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220万例外科手术病例治疗未愈合的骨骼缺陷。骨形态发生蛋白2 （Bone morphogenetic protein 2, BMP2）是骨再生的主要生长因子，在FDA批准的人用剂量下存在显著的不良反应。这些影响包括危及生命的颈椎肿胀和促进脂肪生成或“囊肿样骨空洞”，这是由剂量依赖性过氧化物酶体增殖物激活受体γ (PPAR γ)上调引起的。因此，提高BMP2安全性和有效性的关键障碍是开发有效靶向成骨诱导和脂肪生成抑制以优化骨形成的分子信号策略。目前的新研究者重新提交使用NELL-1（尼尔样分子，I型）解决了这一障碍。NELL-1是一种分泌因子，在多种大小动物模型中诱导显著的体内骨形成。NELL-1抑制bmp2诱导的脂肪生成并增强bmp2诱导的成骨。此外，我们的新数据表明NELL-1激活Wnt/ß-catenin信号传导并抑制PPAR γ信号传导。这导致了我们的中心假设，即NELL-1通过[1]激活Wnt/ß-catenin信号传导，[2]抑制PPAR γ信号传导，提高bmp2诱导骨形成的功效，在四个特定目的中进行了测试。在AIM 1中，我们将确定Wnt/ß-catenin和PPAR γ信号在NELL-1+BMP2介导的骨修复中的作用。利用我们已发表的啮齿动物股骨节段缺损（FSD）模型，我们将精确评估TOPgal Wnt报告小鼠的Wnt和PPAR γ信号。接下来在AIM 2中，我们将评估的必要性

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 γ (PPAR γ) 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/ß-catenin signaling and suppresses PPAR γ signaling. This has led to our central hypothesis that NELL-1 improves the efficacy of BMP2-induced bone formation through: [1] activation of Wnt/ß-catenin signaling, and [2] suppression of PPAR γ signaling, tested in four specific aims. In AIM 1, we will determine the involvement of Wnt/ß-catenin and PPAR γ signaling in NELL-1+BMP2 mediated bone repair. Using our published rodent femoral segmental defect (FSD) model, we will precisely evaluate Wnt and PPAR γ signaling in TOPgal Wnt reporter mice. Next in AIM 2, we will evaluate the necessity of Wnt/ß-catenin signaling in NELL-1+BMP2 regulated bone healing. Here, we will induce Wnt/ß-catenin 'loss of function' by biochemical (Wnt inhibitor) or RNAi (ß-catenin shRNA) methods in our FSD model. In AIM 3, we will determine if increased Wnt/ß-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/ß-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 γ signaling can reproduce NELL-1's effects on promoting BMP2-induced osteogenesis and inhibiting BMP2-induced adipogenesis. PPAR γ "loss of function" will be induced using PPAR γ +/- mice and RNAi (PPAR γ 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.