Both the tumor suppressor p53 and ceramide have been implicated in the cellular response to stress, and specifically, the regulation of apoptosis. Whereas, the ceramide pathway has been shown to be conserved in the yeast Saccharomyces cerevisiae, neither p53 nor any genetic homologs are. This implies that the ceramide stress response evolved at an earlier stage compared to p53. The stress response pathways regulated by these two molecules have common features. These include the ability of both pathways to induce cell cycle arrest, senescence, and apoptosis in response to adverse stimuli. Additionally, these two pathways share some downstream targets that are key regulators of the cell cycle and apoptosis (e.g. the retinoblastoma protein and caspase-3). It was recently demonstrated that p53 functions "upstream" of ceran-tide in the genotoxic stress response leading to apoptosis. It was found that p53 up-regulation in response to y-irradiation led to the gradual accumulation of cellular ceramide occurring over several hours. It was also shown that ceramide can function in a p53-independent manner where it can induce apoptosis even in the absence of p53. Importantly, we recently found that the majority of the accumulated ceramide following p53 up-regulation occurs in a caspase-dependent manner. The work in this proposal aims at investigating the signaling pathways and the molecular events by which p53 up-regulation results in ceramide accumulation. First, p53-activated apoptotic pathways will be investigated in two cell models of p53- dependent function. Based on the preliminary data, the focus will be on those pathways that either directly or indirectly involve caspase activation. p53 functions as a transcription factor in the induction or repression of a large number of genes. Thus, the expression and activity of a number of key modulators of apoptosis in response to p53 up-regulation will be examined. These will include caspases, death receptors (which couple to caspases via adapter proteins), and several p53-induced genes that were recently shown to induce apoptosis when overexpressed (Bax, Noxa, PERP, and PIG3). Second, the order by which the different genes become expressed, or their proteins activated, in response to p53 and in relation to ceramide accumulation will be studied. Candidate regulators of downstream ceramide accumulation will be identified by virtue of their modulated expression or function prior to ceramide accumulation. The functional relationship between p53-activation of specific pathways and ceramide accumulation will be explored. Specific apoptotic modulators that are candidate regulators of ceramide will be individually examined with regard to their ability to induce ceramide accumulation. The essentiality of these candidates with respect to ceramide accumulation and apoptosis will be explored by using various approaches to inhibit their function and examining the effects on ceramide accumulation. Third, the biochemical pathways that are activated to generate ceramide in response to p53 up-regulation will be investigated. Specifically, the contribution of sphingomyelinase activation, which generates ceramide from hydrolysis of membrane sphingomyelin, and de novo synthesis of ceramide by activation of ceramide synthase will be studied. The specific metabolic pathways leading to ceramide accumulation will then be linked to specific apoptotic modulators that are activated by p53. These studies will help us understand the molecular network by which p53 exerts it pro-apoptotic function and may shed some light on how these two major stress response pathways (p53 up-regulation and ceramide accumulation) became linked during evolution.
