Regulation of the sphingosine-recycling pathway for ceramide generation by oxidative stress, and its role in controlling c-Myc/Max function

In the present study, the regulation of the sphingosine-recycling pathway in A549 human lung adenocarcinoma cells by oxidative stress was investigated. The generation of endogenous long-chain ceramide in response to exogenous C ₆-cer (C₆-ceramide), which is FB1 (fumonisin B1)-sensitive, was employed to probe the sphingosine-recycling pathway. The data showed that ceramide formation via this pathway was significantly blocked by GSH and NAC (N-acetylcysteine) whereas it was enhanced by H₂O ₂, as detected by both palmitate labelling and HPLC/MS. Similar data were also obtained using a novel approach that measures the incorporation of 17Sph (sphingosine containing 17 carbons) of 17C₆-cer (C ₆-cer containing a 17Sph backbone) into long-chain 17C ₁₆-cer in cells by HPLC/MS, which was significantly decreased and increased in response to GSH and H₂O₂ respectively. TNF (tumour necrosis factor)-α, which decreases the levels of endogenous GSH, increased the generation of C₁₆-cer in response to C₆-cer, and this was blocked by exogenous GSH or NAC, or by the overexpression of TPxI (thioredoxin peroxidase I), an enzyme that reduces the generation of intracellular ROS (reactive oxygen species). Additional data showed that ROS regulated both the deacylation and reacylation steps of C₆-cer. At a functional level, C₆-cer inhibited the DNA-binding function of the c-Myc/Max oncogene. Inhibition of the generation of long-chain ceramide in response to C₆-cer by FB1 or NAC significantly blocked the modulation of the c-Myc/Max function. These data demonstrate that the sphingosine- recycling pathway for the generation of endogenous long-chain ceramide in response to exogenous C₆-cer is regulated by ROS, and plays an important biological role in controlling c-Myc function.