Pemphigus IgG Internalization and Desmosome Disassembly

描述（由申请人提供）： 寻常天疱疮（PV）是一种严重的自身免疫性水疱性疾病，其特征是口腔粘膜和表皮下层上皮细胞之间的粘附破坏。在这种疾病中，自身抗体靶向的抗原是桥粒钙粘蛋白桥粒芯糖蛋白-3（Dsg 3）。桥粒是细胞间粘附结构，在表皮完整性的建立和维持中起关键作用。Dsg 3自身抗体产生的主要后果是破坏桥粒细胞-细胞粘附，并且PV抗体可能直接干扰Dsg 3介导的细胞-细胞粘附。然而，许多研究还表明，桥粒组分在体内和体外均被暴露于PV血清的角质形成细胞内化。在初步研究中，我们发现PV血清以及针对Dsg 3的致病性单克隆抗体被角质形成细胞内化并进入内吞途径。这些观察结果提高了Dsg 3的内吞作用导致Dsg 3的细胞表面水平显著降低的可能性，并且Dsg 3响应于PV抗体的内化可能与天疱疮疾病发病机制有因果关系。因此，通过抑制Dsg 3对PV IgG连接的内吞作用，可能会减弱PV自身抗体对上皮细胞粘附的影响。为了解决这种可能性，一个复杂的理解膜运输系统如何与桥粒粘附分子的接口是必要的。该R21提案将检验致病性Dsg 3抗体引起该细胞表面粘附分子内化的假设。提出了两个目标。首先，我们将建立一系列的定量测定来监测PV IgG内化，并将这些测定与药理学和显性阴性方法相结合，以确定Dsg 3内化的基本机制。其次，我们将确定PV IgG和Dsg 3是否单独内化，或作为与其他桥粒分子的复合物。然后，我们将测试桥粒的组件调节Dsg 3内化的可能性。这些实验将为后续的R 01应用奠定基础。这些研究的长期目标是揭示新的细胞途径，这些途径可能在治疗上靶向，使患有这种毁灭性疾病的患者的角质形成细胞对PV IgG更具抵抗力。

DESCRIPTION (provided by applicant): Pemphigus vulgaris (PV) is a severe autoimmune blistering disease characterized by the disruption of adhesion between epithelial cells in the oral mucosa and in the lower layers of the epidermis. The antigen targeted by autoantibodies in this disease is the desmosomal cadherin desmoglein-3 (Dsg3). Desmosomes are cell-cell adhesion structures that play critical roles in the establishment and maintenance of epidermal integrity. A major consequence of Dsg3 autoantibody production is the disruption of desmosomal cell-cell adhesion, and it is likely that PV antibodies directly interfere with cell-cell adhesion mediated by Dsg3. However, numerous studies have also demonstrated that desmosomal components are internalized by keratinocytes exposed to PV sera, both in vivo and in vitro. In preliminary studies, we have found that PV sera as well as pathogenic monoclonal antibodies directed against Dsg3 are internalized by keratinocytes and enter the endocytic pathway. These observations raise the possibility that endocytosis of Dsg3 leads to a significant reduction of cell surface levels of Dsg3, and that Dsg3 internalization in response to PV antibodies may be causally related to pemphigus disease pathogenesis. Therefore, by inhibiting Dsg3 endocytosis in response to PV IgG ligation, it might be possible to blunt the impact ofPV autoantibodies on epithelial cellcell adhesion. In order to address this possibility, a sophisticated understanding of how membrane trafficking systems interface with desmosomal adhesion molecules is needed. This R21 proposal will test the hypothesis that pathogenic Dsg3 antibodies cause the internalization of this cell surface adhesion molecule. Two aims are proposed. First, we will establish as series of quantitative assays to monitor PV IgG internalization, and use these assays in combination with pharmacological and dominant negative approaches to determine the basic mechanisms by which Dsg3 is internalized. Secondly, we will determine if PV IgG and Dsg3 are internalized alone, or as a complex with other desmosomal molecules. We will then test the possibility that components of the desmosome regulate Dsg3 internalization. These experiments will form the foundation for a subsequent R01 application. The long-term goal of these studies is to expose new cellular pathways that might be targeted therapeutically to make keratinocytes more resistant to PV IgG in patients suffering from this devastating disease.