Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal.
The early signals generated following cross-linking of Fas/APO-1, a transmembrane receptor whose engagement by ligand results in apoptosis induction, were investigated in human HuT78 lymphoma cells. Fas/APO-1 cross-linking by mAbs resulted in membrane sphingomyelin hydrolysis and ceramide generation by the action of both neutral and acidic sphingomyelinases. Activation of a phosphatidylcholine-specific phospholipase C (PC-PLC) was also detected which appeared to be a requirement for subsequent acidic sphingomyelinase (aSMase) activation, since PC-PLC inhibitor D609 blocked Fas/APO-1-induced aSMase activation, but not Fas/APO-1-induced neutral sphingomyelinase (nSMase) activation. Fas/APO-1 cross-linking resulted also in ERK-2 activation and in phospholipase A2 (PLA2) induction, independently of the PC-PLC/aSMase pathway. Evidence for the existence of a pathway directly involved in apoptosis was obtained by selecting HuT78 mutant clones spontaneously expressing a newly identified death domain-defective Fas/APO-1 splice isoform which blocks Fas/APO-1 apoptotic signalling in a dominant negative fashion. Fas/APO-1 cross-linking in these clones fails to activate PC-PLC and aSMase, while nSMase, ERK-2 and PLA2 activates are induced. These results strongly suggest that a PC-PLC/aSMase pathway contributes directly to the propagation of Fas/APO-1-generated apoptotic signal in lymphoid cells.
Cifone
M G
MG
Department of Experimental Medicine, University of L'Aquila, Italy.
Roncaioli
P
P
De Maria
R
R
Camarda
G
G
Santoni
A
A
Ruberti
G
G
Testi
R
R
D000911:Antibodies, Monoclonal; D017209:Apoptosis; D016718:Arachidonic Acid / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism; D003432:Cross-Linking Reagents; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D008223:Lymphoma; D019950:Mitogen-Activated Protein Kinase 1; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*; D019014:fas Receptor / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002518:Ceramides; D003432:Cross-Linking Reagents; D004075:Diglycerides; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D019014:fas Receptor; D016718:Arachidonic Acid; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase