Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria.
B-cells, triggered via their surface B-cell receptor (BcR), start an apoptotic program known as activation-induced cell death (AICD), and it is widely believed that this phenomenon plays a role in the restriction and focusing of the immune response. Although both ceramide and caspases have been proposed to be involved in AICD, the contribution of either and the exact molecular events through which AICD commences are still unknown. Here we show that in Ramos B-cells, BcR-triggered cell death is associated with an early rise of C16 ceramide that derives from activation of the de novo pathway, as demonstrated using a specific inhibitor of ceramide synthase, fumonisin B1 (FB1), and using pulse labeling with the metabolic sphingolipid precursor, palmitate. There was no evidence for activation of sphingomyelinases or hydrolysis of sphingomyelin. Importantly, FB1 inhibited several specific apoptotic hallmarks such as poly(A)DP-ribose polymerase cleavage and DNA fragmentation. Electron microscopy revealed morphological evidence of mitochondrial damage, suggesting the involvement of mitochondria in BcR-triggered apoptosis, and this was inhibited by FB1. Moreover, a loss of mitochondrial membrane potential was observed in Ramos cells after BcR cross-linking, which was inhibited by the addition of FB1. Interestingly, benzyloxycarbonyl-Val-Ala-dl-Asp, a broad spectrum caspase inhibitor did not inhibit BcR-induced mitochondrial membrane permeability transition but did block DNA fragmentation. These results suggest a crucial role for de novo generated C16 ceramide in the execution of AICD, and they further suggest an ordered and more specific sequence of biochemical events in which de novo generated C16 ceramide is involved in mitochondrial damage resulting in a downstream activation of caspases and apoptosis.
Departments of Pathology and Laboratory Medicine, University Hospital Groningen, 9713 G2 Groningen, The Netherlands.
B J
BJ
Kroesen
B
B
Pettus
C
C
Luberto
M
M
Busman
H
H
Sietsma
L
L
de Leij
Y A
YA
Hannun
D017209:Apoptosis; D001402:B-Lymphocytes / Q000378:metabolism*; D051792:Basic Helix-Loop-Helix Proteins; D015153:Blotting, Western; D002264:Carboxylic Acids / Q000494:pharmacology; D002518:Ceramides / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D053938:DNA Fragmentation; D004268:DNA-Binding Proteins / Q000378:metabolism; D019852:Diacylglycerol Kinase / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D037341:Fumonisins; D006801:Humans; D013058:Mass Spectrometry; D008564:Membrane Potentials; D008854:Microscopy, Electron; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D010088:Oxidoreductases / Q000037:antagonists & inhibitors; D010168:Palmitates / Q000378:metabolism; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D013997:Time Factors; D014157:Transcription Factors
D051792:Basic Helix-Loop-Helix Proteins; D002264:Carboxylic Acids; D002518:Ceramides; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D019852:Diacylglycerol Kinase; D037341:Fumonisins; D010088:Oxidoreductases; D010168:Palmitates; D011947:Receptors, Antigen, B-Cell; D014157:Transcription Factors; C118089:TFPT protein, human; C056933:fumonisin B1; C097760:N-palmitoylsphingosine; C108138:dihydroceramide desaturase