Chen Kate Chao-Wei, Zhou You, Xing Kuiyi, Krysan Kostantyn, Lou Marjorie F
Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE 68583, USA.
Exp Eye Res. 2004 Jun;78(6):1057-67. doi: 10.1016/j.exer.2004.02.004.
Low level of reactive oxygen species (ROS) has been shown to play an important role in host defense and mediating mitogen-stimulated cell signaling in several cell types. This study is to identify the mitogen-induced endogenous ROS generation and the range of exogenous H(2)O(2) that initiate redox signaling and cell proliferation in human lens epithelial cells (HLE B3), using platelet-derived growth factor (PDGF) as a model. To detect ROS generation, serum starved HLE cells (1.6 million) were loaded with fluorescent dye, 2',7'-dichlorofluorescin diacetate (DCFH-DA), before exposing to PDGF (1 ng ml(-1)). The fluorescence generated from the oxidant-sensitive DCFH, the intracellular product of DCFH-DA hydrolysate, was immediately measured in live cells by confocal laser light microscopy (lambda(Ex)=488 nm, lambda(Em)=522 nm, laser power=10%). PDGF-stimulated cells showed strong transient fluorescence during the 60 min while no fluorescence could be seen in the unstimulated cells. The PDGF-induced fluorescence could be suppressed with cells preloaded with N-acetyl-L-cysteine (NAC, 30 mm), catalase (1 mg ml(-1)), or D-mannitol (100mm). The ability of catalase to penetrate and function in HLE cells was confirmed by western blot, enzyme activity and immunofluorescence microscopic analyses. PDGF induced DNA synthesis within one hour as measured by (3)H-thymidine incorporation, and transiently activated the mitogen-activated protein kinases (MAPKs) of ERK1/2 and JNK. PDGF-stimulated DNA synthesis and MAPK activation were eliminated in the presence of catalase or mannitol. Low levels of H(2)O(2) (10-20 microm) mimicked PDGF in both MAPK stimulation and cell proliferation. In conclusion, the mitogenic stimulus function of PDGF in HLE cells appears to be mediated via ROS to activate MAPKs and cell proliferation, which can be mimicked by low levels of H(2)O(2). It is proposed that the physiological function of ROS, the redox signaling, is present in the HLE cells and may play an important role in the development and maintenance of the lens.
低水平的活性氧（ROS）已被证明在宿主防御以及介导几种细胞类型中丝裂原刺激的细胞信号传导方面发挥重要作用。本研究旨在以血小板衍生生长因子（PDGF）为模型，确定丝裂原诱导的内源性ROS生成以及引发人晶状体上皮细胞（HLE B3）氧化还原信号传导和细胞增殖的外源性H₂O₂范围。为了检测ROS生成，在将血清饥饿的HLE细胞（160万个）用荧光染料2',7'-二氯荧光素二乙酸酯（DCFH-DA）加载后，再将其暴露于PDGF（1 ng/ml）。通过共聚焦激光显微镜（激发波长λ(Ex)=488 nm，发射波长λ(Em)=522 nm，激光功率=10%）立即在活细胞中测量由氧化敏感的DCFH（DCFH-DA水解产物的细胞内产物）产生的荧光。PDGF刺激的细胞在60分钟内显示出强烈的瞬时荧光，而未刺激的细胞中未见荧光。预先用N-乙酰-L-半胱氨酸（NAC，30 mM）、过氧化氢酶（1 mg/ml）或D-甘露醇（100 mM）加载的细胞可抑制PDGF诱导的荧光。通过蛋白质印迹、酶活性和免疫荧光显微镜分析证实了过氧化氢酶在HLE细胞中的穿透能力和功能。通过³H-胸苷掺入法测量，PDGF在一小时内诱导DNA合成，并瞬时激活ERK1/2和JNK的丝裂原活化蛋白激酶（MAPK）。在过氧化氢酶或甘露醇存在的情况下，PDGF刺激的DNA合成和MAPK激活被消除。低水平的H₂O₂（10 - 20 μM）在MAPK刺激和细胞增殖方面模拟了PDGF。总之，PDGF在HLE细胞中的促有丝分裂刺激功能似乎是通过ROS介导的，以激活MAPK和细胞增殖，低水平的H₂O₂可以模拟这一过程。有人提出，ROS的生理功能即氧化还原信号传导存在于HLE细胞中，并且可能在晶状体的发育和维持中发挥重要作用。
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