Chondrocyte DNA Double-Strand Breaks in the Pathogenesis of Osteoarthritis

骨关节炎（OA）是关节炎的最常见形式，也是成人残疾的主要原因。虽然衰老是OA发展的重要风险因素，但在衰老背景下导致OA的分子机制仍不清楚。在这里，我们建议测试的假设，即DNA DSB在关节软骨细胞促进细胞周期的重新进入，并诱导IKK β/NF-κ B和IRF信号下游的STING介导的促炎分泌表型，导致软骨退化和炎症周围关节组织。我们发现，IKKBeta/NF-κ B信号在野生型小鼠的关节软骨细胞中随着年龄的增长而增加，并且在24个月时，这些小鼠发展出表现出OA早期标志的膝关节表型。我们还表明，持续IKKBeta激活的年轻小鼠软骨细胞大大加速了这种年龄相关的OA表型的发病可能通过旁分泌作用周围细胞通过促炎分泌程序组成的众多细胞因子，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，并启动先天性免疫反应。已知DNA双链断裂（DSB）可诱导IKK β/NF-κ B信号传导，我们在此提供了老年鼠关节软骨细胞中DNA DSB的证据。我们还提供了基因表达数据，表明IRF和干扰素信号是老年关节软骨细胞中最受影响的途径之一。DNA DSB可以通过干扰素基因刺激因子或STING诱导NF-κ B和IRF信号传导。因此，我们假设STING可能位于NF-κ B和IRF的上游，在具有DSB的软骨细胞中产生促炎分泌表型。在本提案的第一个目的中，我们将使用AcanCreERT 2/+; R26 I-PpoI小鼠测试软骨细胞特异性DNA DSB对软骨细胞功能和OA发展的影响，其中DSB在给予他莫昔芬后在软骨细胞中特异性产生。我们将确定软骨细胞如何应对这种遗传毒性应激，以及持续性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以及与NF-κ B和IRF内源性报告基因组合的体内STING敲除模型，测试STING是否特异性参与诱导DSB后IKK β/NF-κ B和IRF信号传导的激活以及年龄相关性OA的发作。这些研究的结果将为DNA DSB对软骨细胞命运的影响以及对老年软骨细胞中NF-κ B上游激活剂的识别提供有价值的见解，可能导致识别一种抑制IKK β/NF-κ B信号传导和OA软骨细胞炎症的新机制。

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.