Uchida Yoshikazu, Nardo Anna Di, Collins Vanessa, Elias Peter M, Holleran Walter M
Dermatology Service and Research Unit, Department of Veterans Affairs Medical Center, School of Medicine, University of California, San Francisco, USA.
J Invest Dermatol. 2003 Apr;120(4):662-9. doi: 10.1046/j.1523-1747.2003.12098.x.
Ultraviolet irradiation is a major environmental cause of skin cancers, whereas ultraviolet-induced DNA repair and apoptosis are defense mechanisms that rescue and/or protect keratinocytes from this risk. Multiple pathways are involved in ultraviolet-induced keratinocyte apoptosis, including activation of p38-mitogen-activated protein kinase, protein kinase C, and CD95, each of which are associated with caspase activation. Alternatively, ceramides could serve as ultraviolet-induced, second messenger lipids, because they induce cell cycle arrest and apoptosis in a variety of cell types, including keratinocytes. We investigated the role of ceramide versus caspase, and the responsible pathway for ceramide generation in ultraviolet B-induced apoptosis of cultured normal human keratinocytes maintained in low calcium (0.07 mm) medium. Ultraviolet B (40 mJ per cm2) significantly inhibited cultured normal human keratinocyte proliferation, assessed as [3H-methyl]thymidine-thymidine incorporation into DNA, 2 h after irradiation. Terminal nick deoxynucleotide end-labeling-positive apoptotic cells (14.8% at 24 h and 34.4% at 48 h) and trypan blue-positive apoptotic cells (8.4% at 24 h and 28.6% at 48 h) became evident in a time-dependent manner after ultraviolet B irradiation, in parallel with activation of caspase-3. The ceramide content of irradiated cultured normal human keratinocytes increased significantly by 8 h, whereas glucosylceramide only modestly increased, and sphingomyelin content remained unaltered. Metabolic studies with radiolabeled serine, palmitic acid, and phosphorylcholine revealed that the ultraviolet B-induced increase in ceramide results primarily from increased de novo synthesis rather than accelerated sphingomyelin hydrolysis. Increased ceramide synthesis, in turn, could be attributed to increased activity of ceramide synthase (i.e., 1.7-fold increase 8 h after ultraviolet B irradiation), whereas serine palmitoyltransferase activity did not change. Both fumonisin B1, an inhibitor of ceramide synthase, and ISP-1, myriocin an inhibitor of serine palmitoyltransferase, significantly attenuated the ultraviolet B-induced apoptosis in a caspase-3-independent fashion, whereas co-incubation with a caspase-3 inhibitor (Ac-DEVD-chloromethyl-ketone) further attenuated the ultraviolet B-induced apoptosis. Thus, increased de novo ceramide synthesis signals ultraviolet B-induced apoptosis, by a pathway independent of, but in concert with, caspase-3 activation.
紫外线照射是皮肤癌的主要环境诱因，而紫外线诱导的DNA修复和细胞凋亡是使角质形成细胞免受此风险的挽救和/或保护机制。紫外线诱导的角质形成细胞凋亡涉及多种途径，包括p38丝裂原活化蛋白激酶、蛋白激酶C和CD95的激活，每一种都与半胱天冬酶激活相关。另外，神经酰胺可作为紫外线诱导的第二信使脂质，因为它们在包括角质形成细胞在内的多种细胞类型中诱导细胞周期停滞和凋亡。我们研究了神经酰胺与半胱天冬酶的作用，以及在低钙（0.07 mM）培养基中培养的正常人角质形成细胞经紫外线B诱导凋亡过程中神经酰胺生成的相关途径。紫外线B（40 mJ/cm2）照射2小时后，显著抑制了培养的正常人角质形成细胞的增殖，这通过[3H-甲基]胸苷掺入DNA来评估。紫外线B照射后，末端脱氧核苷酸转移酶介导的缺口末端标记阳性凋亡细胞（24小时时为14.8%，48小时时为34.4%）和台盼蓝阳性凋亡细胞（24小时时为8.4%，48小时时为28.6%）呈时间依赖性增加，同时伴有半胱天冬酶-3的激活。照射后的培养正常人角质形成细胞的神经酰胺含量在8小时时显著增加，而葡糖神经酰胺仅适度增加，鞘磷脂含量保持不变。用放射性标记的丝氨酸、棕榈酸和磷酸胆碱进行的代谢研究表明，紫外线B诱导的神经酰胺增加主要源于从头合成增加而非鞘磷脂水解加速。反过来，神经酰胺合成增加可归因于神经酰胺合酶活性增加（即紫外线B照射8小时后增加1.7倍），而丝氨酸棕榈酰转移酶活性未改变。神经酰胺合酶抑制剂伏马菌素B1和丝氨酸棕榈酰转移酶抑制剂伊马菌素（ISP-1）均以不依赖半胱天冬酶-3的方式显著减弱紫外线B诱导的凋亡，而与半胱天冬酶-3抑制剂（Ac-DEVD-氯甲基酮）共同孵育则进一步减弱紫外线B诱导的凋亡。因此，从头神经酰胺合成增加通过一条独立于但与半胱天冬酶-3激活协同的途径发出紫外线B诱导凋亡的信号。
