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)-a, 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.
Sultan
Iyad
I
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA.
Senkal
Can E
CE
Ponnusamy
Suriyan
S
Bielawski
Jacek
J
Szulc
Zdzislaw
Z
Bielawska
Alicja
A
Hannun
Yusuf A
YA
Ogretmen
Besim
B
D000215:Acylation; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002518:Ceramides / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D005786:Gene Expression Regulation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D018384:Oxidative Stress; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013110:Sphingosine / Q000378:metabolism*; D013379:Substrate Specificity; D054481:Thioredoxin Reductase 1; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology
D050976:Basic-Leucine Zipper Transcription Factors; D002518:Ceramides; C067850:Myc associated factor X; D016271:Proto-Oncogene Proteins c-myc; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; C516124:TXNRD1 protein, human; D054481:Thioredoxin Reductase 1; D013880:Thioredoxin-Disulfide Reductase; D013110:Sphingosine