TMEM16A-MARCKS通路调控气道黏液高分泌的分子机制研究

Mucus hypersecretion is one of the important features of chronic obstructive pulmonary disease （COPD）and bronchial asthma. Ca2+ activated Cl- channels (CaCCs) are involved in mucus hypersecretion in the airway, but their molecular identity is unclear and controversial. Recent study demonstrated that transmembrane protein 16A (TMEM16A) is an intrinsic constituent of the CaCC. In our previous work, we found that the expression of TMEM16A mRNA and protein significantly increased with mucus hypersecretion in the human bronchial epithelial 16 (HBE16) cells. In this project, the study will be intended to investigate the molecular mechanism of TMEM16A-MARCKS pathway in the regulation of mucus hypersecretion in the airway. Activation of the CaCC causes depolarization of airway epithelial cells through the efflux of chloride anion. Membrane depolarization induces calcium influx through L-type voltage gated calcium channel (L-VGCC), which mediates exocytosis by regulating myristoylated alanine-rich C kinase substrate (MARCKS) translocation and phosphorylation.In the study, the expression of TMEM16A will be upregulated or downregulated by using TMEM16A plasmid in vitro or adenovirus-expressing TMEM16A in vivo; and by RNAi in vitro or adenovirus-expressing antisense TMEM16A in vivo, respectively. The expression of mucin (MUC)5AC、protein kinase C ε(PKC-ε) and MARCKS will be measured by real-time PCR、western blotting or ELISA. The efflux of chloride anion will be changed by mutating the pore region of TMEM16A. The chloride currents will be recorded by the whole-cell patch -clamp technique.It is expected that a TMEM16A-L-VGCC-MARCKS pathway is responsible for mucus hypersecretion in the airway, which gives a new insight into therapeutic development for mucus hypersecretion in COPD and bronchial asthma.

黏液高分泌是COPD和哮喘病情进展和恶化的独立危险因素，黏液高分泌发生时钙激活氯通道也异常变化。我们的前期工作发现在黏液高分泌模型中，钙激活氯通道的主要成分TMEM16A表达显著上调，其原因、作用和机制引起了我们的高度关注。TMEM16A通过增强氯离子外流引起上皮细胞去极化，而上皮细胞去极化后，激活L型电压门控钙通道（L-VGCC）引起钙离子内流，最终导致MARCKS活化促使黏液分泌。本课题我们分别在体内和体外模型中上调、下调TMEM16A的表达，探讨TMEM16A的表达水平变化对黏蛋白合成和分泌以及L-VGCC、PKC-ε、MARCKS等信号通路的影响；再通过定点突变TMEM16A，改变其电生理特性，研究TMEM16A通过氯离子外流调控黏液分泌的机制。本研究在分子水平阐明TMEM16A-L-VGCC-MARCKS通路在黏液高分泌发生中的重要作用，为黏液高分泌的预防和治疗奠定理论基础。

黏液高分泌是支气管哮喘和慢性阻塞性肺疾病（COPD）进展和恶化的独立危险因素，黏液高分泌发生时钙激活氯离子通道异常变化。TMEM16A作为钙激活氯通道的主要成分参与气道黏液高分泌过程。本课题旨在探讨TMEM16A对黏液分泌的调控及相关机制，并验证TMEM16A-L-VGCC-MARCKS、IL-13-TMEM16A-NF-κB、IL-13-STAT6-TMEM16A-ERK信号通路对黏液的调控。进行以下研究：1.在体内外实验中用IL-13构建黏液高分泌模型，检测TMEM16A、MUC5AC的表达。2.上调或下调TMEM16A的表达，观察TMEM16A和黏液高分泌的关系；3.通过膜片钳技术检测钙激活氯离子通道的电流变化。4.检测TMEM16A上游及下游信号分子的表达，了解TMEM16A调控黏液高分泌的机制。5.构建腺病毒siRNA-TMEM16A气道内滴入，检测气道中MUC5AC的表达、气道反应及杯状细胞数。实验结果有：1.经IL-13处理后，细胞内的TMEM16A与MUC5AC的分泌均明显增加，且两者呈正相关。2.上调TMEM16A增加MUC5AC mRNA和蛋白水平，下调TMEM16A则相反。3.T16Ainh-A01能明显抑制钙激活氯离子通道的电流。4.TMEM16A-NF-κB信号通路实验中，结果显示TMEM16A的高表达导致NF-κB磷酸化水平及荧光酶学活性升高，并且致MUC5AC生成增加；BAY11-7082能抑制NF-κB表达，并抑制了MUC5AC的生成。5.STAT6基因敲除及A771726使HBE16细胞中STAT6磷酸化水平下降，也相应的抑制了TMEM16A及MUC5AC的表达水平；上调TMEM16A基因后，ERK1/2的磷酸化水平明显升高，下调TMEM16A基因后则相反；抑制ERK1/2表达，MUC5AC的生成受到抑制。6.IL-13气道内滴入能够显著提高大鼠气道TMEM16A和MUC5AC的表达，同时提高其气道反应性和杯状细胞百分比，而腺病毒siRNA-TMEM16A滴入则能显著抑制上述变化。综上结果提示TMEM16A在IL-13介导的黏液高分泌中起重要作用，IL-13-STAT6-TMEM16A-ERK/NF-kB信号通路可能是慢性气道炎性疾病黏液高分泌的主要机制，TMEM16A可能作为慢性气道炎性疾病黏液高分泌的治疗靶点。