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），其特点是肠上皮屏障完整性受损。我们之前已经表明，桥粒黏附分子桥粒蛋白2 （Dsg2）对屏障功能至关重要。我们发现，在患有CD的患者的肠道活检中，Dsg2减少并显示出改变的定位。电镜显示，在CD中，桥粒超微结构平行于紧密连接（TJ）发生改变，而粘附连接（adhesion junction）则正常。在培养的肠细胞中，当受到肿瘤坏死因子α的攻击时，Dsg2连接串联肽（TP）稳定了细胞的粘附和屏障功能，这表明Dsg2结合受损可能有助于CD的发病。由于这些数据得到了Dsg2缺陷小鼠Pavel strad合作项目的支持，我们开始通过TP在体内稳定Dsg2的结合。我们在肠细胞表面发现了esmosomal Dsg2。由于Dsg2结合并调节参与屏障调节的EGFR和pi3激酶，我们确定Dsg2是肠细胞中的信号中枢。因此，我们将在第二个资助期研究EGFR和pi3激酶如何控制细胞内聚和TJ功能。在与Mechthild Hatzfeld的合作中，我们将研究桥粒斑块蛋白（如嗜血小板蛋白和血小板红蛋白）在肠细胞缺乏这些蛋白的肠屏障调节中的作用，因为我们发现它们可以改变不同Dsg亚型的分子结合特性。对于桥蛋白，我们将用Carsten Grashoff的新型桥蛋白张力传感器重建肠细胞，我们已经证明它足以诱导桥蛋白体的形成。与与desmocollin 2或EGFR的异亲性相互作用相比，Dsg2的同亲性结合将通过Rudolf Leube进行表征。在该项目的翻译部分，我们观察到神经胶质细胞系衍生的神经营养因子（GDNF）在体外促进肠屏障成熟，在CD活检中减少。在dsg2缺陷细胞中，我们发现GDNF的屏障保护作用是通过稳定dsg2介导的粘附和信号传导来介导的。在dss诱导的结肠炎模型中，体内给药GDNF可减轻炎症诱导的Dsg2损失。GDNF还在体外和体内消除了p38mapk介导的角蛋白8和18的磷酸化以及角蛋白丝的重组，这两者都与CD患者的屏障破坏相似。与Thomas Magin合作，我们观察到角质形成细胞中的角蛋白丝调节p38MAPK的活性。我们将建立缺乏角蛋白8和角蛋白18的肠细胞系，并用拟磷和缺乏角蛋白突变体重建它们，以研究它们对屏障功能的影响。基于第一个资助期收集的新见解，我们将进一步阐明Dsg2粘附特性与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.