Cholesterol-rich plasma membrane domains (lipid rafts) in keratinocytes: Importance in the baseline and UVA-induced generation of reactive oxygen species
Cholesterol-rich plasma membrane domains (lipid rafts) in keratinocytes: Importance in the baseline and UVA-induced generation of reactive oxygen species
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DOI：
10.1046/j.1523-1747.2002.01716.x
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发表时间：
2002-04-01
影响因子：
6.5
通讯作者：
Wulf, HC
中科院分区：
文献类型：
--
作者：
Gniadecki, R;Christoffersen, N;Wulf, HC
紫外线辐射的生物效应，如 DNA 损伤、诱变、细胞衰老和致癌，部分是由活性氧介导的。在未受辐射的细胞中，活性氧的主要已知来源是线粒体呼吸链和与几种膜生长因子受体功能性偶联的膜氧化酶。有证据表明线粒体也在紫外线照射后的氧化应激中发挥作用；然而，尚不清楚质膜水平的生化过程是否有助于调节活性氧的合成。为了阐明这个问题，我们在此研究了微域质膜组织在未照射和紫外线 A (340-400 nm) 照射的 HaCaT 角质形成细胞中氧化应激调节中的重要性。用荧光标记的霍乱毒素 B 亚基 (FITC-CTx) 对汇合的 HaCaT 培养物进行标记，揭示了 GM1 神经节苷脂和富含胆固醇的微域（脂筏）的存在，这些微域在相邻细胞的膜中形成连接样结构，并在其他地方形成斑片状微域。 FITC-CTx 标记水平存在明显的异质性：有些细胞组表现出明显的标记（FITC-CTx（高）），而其他细胞仅弱标记（FITC-CTx（低））。当使用荧光探针羧基-2',7'-二氯二氢荧光素二乙酸酯测量活性氧合成时，我们发现（i）基线和紫外线A诱导的活性氧合成与FITC-CTx标记的幅度相关，并且在FITC-CTx（高）细胞中最高； (ii) 在用甲基-β-环糊精和菲律宾蛋白螯合胆固醇或用 GM1 神经节苷脂处理后，膜域完整性被破坏的细胞中，活性氧合成减少； (iii)胆固醇耗尽的细胞中的活性氧合成在胆固醇补充后完全恢复。我们得出的结论是，质膜参与角质形成细胞氧化应激的调节，其微区结构的破坏会减少基线和紫外线 A 照射后活性氧的合成。我们假设脂筏相关蛋白可能参与活性氧的产生，膜结构的药理调节可能提供一种与光保护和皮肤癌发生相关的新治疗方法。
The biologic effects of ultraviolet radiation such as DNA damage, mutagenesis, cellular aging, and carcinogenesis are in part mediated by reactive oxygen species. In unirradiated cells the major known sources of reactive oxygen species are the mitochondrial respiratory chain and the membrane oxidases functionally coupled to several membrane growth factor receptors. There is evidence that mitochondria also play a role in oxidative stress after ultraviolet irradiation; however, it is unknown whether the biochemical processes at the level of the plasma membrane contribute to the regulation of reactive oxygen species synthesis. In order to elucidate this issue we examined here the importance of the microdomain plasma membrane organization in the regulation of oxidative stress in unirradiated and ultraviolet A (340-400 nm) irradiated HaCaT keratinocytes. Labeling of confluent HaCaT cultures with fluorescently tagged cholera toxin B subunit (FITC-CTx) revealed the presence of GM1 ganglioside and cholesterol-rich microdomains (lipid rafts) that formed junction-like structures in the membranes of adjacent cells and patchy microdomains elsewhere. There was a marked heterogeneity in the level of FITC-CTx labeling: there were groups of cells demonstrating prominent labeling (FITC-CTx(high)) whereas other cells were only weakly labeled (FITC-CTx(low)). When reactive oxygen species synthesis was measured with the fluorescent probe carboxy-2',7'-dichlorodihydrofluorescein diacetate, we found that (i) the baseline and ultraviolet-A-induced reactive oxygen species synthesis correlated with the magnitude of FITC-CTx labeling and was highest in the FITC-CTx(high) cells; (ii) reactive oxygen species synthesis was diminished in cells in which the integrity of membrane domains was disrupted by cholesterol sequestration with methyl-beta-cyclodextrin and filipin, or after treatment with GM1 ganglioside; (iii) reactive oxygen species synthesis in cholesterol-depleted cells was fully restored after cholesterol repletion. We conclude that the plasma membrane takes part in the regulation of oxidative stress in keratinocytes and disruption of its microdomain structure reduces reactive oxygen species synthesis both at the baseline and after ultraviolet A irradiation. We hypothesize that lipid-raft-associated protein(s) may be involved in the generation of reactive oxygen species and that pharmacologic modulation of membrane structure may provide a novel therapeutic approach relevant for photoprotection and cutaneous carcinogenesis.