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Lactosylceramide is required in apoptosis induced by N-Smase

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Abstract

Lactosylceramide (LacCer) is a member of the glycosphingolipid family which has been recently recognized as a signaling intermediate in the regulation of cell proliferation and cell adhesion. In this paper, we present our studies pointing to a potential role of LacCer in inducing apoptosis. In our studies we employed a human osteosarcoma cell line MG-63 (wild type, WT) and a neutral sphingomyelinase (N-SMase) deficient cell line CC derived from MG-63 (mutant) cells. We observed that WT cells were highly sensitive to tumor necrosis factor-α (TNF-α), ceramide and LacCer-induced apoptosis. In contrast, the mutant cells were insensitive to TNF-α-induced apoptosis as they did not generate ceramide and LacCer. However, the exogenous supply of ceramide and/or LacCer rendered the mutant cells apoptotic. Interestingly, preincubation of cells with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of glucosylceramide synthase and lactosylceramide synthase, abrogated ceramide-induced apoptosis but not LacCer-induced apoptosis in both WT cells and the mutant cells. Moreover, TNF-α and LacCer-induced apoptosis required the generation of reactive oxygen species (ROS) in WT cells. However, since mutant cells did not produce significant amounts of LacCer and ROS in response to TNF-α treatment they are insensitive to TNF-α-induced apoptosis. In summary, our studies suggest that TNF-α-induced N-SMase activation and production of ceramide is required to activate the apoptosis pathway in human osteosarcoma cells. But it is not sufficient to induce apoptosis. Rather, the conversion of ceramide to LacCer and ROS generation are critical for apoptosis.

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Abbreviations

LacCer:

LactosylCeramide

TNF-α:

Tumor Necrosis Factor-α

N-SMase:

Neutral Sphingomyelinase

D-PDMP:

D-threo-1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol

ROS:

Reactive Oxygen Species

MnSOD:

Manganese Superoxide Dismutase

NAC:

N-acetyl-L-cysteine

PBS:

Phosphate-Buffered Saline

DTT:

Dithiothreitol

ATP:

Adenosine Tris-Phosphate

DAPI:

4’,6-Diamidine-2’-phenylindole dihydrochloride

DCFH-DA:

diachlorofluorescein diacetate

TNFR1:

TNF-α receptor 1

SM:

sphingomyelin; Wild type

WT:

human osteosarcoma cell line MG-63

Mutant:

N-SMase deficient osteosarcoma cell line CC

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Author notes

  1. Sergio F. Martin

    Present address: INIA-CISA, Valdeolmos (Madrid), Spain

  2. Niesha Williams

    Present address: Department of Biochemistry, Oxford University, England (UK)

Authors and Affiliations

  1. Lipid Research Atherosclerosis Division, Johns Hopkins University, Baltimore, Maryland

    Sergio F. Martin, Niesha Williams & Subroto Chatterjee

  2. Biomedical Engineering Department, Johns Hopkins University, Baltimore, Maryland

    Sergio F. Martin

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  1. Sergio F. Martin
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  2. Niesha Williams
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Correspondence to Subroto Chatterjee.

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Martin, S.F., Williams, N. & Chatterjee, S. Lactosylceramide is required in apoptosis induced by N-Smase. Glycoconj J 23, 147–157 (2006). https://doi.org/10.1007/s10719-006-7920-8

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