Loss of sphingosine kinase-1 activates the intrinsic pathway of programmed cell death: modulation of sphingolipid levels and the induction of apoptosis.
Activation of sphingosine kinase-1 (SK1) by overexpression or agonist stimulation promotes cell proliferation, survival, and anti-apoptosis. Studies on the function of endogenous SK1 are lacking. Endogenous SK1 has been shown to be down-regulated under stress, and knockdown of the enzyme reduces the percentage of viable MCF-7 breast cancer cells (Taha, T. A. et al. 2004. J. Biol. Chem. 279, 20546-20554). In this study, we examined the mechanisms by which SK1 loss affects the growth of cells. Knockdown of the enzyme by small interfering RNA caused cell cycle arrest and induced apoptosis. Cell death involved effector caspase activation, cytochrome c release and Bax oligomerization in the mitochondrial membrane, thus placing SK1 knockdown upstream of the mitochondrial pathway of apoptosis. SK1 knockdown also induced significant increases in ceramide levels in whole cells and in mitochondria enriched fractions of cells. Inhibition of de novo sphingolipid biosynthesis with myriocin significantly attenuated Bax oligomerization and downstream caspase activation after SK1 loss. These studies for the first time implicate endogenous SK1 as an important survival enzyme in MCF-7 cells and link the biological consequences of knocking down the enzyme to its biochemical role as a regulator of sphingolipid metabolism.
Taha
Tarek A
TA
Division of General Internal Medicine, Ralph H. Johnson Veterans Administration Hospital, Charleston, South Carolina, USA.
Kitatani
Kazuyuki
K
El-Alwani
Mazen
M
Bielawski
Jacek
J
Hannun
Yusuf A
YA
Obeid
Lina M
LM
D000230:Adenocarcinoma / Q000473:pathology; D017209:Apoptosis / Q000502:physiology*; D001943:Breast Neoplasms / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle; D045744:Cell Line, Tumor / Q000187:drug effects / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism; D045304:Cytochromes c / Q000502:physiology; D004789:Enzyme Activation; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D005260:Female; D018390:Gene Targeting; D006801:Humans; D008928:Mitochondria / Q000502:physiology; D009363:Neoplasm Proteins / Q000172:deficiency / Q000502:physiology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000172:deficiency / Q000502:physiology*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D034741:RNA, Small Interfering / Q000494:pharmacology; D013107:Sphingolipids / Q000096:biosynthesis / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000502:physiology; D051020:bcl-X Protein / Q000502:physiology; D000099157:Sphingosine Kinase
D020169:Caspases; D002518:Ceramides; D045304:Cytochromes c; D005229:Fatty Acids, Monounsaturated; D009363:Neoplasm Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013107:Sphingolipids; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D000099157:Sphingosine Kinase; C001996:thermozymocidin