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

克罗恩病(CD)是一种以肠上皮屏障完整性受损为特征的炎症性肠病，发病机制复杂。我们先前已经证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CD患者的肠道活检中，Dsg2减少，并显示出局部改变。电子显微镜显示CD的桥粒超微结构改变与紧密连接(TJ)平行，而粘连连接正常。在培养的肠细胞中，当肿瘤坏死因子α攻击时，DSG2连接串联肽(TP)稳定了细胞的黏附和屏障功能，表明DSG2结合受损可能参与了CD的发病。由于这些数据得到了Pavel Strnad在Dsg2基因缺陷小鼠中的合作项目的支持，我们开始在体内通过TP稳定Dsg2结合。肠细胞表面可见桥粒外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是肠细胞中的一个信号枢纽。因此，我们将在第二个资助期研究EGFR和PI3-激酶如何控制细胞凝聚力和TJ功能。与Mechthild Hatzfeld合作，我们将研究桥粒斑块蛋白，如血小板亲和素和蛋白在缺乏这些蛋白的肠细胞中调节肠道屏障的作用，因为我们发现它们改变了不同DSG亚型的分子结合特性。对于桥粒蛋白，我们将用Carsten Grashoff的新的桥粒蛋白张力传感器重建肠细胞，我们已经证明对它来说，它足以诱导桥粒的形成。Rudolf Leube将用Rudolf Leube描述Dsg2与Desmocolin 2或EGFR的同亲结合与异亲作用的比较。在该项目的翻译部分，我们观察到在体外促进肠屏障成熟的胶质细胞系衍生神经营养因子(GDNF)在CD活检中减少。在Dsg2缺乏的细胞中，我们发现GDNF的屏障保护作用是通过稳定Dsg2介导的黏附和信号转导来实现的。在DSS诱导的结肠炎模型中，体内给予GDNF可减轻炎症诱导的Dsg2的丢失。GDNF还抑制了p38MAPK介导的角蛋白8和18的磷酸化以及体内外角蛋白细丝的重组，这两者都与CD患者的屏障破坏相平行。在与Thomas Magin的合作中，我们观察到角质形成细胞中的角蛋白细丝调节p38MAPK的活性。我们将建立缺乏角蛋白8和18的肠上皮细胞系，并将它们与模拟磷酸化和缺陷性角蛋白突变体重组，以研究其对屏障功能的影响。基于在第一个资助期收集的新见解，我们将进一步阐明Dsg2黏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.