Purification and identification of secreted oxidative stress-induced factors from vascular smooth muscle cells
Purification and identification of secreted oxidative stress-induced factors from vascular smooth muscle cells
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DOI：
10.1074/jbc.275.1.189
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发表时间：
2000-01-07
影响因子：
4.8
通讯作者：
Berk, BC
中科院分区：
文献类型：
--
作者：
Liao, DF;Jin, ZG;Berk, BC
活性氧与动脉粥样硬化和高血压的发病机制有关，部分是通过促进血管平滑肌细胞（VSMC）的生长。我们以前已经表明，LY 83583，一种O-2（.-）的产生者，激活细胞外信号调节激酶（ERK 1/2），具有早期（10 min）和晚期（2 h）峰值，并刺激VSMC生长。要调查是否分泌氧化应激诱导因子（SOXF）从VSMC负责晚期ERK 1/2激活响应LY 83583，我们纯化推定SOXF蛋白从条件培养基（LY 83583曝光2小时）的ERK 1/2激活的基础上，通过连续色谱法。经毛细管色谱、电喷雾串联质谱和数据库检索鉴定的蛋白质包括热休克蛋白90-α（HSP 90-alpha）和亲环素B，Western blot分析显示HSP 90-α而非HSP 90-β特异性分泌。人重组HSP 90-α再现了条件培养基对ERK 1/2活化的影响。这些结果表明，短暂的氧化应激导致蛋白质因子从VSMC持续释放，可以刺激ERK 1/2。这些因素可能是活性氧影响血管功能的重要介质。
Reactive oxygen species have been implicated in the pathogenesis of atherosclerosis and hypertension, in part by promoting vascular smooth muscle cell (VSMC) growth, We have previously shown that LY83583, a generator of O-2(.-), activated extracellular signal-regulated kinases (ERK1/2) with early (10 min) and late (2 h) peaks and stimulated VSMC growth. To investigate whether secreted oxidative stress-induced factors (termed SOXF) from VSMC were responsible for late ERK1/2 activation in response to LY83583, we purified putative SOXF proteins from conditioned medium (2 h of LY83583 exposure) by sequential chromatography based on activation of ERK1/2. Proteins identified by capillary chromatography, electrospray ionization tandem mass spectrometry, and data base searching included heat shock protein 90-alpha: (HSP90-alpha) and cyclophilin B, Western blot analysis of conditioned medium showed specific secretion of HSP90-alpha but not HSP90-beta, Immunodepletion of HSP90-alpha from conditioned medium significantly inhibited conditioned medium-induced ERK1/2 activation. Human recombinant HSP90-alpha reproduced the effect of conditioned medium on ERK1/2 activation. These results show that brief oxidative stress causes sustained release of protein factors from VSMC that can stimulate ERK1/2. These factors may be important mediators for the effects of reactive oxygen species on vascular function.