Wnt and PPARy Signaling in Nell-1 and BMP2 Mediated Bone Regeneration

描述（由申请人提供）：每年有超过220万例手术病例涉及非愈合性骨骼缺损。骨形态发生蛋白2（BMP 2）是骨再生的主要生长因子，在FDA批准的人类使用剂量下具有显著的不良反应。这些影响包括危及生命的颈部肿胀和促进脂肪形成或“囊肿样骨空隙”的剂量依赖性过氧化物酶体增殖物激活受体？(PPAR ?)上调。因此，在提高BMP 2的安全性和有效性方面取得进展的关键障碍是开发有效靶向骨生成诱导和脂肪生成抑制以优化骨形成的分子信号传导策略。当前的新研究者重新提交使用NELL-1（Nel样分子，I型）解决了这一障碍。NELL-1是一种分泌因子，在多种小型和大型动物模型中诱导显著的体内骨形成。NELL-1抑制BMP 2诱导的脂肪生成并增强BMP 2诱导的骨生成。此外，我们的新数据表明，NELL-1激活Wnt/β-catenin信号转导和抑制PPAR？发信号。这导致了我们的中心假设，即NELL-1通过以下方式提高BMP 2诱导的骨形成的功效：[1]激活Wnt/β-catenin信号传导，[2]抑制PPAR？信号，在四个具体目标测试。在AIM 1中，我们将 确定Wnt/β-catenin和PPAR的参与？NELL-1+ BMP 2介导的骨修复中的信号传导。使用我们发表的啮齿动物股骨节段性缺损（FSD）模型，我们将精确地评估Wnt和PPAR？TOPgal Wnt报告基因小鼠中的信号传导。接下来在AIM 2中，我们将评估Wnt/β-catenin信号在NELL-1+ BMP 2调节的骨愈合中的必要性。在这里，我们将在我们的FSD模型中通过生物化学（Wnt抑制剂）或RNAi（<$-catenin shRNA）方法诱导Wnt/<$-catenin“功能丧失”。在AIM 3中，我们将确定增加的Wnt/β-catenin信号传导是否足以再现NELL-1促进BMP 2诱导的骨生成和抑制BMP 2诱导的脂肪生成的作用。在这里，我们将使用Axin 2-/-（无效小鼠）通过遗传手段诱导Wnt/<$-连环蛋白“功能获得”，并检查其在我们的FSD模型中的作用。最后，在AIM 4中，我们将确定是否降低了PPAR？信号传导可以再现NELL-1促进BMP 2诱导的骨生成和抑制BMP 2诱导的脂肪生成的作用。PPAR？“功能丧失”将诱导使用过氧化物酶体增殖物激活受体？+/-小鼠和RNAi（PPAR？shRNA）方法在我们的FSD模型中。AIMS的成功完成将提高基于组合治疗NELL-1+ BMP 2的基于BMP 2的骨骼再生的功效并减少不良反应。此外，间充质干细胞诱导成骨和抑制脂肪生成的增加将导致临床骨修复的显著改善。 公共卫生相关性：全球每年有超过220万例手术病例涉及不愈合骨缺损。骨形态发生蛋白2（BMP 2）是最常用的骨生长因子，具有显著的副作用，包括形成质量差、脂肪多的 骨头我们的创新解决方案使用NELL-1，一种新型骨形成分子，可抑制脂肪骨形成，并提高基于BMP 2的骨再生疗法的疗效。

DESCRIPTION (provided by applicant): Non-healing skeletal defects are addressed in over 2.2 million surgical cases each year. Bone morphogenetic protein 2 (BMP2), the main growth factor for bone regeneration, has significant adverse effects at the FDA approved dose for human use. These effects include life-threatening cervical swelling and promotion of adipogenesis or "cyst-like bone voids" from dose-dependent peroxisome proliferator-activated receptor ? (PPAR ?) upregulation. Thus, a critical barrier to progress in improving the safety and efficacy of BMP2 is development of molecular signaling strategies that effectively target osteogenesis induction and adipogenesis suppression to optimize bone formation. The current New Investigator resubmission addresses this barrier using NELL-1 (Nel-like molecule, type I). NELL-1 is a secreted factor that induces significant in vivo bone formation in multiple small and large animal models. NELL-1 represses BMP2-induced adipogenesis and augments BMP2-induced osteogenesis. Moreover, our new data indicate that NELL-1 activates Wnt/¿-catenin signaling and suppresses PPAR ? signaling. This has led to our central hypothesis that NELL-1 improves the efficacy of BMP2-induced bone formation through: [1] activation of Wnt/ ¿ -catenin signaling, and [2] suppression of PPAR ? signaling, tested in four specific aims. In AIM 1, we will determine the involvement of Wnt/ ¿ -catenin and PPAR ? signaling in NELL-1+BMP2 mediated bone repair. Using our published rodent femoral segmental defect (FSD) model, we will precisely evaluate Wnt and PPAR ? signaling in TOPgal Wnt reporter mice. Next in AIM 2, we will evaluate the necessity of Wnt/ ¿ -catenin signaling in NELL-1+BMP2 regulated bone healing. Here, we will induce Wnt/ ¿ -catenin 'loss of function' by biochemical (Wnt inhibitor) or RNAi (¿ -catenin shRNA) methods in our FSD model. In AIM 3, we will determine if increased Wnt/ ¿ -catenin signaling is sufficient to reproduce NELL-1's effects on promoting BMP2-induced osteogenesis and inhibiting BMP2-induced adipogenesis. Here, we will induce Wnt/ ¿ -catenin "gain of function" by genetic means using Axin2-/- (null mice) and examine its effects in our FSD model. Finally in AIM 4, we will determine if decreased PPAR ? signaling can reproduce NELL-1's effects on promoting BMP2-induced osteogenesis and inhibiting BMP2-induced adipogenesis. PPAR ? "loss of function" will be induced using PPAR ? +/- mice and RNAi (PPAR ? shRNA) methods in our FSD model. Successful completion of the AIMS will improve efficacy and reduce adverse effects for BMP2 based skeletal regeneration based on the combination therapeutic NELL-1+BMP2. Moreover, increased induction of osteogenesis and suppression of adipogenesis among mesenchymal stem cells will lead to marked improvements in clinical bone repair. PUBLIC HEALTH RELEVANCE: Non-healing bone defects are addressed in over 2.2 million surgical cases worldwide each year. Bone Morphogenetic Protein2 (BMP2), the most commonly used bone growth factor, has significant adverse effects including formation of poor quality, fatty bone. Our innovative solution uses NELL-1, a novel bone forming molecule to inhibit fatty bone formation and to improve the efficacy of BMP2-based bone regeneration therapies.