Abstract
Ultraviolet light-induced apoptosis can be caused by DNA damage but also involves immediate-early cell death cascades characteristic of death receptor signaling. Here we show that the UV light-induced apoptotic signaling pathway is unique, targeting Bax activation at the mitochondrial membrane independent of caspase-8 or cathepsin D activity. Cells deficient in acid sphingomyelinase (ASMase) do not show UV light-induced Bax activation, cytochrome c release, or apoptosis. In ASMase-deficient cells, the apoptotic UV light response is restored by stable or transient expression of human ASMase. Bax conformational change in ASMase(-/-) cells is also caused by synthetic C(16)-ceramide acting on intact cells or isolated mitochondria. The results suggest that UV light-triggered ASMase activation is essentially required for Bax conformational change leading to mitochondrial release of pro-apoptotic factors like cytochrome c and Smac.
Highlights
Ultraviolet light induces a complex transcriptional cellular response that is similar to that of tumor promotors and mitogens yet includes the induction of growth arrest and apoptosis (1, 2)
The results suggest that UV light-triggered acid sphingomyelinase (ASMase) activation is essentially required for Bax conformational change leading to mitochondrial release of pro-apoptotic factors like cytochrome c and Smac
UV light triggers a variety of signaling pathways, including nuclear DNA damage and activation of the tumor suppressor gene p53 (3) or triggering of cell death receptors either directly (4, 5) or by autocrine release of death ligands leading to mitochondrial damage and cytochrome c release (6, 7), which leads to the activation of executioner caspases and eventually to apoptosis
Summary
Cell Culture and Materials—HeLa, Bcl2-HeLa, and NPD fibroblasts (provided by Dr Sandhoff, Bonn, Germany, 35) were cultured in DMEM and Jurkat, MS1418, and ASM-MS1418 lymphoblasts The supernatant was centrifuged at 16,000 ϫ g for 20 min at 4 °C to remove any residual mitochondria, and the resulting cytosolic fraction was recovered. Cells were incubated with specific primary antibodies for 1 h, washed with 0.1% saponin in PBS, and incubated for 30 min with secondary antibodies conjugated with Alexa Fluor 568 and/or Alexa Fluor 488 (Molecular Probes, Leiden, Netherlands). DNA Fragmentation—For DNA fragmentation assays, 107 Jurkat cells were pelleted at 1000 ϫ g, lysed in 200 l of lysis buffer (100 mM Tris-Cl, pH 8.5, 200 mM NaCl, 0.2% (w/vϪ1) SDS, 5 mM EDTA, and 2 mg mlϪ1 proteinase K), and incubated for 30 min at 56 °C. RNA was removed by addition of 0.2 mg mlϪ1
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