The study of cellular senescence offers a powerful model for aging research in that it is manipulatable and can borrow from the extensive oncology literature on cell-cycle control. Senescence is initiated by dominant internal controls of proliferation which are activated after a predictable number of cell divisions. The principle investigator discovered a dominant acting inhibitor of mitogenesis in senescent fibroblasts, ceramide. Ceramide is substantially elevated in senescent cells and when added to young cells, ceramide can mimic a senescent phenotype. Further, ceramide can specifically inhibit the key mitogenic player, PKC, by blocking phosphatidylcholine phospholipase D (PLD) activation and subsequent diacylglycerol (DAG) generation in young fibroblasts thereby mimicking senescent cells. Sphingolipids (most notably ceramide) have emerged as novel mediators of important cellular pathways. Ceramide has been shown to be important in the induction of the antiproliferative pathways, apoptosis and cell differentiation. Therefore, this proposal sets forth to test the hypothesis that ceramide is a general inhibitor of cell proliferation in senescent cells in part by inhibiting PLD activation.
The specific aims of this proposal are: 1) To examine the relation between ceramide and cell senescence m human umbilical vein endothelial cells (HUVEC's) by: analyzing the production of ceramide and activation of sphingomyelinase in senescent cells and studying the effects of ceramide on critical mitogenic mediators in young cells; 2) To identify the relationship between ceramide and interleukin-1 in HUVEC's by: examining the expression of IL-1 and production of prostacyclin in senescent cells and the effect of lL-1beta treatment on sphingomyelinase activation, ceramide production and growth inhibition; 3) To characterize ceramide induction of the senescent cell histological marker, beta-galactosidase, in HUVEC's by: evaluating the effects of varying ceramide treatments on the expression of beta- galactosidase as compared with the normal onset of cellular senescence; and 4) To determine the effect of senescence on PLD in HUVEC's by: examining the effects of senescence on PLD activation by mitogens as compared with the effect of ceramide and determining the effect of senescence on the levels of PLD1.
