Targeting TNF Receptors to Inhibit Inflammation and to Prompt Bone Regeneration in Type 1 Diabetes

项目摘要 促炎细胞因子TNFα被认为是糖尿病患者骨折愈合延迟的原因。然而，关于TNFα抑制对骨形成的影响没有达成共识，这表明在寻找除纯TNF抑制剂之外的具有独特功能的新试剂用于糖尿病骨折愈合方面的需求尚未得到满足。我们的遗传筛选导致TNFR被鉴定为颗粒蛋白前体（PGRN）的新型受体（Tang等人，Science，2011），其是一种软骨形成因子，已显示出对自身免疫性炎性关节炎具有治疗性。1型糖尿病是最常见的自身免疫性疾病，以慢性炎症和TNFα活性升高为特征。虽然TNFα活性主要通过TNFR 1介导，但我们兴奋地发现PGN刺激的骨再生在很大程度上依赖于TNFR 2。这些矛盾的发现表明，再生PGRN/TNFR 2途径在PGRN刺激的骨折愈合中起着重要作用。此外，14-3-3ε被鉴定为响应PGRN刺激的TNFR 2途径的组分。此外，我们已经开发了一种名为Atsttrin的工程蛋白，其由PGRN的三个TNFR结合结构域组成，并且Atsttrin在炎性关节炎中比PGRN更有效。考虑到升高的TNFα被认为是糖尿病骨折愈合延迟的原因，我们假设PGRN和Atsttrin通过a）抑制TNFα/TNFR 1炎症和骨吸收途径;和主要是B）募集14-3-3ε至TNFR 2，随后激活骨再生途径来刺激糖尿病骨折愈合。具体目的：（1）研究PGRN及其衍生物Atsttrin在糖尿病骨折愈合中的作用。我们将使用系统性和诱导型PGRN敲除小鼠来确定PGRN敲除是否延迟糖尿病骨折愈合，以及重组PGRN和Atsttrin是否可以逆转它（SA#1A）;骨折愈合的哪个阶段需要PGRN以成功完成糖尿病骨折愈合（SA#1B）;以及PGRN，特别是Atsttrin，是否在治疗糖尿病骨折中具有治疗功效（SA#1C）。我们将使用适当的可注射水凝胶局部递送各种剂量的PGRN或Atsttrin。(2)阐明PGRN和Atsttrin促进糖尿病骨折愈合的分子机制。我们将确定PGRN、Atsttrin和TNFα对糖尿病骨髓干细胞的软骨形成、信号传导途径、相互作用和对TNFR和14-3-3ε的依赖性的影响（SA#2A）;两种TNFR是否对介导PGRN在糖尿病骨愈合中的作用重要（SA#2B）;通过诱导型14 - 3 -3ε[-/-]小鼠建立糖尿病骨折模型（SA#2C），探讨PGRN和Atsttrin的保护作用是否依赖于14-3-3ε。拟议的研究不仅将促进我们对糖尿病骨折愈合相关分子事件的理解，而且还可能为糖尿病骨折愈合和其他骨折愈合受损的情况提供新的治疗干预措施。

Project Summary Pro-inflammatory cytokine TNFα is believed to be responsible for the delayed fracture healing observed in diabetes. However, there is no consensus on the effect of TNFα inhibition on the bone formation, indicating the unmet need in searching for new regents with unique features other than pure TNF inhibitors for diabetic fracture healing. Our genetic screen led to the identification of TNFR as the novel receptor of progranulin (PGRN) (Tang, et al, Science, 2011), a chondrogenic factor that has been shown to be therapeutic against autoimmune inflammatory arthritis. Type 1 diabetes is the most common autoimmune disease, characterized by chronic inflammation and elevated TNFα activity. Although TNFα activity is mediated primarily through TNFR1, we were excited to find that PGRN-stimulated bone regeneration largely depends on TNFR2. These paradoxical findings suggest that the regenerative PGRN/TNFR2 pathway plays a major role in PGRN-stimulated fracture healing. In addition, 14-3-3ε was identified as a component of TNFR2 pathway in response to PGRN stimulation. Further, we have developed an engineered protein named Atsttrin which is composed of three TNFR-binding domains of PGRN, and Atsttrin is more effective than PGRN in inflammatory arthritis. Given that elevated TNFα is believed to be responsible for delayed diabetic fracture healing, we hypothesize that PGRN and Atsttrin stimulate diabetic fracture healing through a) inhibition of TNFα/TNFR1 inflammatory and bone resorption pathway; and primarily b) recruitment of 14-3-3ε to TNFR2, followed by activation of bone regeneration pathway. The Specific Aims are: (1) To determine the role of PGRN, especially its derivative Atsttrin, in diabetic fracture healing. We will use both systemic and inducible PGRN knockout mice to determine whether knockout of PGRN delays diabetic fracture healing, and whether recombinant PGRN and Atsttrin can reverse it (SA#1A); which stage of fracture healing requires PGRN for successful completion of diabetic fracture healing (SA#1B); and whether PGRN, especially Atsttrin, has therapeutic efficacy in treating diabetic fracture (SA#1C). We will use an appropriate injectable hydrogel to locally deliver various dosages of PGRN or Atsttrin. (2) To elucidate the molecular mechanisms by which PGRN and Atsttrin stimulate diabetic fracture healing. We will determine the effects of PGRN, Atsttrin, and TNFα on chondrogenesis of diabetic bone marrow stem cells, signaling pathways, interplays and dependence on TNFR and 14-3-3ε (SA#2A); whether both TNFRs are important for mediating PGRN's role in diabetic bone healing (SA#2B); and whether the protective effects of PGRN and Atsttrin depend on 14-3-3ε by establishing diabetic fracture models with inducible 14-3-3ε[-/-] mice (SA#2C). Proposed studies will not only advance our understanding of the molecular events underlying diabetic fracture healing, but could also lead to novel therapeutic interventions for diabetic fracture healing and other conditions in which fracture healing is impaired.