An integrative approach to studying sphingolipid metabolism reveals p53 as a master regulator of the pathway

Sphingolipids (SPLs) are bioactive lipids playing vital functions in cellular stress responses. The tumor suppressor p53 has been implicated in regulating few specific SPL enzymes; however, a comprehensive understanding of p53's overall impact on SPL metabolism is lacking. Here, we employed an integrative biochemical approach combining a novel flux tracing method (using d17dihydrosphingosine) with in situ enzymatic activity assays in the context of treatment with Doxorubicin (Dox), a DNA-damaging agent causing well-established dose-dependent activation of p53. Furthermore, our previous studies established dose-specific modulation of SPLs by sublethal (low dose) versus lethal (high dose) Dox. Here, we exploited this model to focus on the role of p53, and found: i) Both low and high Dox enhanced the rate of synthesis of select dihydroceramide species, d17:0/16:0, d17:0/18:0, and d17:0/20:0, implicating activation of specific ceramide synthases (CerS 1/4 and 5/6), with p53 dependence only at low Dox; ii) Novel p53-dependent suppression of dihydroceramide desaturase (DES) activity at both Dox doses; iii) Both doses of Dox impaired the synthesis of d17hexosylceramide and d17sphingomyelin, with an unanticipated role for p53 only at low Dox. iv) With respect to inhibition of d17sphingomyelin synthesis, an investigation into ceramide transport to the Golgi identified the ceramide transport protein 1 (CERT1) as a novel target of Dox (reduction of protein and activity) and p53 (reduction of activity, particularly at low Dox). These observations underscore p53's prominent role as a master regulator of SPL metabolism, inducing major remodeling of cellular SPL metabolism with extensive and integrated effects on sphingolipid synthesis.