TNF-alpha-mediated apoptosis is initiated in caveolae-like domains

Caveolae-like domains (CLDs) have been hypothesized to mediate apoptosis, s ince they contain sphingomyelin and initiate the conversion of sphingomyeli n to ceramide, To address whether CLDs are directly involved in apoptosis, CLDs from U937 cells were isolated, taking advantage of their detergent ins olubility and low density. The CLDs contained alkaline phosphatase as well as many signaling molecules, including Fyn, protein kinase Ch, Raf-1, phosp holipase C gamma 1, and tyrosine phosphoproteins. Immunoblotting and immuno fluorescent data showed that TNF receptor 1 colocalized with CD36 in CLDs, suggesting that TNF-alpha-initiated apoptosis occurs in CLDs, When cells we re incubated with lipoprotein-deficient medium, the cholesterol concentrati on was greatly decreased in CLDs but not in other fractions, implying that the CLDs were selectively disrupted, In the CLD-disrupted cells, the surfac e expression of TNF receptor 1 and CD36 was significantly reduced, Analysis of cellular morphology, percent DNA fragmentation, DNA laddering, and casp ase-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 req uire CLDs. Taken together, these data strongly imply that TNF-alpha-mediate d apoptosis is initiated in CLDs.