TNFα alters occludin and cerebral endothelial permeability: Role of p38MAPK.
TNFα alters occludin and cerebral endothelial permeability: Role of p38MAPK.
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DOI：
10.1371/journal.pone.0170346
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Lee JC
中科院分区：
文献类型：
--
作者：
Ni Y;Teng T;Li R;Simonyi A;Sun GY;Lee JC
Occludin是脑内皮细胞(CECs)中一个关键的紧密连接(TJ)蛋白，在调节血脑屏障(BBB)功能中起着重要作用。该蛋白(65 KDa)参与多种信号通路和酪氨酸激酶和苏氨酸激酶的磷酸化。尽管机制尚不清楚，但促炎细胞因子和内毒素(脂多糖)可能会改变CECs中的TJ蛋白和BBB功能。在这里，我们展示了封闭素在永生化的人脑内皮细胞系(hCMEC/D3)中对肿瘤坏死因子α(10 ng/mL)、IL-1β(10 ng/mL)和内毒素(100 ng/mL)刺激的反应。细胞暴露于肿瘤坏死因子α后，封闭蛋白的条带发生快速而短暂的向上移动，提示磷酸化程度增加。暴露于IL-1、β对闭锁蛋白条带的影响较小，几乎不影响闭塞蛋白条带的移动。肿瘤坏死因子α还可引起hCMEC/D3细胞瞬时激活p38MAPK和ERK1/2，p38MAPK抑制剂SB202190和MEK1/2-ERK1/2抑制剂U0126可部分抑制α引起的封闭蛋白条带移位。经肿瘤坏死因子α和IL-1β作用24 h后，细胞occludin的表达显著降低(p<；0.001)，p38MAPK抑制剂SB202190可部分阻断这一作用。通过FITC-葡聚糖实验和跨内皮细胞电阻测定，肿瘤坏死因子α处理也改变了细胞形态和细胞内皮细胞层的通透性。然而，单独应用SB202190并不能有效逆转肿瘤坏死因子α引起的形态变化或通透性增加的变化。这些结果表明，尽管肿瘤坏死因子α对p38MAPK介导的封闭蛋白的磷酸化和表达有影响，但这些变化不足以避免肿瘤坏死因子α诱导的细胞形态和通透性的改变。
Occludin is a key tight junction (TJ) protein in cerebral endothelial cells (CECs) playing an important role in modulating blood-brain barrier (BBB) functions. This protein (65kDa) has been shown to engage in many signaling pathways and phosphorylation by both tyrosine and threonine kinases. Despite yet unknown mechanisms, pro-inflammatory cytokines and endotoxin (lipopolysaccharides, LPS) may alter TJ proteins in CECs and BBB functions. Here we demonstrate the responses of occludin in an immortalized human cerebral endothelial cell line (hCMEC/D3) to stimulation by TNFα (10 ng/mL), IL-1β (10 ng/mL) and LPS (100 ng/mL). Exposing cells to TNFα resulted in a rapid and transient upward band-shift of occludin, suggesting of an increase in phosphorylation. Exposure to IL-1β produced significantly smaller effects and LPS produced almost no effects on occludin band-shift. TNFα also caused transient stimulation of p38MAPK and ERK1/2 in hCMEC/D3 cells, and the occludin band-shift induced by TNFα was suppressed by SB202190, an inhibitor for p38MAPK, and partly by U0126, the MEK1/2-ERK1/2 inhibitor. Cells treated with TNFα and IL-1β but not LPS for 24 h resulted in a significant (p < 0.001) decrease in the expression of occludin, and the decrease could be partially blocked by SB202190, the inhibitor for p38MAPK. Treatment with TNFα also altered cell morphology and enhanced permeability of the CEC layer as measured by the FITC-dextran assay and the trans-endothelial electrical resistances (TEER). However, treatment with SB202190 alone could not effectively reverse the TNFα -induced morphology changes or the enhanced permeability changes. These results suggest that despite effects of TNFα on p38MAPK-mediated occludin phosphorylation and expression, these changes are not sufficient to avert the TNFα-induced alterations on cell morphology and permeability.