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220万例外科病例涉及不可愈合的骨骼缺陷。骨形态发生蛋白2(BMP2)是骨再生的主要生长因子，在FDA批准的人类使用剂量下有显著的不良反应。这些效应包括危及生命的颈部肿胀和促进脂肪生成或剂量依赖的过氧化物酶体增殖物激活受体产生的“囊状骨空洞”？(PPAR？)上调。因此，在提高BMP2的安全性和有效性方面取得进展的一个关键障碍是开发有效靶向成骨诱导和抑制脂肪生成的分子信号策略，以优化骨形成。目前的新调查者重新提交使用NEL-1(类NEL分子，类型I)来解决这一障碍。Nell-1是一种分泌因子，可在多种大小动物模型中诱导体内显著的骨形成。Nell-1抑制BMP2诱导的脂肪生成，增强BMP2诱导的成骨。此外，我们的新数据表明，Nell-1激活Wnt/β-catenin信号并抑制PPAR？发信号。这导致了我们的中心假设，Nell-1通过以下途径提高BMP2诱导的骨形成的有效性：[1]激活Wnt/β-catenin信号，[2]抑制PPAR？信号，测试了四个特定的目标。在目标1中，我们将 确定Wnt/β-catenin和PPAR？Nell-1+BMP2介导的骨修复信号转导。使用我们发表的啮齿动物股骨节段性缺损(FSD)模型，我们将准确地评估WNT和PPAR？TOPGAL WNT报告小鼠中的信号。接下来，在AIM 2中，我们将评估Wnt/β-catenin信号在Nell-1+BMP2调控骨愈合中的必要性。在我们的FSD模型中，我们将通过生化(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信号是否足以重现Nell-1‘S促进BMP2诱导的成骨和抑制BMP2诱导的脂肪生成的作用。在这里，我们将使用Axin2-/-(空小鼠)通过遗传手段诱导Wnt/？-catenin“功能获得”，并在我们的FSD模型中检查其效果。最后，在目标4中，我们将确定是否降低了PPAR？信号转导可以重现Nell-1的S促进BMP2诱导的成骨和抑制BMP2诱导的脂肪生成的作用。PPAR？用PPAR？+/-小鼠和RNAi(PPAR？ShRNA)方法在我们的FSD模型中。这些目标的成功完成将提高基于Nell-1+BMP2联合治疗的BMP2骨再生的疗效并减少不良反应。此外，增加骨髓间充质干细胞诱导成骨和抑制脂肪生成将显著改善临床骨修复。

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.