Identification of a defect in the phospholipase D/diacylglycerol pathway in cellular senescence.

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.