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 C₆-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 H₂O₂, 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 (C₆-cer containing a 17Sph backbone) into long-chain 17C₁₆-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 TPx I (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 longchain 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.