Novel approaches to the treatment of bone loss in rheumatoid arthritis

项目摘要 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（OB）构建骨骼的能力。这些糜烂是疼痛的主要来源， 是临床试验的主要终点。重要的是，RA患者发生全身性 目前的治疗剂不能很好地控制骨质减少/骨质疏松症。因此，关键是 在炎症性关节炎的情况下开发新的骨合成代谢剂。我们发现 衔接蛋白Schnurri-3（SHN 3）显著抑制新骨形成。缺乏SHN 3的小鼠发育为 由于通过增强的Wnt信号传导增强OB的活性，骨量逐渐增加。 对于RA患者重要的是，OB中SHN 3的表达被TNF加IL-17 A高度上调，并且SHN 3 缺乏可防止这些细胞因子对OB分化的抑制。因此，SHN 3的抑制是 促进骨形成以预防或治疗全身和/或局部骨丢失的有吸引力的机制， 伴随着RA。最后，间充质干细胞（MSC）是一种有前途的修复机制， 炎症诱导的骨丢失，这些细胞可以通过删除SHN 3进行遗传操作，以增加 它们在这种情况下修复全身性骨丢失的潜力。在此R21应用程序中，我们将测试 假设SHN 3是促进炎症诱导部位骨形成的新靶点 炎症性关节炎的骨丢失，并将确定机制。目标1将检验以下假设： SHN 3缺乏可防止TNF诱导的全身性骨丢失并增强骨愈合。我们将 在部分A中使用MSC中缺乏SHN 3的小鼠确定SHN 3缺乏是否防止全身性骨丢失 以肢体特异性的方式，与TNF转基因小鼠杂交。在部分B中，我们将确定 SHN 3缺陷保护MSC/OB免受诱导的OB分化抑制的机制 由促炎细胞因子引起。此外，使用无偏蛋白质组学和磷酸蛋白质组学，我们将 在TNF α和IL-17 A作用下鉴定MSC/OB中新的SHN 3相互作用蛋白和由SHN 3调节的蛋白 刺激.在C部分，我们将确定SHN 3本身是否调节成纤维细胞因子的产生- 如抑制OB分化/功能的滑膜细胞，包括IL-6和DKK 1。目标2将测试是否 SHN 3缺陷型MSCs可促进炎症性骨丢失的愈合。我们将测试 移植SHN 3缺陷的MSC以恢复TNF转基因小鼠中的全身性骨丢失。合成 与阿仑膦酸盐缀合的拟肽配体（LLP 2A-Ale）将用于将MSC引导至骨。 移植的表达GFP的MSC衍生的OB也将进行转录组分析和靶向分析。 通过多种方法确定的将优先进行验证。这些数据将决定 SHN 3介导促炎细胞因子和Wnt通路之间的信号传导， MSC/OB，并确定新的目标，以促进炎症性关节炎中的合成代谢骨形成。

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.