Venable M E, Lee J Y, Smyth M J, Bielawska A, Obeid L M
Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
J Biol Chem. 1995 Dec 22;270(51):30701-8. doi: 10.1074/jbc.270.51.30701.
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.
最近，鞘磷脂循环，即膜鞘磷脂被活化的鞘磷脂酶水解以生成神经酰胺的过程，已成为白血病及其他细胞类型中细胞分化和凋亡的关键途径。在此，我们研究该途径在WI-38人二倍体成纤维细胞（HDF）衰老过程中的作用。我们发现，随着细胞进入衰老期，神经酰胺的内源性水平显著增加（4倍）且具有特异性（与其他脂质相比）。对神经酰胺增加机制的研究发现，衰老细胞中中性鞘磷脂酶活性升高了8 - 10倍。当HDF因血清剥夺或接触抑制进入静止期时，鞘磷脂酶活性和神经酰胺水平没有变化。因此，HDF中鞘磷脂酶/神经酰胺途径的激活是由于衰老，这支持了衰老代表一种可与静止期区分开来的独特细胞发育程序的假说。进一步的研究揭示了神经酰胺诱导衰老表型的能力。因此，当向年轻的WI-38 HDF施加外源性神经酰胺（15 microM）时，其产生的内源性水平与衰老细胞中观察到的水平相当（通过代谢标记研究确定）。10 - 15 microM的神经酰胺浓度抑制了年轻HDF的生长，并通过其抑制DNA合成和有丝分裂的能力诱导了衰老表型。这些神经酰胺浓度还诱导了年轻HDF中视网膜母细胞瘤的去磷酸化，并抑制了血清诱导的AP-1激活，从而重现了衰老的基本生化和分子变化。因此，鞘磷脂酶和神经酰胺可能作为细胞衰老的介质。
