TNF-alpha-mediated apoptosis is initiated in caveolae-like domains.
Caveolae-like domains (CLDs) have been hypothesized to mediate apoptosis, since they contain sphingomyelin and initiate the conversion of sphingomyelin to ceramide. To address whether CLDs are directly involved in apoptosis, CLDs from U937 cells were isolated, taking advantage of their detergent insolubility and low density. The CLDs contained alkaline phosphatase as well as many signaling molecules, including Fyn, protein kinase Calpha, Raf-1, phospholipase Cgamma1, and tyrosine phosphoproteins. Immunoblotting and immunofluorescent data showed that TNF receptor 1 colocalized with CD36 in CLDs, suggesting that TNF-alpha-initiated apoptosis occurs in CLDs. When cells were incubated with lipoprotein-deficient medium, the cholesterol concentration was greatly decreased in CLDs but not in other fractions, implying that the CLDs were selectively disrupted. In the CLD-disrupted cells, the surface expression of TNF receptor 1 and CD36 was significantly reduced. Analysis of cellular morphology, percent DNA fragmentation, DNA laddering, and caspase-3 activity showed that TNF-alpha-mediated apoptosis was blocked in CLD-disrupted cells, whereas anti-Fas-mediated apoptosis was not. Since Fas was not found in CLDs of Jurkat cells, apoptosis by Fas ligation might not require CLDs. Taken together, these data strongly imply that TNF-alpha-mediated apoptosis is initiated in CLDs.
Ko
Y G
YG
Ilchun Molecular Medicine Institute Medical Research Center, Cancer Research Center, Seoul National University College of Medicine, Korea. ygko@nature.skku.ac.kr
Lee
J S
JS
Kang
Y S
YS
Ahn
J H
JH
Seo
J S
JS
D016475:3T3 Cells; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000378:metabolism; D017209:Apoptosis / Q000276:immunology*; D018955:CD36 Antigens / Q000096:biosynthesis; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism / Q000502:physiology; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D006801:Humans; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000096:biosynthesis / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I; D015398:Signal Transduction / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D020298:U937 Cells; D019014:fas Receptor / Q000502:physiology
D015703:Antigens, CD; D018955:CD36 Antigens; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D008565:Membrane Proteins; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor