Department of Physiology, Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4200-5. doi: 10.1073/pnas.0903923107. Epub 2010 Feb 8.
The intestinal epithelium serves as a major protective barrier between the mammalian host and the external environment. Here we show that the transmembrane serine protease matriptase plays a pivotol role in the formation and integrity of the intestinal epithelial barrier. St14 hypomorphic mice, which have a 100-fold reduction in intestinal matriptase mRNA levels, display a 35% reduction in intestinal transepithelial electrical resistance (TEER). Matriptase is expressed during intestinal epithelial differentiation and colocalizes with E-cadherin to apical junctional complexes (AJC) in differentiated polarized Caco-2 monolayers. Inhibition of matriptase activity using a specific peptide inhibitor or by knockdown of matriptase by siRNA disrupts the development of TEER in barrier-forming Caco-2 monolayers and increases paracellular permeability to macromolecular FITC-dextran. Loss of matriptase was associated with enhanced expression and incorporation of the permeability-associated, "leaky" tight junction protein claudin-2 at intercellular junctions. Knockdown of claudin-2 enhanced the development of TEER in matriptase-silenced Caco-2 monolayers, suggesting that the reduced barrier integrity was caused, at least in part, by an inability to regulate claudin-2 expression and incorporation into junctions. We find that matriptase enhances the rate of claudin-2 protein turnover, and that this is mediated indirectly through an atypical PKCzeta-dependent signaling pathway. These results support a key role for matriptase in regulating intestinal epithelial barrier competence, and suggest an intriguing link between pericellular serine protease activity and tight junction assembly in polarized epithelia.
肠上皮作为哺乳动物宿主与外部环境之间的主要保护屏障。在这里，我们发现跨膜丝氨酸蛋白酶 matriptase 在肠上皮屏障的形成和完整性中起着关键作用。St14 低功能小鼠，其肠 matriptase mRNA 水平降低了 100 倍，其肠上皮跨上皮电阻（TEER）降低了 35%。Matriptase 在肠上皮分化过程中表达，并与分化的极化 Caco-2 单层细胞中的 E-钙粘蛋白共定位到顶端连接复合体（AJC）。使用特异性肽抑制剂抑制 matriptase 活性或通过 siRNA 敲低 matriptase 会破坏形成屏障的 Caco-2 单层细胞中 TEER 的发展，并增加大分子 FITC-右旋糖酐的旁通透性。matriptase 的丢失与通透性相关的“渗漏”紧密连接蛋白 Claudin-2 在细胞间连接处的表达和掺入增加有关。Claudin-2 的敲低增强了 matriptase 沉默的 Caco-2 单层细胞中 TEER 的发展，表明屏障完整性的降低至少部分是由于无法调节 Claudin-2 的表达和掺入到连接处。我们发现 matriptase 增强了 Claudin-2 蛋白周转率，并且这是通过一种非典型的 PKCzeta 依赖的信号通路间接介导的。这些结果支持 matriptase 在调节肠上皮屏障能力方面的关键作用，并提示细胞周丝氨酸蛋白酶活性与极化上皮中紧密连接组装之间存在有趣的联系。
