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

项目摘要 促炎细胞因子肿瘤坏死因子α被认为是糖尿病患者骨折延迟愈合的原因。然而，关于肿瘤坏死因子α抑制对骨形成的影响还没有达成共识，这表明在寻找具有独特功能的新药以替代单纯的肿瘤坏死因子抑制剂用于糖尿病骨折愈合方面的需求尚未得到满足。我们的基因筛选使TNFR被确定为原颗粒蛋白(PGRN)的新受体(Tang等人，科学，2011年)，这是一种软骨化因子，已被证明对自身免疫性炎症性关节炎具有治疗作用。1型糖尿病是最常见的自身免疫性疾病，以慢性炎症和肿瘤坏死因子α活性升高为特征。尽管肿瘤坏死因子α的活性主要通过肿瘤坏死因子受体1介导，但我们兴奋地发现，前列腺素RN刺激的骨再生在很大程度上依赖于肿瘤坏死因子受体2。这些矛盾的发现表明，再生的PGRN/TNFR2通路在PGRN刺激的骨折愈合中发挥着重要作用。此外，14-3-3TNFR2信号通路也参与了对ε刺激的反应。此外，我们还开发了一种名为Atsttrin的工程蛋白，它由PGRN的三个TNFR结合域组成，并且Atsttrin在炎症性关节炎中比PGRN更有效。鉴于肿瘤坏死因子α的升高被认为是糖尿病骨折延迟愈合的原因，我们假设前列腺素RN和爱特林通过a)抑制肿瘤坏死因子α/肿瘤坏死因子受体1的炎症和骨吸收途径，以及主要的b)向肿瘤坏死因子受体2募集14-3-3ε，继而激活骨再生途径来促进糖尿病骨折的愈合。其具体目的是：(1)确定PGRN，特别是其衍生物Atsttrin在糖尿病骨折愈合中的作用。我们将使用全身性和可诱导的PGRN基因敲除小鼠来确定PGRN基因敲除是否会推迟糖尿病骨折的愈合，以及重组PGRN和Atsttrin是否可以逆转这种情况(SA#1A)；成功完成糖尿病骨折愈合需要哪些阶段的PGRN(SA#1B)；以及PGRN，尤其是Atsttrin在治疗糖尿病骨折方面是否有疗效(SA#1C)。我们将使用适当的可注射水凝胶局部输送不同剂量的PGRN或Atsttrin。(2)阐明前列腺素RN和爱特林促进糖尿病骨折愈合的分子机制。我们将通过诱导14-3-3-3α[-/-]小鼠建立糖尿病骨折模型(SA#2C)来确定PGRN、Atsttrin和肿瘤坏死因子ε对糖尿病骨髓干细胞软骨形成的影响、信号转导途径、相互作用和对14-3-3ε(SA#2A)的依赖；这两种TNFR对于介导PGRN在糖尿病骨愈合中的作用是否重要(SA#2B)；以及PGRN和Atsttrin是否依赖14-3-3ε[-/-]小鼠建立糖尿病骨折模型(SA#2C)。拟议的研究不仅将促进我们对糖尿病骨折愈合背后的分子事件的理解，而且还可能导致对糖尿病骨折愈合和其他骨折愈合受损情况的新的治疗干预措施。

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.