Effects of membrane cholesterol depletion and GPI-anchored protein reduction on osteoblastic mechanotransduction.
Effects of membrane cholesterol depletion and GPI-anchored protein reduction on osteoblastic mechanotransduction.
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DOI：
10.1002/jcp.22579
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发表时间：
2011-09
影响因子：
5.6
通讯作者：
You, Jun
中科院分区：
文献类型：
--
作者：
Xing, Yanghui;Gu, Yan;Xu, Li-Chong;Siedlecki, Christopher A.;Donahue, Henry J.;You, Jun
我们以前证明，振荡流体流激活MC 3 T3-E1成骨细胞钙信号通路通过一种机制，涉及ATP释放和P2 Y2嘌呤受体。然而，流体流动启动细胞反应的分子机制仍然不清楚。越来越多的证据表明，脂筏作为细胞膜的重要结构成分之一，在将细胞外液切应力转化为细胞内反应中起着重要作用。由于现有技术的局限性，还没有直接的方法来研究脂筏在传递流体剪切应力中的作用。在这项研究中，我们针对两个重要的膜组件与脂筏，胆固醇和糖基磷脂酰肌醇锚定蛋白，破坏细胞膜结构的完整性。我们首先证明了用甲基-β-环糊精处理的膜胆固醇消耗抑制MC 3 T3-E1成骨细胞中振荡流体流诱导的细胞内钙动员和ERK 1/2磷酸化。其次，我们使用了一种新的方法，通过在MC 3 T3-E1成骨细胞中过表达糖基磷脂酰肌醇特异性磷脂酶D来降低细胞膜上糖基磷脂酰肌醇锚定蛋白的水平。这导致响应于振荡流体流的细胞内钙动员和ERK 1/2磷酸化的显著抑制。最后，我们证明了胆固醇耗竭抑制振荡流体流诱导的ATP释放，这是负责激活MC 3 T3-E1成骨细胞中的钙信号通路。我们的研究结果表明，胆固醇和GPI锚定蛋白，两个膜结构组件相关的脂筏，可能在成骨细胞的机械转导中发挥重要作用。
We previously demonstrated that oscillatory fluid flow activates MC3T3-E1 osteoblastic cell calcium signaling pathways via a mechanism involving ATP releases and P2Y2 puringeric receptors. However, the molecular mechanisms by which fluid flow initiates cellular responses are still unclear. Accumulating evidence suggests that lipid rafts, one of the important membrane structural components, may play an important role in transducing extracellular fluid shear stress to intracellular responses. Due to the limitations of current techniques, there is no direct approach to study the role of lipid rafts in transmitting fluid shear stress. In this study, we targeted two important membrane components associated with lipid rafts, cholesterol and glycosylphosphatidylinositol-anchored proteins, to disrupt the integrity of cell membrane structures. We first demonstrated that membrane cholesterol depletion with the treatment of methyl-β-cyclodextrin inhibits oscillatory fluid flow induced intracellular calcium mobilization and ERK1/2 phosphorylation in MC3T3-E1 osteoblastic cells. Secondly, we used a novel approach to decrease the levels of glycosylphosphatidylinositol-anchored proteins on cell membranes by overexpressing glycosylphosphatidylinositol specific phospholipase D in MC3T3-E1 osteoblastic cells. This resulted in significant inhibition of intracellular calcium mobilization and ERK1/2 phosphorylation in response to oscillatory fluid flow. Finally, we demonstrated that cholesterol depletion inhibited oscillatory fluid flow induced ATP releases, which were responsible for the activation of calcium signaling pathways in MC3T3-E1 osteoblastic cells. Our findings suggest that cholesterol and GPI-anchored proteins, two membrane structural components related to lipid rafts, may play an important role in osteoblastic cell mechanotransduction.
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影响因子：
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作者：
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通讯作者：
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DOI：
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发表时间：
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