Caspase activation by BCR cross-linking in immature B cells: differential effects on growth arrest and apoptosis.
The B cell lymphoma WEHI-231 has been used as a model to study immature B cell tolerance, based on its capacity to undergo growth arrest and programmed cell death on B cell receptor (BCR) cross-linking. Using this model to identify the molecular mechanisms underlying these processes, we found that BCR cross-linking results in the selective activation of caspase 7/Mch3, but not of the other two members of the CPP32 family, caspase 2/Nedd2 and caspase 3/CPP32. This was evidenced by the induction of proteolytic activity against the substrate for the CPP32 subfamily of caspases (z-DVED-AMC) in vitro, as well as PARP proteolysis in vivo and by the processing of the 35 kDa Mch3 into a 32 kDa species, which was later further proteolyzed. The general caspase inhibitor z-VAD-fmk, but not the CPP32 family inhibitor Ac-DEVD-CHO, blocked anti- micro-induced apoptosis, indicating that a caspase not belonging to the CPP32-like family is also implicated in anti- micro-triggered apoptosis. In contrast, z-VAD-fmk was not able to counteract growth arrest induced by anti- micro treatment, suggesting that caspase activation is not necessary for induction of growth arrest. Neither of the inhibitors prevented Mch3 processing; however, z-VAD-fmk prevented proteolysis of the p32 subunit, suggesting that further processing of this subunit is associated with apoptosis. Bcl-2 overexpression prevented anti- micro induction of CPP32-like activity and apoptosis, and blocked further processing of the Mch3 p32 subunit. In contrast, CD40 stimulation completely blocked the appearance of the p32 subunit in addition to blocking CPP32-like activity and apoptosis induced by BCR cross-linking. Moreover, only CD40 stimulation was able to prevent anti- micro-induced growth arrest, which was correlated with inhibition of retinoblastoma and of cyclin A down-regulation. In splenic B cells, Mch3 is also specifically proteolyzed ex vivo after induction of apoptosis by BCR cross-linking, demonstrating the specific involvement of caspase-7/Mch3 in apoptosis induced in B cell tolerance.
Brás
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Departamento de Inmunología, Centro Nacional de Biotecnología, CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain. abras@cnb.uam.es
Ruiz-Vela
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González de Buitrago
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Martinez-A
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D000818:Animals; D017209:Apoptosis; D001402:B-Lymphocytes / Q000166:cytology / Q000201:enzymology* / Q000276:immunology*; D001483:Base Sequence; D023201:CD40 Ligand; D053148:Caspase 3; D053179:Caspase 7; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D003432:Cross-Linking Reagents; D019925:Cyclin A / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation; D015870:Gene Expression; D019254:Genes, bcl-2; D007075:Immunoglobulin M / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000378:metabolism
D061945:Caspase Inhibitors; D003432:Cross-Linking Reagents; D019925:Cyclin A; D017931:DNA Primers; D007075:Immunoglobulin M; D008562:Membrane Glycoproteins; D011947:Receptors, Antigen, B-Cell; D016160:Retinoblastoma Protein; D023201:CD40 Ligand; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases