Role of Claudin-2 in Inflammatory Diseases and Colon Cancer

描述（由申请人提供）：粘膜通透性增加是炎症性肠病（IBD）的一个关键特征。claudin家族蛋白构成紧密连接（TJs），这是完整上皮细胞旁通透性的唯一决定因素。重要的是，从对IBD（克罗恩病和溃疡性结肠炎）患者样本和相关动物模型的分析中得出的一个共同发现是，claudin-2的表达在IBD中高度上调。在claudin家族中，claudin -2是独特的，因为它的表达与上皮渗漏相关。然而，同样值得注意的是，结肠claudin-2在隐窝基部的未分化的结肠细胞中表达，即增殖区。此外，我们的初步研究结果和其他实验室最近发表的研究结果表明，claudin-2的表达与结肠上皮细胞增殖和/或迁移有潜在的相关性。进一步值得注意的是，claudin-2是Wnt/¿-catenin信号传导的靶标。然而，claudin-2在IBD发病机制中的作用尚不清楚。为了进行研究，我们在结肠中产生了过表达claudin-2的绒毛蛋白-claudin-2转基因（Cl-2Tg）小鼠，这种情况类似于IBD。我们对这些小鼠的研究提供了新的结果：1)Cl-2Tg小鼠的结肠上皮通透性与WT窝群相比显著增加（p<0.05）；2)与野生型（WT）相比，Cl-2Tg小鼠的结肠和隐窝长度显著增加（p<0.001）；3)与WT同窝小鼠相比，Cl-2Tg小鼠明显免受实验性结肠炎[使用葡聚糖硫酸钠（DSS，饮用水中4% w/v）诱导]的保护（p<0.001）。我们进一步的数据显示，与WT组相比，dss诱导的Cl-2Tg小鼠中IL-1a、IL-1¿、TNF-a等促炎细胞因子的表达急剧下降。另一方面，与dss处理的WT幼崽相比，dss处理的Cl-2Tg小鼠中与增殖相关的基因表达上调。综上所述，我们推测claudin-2在调节结肠内稳态中起重要作用。我们进一步假设，由于粘膜通透性的组成性增加和/或结肠上皮细胞增殖/凋亡的调节或两者的结合，免疫适应/耐受是Cl-2Tg小鼠免于dss -结肠炎的基础。为了验证我们的假设，我们提出了以下具体目标：1)确定claudin-2 TG小鼠是否具有黏膜炎症和疾病的保护作用；2)确定claudin-2在结肠炎相关再生、修复和结肠炎相关癌变中的作用；3)探讨claudin-2 TG对小鼠结肠炎的保护作用的细胞和分子机制。我们预计我们的研究将提供有价值的和临床相关的信息，这将对理解IBD发病机制及其进展到癌症的调控有直接的影响，并将为治疗干预创造潜在的新机会。

DESCRIPTION (provided by applicant): Increased mucosal permeability is a key characteristic of the inflammatory bowel disease (IBD). The claudin family of proteins constitutes the tight junctions (TJs), which are the sole determinants of the paracellular permeability in an intact epithelium. Importantly, a common finding from the analysis of the IBD (Crohn's and Ulcerative Colitis) patient samples and related animal models is that claudin-2 expression is highly upregulated in the IBD. In the claudin family, Claudin-2 is unique as its expression correlates with the epithelial leakiness. However, it is also noteworthy that the colonic claudin-2 is expressed among the undifferentiated colonocytes at the crypt base, the proliferative zone. Furthermore, outcome from our preliminary studies and recently published studies from other labs suggest potential correlation of claudin-2 expression with the colonic epithelial cell proliferation and/or migration. It is further noteworthy that claudin-2 is a target of the Wnt/¿-catenin signaling. However, role of claudin-2 in the pathogenesis of IBD is not known. To investigate, we generated Villin-claudin-2 transgenic (Cl-2Tg) mice that overexpress claudin-2 in the colon, a condition similar to the IBD. Our studies using these mice have provided novel outcomes: 1) Colonic epithelial permeability in Cl-2Tg mice is significantly increased compared to the WT littermates (p<0.05); 2) the colon and crypt lengths in Cl-2Tg mice are significantly increased compared to the wild type (WT) littermates (p<0.001 for both); and 3) Cl-2Tg mice are significantly protected from experimental-colitis [induced using dextran sodium sulfate (DSS, 4% w/v in drinking water)] compared to WT littermates (p<0.001). Our further data show sharp decreases in the DSS-induced expressions of proinflammatory cytokines including IL-1a, IL- 1¿, TNF-a in Cl-2Tg mice compared to the WT littermates. On the other hand, gene expressions related with proliferation were upregulated in the DSS-treated Cl-2Tg mice compared to the DSS-treated WT littermates. Taken together, we postulate that claudin-2 plays an important role in the regulation of colonic homeostasis. We further hypothesize that immune adaptation/tolerance due to the constitutive increase in the mucosal permeability and/or modulations of the colonic epithelial cell proliferation/apoptosis or combination of both underlie the protection from DSS-colitis in Cl-2Tg mice. To test our hypothesis, we have proposed following specific aims: 1) To determine whether claudin-2 TG mice are protected against mucosal inflammation and disease; 2) To determine the role of claudin-2 in colitis-associated regeneration, repair and colitis-associated cancer; and 3) To determine the cellular and molecular mechanism/s underlying protection from colitis in claudin-2 TG mice. We anticipate that our studies will provide valuable and clinically relevant information that will have direct impact upon the understanding of the regulation of IBD pathogenesis and its progression to cancer, and will create potential new opportunities for therapeutic interventions. PUBLIC HEALTH RELEVANCE: We will investigate the role of mucosal permeability in colonic inflammation using mice engineered to over- express claudin-2, a tight junction integral protein. We will further determine the role of claudin-2 in the regulation of colonic homeostasis, epithelial cell proliferation and a causal role in the IBD-associated cancer.