Role of claudin-7 in intestinal structure and inflammation

项目摘要 肠粘膜的完整性对于营养吸收和抵抗病原体至关重要。 肠上皮细胞稳态的破坏导致多种肠道病症，例如慢性肠道炎症。 炎症、粘膜溃疡和癌症。紧密连接蛋白是紧密连接（TJ）的主要组成膜 哺乳动物中至少有24个成员的蛋白质。尽管有10多名claudin家族成员表示 在人和小鼠肠中，已知只有紧密连接蛋白-7和-15是必需，因为它们的缺失导致 严重的肠道表型。然而，很少有人知道他们的运作机制， 健康和疾病中的上皮损伤、修复和再生。 我们的实验室已经产生了第一个claudin-7基因敲除小鼠模型。肠道Claudin-7缺失 导致严重的粘膜溃疡、上皮细胞脱落和炎症。除了传统的 与TJ相关，claudin-7也与上皮基底外侧区室中的整合素α2相互作用。 肠claudin-7的缺失减少整合素α2的表达，上调MMP和细胞因子，并引起 炎症反应。此外，肠上皮干细胞（IESC）增殖之间的平衡 并且分化在Cldn 7-/-肠中被破坏。这些发现导致了一种假设，即claudin-7 在维持上皮细胞-基质相互作用中具有一种新的、先前未鉴定的非TJ功能， 通过调节炎症和干细胞信号传导来维持肠道内稳态。该项目的目标是 研究claudin-7如何作为肠道功能所需的关键元素。我们提出两个目标。目的 1将确定claudin-7缺失是否通过破坏TJ屏障诱导肠道炎症 功能或由MMPs介导的细胞-基质粘附。这一目标将通过确定（1）是否 TJ屏障功能被与炎症反应相关的claudin-7缺失破坏，和（2） MMPs的增加是否促进或抑制MMPs减弱炎症反应， 文化和动物。目的2将确定claudin-7缺失是否会诱导肠道炎症，导致肠道炎症反应。 破坏IESC增殖和分化。基于基因和蛋白质表达谱显示 IESC标志物Olfm-4的显著下调，该目的将确定（1）Olfm 4在 在有或没有claudin-7的肠上皮细胞中LPS或TNFα处理后的炎症反应 表达，以及（2）炎症信号传导是否增加干细胞增殖并减少干细胞增殖。 肠上皮细胞分化该项目将阐明claudin-7缺失是否会引起炎症 通过破坏典型的TJ屏障或细胞-基质粘附以及claudin-7如何诱发炎症， 影响IESC的命运。总体而言，该项目的广泛影响将是鉴定“claudin-7-MMPs-IESC 作为潜在的靶点，claudin-7通过其控制肠道内稳态对抗炎症。

Project Summary The integrity of intestinal mucosa is vital for nutrition absorption and defense against pathogens. Disruption of intestinal epithelial homeostasis leads to a variety of intestinal disorders, such as chronic inflammation, mucosal ulceration, and cancer. Claudins are the major tight junction (TJ) integral membrane proteins with at least 24 members in mammals. Although more than 10 claudin family members are expressed in human and mouse intestines, only claudin-7 and -15 are known to be essential since their deletion causes severe intestinal phenotypes. However, little is known about the mechanisms by which they function in epithelial injury, repair, and regeneration in health and diseases. Our laboratory has generated the first claudin-7 knockout mouse model. Claudin-7 deletion in intestines leads to severe mucosal ulcerations, epithelial cell sloughing, and inflammation. Besides its conventional association with TJ, claudin-7 also interacts with integrin α2 in the epithelial basolateral compartment of intestines. Deletion of claudin-7 reduces integrin α2 expression, upregulates MMPs and cytokines, and evokes inflammatory responses. In addition, the balance between the intestinal epithelial stem cell (IESC) proliferation and differentiation is disrupted in Cldn7-/- intestines. These findings have led to the hypothesis that claudin-7 has a novel, previously unidentified non-TJ function in maintaining epithelial cell-matrix interactions and intestinal homeostasis through modulating inflammatory and stem cell signaling. The goal of this project is to investigate how claudin-7 serves as a key element required for intestinal functions. We propose two aims. Aim 1 will determine whether claudin-7 deletion induces intestinal inflammation through disruption of the TJ barrier function or cell-matrix adhesion mediated by MMPs. This aim will be achieved by determining (1) whether the TJ barrier function is disrupted with claudin-7 deletion associated with inflammatory responses, and (2) whether the increase of MMPs promotes or the inhibition of MMPs attenuates the inflammatory response in cultures and in animals. Aim 2 will determine if claudin-7 deletion induces intestinal inflammation, leading to the disruption of IESC proliferation and differentiation. Based on the gene and protein expression profiling showing the striking down-regulation of IESC marker Olfm-4, this aim will determine (1) the role of Olfm4 in inflammatory response upon LPS or TNFα treatment in intestinal epithelial cells with or without claudin-7 expression, and (2) whether the inflammatory signaling increases the stem cell proliferation and reduces the intestinal epithelial cell differentiation. This project will shed light on if claudin-7 deletion induces inflammation through disruption of the canonical TJ barrier or cell-matrix adhesion and how claudin-7 elicits inflammation to affect the fate of IESCs. Overall, the broad impact of this project will be identifying “claudin-7-MMPs-IESCs functions” as potential targets through which claudin-7 controls intestinal homeostasis against inflammation.