Folate Stress Induces Apoptosis via p53-dependent de Novo Ceramide Synthesis and Up-regulation of Ceramide Synthase 6*

Background: Sphingolipid ceramide regulates cellular responses to stress stimuli. Results: Aldh1l1, the enzyme regulating folate metabolism, leads to CerS6 up-regulation and C16-ceramide accumulation in a p53-dependent manner as a proapoptotic signal. Conclusion: Ceramide mediates the cellular response to nongenotoxic folate stress. Significance: We have demonstrated the interaction between two major metabolic pathways, folate and sphingolipids, in regulation of cellular homeostasis. We have investigated the role of ceramide in the cellular adaptation to folate stress induced by Aldh1l1, the enzyme involved in the regulation of folate metabolism. Our previous studies demonstrated that Aldh1l1, similar to folate deficiency, evokes metabolic stress and causes apoptosis in cancer cells. Here we report that the expression of Aldh1l1 in A549 or HCT116 cells results in the elevation of C16-ceramide and a transient up-regulation of ceramide synthase 6 (CerS6) mRNA and protein. Pretreatment with ceramide synthesis inhibitors myriocin and fumonisin B1 or siRNA silencing of CerS6 prevented C16-ceramide accumulation and rescued cells supporting the role of CerS6/C16-ceramide as effectors of Aldh1l1-induced apoptosis. The CerS6 activation by Aldh1l1 and increased ceramide generation were p53-dependent; this effect was ablated in p53-null cells. Furthermore, the expression of wild type p53 but not transcriptionally inactive R175H p53 mutant strongly elevated CerS6. Also, this dominant negative mutant prevented accumulation of CerS6 in response to Aldh1l1, indicating that CerS6 is a transcriptional target of p53. In support of this mechanism, bioinformatics analysis revealed the p53 binding site 3 kb downstream of the CerS6 transcription start. Interestingly, ceramide elevation in response to Aldh1l1 was inhibited by silencing of PUMA, a proapoptotic downstream effector of p53 whereas the transient expression of CerS6 elevated PUMA in a p53-dependent manner indicating reciprocal relationships between ceramide and p53/PUMA pathways. Importantly, folate withdrawal also induced CerS6/C16-ceramide elevation accompanied by p53 accumulation. Overall, these novel findings link folate and de novo ceramide pathways in cellular stress response.