Claudin-4 is required for modulation of paracellular permeability by muscarinic acetylcholine receptor in epithelial cells
Claudin-4 is required for modulation of paracellular permeability by muscarinic acetylcholine receptor in epithelial cells
复制标题
Claudin-4 是上皮细胞中毒蕈碱乙酰胆碱受体调节细胞旁通透性所必需的
DOI：
10.1242/jcs.165878
复制
发表时间：
2015-06-15
影响因子：
4
通讯作者：
Yu, Guang-Yan
中科院分区：
文献类型：
--
作者：
Cong, Xin;Zhang, Yan;Yu, Guang-Yan
上皮胆碱能系统在水、离子和溶质的转运中起重要作用。以往的研究表明，激活毒蕈碱乙酰胆碱受体（mAChRs）调节上皮细胞的细胞旁转运，然而，其潜在的机制仍然是未知的。在这里，我们发现，mAChR激活卡巴胆碱和西维美林降低跨上皮电阻（TER），并增加大鼠唾液腺上皮SMG-C6细胞的细胞旁示踪剂的渗透性。卡巴胆碱诱导claudin-4的下调和重新分布，但不诱导occludin或ZO-1（也称为TJP 1）。小发夹RNA（shRNA）介导的claudin-4敲低抑制，而claudin-4过表达保留，TER反应卡巴胆碱。从机制上讲，mAChR调节的密蛋白-4特性和细胞旁渗透性是通过ERK 1/2（也分别称为MAPK 3和MAPK 1）的密蛋白-4磷酸化触发的。突变实验表明，卡巴胆碱作用的靶基因是claudin-4的S195，而不是S199、S203和S207。随后，磷酸化的claudin-4与β-arrestin 2相互作用并通过网格蛋白依赖性途径触发claudin-4内化。内化的claudin-4通过泛素化进一步降解。总之，这些发现表明，密蛋白-4是通过ERK 1/2、β-arrestin 2、网格蛋白和泛素依赖性信号通路调节上皮细胞的mAChR细胞旁通透性所必需的。突出显示的文章：上皮胆碱能系统通过调节紧密连接蛋白claudin-4的含量和分布来激活调节细胞旁通透性的信号通路。
ABSTRACT The epithelial cholinergic system plays an important role in water, ion and solute transport. Previous studies have shown that activation of muscarinic acetylcholine receptors (mAChRs) regulates paracellular transport of epithelial cells; however, the underlying mechanism is still largely unknown. Here, we found that mAChR activation by carbachol and cevimeline reduced the transepithelial electrical resistance (TER) and increased the permeability of paracellular tracers in rat salivary epithelial SMG-C6 cells. Carbachol induced downregulation and redistribution of claudin-4, but not occludin or ZO-1 (also known as TJP1). Small hairpin RNA (shRNA)-mediated claudin-4 knockdown suppressed, whereas claudin-4 overexpression retained, the TER response to carbachol. Mechanistically, the mAChR-modulated claudin-4 properties and paracellular permeability were triggered by claudin-4 phosphorylation through ERK1/2 (also known as MAPK3 and MAPK1, respectively). Mutagenesis assay demonstrated that S195, but not S199, S203 or S207, of claudin-4, was the target for carbachol. Subsequently, the phosphorylated claudin-4 interacted with β-arrestin2 and triggered claudin-4 internalization through the clathrin-dependent pathway. The internalized claudin-4 was further degraded by ubiquitylation. Taken together, these findings suggested that claudin-4 is required for mAChR-modulated paracellular permeability of epithelial cells through an ERK1/2, β-arrestin2, clathrin and ubiquitin-dependent signaling pathway. Highlighted Article: The epithelial cholinergic system activates a signaling pathway that regulates paracellular permeability by modulating the content and distribution of the tight junction protein claudin-4.