Venable M E, Blobe G C, Obeid L M
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1994 Oct 21;269(42):26040-4.
Normal cells become senescent in culture after a limited number of population doublings becoming unable to respond to mitogens. This raises the possibility of defects in mitogenic signaling pathways in cellular senescence. In contrast to young human diploid fibroblasts (HDF), their senescent counterparts failed to undergo protein kinase C translocation in response to serum stimulation. On the other hand, phorbol 12-myristate 13-acetate was equally active in inducing protein kinase C translocation in young and senescent HDF. This suggested a defect in generation of the endogenous activator of protein kinase C, diacylglycerol. Stimulation of young HDF with serum resulted in 3-4-fold generation of diacylglycerol (DAG). In contrast, senescent cells displayed insignificant DAG formation in response to serum. The mechanism of DAG generation was investigated next. In young HDF, serum induced a 5-fold activation of the phospholipase D (PLD) pathway as measured by the incorporation of exogenous ethanol into phosphatidylethanol, which is a measure of the transphosphatidylation reaction of PLD. In contrast, PLD in senescent cells was not activated by serum. Since senescent cells demonstrate significant elevations in the level of endogenous ceramide, the impact of ceramide on the PLD/DAG pathway was also investigated. A soluble analog of ceramide, C6-ceramide, was found to inhibit serum-stimulated DAG accumulation and PLD activation in young cells. These data demonstrate for the first time a defect in PLD activation in cellular senescence and suggest that ceramide may be responsible for the inhibition of this pathway.
正常细胞在培养中经过有限次数的群体倍增后会进入衰老状态，变得无法对有丝分裂原作出反应。这增加了细胞衰老过程中有丝分裂信号通路存在缺陷的可能性。与年轻的人二倍体成纤维细胞（HDF）相比，其衰老对应细胞在血清刺激下未能发生蛋白激酶C易位。另一方面，佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯在诱导年轻和衰老HDF中的蛋白激酶C易位方面同样有效。这表明蛋白激酶C的内源性激活剂二酰基甘油的生成存在缺陷。用血清刺激年轻HDF会导致二酰基甘油（DAG）生成增加3 - 4倍。相比之下，衰老细胞对血清刺激显示出微不足道的DAG形成。接下来研究了DAG生成的机制。在年轻HDF中，血清诱导磷脂酶D（PLD）途径激活5倍，这通过将外源性乙醇掺入磷脂酰乙醇来衡量，而这是PLD转磷脂酰化反应的一种度量。相比之下，衰老细胞中的PLD未被血清激活。由于衰老细胞内源性神经酰胺水平显著升高，因此也研究了神经酰胺对PLD/DAG途径的影响。发现神经酰胺的一种可溶性类似物C6 - 神经酰胺可抑制年轻细胞中血清刺激的DAG积累和PLD激活。这些数据首次证明了细胞衰老过程中PLD激活存在缺陷，并表明神经酰胺可能是该途径抑制的原因。
