The role of the disintegrin metalloproteinase ADAM15 in the apoptosis resistance of synovial fibroblasts in rheumatoid arthritis

风湿性关节炎（RA）是最常见的慢性炎症性关节疾病。在各自的遗传易感性的背景下，失调的免疫效应物途径助长了关节中的慢性炎症过程，导致蛋白水解过程的激活，这代表了对关节软骨和骨的结构完整性的持续威胁。参与炎性组织破坏的关键效应细胞群是滑膜中的成纤维细胞，其在RA关节中表现出持续活化的表型，并且是基质降解蛋白酶局部释放的主要来源。典型的RA滑膜成纤维细胞（RASF）对凋亡诱导的抵抗力显著增加，但迄今尚未完全了解。在我们的初步研究中，我们可以发现RASF中去整合素金属蛋白酶ADAM 15的强烈上调。这一发现在RASF细胞凋亡抗性增加的背景下具有潜在的相关性，因为我们还可以证明去整合素-金属蛋白酶在基因毒性应激或死亡受体Fas/CD 95活化后诱导细胞凋亡时具有保护性抗细胞凋亡作用。该项目的目的是阐明ADAM 15是否对RASF具有更普遍的细胞保护作用，或者其抗凋亡功能是否更局限于少数不同的死亡诱导刺激。该项目进一步旨在揭示依赖于其ADAM 15表达的凋亡诱导后在RASF中被激活的信号通路。基于研究结果，我们旨在阐明选择性阻断这些ADAM 15依赖性效应级联的可能性，以便通过药理学手段在RASF中重建对凋亡诱导的敏感性。要解开所涉及的分子机制，我们将表征ADAM 15配体和它们潜在的相关贡献的信号转导通路的哺乳动物双杂交，免疫沉淀，蛋白结合，和磷酸化的研究模型中的滑膜成纤维细胞系K4 IM。在该建立的细胞系模型中，将通过SILAC质谱法和随后的siRNA转染研究，对凋亡诱导后抗凋亡效应物的ADAM 15依赖性活化进行定量，并就所涉及的抗凋亡效应物蛋白进行功能分析。将对原发性RASF和RA滑膜组织的免疫组织学进行结果验证。阐明ADAM 15对RASF的抗凋亡作用旨在鉴定新的潜在靶结构，用于治疗性调节这种侵袭性细胞群体的病理学上至关重要的凋亡抗性。

Rheumatoid arthritis (RA) is the most frequent chronic inflammatory joint disease. On the background of respective genetic predispositions dysregulated immunological effector pathways fuel a chronic inflammatory process in the joints leading to the activation of proteolytic processes that represent a constant threat to the structural integrity of articular cartilage and bone. A critical effector cell population involved in the inflammatory tissue destruction is the fibroblast in the synovial membrane that exhibits a persistently activated phenotype in the RA joint and is a major source for the local liberation of matrix degrading proteinases. Characteristically the synovial fibroblast in RA (RASF) exhibits a considerably increased resistance to apoptosis induction that is so far not fully understood. In our preliminary studies we could uncover a strong upregulation of the disintegrin metalloproteinase ADAM15 in RASF. This finding is of potential relevance in the context of the increased apoptosis resistance of RASF since we could also demonstrate that the disintegrin-metalloproteinase has a protective anti-apoptotic effect upon apoptosis induction by genotoxic stress or following activation of the death receptor Fas/CD95. Aim of the project is to elucidate whether ADAM15 has a more general cytoprotective effect on RASF or if its anti-apoptotic function is more confined to few distinct death inducing stimuli. The project further aims at uncovering the signaling pathways that get activated in the RASF upon apoptosis induction in dependency on their ADAM15 expression Based on the study results we aim at elucidating possibilities to selectively block these ADAM15 dependent effector cascades in order to reestablish sensitivity to apoptosis induction in the RASF by pharmacological means. To unravel the involved molecular mechanisms we will characterize ADAM15 ligands and their potentially associated contributions to signal transduction pathways by mammalian two-hybrid-, immunoprecipitation-, protein-binding-, and phosphorylation studies in the model of the synovial fibroblast cell line K4IM. In this established cell line model the ADAM15 dependent activation of anti-apoptotic effectors upon apoptosis induction will be quantified and functionally analyzed with regard to the involved anti-apoptotic effector proteins by SILAC mass spectrometry and subsequent siRNA-transfection studies. A validation of the results will be performed on primary RASF and by immunohistology of RA synovial tissue. The elucidation of the anti-apoptotic action of ADAM15 on RASF aims at identifying new potential target structures for a therapeutic modulation of the pathogenetically crucial apoptosis resistance of this aggressive cell population.