| The role of ceramide neo-genesis in cellular stress response signaling is gaining increasing attention with recent progress in elucidating the novel roles and biochemical properties of the ceramide synthase (CerS) proteins. Selective tissue and subcellular distribution of the six mammalian CerS isoforms, combined with distinct fatty acyl chain length substrate preferences, implicate differential functions of specific ceramide species in cellular signaling. We report here that ionizing radiation (IR) induces de novo synthesis of ceramide to influence HeLa cell apoptosis by specifically activating CerS isoforms 2, 5, and 6 to generate opposing anti- and pro-apoptotic ceramides in mitochondrial membranes. Overexpression of CerS2 resulted in partial protection from IR-induced apoptosis whereas overexpression of CerS5 yielded elevated levels of apoptosis in HeLa cells. Knockdown studies determined that CerS2 is responsible for all observable IR-induced C24:0 CerS activity, and although CerS5 and CerS6 each confer ∼50% of the C16:0 CerS baseline activity, both are required for IR-induced activity. Additionally, co-immunoprecipitation studies suggest that CerS2, 5, and 6 exist as heterocomplexes in HeLa cells, providing further insight into regulation of CerS proteins. Furthermore, genetic loss-of-function of sensors and transducers of DNA double strand break repair confer the CerS-mediated lethal pathway in intestines via apoptosis of crypt stem cell clonogens of sv129/B6Mre11 ATLD1/ATLD1 and C57BL/6Prkdc/SCID (SCID) mice exposed to low-dose IR, providing in vivo models to study CerS in the context of DNA damage repair in intact tissue. These data add to the growing body of evidence demonstrating the important and distinct roles of the CerS proteins both in vitro and in vivo, as well as the interplay among them in a stress stimulus-, cell- and tissue-type-, and subcellular compartment-specific manner. |
