Chondrocyte DNA Double-Strand Breaks in the Pathogenesis of Osteoarthritis

软骨细胞 DNA 双链断裂在骨关节炎发病机制中的作用

基本信息

批准号：
10624247
负责人：
Jennifer Harrell Jonason
金额：
$ 33.88万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-07 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10624247
关键词：
Acceleration Adaptor Signaling Protein Adult Affect Age Age Months Age of Onset Aging Arthritis Automobile Driving Biological Assay Bleomycin Bromodeoxyuridine Cartilage Cell Aging Cell Culture Techniques Cell Cycle Cell Survival Cells Chondrocytes Chronic Clinic Clinical Coculture Techniques DNA Damage DNA Double Strand Break Data Degenerative polyarthritis Development Disease Disease Progression Endogenous Factors Exhibits Extracellular Matrix Gene Expression Gene Expression Profiling Generations Genotoxic Stress Growth Factor Harvest Histology Homing Human Immune In Vitro Inflammation Inflammatory Inflammatory Response Innate Immune Response Interferons Investigation Joints Knee joint Knock-out Mass Spectrum Analysis Matrix Metalloproteinases Mediating Methods Modeling Molecular Mus NF-kappa B Nuclear Pathogenesis Pathway interactions Phenotype Population Proliferating Protein Inhibition Proteins Public Health Reporter Risk Factors Role STING agonists Signal Pathway Signal Transduction Source Stimulator of Interferon Genes Tamoxifen Testing Therapeutic Tissues Toxic effect Western Blotting Wild Type Mouse age related aged articular cartilage cartilage degradation cartilage regeneration chemokine cytokine design disability endonuclease experience experimental study in vivo insight joint destruction loss of function microCT migration novel paracrine postnatal prevent programs response senescence small molecule inhibitor therapeutic development transcriptome sequencing

项目摘要

Osteoarthritis (OA) is the most common form of arthritis and a leading cause of disability in adults. While aging is an important risk factor for the development of OA, the molecular mechanisms responsible for OA in the context of aging remain unclear. Here, we propose to test the hypothesis that DNA DSBs in articular chondrocytes promote cell cycle re-entry and induce both IKKBeta/NF-kappaB and IRF signaling downstream of STING to mediate a pro-inflammatory secretory phenotype that leads to cartilage degeneration and inflammation in surrounding joint tissues. We show that IKKBeta/NF-kappaB signaling increases with age in the articular chondrocytes of wild type mice and that, by 24 months, these mice develop a knee joint phenotype exhibiting early hallmarks of OA. We also show that sustained IKKBeta activation in the chondrocytes of young mice greatly accelerates the onset of this age-related OA phenotype likely through paracrine actions on surrounding cells via a proinflammatory secretory program consisting of numerous cytokines, chemokines, growth factors, and MMPs that can alter proliferation and viability of neighboring cells, remodel the cartilage ECM, and initiate an innate immune response. DNA double-strand breaks (DSBs) are known to induce IKKBeta/NF-kappaB signaling and we provide evidence here of DNA DSBs in aged murine articular chondrocytes. We also provide gene expression data showing that IRF and Interferon signaling are among the most significantly affected pathways in aged articular chondrocytes. DNA DSBs can induce both NF-kappaB and IRF signaling through Stimulator of interferon genes, or STING. Thus, we hypothesize that STING may be upstream of both NF-kappaB and IRF to produce a proinflammatory secretory phenotype in chondrocytes with DSBs. In the first Aim of this proposal, we will test the effects of chondrocyte-specific DNA DSBs on chondrocyte function and OA development using AcanCreERT2/+; R26I-PpoI mice where DSBs are produced specifically in chondrocytes upon administration of tamoxifen. We will determine how chondrocytes respond to this genotoxic stress and also whether persistent DSBs are capable of promoting OA. In the second Aim, we will test whether STING is specifically involved in the activation of IKKBeta/NF-kappaB and IRF signaling following induction of DSBs and in the onset of age-related OA using in vitro cell culture methods as well as an in vivo STING knockout model in combination with NF-kappaB and IRF endogenous reporters. Results from these studies will provide valuable insight to the effects of DNA DSBs on chondrocyte fate and also on the identity of upstream activators of NF-kappaB in aged chondrocytes, potentially leading to the identification of a novel mechanism in which to inhibit IKKBeta/NF-kappaB signaling and inflammation in OA chondrocytes.

骨关节炎 (OA) 是最常见的关节炎形式，也是成人残疾的主要原因。虽然衰老是 OA 发生的重要危险因素，但衰老背景下 OA 的分子机制仍不清楚。在这里，我们建议检验以下假设：关节软骨细胞中的 DNA DSB 促进细胞周期重入，并诱导 STING 下游的 IKKBeta/NF-kappaB 和 IRF 信号传导，介导促炎分泌表型，从而导致软骨退化和炎症。周围的关节组织。我们发现，野生型小鼠关节软骨细胞中的 IKKβ/NF-kappaB 信号随着年龄的增长而增加，并且到 24 个月时，这些小鼠形成了表现出 OA 早期特征的膝关节表型。我们还表明，年轻小鼠软骨细胞中持续的 IKKBeta 激活极大地加速了这种与年龄相关的 OA 表型的发生，这可能是通过促炎性分泌程序对周围细胞产生旁分泌作用，该程序由多种细胞因子、趋化因子、生长因子和 MMP 组成，这些细胞因子、趋化因子、生长因子和 MMP 可以改变邻近细胞的增殖和活力，重塑软骨 ECM，并启动先天免疫反应。已知 DNA 双链断裂 (DSB) 会诱导 IKKBeta/NF-kappaB 信号传导，我们在此提供了衰老小鼠关节软骨细胞中 DNA 双链断裂的证据。我们还提供基因表达数据，显示 IRF 和干扰素信号传导是衰老关节软骨细胞中受影响最显着的途径之一。 DNA DSB 可以通过干扰素基因刺激器 (STING) 诱导 NF-kappaB 和 IRF 信号传导。因此，我们假设 STING 可能位于 NF-kappaB 和 IRF 的上游，在具有 DSB 的软骨细胞中产生促炎分泌表型。在该提案的第一个目标中，我们将使用 AcanCreERT2/+ 测试软骨细胞特异性 DNA DSB 对软骨细胞功能和 OA 发育的影响； R26I-PpoI 小鼠，在给予他莫昔芬后，软骨细胞中特异产生 DSB。我们将确定软骨细胞如何应对这种基因毒性应激，以及持久的 DSB 是否能够促进 OA。在第二个目标中，我们将使用体外细胞培养方法和体内细胞培养方法来测试 STING 是否特异性参与 DSB 诱导后 IKKBeta/NF-kappaB 和 IRF 信号传导的激活以及年龄相关 OA 的发生。 STING 敲除模型与 NF-kappaB 和 IRF 内源报告基因相结合。这些研究的结果将为了解 DNA DSB 对软骨细胞命运的影响以及对衰老软骨细胞中 NF-κB 上游激活剂的影响提供有价值的见解，从而可能导致鉴定一种抑制 IKKβ/NF- 的新机制。 OA 软骨细胞中的 kappaB 信号传导和炎症。