ULTRAVIOLET-LIGHT INDUCES APOPTOSIS VIA DIRECT ACTIVATION OF CD95 (FAS APO-1) INDEPENDENTLY OF ITS LIGAND CD95L/

Induction of apoptosis in keratinocytes by UV light is a critical even t in photocarcinogenesis. Although p53 is of importance in this proces s, evidence exists that other pathways play a role as well. Therefore, we studied whether the apoptosis-related surface molecule CD95 (Fas/A PO-1) is involved. The human keratinocyte cell line HaCaT expresses CD 95 and undergoes apoptosis after treatment with UV light or with the l igand of CD95 (CD95L). Incubation with a neutralizing CD95 antibody co mpletely prevented CD95L-induced apoptosis but not UV-induced apoptosi s, initially suggesting that the CD95 pathway may not be involved. How ever, the protease CPP32, a downstream molecule of the CD95 pathway, w as activated in UV-exposed HaCaT cells, and UV-induced apoptosis was b locked by the ICE protease inhibitor zVAD, implying that at least simi lar downstream events are involved in CD95- and UV-induced apoptosis. Activation of CD95 results in recruitment of the Pas-associated protei n with death domain (FADD) that activates ICE proteases. Immunoprecipi tation of UV-exposed HaCaT cells revealed that UV light also induces r ecruitment of FADD to CD95. Since neutralizing anti-CD95 antibodies fa iled to prevent UV-induced apoptosis, this suggested that UV light dir ectly activates CD95 independently of the ligand CD95L. Confocal laser scanning microscopy showed that UV light induced clustering of CD95 i n the same fashion as CD95L. Prevention of UV-induced CD95 clustering by irradiating cells at 10 degrees C was associated with a significant ly reduced death rate. Together, these data indicate that UV light dir ectly stimulates CD95 and thereby activates the CD95 pathway to induce apoptosis independently of the natural ligand CD95L. These findings f urther support the concept that UV light can affect targets at the pla sma membrane, thereby even inducing apoptosis.