Claudin-3：肿瘤坏死因子α调控下颌下腺紧密连接的分子靶点

Tight junction is a protein complex for materials transport through paracellular route in epithelial cells; however, its exact role in submandibular gland is still unclear. Based on pro-inflammatory cytokine tumor necrosis factor-α (TNF-α)-induced inflammatory response, our present study focuses on the effect of the tight junction protein claudin-3 in submandibular gland. By using immunoblotting, immunofluorescence, gene knockdown and overexpression, we will explore the role of claudin-3 in submandibular tight junction function. On the TNF-α-induced rat submandibular inflammation and cellular model, we will detect the changes of expression, distribution and function of tight junction proteins, aiming to determine that claudin-3 is the potential target for TNF-α from both organic and cellular levels. We will further investigate the signaling pathway connecting TNF-α and claudin-3, especially focusing on ERK1/2 and NF-κB signaling pathways, together with the downstream transcriptional factors Egr-1 and slug. This study will identify the physiological and pathophysiological significance of claudin-3 in submandibular gland, explore the molecular mechanism involved in the TNF-α-regulated claudin-3, and further provide a novel insight into a future therapeutic target for inflammatory diseases of submandibular gland.

紧密连接是介导上皮细胞物质经旁细胞途径转运的蛋白复合体，但对其在下颌下腺中的作用了解甚少。本项目以肿瘤坏死因子α（TNF-α）诱导的下颌下腺炎症反应为主线，紧密连接蛋白claudin-3为重点，采用免疫印迹、免疫荧光、基因敲低或过表达等方法证实claudin-3在下颌下腺紧密连接中的作用；在TNF-α诱导的大鼠下颌下腺炎和SMG-C6细胞上观察紧密连接蛋白表达、分布和功能的变化，在器官、细胞和分子水平证实claudin-3是TNF-α特异性调控的紧密连接靶分子；以ERK1/2和NF-κB通路为中心，探讨TNF-α调控转录因子Egr-1和slug进而影响claudin-3表达和功能的信号转导途径。本研究将丰富紧密连接介导下颌下腺分泌的理论，揭示claudin-3在下颌下腺的生理功能，明确TNF-α调控claudin-3的分子机制，并为探讨以紧密连接为靶点防治下颌下腺炎症性疾病提供新的思路。

紧密连接是介导上皮细胞物质经旁细胞途径转运的蛋白复合体。肿瘤坏死因子α（TNF-α）在唾液腺炎性疾病的发病中起重要作用，但对其在下颌下腺中调节紧密连接屏障的作用了解甚少。我们发现TNF-α可降低大鼠下颌下腺细胞系SMG-C6的跨上皮电阻（TER），增加FITC-葡聚糖渗出量。TNF-α引起紧密连接蛋白claudin-3表达减少和膜分布降低，而claudin-1、claudin-4和β-catenin的表达和分布不受影响。在SMG-C6细胞过表达claudin-3可增加TER值，降低FITC-dextran的通过率；而采用shRNA敲低claudin-3表达，则降低TER值，增加FITC-dextran的通过率。TNF-α作用于过表达claudin-3的细胞可降低claudin-3的表达，升高旁细胞途径通透性，提示claudin-3是构成下颌下腺上皮细胞屏障功能的重要分子，在TNF-α介导的下颌下腺旁细胞通透性升高中claudin-3发挥了关键作用。TNF-α增加细胞外信号调节激酶（ERK1/2）磷酸化和转录因子slug的表达，预孵育ERK1/2激酶抑制剂PD98059和U0126可以抑制TNF-α引起的TER下降和FITC-dextran通过率升高，并逆转TNF-α引起的claudin-3表达下降和膜分布改变，抑制TNF-α引起的slug表达。在SMG-C6中过表达转录因子slug可以降低claudin-3的表达，使用shRNA敲低slug，claudin-3表达升高，荧光素酶报告基因实验显示，slug可以结合到claudin-3启动子区域的E-box结合位点，发挥转录抑制作用。CHIP实验显示，TNF-α可以通过ERK1/2途径促进slug对claudin-3的转录抑制作用。对人正常下颌下腺组织进行体外孵育，TNF-α可降低claudin-3的表达，升高slug的表达，提示TNF-α亦可经转录因子slug对人下颌下腺紧密连接蛋白claudin-3进行调控。我们的研究表明TNF-α经ERK1/2通路和转录因子slug选择性调控紧密连接蛋白claudin-3表达及分布，从而增加旁细胞通透性。本研究丰富了紧密连接介导下颌下腺分泌的理论，揭示claudin-3在下颌下腺分泌调控中的作用，并为以紧密连接蛋白为靶点防治下颌下腺炎症性疾病提供了新的思路。