Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China.
Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
Respir Res. 2017 Sep 20;18(1):174. doi: 10.1186/s12931-017-0657-z.
Mucus overproduction is an important feature of asthma. Interleukin (IL)-4 is required for allergen-induced airway inflammation and mucus production. MUC5AC gene expression is regulated by transcript factors NF-κB. The intracellular Ca ([Ca]) signal is required for activation of NF-κB. The transient receptor potential canonical 1 (TRPC1) channel has been shown to contribute for agonist-stimulated Ca influx in some types of cells. However, the relationships among IL-4, TRPC1 and mucus overproduction in bronchial epithelial cells (BECs) in asthma are poorly understood.
BECs were isolated from large bronchial airway of rats and used as cell model. To present changes of lipid raft, caveolin-1 and TRPC1, immunofluorescence staining and sucrose gradient centrifugation were performed. [Ca] was measured after loading with Fura-2. NF-κB activities were measured by an ELISA-based assay. MUC5AC mRNA and protein levels were detected by real-time quantitative RT-PCR, ELISA analysis and immunofluorescence staining respectively.
IL-4 induced Ca influx in BECs, and this was blocked by a Ca influx inhibitor (2-APB). 2-APB also prevented MUC5AC protein synthesis induced by IL-4. Depletion of extracellular Ca resulted in partial decrease in expression of MUC5AC in IL-4 treated cells. NF-κB rather than STAT6 activation mediated IL-4-induced MUC5AC protein synthesis. Then the mechanism of Ca influx was investigated. Immunofluorescence staining and sucrose gradient centrifugation revealed that caveolin-1-containing lipid rafts aggregation was involved in TRPC1 activation and Ca influx in BECs. Lastly, the data revealed that blocking lipid rafts aggregation exactly prevented Ca influx, NF-κB activation and MUC5AC synthesis induced by IL-4.
Our results indicate that IL-4-induced caveolin-1-containing lipid rafts aggregation at least partly contributes to MUC5AC synthesis in BECs.
黏液过度产生是哮喘的一个重要特征。白细胞介素（IL）-4 是变应原诱导气道炎症和黏液产生所必需的。MUC5AC 基因的表达受转录因子 NF-κB 调节。细胞内 Ca（[Ca]）信号对于 NF-κB 的激活是必需的。瞬时受体电位经典通道 1（TRPC1）通道已被证明有助于一些类型细胞中激动剂刺激的 Ca 内流。然而，在哮喘的支气管上皮细胞（BEC）中，IL-4、TRPC1 和黏液过度产生之间的关系尚不清楚。
从大鼠大支气管气道中分离 BECs 作为细胞模型。通过免疫荧光染色和蔗糖梯度离心来研究脂筏、窖蛋白-1 和 TRPC1 的变化。用 Fura-2 负载后测量 [Ca]。通过 ELISA 测定 NF-κB 活性。通过实时定量 RT-PCR、ELISA 分析和免疫荧光染色分别检测 MUC5AC mRNA 和蛋白水平。
IL-4 诱导 BECs 中的 Ca 内流，而 Ca 内流抑制剂（2-APB）可阻断该过程。2-APB 还可阻止 IL-4 诱导的 MUC5AC 蛋白合成。在 IL-4 处理的细胞中，细胞外 Ca 缺失导致 MUC5AC 的表达部分减少。NF-κB 而不是 STAT6 激活介导 IL-4 诱导的 MUC5AC 蛋白合成。然后研究了 Ca 内流的机制。免疫荧光染色和蔗糖梯度离心显示，窖蛋白-1 包含的脂筏聚集参与了 BECs 中 TRPC1 的激活和 Ca 内流。最后，数据表明，阻断脂筏聚集可确切地阻止 IL-4 诱导的 Ca 内流、NF-κB 激活和 MUC5AC 合成。
我们的结果表明，IL-4 诱导的含窖蛋白-1 的脂筏聚集至少部分有助于 BEC 中 MUC5AC 的合成。
