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

类风湿性关节炎（RA）是最常见的慢性炎症性关节疾病。在各自遗传易感性的背景下，失调的免疫效应通路在关节中引发慢性炎症过程，导致蛋白水解过程的激活，这对关节软骨和骨的结构完整性构成了持续的威胁。参与炎症组织破坏的关键效应细胞群是滑膜中的成纤维细胞，它在RA关节中表现出持续激活的表型，并且是局部释放基质降解蛋白酶的主要来源。RA中的滑膜成纤维细胞（RASF）表现出显著增加的对细胞凋亡诱导的抗性，迄今尚未完全了解。在我们的初步研究中，我们可以发现RASF中崩解素金属蛋白酶ADAM15的强烈上调。这一发现与RASF增加的细胞凋亡抗性具有潜在的相关性，因为我们也可以证明崩解素-金属蛋白酶在基因毒性应激或死亡受体Fas/CD95激活诱导细胞凋亡时具有保护性抗凋亡作用。该项目的目的是阐明ADAM15是否对RASF具有更普遍的细胞保护作用，还是其抗凋亡功能更局限于几种不同的死亡诱导刺激。本项目进一步旨在揭示在诱导凋亡时，在RASF中激活的依赖于ADAM15表达的信号通路，基于研究结果，我们旨在阐明选择性阻断这些ADAM15依赖性效应级联的可能性，从而通过药理学手段重建RASF对诱导凋亡的敏感性。为了揭示所涉及的分子机制，我们将在滑膜成纤维细胞系K4IM模型中通过哺乳动物双杂交、免疫沉淀、蛋白质结合和磷酸化研究来表征ADAM15配体及其对信号转导途径的潜在相关贡献。在这个已建立的细胞系模型中，我们将通过SILAC质谱法和随后的sirna转染研究，对ADAM15依赖性的抗凋亡效应蛋白在诱导凋亡过程中的激活进行量化和功能分析。结果的验证将在原发性RASF和RA滑膜组织的免疫组织学上进行。阐明ADAM15对RASF的抗凋亡作用，旨在确定新的潜在靶标结构，用于治疗调节这种侵袭性细胞群的病理关键细胞凋亡抗性。

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.