Sterol carrier protein-2 (SCP-2) involvement in cholesterol hydroperoxide cytotoxicity as revealed by SCP-2 inhibitor effects.
Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. The purpose of this study was to determine whether SCP-2, under oxidative stress conditions, might also traffic hydroperoxides of cholesterol, thereby disseminating their cytotoxic effects. Two inhibitors, SCPI-1 and SCPI-3, known to block cholesterol binding by an insect SCP-2, were used to investigate this. A mouse fibroblast transfectant clone (SC2F) overexpressing SCP-2 was found to be substantially more sensitive to apoptotic killing induced by liposomal 7α-hydroperoxycholesterol (7α-OOH) than a wild-type control. 7α-OOH uptake by SC2F cells and resulting apoptosis were both inhibited by SCPI-1 or SCPI-3 at a subtoxic concentration. Preceding cell death, reactive oxidant accumulation and loss of mitochondrial membrane potential were also strongly inhibited. Similar SCPI protection against 7α-OOH was observed with two other types of SCP-2-expressing mammalian cells. In striking contrast, neither inhibitor had any effect on H(2)O(2)-induced cell killing. To learn whether 7α-OOH cytotoxicity is due to uptake/transport by SCP-2, we used a fluorescence-based competitive binding assay involving recombinant SCP-2, NBD-cholesterol, and SCPI-1/SCPI-3 or 7α-OOH. The results clearly showed that 7α-OOH binds to SCP-2 in SCPI-inhibitable fashion. Our findings suggest that cellular SCP-2 not only binds and translocates cholesterol but also cholesterol hydroperoxides, thus expanding their redox toxicity and signaling ranges under oxidative stress conditions.
Kriska
Tamas
T
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA.
Pilat
Anna
A
Schmitt
Jared C
JC
Girotti
Albert W
AW
D000083:Acetanilides / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D048588:Benzoxazines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D002460:Cell Line; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000633:toxicity; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism / Q000494:pharmacology
D000083:Acetanilides; D048588:Benzoxazines; D002352:Carrier Proteins; C532816:N-(4-((4-(3,4-dichlorophenyl)-1,3-thiazol-2-yl)amino)phenyl)acetamide; D017382:Reactive Oxygen Species; D013844:Thiazoles; C072087:sterol carrier proteins; C041011:cholesterol 7-hydroperoxide; C002843:cholesterol hydroperoxide; D002784:Cholesterol; D006861:Hydrogen Peroxide