Role of Dsg2-dependent adhesion and signalling in Crohn s disease

克罗恩病（CD）是一种发病机制复杂的炎症性肠病（IBD），其特征是肠上皮屏障完整性受损。我们之前已经证明桥粒粘附分子桥粒芯糖蛋白2（Dsg 2）对于屏障功能至关重要。我们发现，在患者的肠活检与CD Dsg 2减少，并显示改变定位。电子显微镜显示，桥粒超微结构的改变平行于紧密连接（TJ），而粘附连接出现正常的CD。在培养的肠上皮细胞中，当用肿瘤坏死因子α攻击时，Dsg 2连接串联肽（TP）稳定细胞粘附和屏障功能，表明受损的Dsg 2结合可能有助于CD发病机制。由于这些数据得到了Pavel Strnad在Dsg 2缺陷小鼠中的合作项目的支持，我们开始通过TP在体内稳定Dsg 2结合。我们在肠上皮细胞表面发现了桥粒外Dsg 2。由于Dsg 2结合并调节EGFR和PI 3-激酶，它们参与屏障调节，我们将Dsg 2鉴定为肠细胞中的信号传导枢纽。因此，我们将研究EGFR和PI 3-激酶如何控制细胞凝聚力和TJ功能在第二个资助期。在与Mechthild Hatzfeld的合作中，我们将研究桥粒斑块蛋白如斑嗜蛋白和斑珠蛋白在缺乏这些蛋白的肠细胞中对肠屏障调节的作用，因为我们发现它们修饰不同Dsg亚型的分子结合特性。对于桥粒斑蛋白，我们将用Carsten Grashoff的新的桥粒斑蛋白张力传感器重建肠细胞，我们已经证明它足以诱导桥粒形成。与桥粒胶蛋白2或EGFR两者的嗜异性相互作用相比，Dsg 2的嗜同性结合将用Rudolf Leube表征。在该项目的翻译部分，我们观察到，胶质细胞源性神经营养因子（GDNF），在体外增强肠屏障成熟，减少CD活检。在Dsg 2缺陷细胞中，我们发现GDNF的屏障保护作用是通过稳定Dsg 2介导的粘附和信号传导介导的。GDNF在DSS诱导的结肠炎模型中的体内施用减弱了炎症诱导的Dsg 2损失。GDNF还在体外和体内消除了p38 MAPK介导的角蛋白8和18的磷酸化以及角蛋白丝重组，这两者都阻止了CD患者的屏障破坏。在与托马斯Magin合作，我们观察到角质形成细胞中的角蛋白丝调节p38 MAPK活性。我们将建立肠上皮细胞株缺乏角蛋白8和18，并将重建他们与磷酸模拟和缺乏角蛋白突变体，以研究屏障功能的影响。基于在第一个资助期间收集的新见解，我们将进一步阐明Dsg 2粘附特性和信号通路之间的相互作用。

Crohn’s disease (CD) is an inflammatory bowel disease (IBD) with complex pathogenesis which is characterized by impaired intestinal epithelial barrier integrity. We had previously shown that the desmosomal adhesion molecule desmoglein 2 (Dsg2) is crucial for barrier function. We found that in patients’ intestinal biopsies with CD Dsg2 is reduced and displays altered localization. Electron microscopy revealed that desmosome ultrastructure is altered in parallel to tight junctions (TJ) whereas adherens junctions appeared normal in CD. In cultured enterocytes, a Dsg2-linking tandem peptide (TP) stabilized cell adhesion and barrier function when challenged with tumor necrosis factor α indicating that impaired Dsg2 binding may contribute to CD pathogenesis. Because these data are supported by the cooperation project of Pavel Strnad in Dsg2-deficient mice, we started to stabilize Dsg2 binding via TP in vivo. We found extradesmosomal Dsg2 on the surface of enterocytes. Since Dsg2 binds to and modulates EGFR and PI3-kinase, which are involved in barrier regulation we identified Dsg2 as a signaling hub in enterocytes. Therefore we will study how EGFR and PI3-kinase control cell cohesion and TJ function in the second funding period. In cooperation with Mechthild Hatzfeld we will study the role of desmosomal plaque proteins such as plakophilins and plakoglobin for intestinal barrier regulation in enterocytes deficient for these proteins because we found that they modify molecular binding properties of different Dsg isoforms. For desmoplakin, we will reconstitute enterocytes with the new desmoplakin tension sensor of Carsten Grashoff for which we have shown that it is sufficient to induce desmosome formation. Homophilic binding of Dsg2 in comparison to heterophilic interaction with both desmocollin 2 or EGFR will be characterized with Rudolf Leube. In the translational part of the project, we observed that glial cell line-derived neurotrophic factor (GDNF), which enhanced intestinal barrier maturation in vitro, was reduced in CD biopsies. In Dsg2-deficient cells, we found that the barrier-protective effects of GDNF are mediated via stabilisation of Dsg2-mediated adhesion and signalling. GDNF administration in vivo in a model of DSS-induced colitis attenuated inflammation-induced loss of Dsg2. GDNF also abolished p38MAPK-mediated phosphorylation of keratins 8 and 18 as well as keratin filament reorganization in vitro and in vivo, both of which paralleled barrier breakdown in CD patients. In cooperation with Thomas Magin, we observed that keratin filaments in keratinocytes regulate p38MAPK activity. We will establish enterocyte cell lines deficient for keratin 8 and 18 and will reconstitute them with phospho-mimetic and –deficient keratin mutants to investigate the effects on barrier function. Based on new insights gathered during the first funding period, we will further elucidate the interplay between Dsg2 adhesion properties and signalling pathways.