Extracellular heat shock protein 90α mediates HDM-induced bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling.
Extracellular heat shock protein 90α mediates HDM-induced bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling.
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细胞外热休克蛋白 90α 通过激活 RhoA/MLC 信号传导介导 HDM 诱导的支气管上皮屏障功能障碍
DOI：
10.1186/s12931-017-0593-y
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发表时间：
2017-05-30
影响因子：
5.8
通讯作者：
Cai SX
中科院分区：
文献类型：
--
作者：
Dong HM;Le YQ;Wang YH;Zhao HJ;Huang CW;Hu YH;Luo LS;Wan X;Wei YL;Chu ZQ;Li W;Cai SX
支气管上皮屏障的破坏和通透性增高与哮喘的发病密切相关。屋尘螨（HDM）是最重要的过敏原之一，可使气道上皮通透性增加.热休克蛋白（Hsp）90α也通过破坏RhoA信号传导参与肺内皮屏障功能障碍。然而，细胞外热休克蛋白90 α（eHsp 90 α）对HDM引起的支气管上皮屏障破坏的影响尚未见报道。为探讨eHsp 90 α在HDM诱导的支气管上皮屏障破坏中的作用，分别用HDM、人重组（hr）Hsp 90 α和hrHsp 90 β处理正常人支气管上皮细胞系16 HBE 14 o-（16 HBE），并用特异性抗分泌型Hsp 90 α单克隆抗体1G 6-D 7预处理。同时构建了Hsp 90 α沉默细胞。为了进一步评估RhoA信号传导在该过程中的作用，用Rho激酶抑制剂GSK 429286 A和Y27632 2 HCl预处理细胞。检测跨上皮电阻（TEER）和FITC-葡聚糖通量（FITC-DX）作为上皮屏障功能。分别采用免疫印迹法和免疫荧光法检测粘附连接蛋白E-钙粘蛋白和β-连环蛋白的表达和定位。通过条件培养基的浓缩和纯化来研究eHsp 90 α的水平。通过使用Rho G-LISA® RhoA活化测定试剂盒TM生物化学试剂盒测定RhoA活性，并通过蛋白质印迹法评估RhoA下游信号分子肌球蛋白轻链（MLC）的磷酸化。HDM处理16 HBE细胞后，上皮屏障破坏，细胞膜粘附连接蛋白E-cadherin和β-catenin缺失，并伴随eHsp 90 α分泌的增加。所有这些都在Hsp 90 α沉默细胞中或通过用1G 6-D 7预处理16 HBE细胞而被拯救。此外，1G 6-D 7抑制HDM诱导的RhoA活性和MLC磷酸化。此外，Rho激酶抑制剂预防和恢复气道屏障破坏。hrHsp 90 α促进了16 HBE细胞的屏障功能障碍并激活了RhoA/MLC信号通路。eHsp 90 α通过激活RhoA/MLC信号通路介导HDM诱导的人支气管上皮屏障功能障碍，提示eHsp 90 α是治疗哮喘的潜在靶点。
The disruption and hyperpermeability of bronchial epithelial barrier are closely related to the pathogenesis of asthma. House dust mite (HDM), one of the most important allergens, could increase the airway epithelial permeability. Heat shock protein (Hsp) 90α is also implicated in the lung endothelial barrier dysfunction by disrupting RhoA signaling. However, the effect of extracellular Hsp90α (eHsp90α) on the bronchial epithelial barrier disruption induced by HDM has never been reported. To investigate the involvement of eHsp90α in the bronchial epithelial barrier disruption induced by HDM, normal human bronchial epithelial cell line 16HBE14o- (16HBE) cells were treated by HDM, human recombinant (hr) Hsp90α and hrHsp90β respectively and pretreated by1G6-D7, a specific anti-secreted Hsp90α monoclonal antibody (mAb). Hsp90α-silencing cells were also constructed. To further evaluate the role of RhoA signaling in this process, cells were pretreated by inhibitors of Rho kinase, GSK429286A and Y27632 2HCl. Transepithelial electrical resistance (TEER) and FITC-dextran flux (FITC-DX) were examined as the epithelial barrier function. Expression and localization of adherens junctional proteins E-cadherin and β-catenin were evaluated by western blotting and immunofluorescence respectively. The level of eHsp90α was investigated by concentration and purification of condition media. RhoA activity was determined by using a Rho G-LISA® RhoA activation assay kitTM biochem kit, and the phosphorylation of myosin light chain (MLC), the downstream signal molecule of RhoA, was assessed by western blotting. The epithelial barrier disruption and the loss of adherens junctional proteins E-cadherin and β-catenin in cytomembrane were observed in HDM-treated 16HBE cells, paralleled with the increase of eHsp90α secretion. All of which were rescued in Hsp90α-silencing cells or by pretreating 16HBE cells with 1G6-D7. Also, 1G6-D7 suppressed RhoA activity and MLC phosphorylation induced by HDM. Furthermore, inhibitors of Rho kinase prevented and restored the airway barrier disruption. Consistently, it was hrHsp90α instead of hrHsp90β that promoted barrier dysfunction and activated RhoA/MLC signaling in 16HBE cells. The eHsp90α mediates HDM-induced human bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling, suggesting that eHsp90α is a potential therapeutic target for treatment of asthma.