Protease Activated Receptor Signaling Is Required for African Trypanosome Traversal of Human Brain Microvascular Endothelial Cells
Protease Activated Receptor Signaling Is Required for African Trypanosome Traversal of Human Brain Microvascular Endothelial Cells
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DOI：
10.1371/journal.pntd.0000479
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发表时间：
2009-07-01
影响因子：
3.8
通讯作者：
Dumler, J. Stephen
中科院分区：
文献类型：
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作者：
Grab, Dennis J.;Garcia-Garcia, Jose C.;Dumler, J. Stephen
背景：利用人脑微血管内皮细胞作为非洲锥虫穿越血脑屏障的体外模型，我们最近报道，寄生虫通过寄生虫半胱氨酸蛋白酶，特别是组织蛋白酶L(Bruciain)的活性，在HBMECs中产生钙激活信号，从而穿过血脑屏障。在目前的研究中，我们研究了一类被称为蛋白酶激活的受体(PARs)的蛋白酶刺激的HBMEC G蛋白偶联受体(GPCRs)的可能作用，该受体可能参与非洲锥体的钙信号转导。方法/主要发现：利用RNA干扰(RNAi)，我们发现在体外HBMEC单层中PAR-2基因(F2RL1)的表达可以降低95%以上。我们还发现，布氏锥虫跨越F2RL1沉默的HBMEC单层的能力降低(39%-49%)，在存在寄生虫的情况下，沉默F2RL1的HBMEC维持屏障功能的控制水平。与PAR-2的作用一致，我们发现用多杀性巴氏杆菌毒素(PMT)阻断Gα(Q)后，HBMEC的屏障功能也保持不变。PAR-2信号在其他系统中已经被证明具有神经炎症和神经保护作用，我们的数据暗示了蛋白酶(即布鲁氏痛)和PAR-2在非洲锥虫/HBMEC相互作用中的作用。使用基因图谱方法询问由Bruciain特异性触发的候选HBMEC通路，确定了几条可能与CNS HAT相关的病理生理过程相关联的通路。结论/意义：这些数据部分支持GPCRs作为寄生虫蛋白水解酶的分子靶标，导致Gα(Q)介导的钙信号的激活。据预测，这些事件的后果是血脑屏障对寄生虫移行的通透性增加，并引发神经炎症，这些事件是中枢神经系统疾病的先兆。
Background: Using human brain microvascular endothelial cells (HBMECs) as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB) we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain). In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs) known as protease activated receptors (PARs) that might be implicated in calcium signaling by African trypanosomes.Methodology/Principal Findings: Using RNA interference (RNAi) we found that in vitro PAR-2 gene (F2RL1) expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%-49%) and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of G alpha(q) with Pasteurella multocida toxin (PMT). PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain) and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified.Conclusions/Significance: Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of G alpha(q)-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.