Lo I-Chung, Shih Jun-Ming, Jiang Meei Jyh
Department of Cell Biology and Anatomy, National Cheng Kung University Medical College, Taiwan.
J Biomed Sci. 2005;12(2):377-88. doi: 10.1007/s11373-005-1703-2.
Monocyte chemotactic protein-1 (MCP-1), a potent chemoattractant for monocytes, is thought to play a major role in atherosclerosis, but whether its atherogenic effects involve the direct modulation of vascular smooth muscle cell (SMC) functions remains unclear. This study examined the effects of MCP-1 on the migration of cultured A7r5 SMCs and the signaling pathways involved. Addition of recombinant MCP-1 stimulated SMC migration in modified Boyden chambers coated with type I collagen in a concentration-dependent manner, with 10(-9) M being maximally effective. Using untreated A7r5 cells, two MCP-1 receptors, CCR2 and CCR4, were detected and MCP-1 secretion was significantly increased by stimulation with platelet-derived growth factor. MCP-1-stimulated A7r5 migration was completely blocked by the NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI), and dose-dependently inhibited by polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), suggesting a role for reactive oxygen species (ROS) in this process. During MCP-1 stimulation, ROS production increased rapidly, then gradually decayed over 60 min, and this effect was markedly decreased by pretreatment with DPI or PEG-SOD. Interestingly, U0126 and PD98059, which inhibit activation of extracellular signal-regulated kinases 1/2 (ERK 1/2), significantly inhibited MCP-1-activated ROS generation. Furthermore, transfection of an active mutant of MEK1 (ERK 1/2 kinase) markedly increased superoxide production in rat aortic smooth muscle cells, as detected by dihydroethydium staining, suggesting that ERK 1/2 activation stimulates ROS generation. ERK 1/2 activation was increased for at least 30 min in cells incubated with MCP-1, and this effect was abolished by U0126 or DPI pretreatment. These results demonstrate that MCP-1 is a chemoattractant for SMCs and that MCP-1-stimulated migration requires both ROS production and ERK 1/2 activation in a positive activation loop, which may contribute to the atherogenic effects of MCP-1.
单核细胞趋化蛋白-1（MCP-1）是一种对单核细胞具有强大趋化作用的物质，被认为在动脉粥样硬化中起主要作用，但其致动脉粥样硬化作用是否涉及对血管平滑肌细胞（SMC）功能的直接调节仍不清楚。本研究检测了MCP-1对培养的A7r5 SMC迁移的影响以及相关的信号通路。添加重组MCP-1以浓度依赖的方式刺激了涂有I型胶原的改良博伊登小室中的SMC迁移，10^(-9) M时效果最佳。使用未处理的A7r5细胞，检测到两种MCP-1受体，CCR2和CCR4，并且血小板衍生生长因子刺激后MCP-1分泌显著增加。MCP-1刺激的A7r5迁移被NAD(P)H氧化酶抑制剂二苯基碘鎓（DPI）完全阻断，并被聚乙二醇偶联的超氧化物歧化酶（PEG-SOD）剂量依赖性抑制，表明活性氧（ROS）在此过程中起作用。在MCP-1刺激期间，ROS产生迅速增加，然后在60分钟内逐渐衰减，并且用DPI或PEG-SOD预处理可显著降低这种效应。有趣的是，抑制细胞外信号调节激酶1/2（ERK 1/2）激活的U0126和PD98059显著抑制了MCP-1激活的ROS产生。此外，通过二氢乙锭染色检测到，MEK1（ERK 1/2激酶）的活性突变体转染显著增加了大鼠主动脉平滑肌细胞中的超氧化物产生，表明ERK 1/2激活刺激ROS产生。在用MCP-1孵育的细胞中，ERK 1/2激活至少增加30分钟，并且这种效应被U0126或DPI预处理消除。这些结果表明，MCP-1是SMC的趋化剂，并且MCP-1刺激的迁移在一个正反馈激活环中需要ROS产生和ERK 1/2激活，这可能有助于MCP-1的致动脉粥样硬化作用。
