Chondrocyte DNA Double-Strand Breaks in the Pathogenesis of Osteoarthritis

骨关节炎(OA)是最常见的关节炎形式，也是导致成年人残疾的主要原因。虽然衰老是骨性关节炎发生发展的重要危险因素，但在衰老的背景下导致骨性关节炎的分子机制仍不清楚。在这里，我们提出了一种假设，即关节软骨细胞中的DNA DSB促进细胞周期的重新进入，并诱导IKKbeta/NF-kappaB和STING下游的IRF信号介导促炎分泌表型，从而导致周围关节组织的软骨退化和炎症。我们发现，野生型小鼠关节软骨细胞中的IKKbeta/NF-kappaB信号随着年龄的增长而增加，24个月后，这些小鼠发展成膝关节表型，表现出早期骨性关节炎的特征。我们还表明，幼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表型的开始，可能是通过对周围细胞的旁分泌作用来实现的，该旁分泌程序由大量细胞因子、趋化因子、生长因子和MMPs组成，可以改变邻近细胞的增殖和活性，重塑软骨ECM，并启动先天免疫反应。DNA双链断裂(DSB)可诱导IKKbeta/NF-kappaB信号转导，我们在此提供了衰老小鼠关节软骨细胞中存在DNA双链断裂的证据。我们还提供了基因表达数据，表明IRF和干扰素信号通路在老年关节软骨细胞中受到的影响最大。DNADSB可通过干扰素基因刺激物或STIN诱导核因子-kappaB和IRF信号转导。因此，我们假设刺痛可能位于核因子-kappaB和IRF的上游，从而在患有DSB的软骨细胞中产生促炎分泌表型。在这项建议的第一个目标中，我们将使用AcanCreERT2/+；R26I-PpoI小鼠来测试软骨细胞特异性DNA DSB对软骨细胞功能和骨关节炎发育的影响，其中DSB是在给予他莫昔芬后在软骨细胞中特异性产生的。我们将确定软骨细胞如何对这种基因毒性应激做出反应，以及持久性DSB是否能够促进骨关节炎。在第二个目标中，我们将使用体外细胞培养方法和体内STING基因敲除模型，结合NF-kappaB和IRF内源性报告，测试STING是否特异性地参与了DSB诱导后IKKbeta/NF-kappaB和IRF信号的激活以及与年龄相关的骨性关节炎的发病。这些研究结果将为DNADSB对软骨细胞命运的影响以及对衰老软骨细胞中NF-kappaB上游激活物的识别提供有价值的见解，可能导致识别一种新的机制，其中抑制IKKbeta/NF-kappaB信号转导和OA软骨细胞的炎症。

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.