DIFFERENTIAL ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASES BY H2O2 AND O-2(-) IN VASCULAR SMOOTH-MUSCLE CELLS
DIFFERENTIAL ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASES BY H2O2 AND O-2(-) IN VASCULAR SMOOTH-MUSCLE CELLS
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DOI：
10.1161/01.res.77.1.29
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发表时间：
1995-07-01
影响因子：
20.1
通讯作者：
BERK, BC
中科院分区：
文献类型：
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作者：
BAAS, AS;BERK, BC
过氧化氢和O-2(-)等活性氧物种的增加可能在动脉粥样硬化和再狭窄相关的血管平滑肌细胞生长中起重要作用。在以前的工作中，我们发现过氧化氢可以刺激血管平滑肌细胞的生长和原癌基因的表达。在本研究中，我们比较了H_2O_2和O_2(-)对培养的大鼠主动脉血管平滑肌细胞生长和信号转导的影响。O-2(-)由萘喹啉二酮LY83583以浓度依赖的方式产生。用[H-3]胸腺嘧啶核苷掺入法检测，200 mU/L过氧化氢(比0.1%血清增加110%)和1 mU/L LY83583(增加175%)刺激血管平滑肌细胞生长，其水平与10 mU g/mL血小板衍生生长因子(增加210%)相当。由于丝裂原活化蛋白激酶(MAPK)的激活是最早的生长因子信号事件之一，因此通过蛋白质印迹迁移率的变化和髓鞘碱性蛋白的磷酸化来衡量MAP激酶的活性。LY83583(最大浓度为10mU/L)可浓度依赖性地增加MAP-K活性，但不能被H_2O_2诱导。LYS3583激活MAP的时间进程在5~10min达到最大值，20min恢复到基线水平。LY83583对MAP激酶的激活依赖于蛋白激酶C。MKP-1是一种转录调控的氧化还原敏感蛋白酪氨酸/苏氨酸磷酸酶。虽然H_2O_2对MKP-1mRNA的诱导作用强于LY83583，但MKP-1的表达增加并不能解释H_2O_2不能刺激MAP的原因，因为直到60分钟才检测到MKP-1的表达。研究发现，O-2(-)和H_2O_2都能刺激血管平滑肌细胞生长，但只有O-2(-)能迅速激活MAP激酶，这表明H_2O_2的促有丝分裂作用需要额外的信号事件。
Increased generation of active oxygen species such as H2O2 and O-2(-) may be important in vascular smooth muscle cell growth associated with atherosclerosis and restenosis. In previous work, we showed that H2O2 stimulated vascular smooth muscle cell growth and proto-oncogene expression. In the present study, we compared the effects of H2O2 and O-2(-) on cultured rat aortic vascular smooth muscle cell growth and signal transduction. O-2(-) was generated in a concentration-dependent manner by the naphthoquinolinedione LY83583. Vascular smooth muscle cell growth, as measured by [H-3]thymidine incorporation, was stimulated by 200 mu mol/L H2O2 (110% increase versus 0.1% serum) and 1 mu mol/L LY83583 (175% increase) to levels comparable to 10 mu g/mL platelet-derived growth factor (210% increase). Since activation of mitogen-activated protein kinase (MAP kinase) is one of the earliest growth factor signal events, the activity of MAP kinase was measured by changes in mobility on Western blot and by phosphorylation of myelin basic protein. There was a concentration-dependent increase in MAP kinase activity by LY83583 (maximum, 10 mu mol/L) but not by H2O2. The time course for activation of MAP kinase by LYS3583 showed a maximum at 5 to 10 minutes with return to baseline by 20 minutes. Activation of MAP kinase by LY83583 was protein kinase C dependent. Expression of MAP kinase phosphatase-1 (MKP-1), a transcriptionally regulated redox-sensitive protein tyrosine/threonine phosphatase, was also measured. Although H2O2 induced MKP-1 mRNA to a greater extent than did LY83583, the increased MKP-1 expression could not explain the inability of H2O2 to stimulate MAP kinase, because mRNA levels were not detected until 60 minutes. The findings that both O-2(-) and H2O2 stimulate vascular smooth muscle cell growth but only O-2(-) rapidly activates MAP kinase suggest that additional signal events are required for mitogenic effects of H2O2.