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

解整合素金属蛋白酶ADAM15在类风湿性关节炎滑膜成纤维细胞凋亡抵抗中的作用

基本信息

批准号：
252699675
负责人：
Professor Dr. Harald Burkhardt
金额：
--
依托单位：
Klinik für Orthopädie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2014
资助国家：
德国
起止时间：
2013-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/252699675?language=en
关键词：
role disintegrin metalloproteinase ADAM15 apoptosis

项目摘要

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.

类风湿性关节炎（RA）是最常见的慢性炎症性关节疾病。在各自遗传倾向的背景下，免疫效应途径失调会加剧关节中的慢性炎症过程，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持续威胁。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，其在 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 (RASF) 中滑膜成纤维细胞的特征是对细胞凋亡诱导的抵抗力显着增加，但迄今为止尚未完全了解这一点。在我们的初步研究中，我们发现 RASF 中解整合素金属蛋白酶 ADAM15 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相关性，因为我们还可以证明解整合素金属蛋白酶对基因毒性应激或死亡受体 Fas/CD95 激活后诱导细胞凋亡具有保护性抗细胞凋亡作用。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，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 RASF 中依赖于 ADAM15 表达的凋亡诱导激活的信号通路。根据研究结果，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，以便重新建立对凋亡诱导的敏感性通过药理学手段进入RASF。为了阐明所涉及的分子机制，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。在此建立的细胞系模型中，将通过 SILAC 质谱法和随后的 siRNA 转染研究对所涉及的抗凋亡效应蛋白进行量化和功能分析。结果的验证将通过原发性 RASF 和 RA 滑膜组织的免疫组织学进行。阐明 ADAM15 对 RASF 的抗凋亡作用旨在确定新的潜在靶结构，用于治疗性调节这种侵袭性细胞群的致病性关键凋亡抵抗。