Novel approaches to the treatment of bone loss in rheumatoid arthritis

项目总结 类风湿性关节炎(RA)通过激活破骨细胞吸收骨而产生关节侵蚀 抑制成骨细胞(OBS)构建骨骼的能力。这些腐蚀物是疼痛的主要来源 在类风湿关节炎中的残疾，是临床试验的主要终点。重要的是，类风湿关节炎患者发展为全身性 骨量减少/骨质疏松症，目前的治疗药物不能很好地控制它。因此，至关重要的是 在炎症性关节炎的背景下，开发对骨骼合成代谢的新试剂。我们发现， 适配蛋白SchNurri-3(SHN3)能显著抑制新骨形成。缺乏SHN3的小鼠会患上 骨量的进行性增加是由于OBS的活性通过增强的Wnt信号而增强。 对于RA患者来说，重要的是，SHN3在OBS中的表达被肿瘤坏死因子和IL-17A以及SHN3高度上调 缺乏这些细胞因子可防止OB分化受到抑制。因此，对SHN3的抑制是 一种诱人的促进骨形成的机制，以预防或治疗全身和/或局部骨丢失 伴随着RA。最后，间充质干细胞(MSCs)是一种很有前途的修复 炎症诱导的骨丢失，这些细胞可能通过删除SHN3来增强 它们在这种情况下修复全身骨丢失的潜力。在此R21应用程序中，我们将测试 假设SHN3是促进炎症诱导部位骨形成的新靶点 骨丢失在炎症性关节炎中的作用机制将被确定。目标1将检验假设 SHN3缺乏可防止肿瘤坏死因子诱导的全身性骨丢失，并促进骨愈合。我们会 利用骨髓间充质干细胞中缺乏SHN3的小鼠，在A部分确定SHN3缺乏是否可以防止全身骨丢失 以一种肢体特异的方式，与肿瘤坏死因子转基因小鼠杂交。在B部分，我们将确定分子 SHN3缺乏保护MSCs/OBS抑制成骨细胞分化的机制 通过促炎细胞因子。此外，使用无偏见的蛋白质组学和磷酸蛋白质组学，我们将 鉴定新的SHN3相互作用蛋白及受SHN3调控的MSCs/OBS对TNFa和IL-17A的影响 刺激。在C部分，我们将确定SHN3本身是否调节成纤维细胞产生因子- 像滑膜细胞一样，抑制OB分化/功能，包括IL-6和Dkk1。Aim 2将测试 SHN3基因缺陷的骨髓间充质干细胞可促进炎症诱导的骨丢失愈合。我们将测试他们的能力 移植SHN3基因缺陷的间充质干细胞修复肿瘤坏死因子转基因小鼠全身骨丢失。一种合成纤维 拟肽配体-阿伦磷酸酯(LLP2A-ALE)将被用于将MSCs定向到骨组织中。 移植的表达绿色荧光蛋白的骨髓间充质干细胞来源的OB也将接受转录组分析和靶标。 将对多个方法确定的优先顺序进行验证。这些数据将决定这些机制 SHN3介导促炎细胞因子和Wnt通路之间的信号转导 MSCs/OBS，并确定促进炎症性关节炎合成代谢骨形成的新靶点。

PROJECT SUMMARY Rheumatoid arthritis (RA) produces articular erosions by activating osteoclasts to resorb bone while suppressing the ability of osteoblasts (OBs) to build bone. These erosions are a major source of pain and disability in RA and are a primary endpoint in clinical trials. Importantly, patients with RA develop systemic osteopenia/osteoporosis that is not well controlled by current therapeutic agents. Therefore, it is critical to develop new agents that are anabolic for bone in the setting of inflammatory arthritis. We discovered that the adaptor protein Schnurri-3 (SHN3) profoundly suppresses new bone formation. Mice lacking SHN3 develop a progressive increase in bone mass due to augmented activity of OBs through enhanced Wnt signaling. Importantly for patients with RA, SHN3 expression in OBs is highly upregulated by TNF plus IL-17A, and SHN3 deficiency protects from suppression of OB differentiation by these cytokines. Therefore, inhibition of SHN3 is an attractive mechanism to promote bone formation to prevent or treat the systemic and/or local bone loss that accompanies RA. Finally, mesenchymal stem cells (MSCs) are a promising mechanism for repair of inflammation-induced bone loss and these cells may be genetically manipulated by deleting SHN3 to augment their potential to repair systemic bone loss in this setting. In this R21 application, we will test the hypothesis that SHN3 is a novel target to promote bone formation at sites of inflammation-induced bone loss in inflammatory arthritis and will determine mechanism. Aim 1 will test the hypothesis that SHN3 deficiency prevents TNF-induced systemic bone loss and augments bone healing. We will determine in Part A whether SHN3 deficiency prevents systemic bone loss using mice lacking SHN3 in MSCs in a limb-specific manner, crossed with TNF-transgenic mice. In Part B we will determine the molecular mechanisms by which SHN3 deficiency protects MSCs/OBs from the suppression of OB differentiation induced by pro-inflammatory cytokines. Additionally, using unbiased proteomics and phosphoproteomics, we will identify novel SHN3-interacting proteins and proteins regulated by SHN3 in MSCs/OBs upon TNFa and IL-17A stimulation. In Part C, we will determine whether SHN3 itself regulates the production of factors by fibroblast- like synoviocytes that inhibit OB differentiation/function, including IL-6 and DKK1.! Aim 2 will test whether SHN3-deficient MSCs can promote healing of inflammation-induced bone loss. We will test the ability of transplanted SHN3-deficient MSCs to restore systemic bone loss in TNF-transgenic mice. A synthetic peptidomimetic ligand-conjugated with alendronate (LLP2A-Ale) will be utilized to direct MSCs to the bone. Transplanted GFP-expressing MSC-derived OBs will also be subjected to transcriptome analysis and targets identified by multiple approaches will be prioritized for validation. These data will determine the mechanisms by which SHN3 mediates signaling between pro-inflammatory cytokines and the Wnt pathway in MSCs/OBs and identify new targets to promote anabolic bone formation in inflammatory arthritis.