1-磷酸鞘氨醇诱导平滑肌细胞迁移过程中氧自由基的产生需要Gα12/13蛋白介导的磷脂酶C激活。
Sphingosine-1-phosphate-induced oxygen free radical generation in smooth muscle cell migration requires Galpha12/13 protein-mediated phospholipase C activation.
作者信息
Roztocil Eliza, Nicholl Suzanne M, Davies Mark G
机构信息
Vascular Biology and Therapeutics Program, Department of Surgery, University of Rochester, Rochester, NY 14642, USA.
出版信息
J Vasc Surg. 2007 Dec;46(6):1253-1259. doi: 10.1016/j.jvs.2007.08.013.
BACKGROUND
Sphingosine-1-phosphate (S-1-P) is a bioactive sphingolipid that stimulates the migration of vascular smooth muscle cell (VSMC) through G-protein coupled receptors; it has been shown to activate reduced nicotinamide dinucleotide phosphate hydrogen (NAD[P]H) oxidase. The role of phospholipase C (PLC) in oxygen free radical generation, and the regulation of VSMC migration in response to S-1-P, are poorly understood.
METHODS
Rat arterial VSMC were cultured in vitro. Oxygen free radical generation was measured by fluorescent redox indicator assays in response to S-1-P (0.1microM) in the presence and absence of the active PLC inhibitor (U73122; U7, 10nM) or its inactive analog U73343 (InactiveU7, 10nM). Activation of PLC was assessed by immunoprecipitation and Western blotting for the phosphorylated isozymes (beta and gamma). Small interfering (si) RNA to the G-proteins Galphai, Galphaq, and Galpha12/13 was used to downregulate specific proteins. Statistics were by one-way analysis of variance (n = 6).
RESULTS
S-1-P induced time-dependent activation of PLC-beta and PLC-gamma; PLC-beta but not PLC-gamma activation was blocked by U7 but not by InactiveU7. PLC-beta activation was Galphai-independent (not blocked by pertussis toxin, a Galphai inhibitor, or Galphai2 and Galphai3 siRNA) and Galphaq-independent (not blocked by glycoprotein [GP] 2A, a Galphaq inhibitor, or Galphaq siRNA). PLC-beta activation and cell migration was blocked by siRNA to Galpha12/13. Oxygen free radical generation induced by S-1-P, as measured by dihydroethidium staining, was significantly inhibited by U7 but not by InactiveU7. Inhibition of oxygen free radicals with the inhibitor diphenyleneiodonium resulted in decreased cell migration to S-1-P. VSMC mitogen-activated protein kinase activation and VSMC migration in response to S-1-P was inhibited by PLC- inhibition.
CONCLUSION
S-1-P induces oxygen free radical generation through a Galpha12/13, PLC-beta-mediated mechanism that facilitates VSMC migration. To our knowledge, this is the first description of PLC-mediated oxygen free radical generation as a mediator of S-1-P VSMC migration and illustrates the need for the definition of cell signaling to allow targeted strategies in molecular therapeutics for restenosis.
背景
1-磷酸鞘氨醇（S-1-P）是一种生物活性鞘脂，可通过G蛋白偶联受体刺激血管平滑肌细胞（VSMC）迁移；已证明它能激活还原型烟酰胺腺嘌呤二核苷酸磷酸（NAD[P]H）氧化酶。磷脂酶C（PLC）在氧自由基生成中的作用以及对S-1-P应答时VSMC迁移的调节尚不清楚。
方法
体外培养大鼠动脉VSMC。在存在和不存在活性PLC抑制剂（U73122；U7，10nM）或其无活性类似物U73343（无活性U7，10nM）的情况下，通过荧光氧化还原指示剂测定法测量对S-1-P（0.1μM）应答时的氧自由基生成。通过免疫沉淀和针对磷酸化同工酶（β和γ）的蛋白质印迹法评估PLC的激活。针对G蛋白Gαi、Gαq和Gα12/13的小干扰（si）RNA用于下调特定蛋白质。采用单因素方差分析进行统计学分析（n = 6）。
结果
S-1-P诱导PLC-β和PLC-γ的时间依赖性激活；U7可阻断PLC-β而非PLC-γ的激活，无活性U7则不能。PLC-β的激活不依赖Gαi（不受Gαi抑制剂百日咳毒素或Gαi2和Gαi3 siRNA的阻断）且不依赖Gαq（不受Gαq抑制剂糖蛋白[GP] 2A或Gαq siRNA的阻断）。针对Gα12/13的siRNA可阻断PLC-β的激活和细胞迁移。通过二氢乙锭染色测量，S-1-P诱导的氧自由基生成被U7显著抑制，而无活性U7则不能。用抑制剂二苯碘鎓抑制氧自由基导致细胞对S-1-P的迁移减少。PLC抑制可抑制VSMC丝裂原活化蛋白激酶的激活以及VSMC对S-1-P的迁移。
结论
S-1-P通过Gα12/13、PLC-β介导的机制诱导氧自由基生成，该机制促进VSMC迁移。据我们所知，这是首次将PLC介导的氧自由基生成描述为S-1-P诱导VSMC迁移的介质，并说明了定义细胞信号传导以实现再狭窄分子治疗靶向策略的必要性。
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