Role of ceramide in cellular senescence.
Recently the sphingomyelin cycle, involving the hydrolysis of membrane sphingomyelin by an activated sphingomyelinase to generate ceramide, has emerged as a key pathway in cell differentiation and apoptosis in leukemic and other cell types. Here we investigate a role for this pathway in the senescence of WI-38 human diploid fibroblasts (HDF). We found that endogenous levels of ceramide increased considerably (4-fold) and specifically (compared with other lipids) as cells entered the senescent phase. Investigation of the mechanism of increased ceramide led to the discovery that neutral sphingomyelinase activity is elevated 8-10 fold in senescent cells. There were no changes in sphingomyelinase activity or ceramide levels as HDF entered quiescence following serum withdrawal or contact inhibition. Thus, the activation of the sphingomyelinase/ceramide pathway in HDF is due to senescence and supports the hypotheses that senescence represents a distinct program of cell development that can be differentiated from quiescence. Additional studies disclosed the ability of ceramide to induce a senescent phenotype. Thus, when exogenous ceramide (15 microM) was administered to young WI-38 HDF, it produced endogenous levels comparable to those observed in senescent cells (as determined by metabolic labeling studies). Ceramide concentrations of 10-15 microM inhibited the growth of young HDF and induced a senescent phenotype by its ability to inhibit DNA synthesis and mitogenesis. These concentrations of ceramide also induced retinoblastoma dephosphorylation and inhibited serum-induced AP-1 activation in young HDF, thus recapitulating basic biochemical and molecular changes of senescence. Sphingomyelinase and ceramide may thus be implicated as mediators of cellular senescence.
Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
M E
ME
Venable
J Y
JY
Lee
M J
MJ
Smyth
A
A
Bielawska
L M
LM
Obeid
D001483:Base Sequence; D001665:Binding Sites; D016923:Cell Death; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D016922:Cellular Senescence / Q000187:drug effects / Q000502:physiology*; D002518:Ceramides / Q000378:metabolism / Q000494:pharmacology* / Q000502:physiology*; D003260:Contact Inhibition; D016895:Culture Media, Serum-Free; D004247:DNA / Q000096:biosynthesis; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013936:Thymidine / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism
D002518:Ceramides; D016895:Culture Media, Serum-Free; D009838:Oligodeoxyribonucleotides; D018808:Transcription Factor AP-1; D004247:DNA; D013108:Sphingomyelin Phosphodiesterase; D013936:Thymidine