Roles Of Ceramide And Apoptosis-inducing Factor In UVA-induced Apoptosis

BACKGROUND: Ultraviolet radiation (UV)-mediated apoptosis is triggered by at least two independent pathways, i.e. UV-induced DNA damage and UV-induced cell death receptor activation. DNA damage activates the intrinsic apoptosis pathway resulting in the release of a number of pro-apoptotic factors (e.g. cytochrome c, AIF and Smac/DIABLO) from the intermembrane space of mitochondria. In the presence of ATP/dATP, cytochrome c binds to the WD40 repeat domain of Apaf-1 and promotes Apaf-1 oligomerization, leading to the formation of a Apaf-1—cytochrome c multimeric complex. This complex recruits and interacts with multiple procaspase-9 at a 1:1 ratio through protein-protein interaction between N-terminal CARD domains of Apaf-1 and that of procaspase-9. Procaspase-9 molecules approach each other and are activated by autocleavage. Activated caspase-9 initiates a cascade by cleaving procaspase-3 , -6 and -7. Active caspase-3, -6 and -7 specifically cleavage a set of protein substrates and then lead to apoptosis. The death receptors (e.g., Fas, tumor necrosis factor receptor, etc.) activate the extrinsic apoptosis signaling pathway: Fas ligand (FasL) is a homotrimeric molecule and each FasL trimer binds three Fas molecules. Upon binding to the trimeric FasL, Fas recruits a cytosolic adapter protein FADD through a homotypic interaction between the Fas intracellular DD and the FADD C- terminal DD. FADD contains the DED in its N-terminal region. This domain interacts with the DED in the prodomain of procaspase-8 and recruits procaspase-8 to Fas. Fas, FADD and procaspase-8 form death-inducing signaling complex (DISC). Procaspase-8 has weak proteolytic activity, which is enhanced by oligomerization within the DISC. Caspase-8 activated by autocatalysis is releasedinto the cytoplasm and initiates the caspase cascades that activate downstream effector caspases which cause apoptosis. In one word, UV-induced apoptosis is caspase-dependent.It has been reported that UV can produce ceramide via a variety of pathways. Ceramide is one member of sphingolipids and the second messenger that exerts many biological effects including apoptosis, cell cycle arrest and aging. Yet, several issues are to be solved such as whether ceramide is produced, and what are the role and the downstream molecules of ceramide during UVA-induced apoptosis.What is released from mitochondria together with cytochrome c is apoptosis-inducing factor (AIF), which is a caspase-independent apoptosis effector. Upon apoptosis stimulation, AIF translocates from the mitochondria into the nucleus where AIF causes peripheral chromatin condensation and large scale DNA fragmentation (approximately 50kbp). It has yet not been reported whether UVA-induced apoptosis is caspase-independent and what effects AIF exerts on UVA-induced apoptosis.OBJECTIVE AND SIGNIFICANCE: Epidemiological studies suggest that solar UV is responsible for skin tumor development via gene mutations and immunosuppression. Simultaneously, UV-induced apoptosis provides a defensive mechanism that removes unwanted cells and prevents cells from malignant transformation. UVB (290-320nm) and UVC (180-290 nm)- induced apoptosis has been well studied, but little attention was paid to the apoptosis induced by UVA (320-400nm) which comprises over 90% of the solar UV. The objective of this study is to investigate the molecular mechanism of UVA-induced apoptosis, particularly, the role of ceramide and AIF in UVA-induced apoptosis. The results will help to further clarify the molecular mechanism of UVA-induced apoptosis.METERIALS AND METHODS: Lymphoblasts JY cells and MS1418 cells were used in this study. Following various doses of UVA radiation, the rate of cell death was measured by MTT reduction assay, oligonucleosomal DNA fragmentation was detected by DNA ladder assay, apoptotic cells stained by hoechst33258 were observed and counted through fluorescent microscope, western blotting was used toexamine signal transduction pathway, the subcellular location of AIF was observed through immunoelectron microscopy, enforced expression of AIF was used to observe the characteristics of AIF-induced apoptosis, and down-regulation of AIF by RNA interference was used to investigate the effect of AIF on UVA-induced apoptosis.RESULTS AND DISCUSSION.-1. The rate of apoptosis induced by UVA is higher in JY cells than in MS1418. MTT reduction assay and DNA ladder assay show that the rate of apoptosis induced by UVA is higher in JY cells than in MS 1418 at the same dose of UVA. Both JY and MS 1418 cells are human lymphoblasts, and their difference is that JY cells are from normal human and MS 1418 are from Niemann-Pick patients with inherited deficiency of acid sphingomyelinase (ASM). ASM is one of leading enzymes which produce ceramide. Therefore, we inferred that UVA induces apoptosis via activating ASM and producing ceramide.2. In the presence of specific inhibitor of ASM, there was no significant difference of the rate of apoptosis induced by UVA between JY and MS1418 cells. Moreover, extrinsic ceramide-induced cell death in JY cells or MS1418 is not markedly different. NB6, the specific inhibitor of ASM, can significantly lighten the UVA-induced apoptosis in JY cells, however, has no effect on UVA-induced apoptosis in MS 1418 cells; Therefore, there was no significant difference of the rate of apoptosis induced by UVA between JY and MS 1418 cells. Administering extrinsic C2-ceramide, not exposed to UVA, cell death rate in JY cells or MSI418 is not markedly different. These evidences further support the putative conclusion that UVA induces apoptosis via activating ASM and producing ceramide.3. Ceramide activates MAPK signaling pathways during UVA-induced apoptosis. MAPK signaling pathways contain ERK pathway, JNK pathway and p38 pathway. In comparison with the cells unexposed to UVA radiation, the phosphorylation of ERK level increased in MS1418 cells, and decreasedin JY cells; the phosphorylation of JNK level increased in JY cells, and remained unchanged in MS1418 cells; and the phosphorylation of p38 level slightly increased in JY and MS 1418 cells. In the presence of specific inhibitor of ASM, these differences in between JY and MS 1418 cells disappeared. Therefore, UVA-induced apoptosis depends on activating ASM and producing ceramide that activates JNK signaling pathway and inhibits ERK signaling pathway.4. UVA-indued apoptosis cannot be completely blocked by pan-caspase inhibitor, and AIF translocated from mitochondria into the nucleus and caused peripheral chromatin condensation during the early stage of UVA-induced apoptosis. The results indicate that there is a caspase-independent apoptosis signaling pathway, and it has been known that AIF-induced apoptosis is caspase-independent. Moreover, western blotting and immunoelectron microscopy found that AIF translocated from mitochondria into nucleus and caused peripheral chromatin condensation, which is one of characteristics of AIF-induced apoptosis. The evidence accords with AIF-induced apoptosis and indicates that AIF participated in UVA-induced apoptosis.5. Overexpression of recombinant AIF protein induced caspase-independent apoptosis. Under normal circumstances, transcription and translation of aif gene give rise to a -67 kDa precursor molecule with a 102 amino acid mitochondrial localization sequence (MLS) in its NH2 terminus, which cannot induce apoptosis. Upon into mitochondria, the MLS is removed and forms a ~57 kDa mature molecule. Upon apoptosis stimulation, AIF translocates from the mitochondria into the cytoplasm and the nucleus where AIF causes peripheral chromatin condensation and large scale DNA fragmentation (approximately 50kbp) in a caspase- independent manner. In order to study apoptotic activity of AIF, the segment of aif gene (mature AIF molecule) was cloned and expressed, which has not been reported so far. Overexpression of mature AIF induced caspase-independent apoptosis.6. Down-regulation of AIF by RAN interference (RNAi) alleviatedUVA-induced apoptosis. Finally, to determine whether AIF function is necessary for UVA-induced apoptosis, AIF protein level was downregulated by RNAi and down-regulation of AIF reduced UVA-induced apoptosis. Down-regulation of AIF combined with caspase inhibitor almost completely abolished UVA-induced apoptosis. The results suggest that AIF and caspase are involved in two different pathways and UVA-induced apoptosis is dependent on AIF and caspase.CONCLUSIONS:1. UVA-induced apoptosis is dependent on ceramide produced by activation of ASM;2. UVA-induced apoptosis is dependent on inhibition of ERK signaling pathway and activation of JNK signaling pathway;3. Ceramide is on the upstream of ERK and JNK signaling pathways;4. AIF -induced apoptosis is caspase-independent, AIF and caspase are involved in two different apoptosis signaling pathways;5. UVA-induced apoptosis is dependent on caspases and AIF.