Differential protection by wildtype vs. organelle-specific Bcl-2 suggests a combined requirement of both the ER and mitochondria in ceramide-mediated caspase-independent programmed cell death.
BACKGROUND
Programmed cell death (PCD) is essential for development and homeostasis of multicellular organisms and can occur by caspase-dependent apoptosis or alternatively, by caspase-independent PCD (ciPCD). Bcl-2, a central regulator of apoptosis, localizes to both mitochondria and the endoplasmic reticulum (ER). Whereas a function of mitochondrial and ER-specific Bcl-2 in apoptosis has been established in multiple studies, corresponding data for ciPCD do not exist.


METHODS
We utilized Bcl-2 constructs specifically localizing to mitochondria (Bcl-2 ActA), the ER (Bcl-2 cb5), both (Bcl-2 WT) or the cytosol/nucleus (Bcl-2 DeltaTM) and determined their protective effect on ceramide-mediated ciPCD in transiently and stably transfected Jurkat cells. Expression of the constructs was verified by immunoblots. Ceramide-mediated ciPCD was induced by treatment with human recombinant tumor necrosis factor and determined by flow cytometric measurement of propidium iodide uptake as well as by optical analysis of cell morphology.


RESULTS
Only wildtype Bcl-2 had the ability to efficiently protect from ceramide-mediated ciPCD, whereas expression of Bcl-2 solely at mitochondria, the ER, or the cytosol/nucleus did not prevent ceramide-mediated ciPCD.


CONCLUSIONS
Our data suggest a combined requirement for both mitochondria and the ER in the induction and the signaling pathways of ciPCD mediated by ceramide.
Deerberg
Andrea
A
Institut für Immunologie, Christian-Albrechts-Universität Kiel, 24105 Kiel, Germany. dadam@email.uni-kiel.de
Sosna
Justyna
J
Thon
Lutz
L
Belka
Claus
C
Adam
Dieter
D
D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002518:Ceramides / Q000633:toxicity; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D019169:Jurkat Cells; D008928:Mitochondria / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
D002518:Ceramides; D019253:Proto-Oncogene Proteins c-bcl-2; D020169:Caspases