activation via platelet endothelial cell adhesion molecule-1: involvement of rapid phospholipase C gamma 2 activation by Src family kinases. , 2001, Biochemistry.

[6]  C. Riccardi,et al.  Modulation of T-cell activation by the glucocorticoid-induced leucine zipper factor via inhibition of nuclear factor kappaB. , 2001, Blood.

[7]  D. Faller,et al.  The recruitment of Fas-associated death domain/caspase-8 in Ras-induced apoptosis. , 2001, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[8]  K. Schulze-Osthoff,et al.  Role of the CD95/CD95 Ligand System in Glucocorticoid-Induced Monocyte Apoptosis1 , 2001, The Journal of Immunology.

[9]  T. Nakada,et al.  STRESS AND TESTICULAR GERM CELL APOPTOSIS , 2001, Archives of andrology.

[10]  P. Krammer,et al.  CD95's deadly mission in the immune system , 2000, Nature.

[11]  D. Wood,et al.  Involvement of caspase 3- and 8-like proteases in ceramide-induced apoptosis of cardiomyocytes. , 2000, Journal of cardiac failure.

[12]  R. Kofler,et al.  The molecular basis of glucocorticoid-induced apoptosis of lymphoblastic leukemia cells , 2000, Histochemistry and Cell Biology.

[13]  P. Henkart,et al.  Assessment of Caspase Activities in Intact Apoptotic Thymocytes Using Cell-Permeable Fluorogenic Caspase Substrates , 2000, The Journal of experimental medicine.

[14]  R. Flower,et al.  Glucocorticoids act within minutes to inhibit recruitment of signalling factors to activated EGF receptors through a receptor‐dependent, transcription‐independent mechanism , 2000, British journal of pharmacology.

[15]  S. Peters,et al.  Effect of IL‐5, glucocorticoid, and Fas ligation on Bcl‐2 homologue expression and caspase activation in circulating human eosinophils , 2000, Clinical and experimental immunology.

[16]  C. Riccardi,et al.  Dexamethasone-induced thymocyte apoptosis: apoptotic signal involves the sequential activation of phosphoinositide-specific phospholipase C, acidic sphingomyelinase, and caspases. , 1999, Blood.

[17]  E. Solary,et al.  Fas Ligand-independent, FADD-mediated Activation of the Fas Death Pathway by Anticancer Drugs* , 1999, The Journal of Biological Chemistry.

[18]  John Calvin Reed,et al.  Investigation of glucocorticoid-induced apoptotic pathway: Processing of Caspase-6 but not Caspase-3 , 1998, Cell Death and Differentiation.

[19]  Xiaodong Wang,et al.  Bid, a Bcl2 Interacting Protein, Mediates Cytochrome c Release from Mitochondria in Response to Activation of Cell Surface Death Receptors , 1998, Cell.

[20]  Junying Yuan,et al.  Cleavage of BID by Caspase 8 Mediates the Mitochondrial Damage in the Fas Pathway of Apoptosis , 1998, Cell.

[21]  Dean P. Jones,et al.  Mitochondrial control of apoptosis: the role of cytochrome c. , 1998, Biochimica et biophysica acta.

[22]  José Luis de la Pompa,et al.  Differential Requirement for Caspase 9 in Apoptotic Pathways In Vivo , 1998, Cell.

[23]  Keisuke Kuida,et al.  Reduced Apoptosis and Cytochrome c–Mediated Caspase Activation in Mice Lacking Caspase 9 , 1998, Cell.

[24]  A. Parfitt,et al.  Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. , 1998, The Journal of clinical investigation.

[25]  T. Kuwana,et al.  Apoptosis Induction by Caspase-8 Is Amplified through the Mitochondrial Release of Cytochrome c * , 1998, The Journal of Biological Chemistry.

[26]  Huiling He,et al.  In VitroThymocyte Maturation Is Associated with Reduced Cellular Susceptibility to Fas-Mediated Apoptosis , 1998 .

[27]  J. Mountz,et al.  Fas/Fas ligand signaling during gestational T cell development. , 1998, Journal of immunology.

[28]  V. Dixit,et al.  Activation of caspases triggered by cytochrome c in vitro 1 , 1998, FEBS letters.

[29]  D. Goeddel,et al.  FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis. , 1998, Science.

[30]  M. Peter,et al.  Two CD95 (APO‐1/Fas) signaling pathways , 1998, The EMBO journal.

[31]  D. Kufe,et al.  Cytochrome c-dependent and -independent Induction of Apoptosis in Multiple Myeloma Cells* , 1997, The Journal of Biological Chemistry.

[32]  S. Srinivasula,et al.  Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade , 1997, Cell.

[33]  R. Sékaly,et al.  Specific Activation of the Cysteine Protease CPP32 during the Negative Selection of T Cells in the Thymus , 1997, The Journal of experimental medicine.

[34]  N. Thornberry,et al.  A Combinatorial Approach Defines Specificities of Members of the Caspase Family and Granzyme B , 1997, The Journal of Biological Chemistry.

[35]  Michael J. Bolt,et al.  1,25 dihydroxyvitamin D3 stimulates phospholipase C-gamma in rat colonocytes: role of c-Src in PLC-gamma activation. , 1997, The Journal of clinical investigation.

[36]  A. Goldberg,et al.  Proteasomes play an essential role in thymocyte apoptosis. , 1996, The EMBO journal.

[37]  J. M. Cousin,et al.  Opposing effects of glucocorticoids on the rate of apoptosis in neutrophilic and eosinophilic granulocytes. , 1996, Journal of immunology.

[38]  G. Chrousos,et al.  Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids. , 1996, Endocrine reviews.

[39]  P. Amiri,et al.  Requirement of an ICE-Like Protease for Induction of Apoptosis and Ceramide Generation by REAPER , 1996, Science.

[40]  J. Hanke,et al.  Discovery of a Novel, Potent, and Src Family-selective Tyrosine Kinase Inhibitor , 1996, The Journal of Biological Chemistry.

[41]  Seamus J. Martin,et al.  Proteolysis of Fodrin (Non-erythroid Spectrin) during Apoptosis (*) , 1995, The Journal of Biological Chemistry.

[42]  A. Mendez.  Monensin and Brefeldin A Inhibit High Density Lipoprotein- mediated Cholesterol Efflux from Cholesterol-enriched Cells , 1995, The Journal of Biological Chemistry.

[43]  M. Karin,et al.  Glucocorticoid‐induced apoptosis of human leukemic cells is caused by the repressive function of the glucocorticoid receptor. , 1995, The EMBO journal.

[44]  I. Adcock,et al.  Anti-inflammatory actions of steroids: molecular mechanisms. , 1993, Trends in pharmacological sciences.

[45]  N. Davidson,et al.  Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. , 1993, Cancer research.

[46]  J. Bach,et al.  Immunosuppressive therapy of autoimmune diseases. , 1993, Trends in pharmacological sciences.

[47]  Hoffman Gs.  Immunosuppressive therapy for autoimmune diseases. , 1993 .

[48]  C. Riccardi,et al.  Interleukin-4 protects double-negative and CD4 single-positive thymocytes from dexamethasone-induced apoptosis , 1993 .

[49]  C. Unger,et al.  Tumor necrosis factor induces rapid production of 1'2'diacylglycerol by a phosphatidylcholine-specific phospholipase C , 1991, The Journal of experimental medicine.

[50]  H. Akil,et al.  Localization of the 90-kDa heat shock protein-binding site within the hormone-binding domain of the glucocorticoid receptor by peptide competition. , 1991, The Journal of biological chemistry.

[51]  G. Melnykovych,et al.  Apoptosis: mode of cell death induced in T cell leukemia lines by dexamethasone and other agents , 1991, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[52]  R. Smith,et al.  Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. , 1990, The Journal of pharmacology and experimental therapeutics.

[53]  J. Cohen,et al.  Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. , 1984, Journal of immunology.

[54]  A. Fauci,et al.  Corticosteroid‐Mediated Immunoregulation in Man , 1982, Immunological reviews.

[55]  A. Wyllie.  Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation , 1980, Nature.

[56]  D. Young,et al.  Effect of glucocorticoid hormones in vitro on the structural integrity of nuclei in corticosteroid-sensitive and -resistant lines of lymphosarcoma P1798. , 1978, Cancer research.

[57]  Seymour,et al.  Glucocorticoid-binding proteins in human acute lymphoblastic leukemic blast cells. , 1973, The Journal of clinical investigation.

[58]  M. Cohn,et al.  Glucocorticoid Receptors in Lymphoma Cells in Culture: Relationship to Glucocorticoid Killing Activity , 1971, Science.

[59]  G. Salvesen,et al.  Caspase activation. , 2003, Biochemical Society symposium.

[60]  C. Riccardi,et al.  Modulation of T-cell activation by the glucocorticoid-induced leucine zipper factor via inhibition of nuclear factor kB , 2001 .

[61]  S. Scherer,et al.  On the molecular architecture of myelinated fibers , 2000, Histochemistry and Cell Biology.

[62]  P. Hershberger,et al.  In vitro thymocyte maturation is associated with reduced cellular susceptibility to Fas-mediated apoptosis. , 1998, Cellular immunology.

[63]  E. Vreugdenhil,et al.  Corticosteroid hormones and neuronal vulnerability: towards identification of candidate vulnerability genes. , 1998, Progress in brain research.

[64]  E. Alnemri,et al.  Baculovirus P35 inhibits the glucocorticoid-mediated pathway of cell death. , 1997, Cancer research.

[65]  J. Joffe.  Cancer chemotherapy and biological response modifiers annual 16, H.M. Pinedo, D.L. Longo, B.A. Chabner (Eds.). Elsevier, Leuven (1996), ISBN: 0-444-82326-3 , 1996 .

[66]  G. Migliorati,et al.  Dexamethasone induces apoptosis in mouse natural killer cells and cytotoxic T lymphocytes. , 1994, Immunology.

[67]  C. Riccardi,et al.  Interleukin-4 protects double-negative and CD4 single-positive thymocytes from dexamethasone-induced apoptosis. , 1993, Blood.

[68]  D. Longo,et al.  Cancer chemotherapy and biological response modifiers annual , 1987 .