The Antiapoptotic Effect of Low-dose UVB Irradiation in NIH3T3 Cells Involves Caspase Inhibition¶
The Antiapoptotic Effect of Low-dose UVB Irradiation in NIH3T3 Cells Involves Caspase Inhibition¶
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低剂量 UVB 照射对 NIH3T3 细胞的抗凋亡作用涉及 Caspase 抑制¶
DOI：
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
R. Goto
中科院分区：
文献类型：
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作者：
Y. Ibuki;R. Goto
UVB辐射是一种众所周知的凋亡诱导因子。然而，我们以前已经发现，低剂量的UVB照射抑制细胞凋亡诱导的血清饥饿和缺乏细胞外基质，涉及一个显着的抑制caspase-3/7激活。在这项研究中，我们报告的UVB诱导的抗凋亡作用和caspase-3/7抑制活性氧（ROS）之间的关系。抗氧化剂N-乙酰半胱氨酸可部分阻止UVB诱导的抗凋亡作用。活性氧产生剂甲萘醌和促氧化剂H2 O2也表现出与UVB诱导的抗细胞凋亡作用相似的作用，表明活性氧参与了抗细胞凋亡作用。UVB照射显着抑制caspase-3/7的激活，这是由蛋白水解的抑制，而不是由酶活性本身的抑制。蛋白水解的预防也通过以下两个结果证实：一个是在细胞色素c和dATP存在下暴露于UVB的细胞裂解物中抑制体外caspase-3/7和-9活化，这是由ROS的产生引起的，另一个是在活性caspase-9存在下抑制体外caspase-3/7活化。这些结果表明，抑制caspase级联下游线粒体ROS的产生，导致一个显着的抑制caspase-3/7的激活，是小剂量UVB照射的抗凋亡作用的原因之一。细胞凋亡的特征是形态学改变如细胞皱缩、染色质浓缩和膜起泡，以及生化改变如DNA片段化、半胱天冬酶的激活和半胱天冬酶包括多聚半胱天冬酶对各种底物的切割。（ADP-核糖）聚合酶等。胱天蛋白酶是关键蛋白，特别是，据报道，胱天蛋白酶-3通常被许多死亡信号激活，并且与DNA片段化过程和与凋亡相关的其它形态学变化有关（1）。胱天蛋白酶-3通过蛋白水解激活，在天冬氨酸残基处切割32 kDa前体，产生17和12 kDa亚基的活性异源二聚体（2）。蛋白水解过程需要减少半胱氨酸残基在procaspase-3以及周围的催化位点的酶活性（3，4）。因此，除了形成与细胞色素c（cyt c）、凋亡蛋白酶激活因子-1（apaf-1）、半胱天冬酶-9、dATP和半胱天冬酶原-3复合的溶酶体外，半胱天冬酶-3激活还需要硫氧还蛋白和谷胱甘肽（GSH）等抗氧化分子维持的细胞还原环境，以诱导细胞凋亡（5，6）。巯基烷基化剂和自发巯基氧化抑制半胱天冬酶-3活性（7，8），半胱氨酸残基的还原增强半胱天冬酶-3活化（9，10）。另一方面，有很多证据表明活性氧（ROS）参与了细胞凋亡的过程。众所周知，促氧化剂H2 O2可诱导细胞凋亡（11）。此外，在一些凋亡过程中观察到脂质过氧化（12，13）、ROS的产生（14-17）和抗氧化防御的下调，其特征在于GSH水平降低（16-18）和过氧化氢酶、超氧化物歧化酶和硫氧还蛋白转录水平的逐渐下降（19，20）。这些发现与半胱氨酸残基的还原是半胱天冬酶-3活化所必需的事实不一致。一些报道显示，早期半胱天冬酶激活诱导ROS从线粒体爆发，包括细胞色素c释放，其主要促成细胞凋亡过程中的细胞间氧化（17，21），并且氧化具有相反的功能，例如通过半胱天冬酶的失活阻断细胞凋亡（22，23）。尽管UVB照射也诱导涉及半胱天冬酶活化的细胞凋亡（24），但尚未讨论UV诱导的细胞内分子氧化与导致细胞凋亡的半胱天冬酶活化之间的关系。紫外线诱导的细胞凋亡的一个标志性事件是表皮内晒伤细胞的发生。紫外线损伤的角质形成细胞不能修复损伤，就像晒伤细胞一样发生凋亡，从而导致皮肤老化。* 信件应寄往：日本静冈市矢田市静冈大学营养与环境科学研究生院放射生物学实验室，邮编422-8526。传真：81-54-264-5795;电子邮件：ibuki@sea.u-shizuoka-ken.ac.jp Ac-DEVD-MCA，乙酰基-Asp-Glu-Val-Asp-a-（4-甲基香豆酰-7-酰胺）; Ac-LEHD-MCA，乙酰基-Leu-Glu-His-Asp-a-（4-甲基-香豆酰-7-酰胺）; BSA，牛血清白蛋白; CDCFH-DA，6-羧基-2，79-二氯二氢荧光素二乙酸酯，二（乙酰氧基甲酯）; CS，小牛血清; cyt c，细胞色素c; DTT，二硫苏糖醇; DMEM，达尔伯克改良伊格尔培养基; ECM，细胞外基质; EGTA，乙二醇双（2-氨基乙醚）-N，N，N9，N9-四乙酸; FCM，流式细胞仪; FDA，二乙酸荧光素; GSH，谷胱甘肽; HEPES，N-（2-羟乙基）哌嗪-N9-（2-乙磺酸）; Hoechst 33342，双苯甲酰亚胺; NAC，N-乙酰半胱氨酸; PAGE，聚丙烯酰胺凝胶电泳; PBS，磷酸盐缓冲盐水; PI 3-激酶，磷脂酰肌醇3-激酶; ROS，活性氧; SDS，十二烷基硫酸钠。2003年美国光生物学会0031-8655/01 $5.0010.00
UVB irradiation is a well-known apoptosis induction factor. However, we have previously found that low doses of UVB irradiation inhibited apoptosis induced by both serum starvation and lack of extracellular matrix, involving a significant inhibition of caspase-3/7 activation. In this study, we report on the relationship between the UVB-induced antiapoptotic effect and caspase-3/7 inhibition by reactive oxygen species (ROS). The UVB-induced antiapoptotic effect was partially prevented by an antioxidant agent, N-acetylcysteine. A ROS-generating agent, menadione and a pro-oxidant agent, H2O2 also showed an effect that was similar to the UVBinduced antiapoptotic effect, indicating that ROS contributed to the antiapoptotic effect. UVB irradiation significantly suppressed caspase-3/7 activation, which was caused by the inhibition of proteolysis and not by the inhibition of enzymatic activity itself. The prevention of proteolysis was also confirmed by both the following results: one is the inhibition of in vitro caspase-3/7 and -9 activation in cell lysates exposed to UVB in the presence of cytochrome c and dATP, which was caused by the production of ROS, and the other is the inhibition of in vitro caspase-3/7 activation in the presence of active caspase-9. These results showed that the inhibition of the caspase cascade downstream mitochondria by ROS production, leading to a significant inhibition of caspase-3/7 activation, was one of the causes of the antiapoptotic effect by small doses of UVB irradiation. INTRODUCTION Apoptosis is characterized both by morphological changes such as cell shrinkage, chromatin condensation and membrane blebbing and by biochemical changes such as DNA fragmentation, activation of caspases and cleavage of various substrates by caspases including poly(ADP-ribose)polymerase, etc. Caspases are key proteins, in particular, caspase-3 is reported to be commonly activated by numerous death signals and is related to DNA fragmentation processes and other morphological changes associated with apoptosis (1). Caspase-3 is activated by proteolysis that cleaves the 32 kDa precursor at aspartic acid residues to generate an active heterodimer of 17 and 12 kDa subunits (2). The proteolytic process requires the reduction of cysteine residues in procaspase-3 as well as those around the catalytic site for enzymatic activities (3,4). Therefore, a cellular-reducing environment maintained by antioxidant molecules, such as thioredoxin and glutathione (GSH), is required for caspase-3 activation to induce apoptosis in addition to the formation of apoptosome that is complex with cytochrome c (cyt c), apoptosis protease–activating factor-1 (apaf-1), caspase-9, dATP and procaspase-3 (5,6). Thiolalkylating agents and spontaneous thiol oxidation inhibit caspase-3 activity (7,8), and a reduction of cysteine residue enhances caspase-3 activation (9,10). On the other hand, there is much evidence regarding the involvement of reactive oxygen species (ROS) in the process of apoptosis. The pro-oxidant agent, H2O2, is well known to induce apoptosis (11). Furthermore, lipid peroxidation (12,13), production of ROS (14–17) and down-regulation of antioxidant defenses characterized by a reduced GSH level (16–18) and a progressive decline in the transcript levels for catalase, superoxide dismutase and thioredoxin (19,20) have been observed in some apoptotic processes. These findings are inconsistent with the fact that the reduction of cysteine residues is required for caspase-3 activation. Some reports showed that early caspase activation induced ROS burst from the mitochondria including cyt c release, which mainly contributed to intercellular oxidation in the process of apoptosis (17,21), and that the oxidation has opposite functions such as blocking of apoptosis via the inactivation of caspases (22,23). Although UVB irradiation also induces apoptosis involving caspase activation (24), the relationship between UV-induced oxidation of intracellular molecules and caspase activation leading to apoptosis has not been discussed. A hallmark event of UV-induced apoptosis is the occurrence of sunburn cells within the epidermis. UV-damaged keratinocytes that fail to repair the damage undergo apoptosis as sunburn cells to {Posted on the website on 1 February 2003. *To whom correspondence should be addressed at: Laboratory of Radiation Biology, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1, Yada, Shizuoka-shi 422-8526, Japan. Fax: 81-54-264-5795; e-mail: ibuki@sea.u-shizuoka-ken.ac.jp Abbreviations: Ac-DEVD-MCA, acetyl-Asp-Glu-Val-Asp-a-(4-methylcoumaryl-7-amide); Ac-LEHD-MCA, acetyl-Leu-Glu-His-Asp-a-(4methyl-coumaryl-7-amide); BSA, bovine serum albumin; CDCFH-DA, 6-carboxy-2,79-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester); CS, calf serum; cyt c, cytochrome c; DTT, dithiothreitol; DMEM, Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N9,N9-teteraacetic acid; FCM, flowcytometer; FDA, fluorescein diacetate; GSH, glutathione; HEPES, N-(2-hydroxyethyl)piperazine-N9-(2-ethanesulphonic acid); Hoechst33342, bis-benzimide; NAC, N-acetylcysteine; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PI3-kinase, phosphatidylinositol 3-kinase; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate. 2003 American Society for Photobiology 0031-8655/01 $5.0010.00
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影响因子：
7.4
作者：
Zhang, XS;Rosenstein, BS;Wei, HC
通讯作者：
Wei, HC
DOI：
10.1006/bbrc.1999.1908
发表时间：
2000-02-05
影响因子：
3.1
作者：
Baker, A;Dos Santos, B;Powis, G
通讯作者：
Powis, G
影响因子：
6
作者：
Pierce, RH;Campbell, JS;Fausto, N
通讯作者：
Fausto, N
影响因子：
56.9
作者：
KANE, DJ;SARAFIAN, TA;BREDESEN, DE
通讯作者：
BREDESEN, DE