Hypoxia-induced neuronal apoptosis is mediated by de novo synthesis of ceramide through activation of serine palmitoyltransferase.
Cellular hypoxia can lead to cell death or adaptation and has important effects on development, physiology, and pathology. Here, we investigated the role and regulation of ceramide in hypoxia-induced apoptosis of SH-SY5Y neuroblastoma cells. Hypoxia increased the ceramide concentration; subsequently, we observed biochemical changes indicative of apoptosis, such as DNA fragmentation, nuclear staining, and poly ADP-ribose polymerase (PARP) cleavage. The hypoxic cell death was potently inhibited by a caspase inhibitor, zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone). l-Cycloserine, a serine palmitoyltransferase (SPT) inhibitor, and fumonisin B(1) (FB(1)), a ceramide synthase inhibitor, inhibited the hypoxia-induced increase in ceramide, indicating that the increase occurred via the de novo pathway. Hypoxia increased the activity and protein levels of SPT2, suggesting that the hypoxia-induced increase in ceramide is due to the transcriptional up-regulation of SPT2. Specific siRNA of SPT2 prevented hypoxia-induced cell death and ceramide production. However, hypoxia also increased the cellular level of glucosylceramide, which was inhibited by a glucosylceramide synthase (GCS) inhibitor and specific siRNA, but not a ceramidase inhibitor. The increase in glucosylceramide was accompanied by increases in both PARP cleavage and DNA fragmentation. Together, the current results suggest that both SPT and GCS may regulate the cellular level of ceramide, and thus may be critical enzymes for deciding the fate of the cells exposed to hypoxia.
Kang
Mi Sun
MS
Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University, 221 Huksuk-Dong, Dongjak-Ku, Seoul 156-756, South Korea.
Ahn
Kyong Hoon
KH
Kim
Seok Kyun
SK
Jeon
Hyung Jun
HJ
Ji
Jung Eun
JE
Choi
Jong Min
JM
Jung
Kwang Mook
KM
Jung
Sung Yun
SY
Kim
Dae Kyong
DK
D017209:Apoptosis; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002518:Ceramides / Q000378:metabolism*; D063388:Endocannabinoids; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004983:Ethanolamines / Q000494:pharmacology; D005963:Glucosylceramides / Q000378:metabolism; D005964:Glucosyltransferases / Q000378:metabolism; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009829:Oleic Acids; D051102:Serine C-Palmitoyltransferase / Q000378:metabolism*; D013110:Sphingosine / Q000378:metabolism
D002518:Ceramides; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D004983:Ethanolamines; D005963:Glucosylceramides; D009025:Morpholines; D009829:Oleic Acids; C033595:N-oleoylethanolamine; C033110:RV 538; D051102:Serine C-Palmitoyltransferase; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; D013110:Sphingosine