紧密连接蛋白claudin-4—毒蕈碱乙酰胆碱受体调控颌下腺分泌的新靶点

Paracellular pathway via tight junction is an important route for materials transport. However, whether the muscarinic cholinergic receptor (M receptor) regulates tight junction of submandibular gland (SMG), as well as the related signaling pathway, is still unknown. On basis of our preliminary experiments, we hypothesize that tight junction protein claudin-4 is a novel target regulated by M receptor. By using RT-PCR, Western blot, and immunofluorescence, we will detect the effect of M receptor on the expression and distribution of tight junction components in rat isolated SMG tissues and SMG-C6 cells, aiming to determine that claudin-4 is the potential target for M receptor from organic and cellular levels. By using transepithelial electrical resistance (TER) measurement, siRNA and plasmid transfection, we will determine a crucial role of claudin-4 in the paracellular permeability regulated by M receptor. We will further investigate the signaling pathway connecting M receptor with claudin-4, especially focusing on ERK/β-arrestin/clathrin/ubiquitin signaling pathway. This study will reveal the molecular mechanism involved in the M receptor-modulated paracellular permeability via claudin-4, providing a novel insight into a future therapeutic target for hyposecretion of SMG.

紧密连接是介导物质经旁细胞通路转运的重要途径，但毒蕈碱乙酰胆碱受体(M受体)是否调控颌下腺紧密连接及其作用机制尚缺乏了解。本课题在预实验基础上提出紧密连接蛋白claudin-4是M受体调控颌下腺分泌的新分子的科学设想，拟采用RT-PCR、Western blot和免疫荧光等方法检测激活M受体对大鼠颌下腺多种紧密连接蛋白表达和分布的影响，在器官水平和细胞水平上证实claudin-4是受M受体特异性调控的靶分子；采用跨上皮细胞电阻、siRNA及质粒转染等方法证实claudin-4在介导M受体调控旁细胞通透性中起关键作用；围绕ERK/β-arrestin/clathrin/泛素化降解途径，揭示M受体调控claudin-4的信号转导途径。该研究将揭示M受体以claudin-4为新靶点调控旁细胞途径通透性的分子机制，为探讨以紧密连接为靶点改善颌下腺分泌提供新思路。

由紧密连接介导的旁细胞通路是物质跨上皮转运的重要途径，但毒蕈碱乙酰胆碱受体（M受体）是否调控颌下腺紧密连接及调控机制尚不清楚。本项目在细胞水平上证实激活M受体可增加颌下腺旁细胞途径的通透性，选择性下调claudin-4的蛋白表达，引起claudin-4由胞膜向胞浆的重分布。Claudin-4敲低和过表达实验证实激活M受体促进颌下腺旁细胞途径通透性的作用是经特异性地调控claudin-4而实现的。进一步机制研究结果显示，激活M受体可经ERK1/2磷酸化claudin-4，点突变实验发现claudin-4第195位丝氨酸是受M受体特异性调控的位点。claudin-4被磷酸化后可选择性与β-arrestin2结合，通过β-arrestin2的招募作用引起claudin-4以clathrin包被小体形式发生内化，内化后的claudin-4可在胞浆中发生泛素化降解，最终引起旁细胞途径的通透性增加。本项目还建立了家兔自体颌下腺移植术后远期泪溢模型，检测M受体表达恢复至正常水平，紧密连接宽度以及腺泡顶侧膜周F-actin含量均明显增加。此外，本项目还探讨了激活M受体对颌下腺血管内皮细胞间紧密连接功能的影响，在体水平证实M受体激动剂可引起小鼠颌下腺血管内皮细胞对旁细胞示踪分子4和40kDa FITC-dextran的通透性增加，血管内皮细胞特异性表达的紧密连接分子claudin-5发生从顶侧膜向基底侧膜及胞浆中的重分布。在培养的人正常颌下腺组织块中给予M受体激动剂以及泪溢患者减量标本中也均观察到claudin-5重分布现象。机制研究发现与调控紧密连接分布相关的重要信号分子p-MLC2的含量在M受体激动剂刺激的小鼠颌下腺以及泪溢患者减量标本的血管中均增加，p-MLC2下游分子细胞骨架F-actin也发生从靠近顶侧膜向胞浆的重分布。本项目的研究成果为阐明激活M受体促进颌下腺分泌的分子机制提供了新见解，为探寻人工调控唾液分泌的靶点提供了新思路。