Center for Salivary Gland Diseases of Peking University School and Hospital of Stomatology, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing, China.
J Cell Sci. 2013 Mar 1;126(Pt 5):1109-21. doi: 10.1242/jcs.111781. Epub 2013 Jan 23.
Occludin plays an important role in maintaining tight junction barrier function in many types of epithelia. We previously reported that activation of transient receptor potential vanilloid subtype 1 (TRPV1) in rabbit submandibular gland promoted salivary secretion, partly by an increase in paracellular permeability. We have now explored the role of occludin in TRPV1-modulated paracellular permeability in a rat submandibular gland cell line SMG-C6. Both TRPV1 and occludin were expressed in SMG-C6 cells, and capsaicin induced redistribution of occludin, but not claudin-3, claudin-4 or E-cadherin, from the cell membrane into the cytoplasm. Capsaicin also decreased transepithelial electrical resistance (TER) and increased the Trypan Blue and FITC-dextran flux. Capsazepine (CPZ), a TRPV1 antagonist, inhibited the capsaicin-induced occludin redistribution and TER decrease. Moreover, occludin knockdown by shRNA suppressed, whereas occludin re-expression restored, the TER response to capsaicin. Mechanistically, TRPV1 activation increased ERK1/2 and MLC2 phosphorylation. PD98059, an ERK1/2 kinase inhibitor, abolished the capsaicin-induced MLC2 phosphorylation, whereas ML-7, an MLC2 kinase inhibitor, did not affect ERK1/2 phosphorylation, suggesting that ERK1/2 is the upstream signaling molecule of MLC2. Capsaicin also induced F-actin reorganization, which was abolished by CPZ, PD98059 and ML-7, indicating that TRPV1 activation altered F-actin organization in an ERK1/2- and MLC2-dependent manner. Furthermore, either PD98059 or ML-7 could abolish the capsaicin-induced TER response and occludin redistribution, whereas knockdown of ERK1/2 further confirmed that the TRPV1-modulated paracellular permeability was ERK1/2 dependent. Taken together, these results identified a crucial role of occludin in submandibular epithelial cells, and more importantly, demonstrated that occludin was required to mediate TRPV1-modulated paracellular permeability.
紧密连接蛋白在许多类型的上皮组织中对于维持紧密连接屏障功能起着重要作用。我们之前曾报道，兔颌下腺中瞬时受体电位香草酸亚型 1（TRPV1）的激活促进了唾液分泌，部分是通过增加细胞旁通透性实现的。现在，我们在大鼠颌下腺细胞系 SMG-C6 中探索了紧密连接蛋白在 TRPV1 调节的细胞旁通透性中的作用。SMG-C6 细胞中表达 TRPV1 和紧密连接蛋白，辣椒素诱导紧密连接蛋白（而非 Claudin-3、Claudin-4 或 E-cadherin）从细胞膜重新分布到细胞质中。辣椒素还降低了跨上皮电阻（TER）并增加了台盼蓝和 FITC-葡聚糖的通量。TRPV1 拮抗剂辣椒素锌（CPZ）抑制了辣椒素诱导的紧密连接蛋白重新分布和 TER 降低。此外，通过 shRNA 敲低紧密连接蛋白抑制了辣椒素诱导的 TER 反应，而重新表达紧密连接蛋白则恢复了该反应。从机制上讲，TRPV1 激活增加了 ERK1/2 和 MLC2 的磷酸化。ERK1/2 激酶抑制剂 PD98059 消除了辣椒素诱导的 MLC2 磷酸化，而 MLC2 激酶抑制剂 ML-7 则不影响 ERK1/2 的磷酸化，这表明 ERK1/2 是 MLC2 的上游信号分子。辣椒素还诱导 F-肌动蛋白重组，CPZ、PD98059 和 ML-7 均可消除该重组，表明 TRPV1 激活以 ERK1/2 和 MLC2 依赖的方式改变 F-肌动蛋白组织。此外，PD98059 或 ML-7 均可消除辣椒素诱导的 TER 反应和紧密连接蛋白重新分布，而 ERK1/2 的敲低进一步证实 TRPV1 调节的细胞旁通透性依赖于 ERK1/2。总之，这些结果确定了紧密连接蛋白在颌下腺上皮细胞中的关键作用，更重要的是，证明了紧密连接蛋白是介导 TRPV1 调节的细胞旁通透性所必需的。
