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首页> 外文期刊>Cellular Signalling >Cell confluence induces switching from proliferation to migratory signaling by site-selective phosphorylation of PDGF receptors on lipid raft platforms
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Cell confluence induces switching from proliferation to migratory signaling by site-selective phosphorylation of PDGF receptors on lipid raft platforms

机译：细胞融合通过脂质筏平台上PDGF受体的位点选择性磷酸化诱导从增殖信号转移到迁移信号

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Platelet derived growth factor receptors (PDGFR) play an important role in tumor pathogenesis and are frequently overexpressed in glioblastoma. Earlier we have shown that only confluent glioblastoma cell cultures exhibit a biphasic calcium transient upon PDGF stimulation. Here, we examined how the change in cell density leads to differential cellular responses to the same PDGF stimulus. PDGF beta receptors and their specific phosphotyrosine residues were fluorescently co-labeled on A172 and T98G glioblastoma cells. The distribution in cell membrane microdomains (lipid rafts) and the phosphorylation state of PDGFR was measured by confocal microscopy and quantitated by digital image processing. Corresponding bulk data were obtained by Western blotting. Activation of relevant downstream signaling pathways was assessed by immunofluorescence in confocal microscopy and by Western blot analysis. Functional outcomes were confirmed with bulk and single cell proliferation assays and motility measurements. In non-confluent (sparse) cultures PDGF-BB stimulation significantly increased phosphorylation of Tyr716 specific for the Ras/MAPK pathway and Tyr751 specific for the phosphoinositide 3-kinase/Akt pathway. As cell monolayers reached confluence, Tyr771 and Tyr1021 were the prominently phosphorylated residues. Tyr771 serves as adaptor for Ras-GAP, which inactivates the MAPK pathway, and Tyr1021 feeds into the phospholipase C-gamma/PKC pathway. Coherent with this, MAPK phosphorylation, Ki-67 positivity and proliferation dominated in dispersed cells, and could be abolished with inhibitors of the MAPK pathway. At the same time, RhoA activation, redistribution of cortactin to leading edges, and increased motility were the prominent output features in confluent cultures. Importantly, the stimulus-evoked confluence-specific changes in the phosphorylation of tyrosine residues occurred mainly in GM1-rich lipid microdomains (rafts). These observations suggest that the same stimulus is able to promote distinctly relevant signaling outputs through a confluence dependent, lipid raft-based regulatory mechanism. In particular, cell division and survival in sparse cultures and inhibition of proliferation and promotion of migration in confluent monolayers. In our model, the ability to switch the final output of the same stimulus as a function of cell density could be a key to the balance of proliferation and invasion in malignant glioblastoma. (C) 2015 Elsevier Inc All rights reserved.
机译：血小板衍生的生长因子受体（PDGFR）在肿瘤发病机理中起重要作用，并且在胶质母细胞瘤中经常过表达。先前我们已经证明，只有融合的胶质母细胞瘤细胞培养物在PDGF刺激后显示出两相钙瞬变。在这里，我们检查了细胞密度的变化如何导致对相同PDGF刺激的不同细胞反应。 PDGFβ受体及其特定的磷酸酪氨酸残基在A172和T98G胶质母细胞瘤细胞上进行了荧光共标记。通过共聚焦显微镜测量细胞膜微区（脂质筏）中的分布和PDGFR的磷酸化状态，并通过数字图像处理进行定量。通过蛋白质印迹获得相应的大量数据。通过共聚焦显微镜中的免疫荧光和蛋白质印迹分析评估相关下游信号通路的激活。通过大量和单细胞增殖测定以及运动性测定来确认功能结果。在非融合（稀疏）培养物中，PDGF-BB刺激显着增加了Ras / MAPK途径特异的Tyr716和磷酸肌醇3-激酶/ Akt途径特异的Tyr751的磷酸化。当细胞单层达到汇合时，Tyr771和Tyr1021是突出的磷酸化残基。 Tyr771充当Ras-GAP的衔接子，后者使MAPK通路失活，而Tyr1021则进入磷脂酶C-γ/ PKC通路。与此相关的是，MAPK磷酸化，Ki-67阳性和增殖在分散的细胞中占主导地位，并且可以通过MAPK途径的抑制剂消除。同时，在融合培养中，RhoA激活，cortactin在前缘的重新分布以及运动性增强是突出的输出特征。重要的是，酪氨酸残基磷酸化的刺激诱发的汇合特异性变化主要发生在富含GM1的脂质微区（筏）中。这些观察结果表明，相同的刺激能够通过汇合依赖性，基于脂质筏的调节机制来促进明显相关的信号输出。特别是在稀疏培养中的细胞分裂和存活，以及在汇合单层中抑制增殖和促进迁移。在我们的模型中，根据细胞密度切换相同刺激的最终输出的能力可能是恶性胶质母细胞瘤增殖与侵袭平衡的关键。 （C）2015 Elsevier Inc保留所有权利。

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