Epigenetic Regulation of Bone Regeneration in Inflammatory Disease

摘要 发炎的骨折是一个重要的临床问题。在美国，大约有160万人 骨折每年都会出现长时间愈合或骨不连的情况，其中， 有这些临床并发症的是炎症性疾病的患者，如老年患者、吸烟、糖尿病 或类风湿性关节炎(RA)患者。在这些患者中，由于骨骼质量差，骨折风险增加， 强调慢性全身炎症在骨折修复中的潜在有害作用。 这一提议的首要假设是，在炎症条件下，主要的NF-κB 炎症介质，通过下调Dnmt3b及其κ甲基化诱导RbpjDNA表达 活动。我们进一步假设，Dnmt3bGOF或Rbpjκ抑制恢复了MPC的分化和 在骨折修复过程中因炎症而减少的软骨细胞成熟。这一假设得到了 我们的初步数据显示，在骨折修复过程中，DNMT3b在骨折骨痂中高度表达 Dnmt3b是MPC和软骨细胞对细胞因子反应的主要DNA甲基转移酶(Dnmt)。 与我们的建议相关，我们提供的证据表明，炎症信号抑制MPC和MPC中的Dnmt3b 软骨细胞以核因子-κB依赖的方式表达。一直以来，携带Dnmt3b的小鼠在MPC中功能丧失(LOF) 软骨细胞显示骨折修复延迟；MPC或软骨细胞中Dnmt3b功能增强(GOF) 在体外显示抗炎保护作用，并加速小鼠骨折修复。从机械上讲，MPC 炎症和DNMT3b LOF介导的分化缺陷与MPC中Rbpjκ的上调一致 抑制RBPJκ可恢复体外分化能力。 体外机制研究和体内LOF和GOF方法将被用于调节IKK2，Dnmt3b 以及RBPJκ在骨髓基质细胞和软骨细胞中的表达，分析其在骨折修复过程中的作用。三大主线 提出了具体的目标。特定目标1将描述结构性激活的NF-κB信号的作用 (IKK2ca)作为炎症的主要分子驱动因子，对Dnmt3b的表达和骨折修复起着重要的作用。特定的 目的研究Dnmt3b GOF在骨髓基质细胞和软骨细胞促进骨折修复中的作用。 具体目标3将描述DNMT3B在以下过程中调节下游靶标Rbpjκ的机制 骨折修复。这项工作将加强我们对全身炎症(通过 核因子-κB途径)通过DNMT3b影响骨折愈合过程，并识别 DNMT3b(如Rbpjκ)是治疗干预的新候选基因。

ABSTRACT Inflamed bone fracture poses a significant clinical problem. In the United States, approximately 1.6 million bone fractures encounter prolonged healing or non-union each year, among which, the major population bearing with these clinical complications are patients with inflammatory conditions, e.g, elder patients, smoking, diabetic or rheumatoid arthritis (RA) patients. In these patients, the fracture risk is increased due to the poor bone quality, highlighting the potential deleterious role of chronic systemic inflammation in fracture repair. The overarching hypothesis of this proposal is that under inflammatory conditions, NF-κB, the principal mediator of inflammation, induces Rbpjκ expression through downregulating Dnmt3b and its DNA methylation activity. We further hypothesize that Dnmt3b GOF or Rbpjκ inhibition restores MPC differentiation and chondrocyte maturation that are reduced by inflammation during fracture repair. This hypothesis is supported by our preliminary data wherein we show that Dnmt3b is highly expressed in fracture callus during fracture repair and Dnmt3b is the major DNA methyltransferase (Dnmt) responsive to cytokine in MPCs and chondrocytes. Relevant to our proposal, we provide evidence that inflammatory signals inhibit Dnmt3b in MPCs and chondrocytes in an NF-κB-dependent manner. Consistently, mice with Dnmt3b loss-of-function (LOF) in MPCs and chondrocytes display delayed fracture repair; and Dnmt3b gain-of-function (GOF) in MPCs or chondrocytes shows protective effect from inflammation in vitro and accelerates fracture repair in mice. Mechanistically, MPC differentiation defect mediated by inflammation and Dnmt3b LOF coincide with upregulation of Rbpjκ in MPCs and Rbpjκ inhibition can restore differentiation capacity in vitro. In vitro mechanistic studies and in vivo LOF and GOF approaches will be used to modulate IKK2, Dnmt3b and Rbpjκ expression in MPCs and chondrocytes to dissect its effects during fracture repair process. Three main Specific Aims are proposed. Specific Aim 1 will delineate the effect of constitutively active NF-κB signaling (IKK2ca), as the principal molecular driver of inflammation, on Dnmt3b expression and fracture repair. Specific Aim 2 will establish the effect of Dnmt3b GOF in MPCs and chondrocytes on accelerating fracture repair. Specific Aim 3 will delineate the mechanism by which Dnmt3b regulates downstream target, Rbpjκ, during fracture repair. This work will enhance our understanding of mechanisms by which systemic inflammation (via the NF-κB pathway) affects the fracture healing process through Dnmt3b and identify downstream targets of Dnmt3b (such as Rbpjκ) as novel candidates for therapeutic intervention.