Epigenetic Regulation of Bone Regeneration in Inflammatory Disease

摘要 发炎性骨折是一个重要的临床问题。在美国，大约有160万人 骨折愈合时间延长或骨不连，其中， 具有这些临床并发症的是具有炎性病症的患者，例如，老年患者、吸烟者、糖尿病患者 或类风湿性关节炎（RA）患者。在这些患者中，由于骨质差，骨折风险增加， 突出了慢性全身性炎症在骨折修复中的潜在有害作用。 该建议的主要假设是，在炎症条件下，NF-κB，主要的 炎症介质，通过下调Dnmt 3b及其DNA甲基化诱导Rbpjκ表达 活动我们进一步假设Dnmt 3b GOF或Rbpjκ抑制恢复MPC分化， 在骨折修复过程中，炎症会降低软骨细胞的成熟。这一假设得到了 我们的初步数据表明，在骨折修复过程中，Dnmt 3b在骨折骨痂中高度表达， Dnmt 3b是MPC和软骨细胞中对细胞因子应答的主要DNA甲基转移酶（Dnmt）。 与我们的提议相关，我们提供了炎症信号抑制MPC中Dnmt 3b的证据， NF-κ B依赖性的软骨细胞。一致地，MPC中Dnmt 3b功能丧失（LOF）的小鼠 和软骨细胞显示延迟的骨折修复;和Dnmt 3b在MPC或软骨细胞中的功能获得性（GOF） 在体外对炎症具有保护作用，并加速小鼠骨折修复。从机制上讲，MPC 炎症和Dnmt 3b LOF介导的分化缺陷与MPC中Rbpjκ的上调一致 Rbpjκ抑制可恢复体外分化能力。 体外机制研究和体内LOF和GOF方法将用于调节IKK 2、Dnmt 3b和Dnmt 3b。 及Rbpjκ在MPCs和软骨细胞中的表达，探讨其在骨折修复过程中的作用。三个主要 提出了具体目标。特异性Aim 1将描述组成性激活的NF-κB信号传导的作用 IKK 2ca作为炎症的主要分子驱动因子，对Dnmt 3b表达和骨折修复的影响。具体 目的2将确定MPC和软骨细胞中的Dnmt 3b GOF对加速骨折修复的作用。 特异性目的3将描述Dnmt 3b调节下游靶点Rbpjκ的机制， 骨折修复这项工作将增强我们对全身性炎症（通过 NF-κB通路）通过Dnmt 3 B影响骨折愈合过程，并鉴定了 Dnmt 3b（如Rbpjκ）作为治疗干预的新候选物。

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.