Wnt signaling in skeletal tissue regeneration

描述(申请人提供)：危害或削弱骨骼功能的疾病构成了美国最普遍的慢性损伤，每年相关的直接和间接成本为8490亿美元。随着婴儿潮一代的老龄化，这一数字预计将在未来10-20年内上升。因此，迫切需要刺激骨骼修复和再生的新策略。目前的文献表明，Wnt途径是促进骨骼修复和再生的最有吸引力的候选途径之一。Wnt通路的中断与多种人类肌肉骨骼疾病有关，Wnt信号是人体对骨骼损伤的自然反应的一部分。因此，这项建议的目的是研究Wnt信号影响骨再生的基本机制(S)，并利用这一信息制定骨修复的治疗策略。在目标1中，我们将研究Wnt抑制剂Axin2的缺失如何影响细胞中的Wnt信号。我们假设，失去Axin2，将导致WNT反应的持续时间和幅度增加。我们将检测从Axin2突变小鼠分离的原代骨祖细胞中的Wnt反应，该突变小鼠已被Wnt报告慢病毒感染，其中荧光素酶活性是Wnt信号的读数。在目标2中，我们将在体外和体内检测Axin2突变对骨髓细胞成骨能力的影响。我们假设Axin2的缺失增加了骨髓基质细胞(BMSC)的成骨能力。在我们的第一组实验中，我们将评估野生型和Axin2突变的BMSCs在有或没有纯化的Wnt3a蛋白的情况下的成骨分化。此外，我们将通过将Axin2小鼠的骨髓细胞移植到颅骨的临界大小缺损处来测试它们的体内成骨能力。我们将检查损伤部位的细胞增殖、成骨分化和基质沉积。在目标3中，我们将使用Wnt蛋白本身作为治疗剂，并确定其对骨愈合和再生的影响。与AIM 2类似，WNT3a或PBS脂质体处理后的整个骨髓将被移植到颅骨的临界大小缺损处。我们将使用原位杂交、u-CT、组织学和免疫组织化学来表征治疗对愈合组织中细胞增殖、成骨细胞分化和基质沉积的影响。综上所述，这些拟议的实验将阐明Axin2在Wnt信号中的作用，并将直接测试使用Wnt刺激骨再生的可行性。我们预见，这些信息将在治疗肌肉骨骼疾病的治疗方法中具有相当大的价值。

DESCRIPTION (provided by applicant): Diseases that jeopardize or weaken skeletal function constitute the most prevalent chronic impairments in the U.S with associated annual direct and indirect costs of $849 billion. As the baby boomer generation ages, this figure is expected to escalate in the next 10-20 years. Therefore, new strategies that stimulate skeletal repair and regeneration are badly needed. The current literature indicates that the Wnt pathway is among the most attractive candidates for enhancing skeletal repair and regeneration. Disruptions in the Wnt pathway are associated with multiple human musculoskeletal diseases and Wnt signaling is part ofthe body's natural response to skeletal damage. Thus, the objective of this proposal is to examine the basic mechanism(s) by which Wnt signaling effect bone regeneration and to use this information to formulate therapeutic strategies for bone repair. In Aim 1, we will examine how the loss of the Wnt inhibitor, Axin2, affects Wnt signaling in a cell. We hypothesize that the loss of Axin2, will result in an increased in the duration and magnitude of Wnt response. We will examine the Wnt response in primary osteoprogenitor cells isolated from Axin2 mutant mice that has been transfected with Wnt reporter lentivirus where luciferase activity is the readout of Wnt signaling. In Aim 2, we will examine the effect of Axin2 mutation on the osteogenic potential of bone marrow cells both in vitro and in vivo. We hypothesize that loss of Axin2 increases the osteogenic potential of bone marrow stromal cells (BMSC). In our first set of experiments we will evaluate osteoblast differentiation of wild-type and Axin2 mutant BMSCs with or without purified Wnt3a protein. In addition, we will test the osteogenic potential of bone marrow cells from Axin2 mice in vivo by transplanting them into critical size defects in calvaria. We will examine cell proliferation, osteogenic differentiation, and matrix deposition at the injury site. In Aim 3, we will use Wnt protein itself as a therapeutic agent and we will determine its affect on bone healing and regeneration. Similar to Aim 2, Wnt3a or PBS liposomes treated whole bone marrow will be grafted into critical size defects in the calvaria. We will use in situ hybridization, u-CT, histology and immunohistochemistry to characterize the effects of the treatment on cell proliferation, osteoblast differentiation, and matrix deposition in the healing tissue. In summary, these proposed experiments will clarify the function of Axin2 in Wnt signaling, and will directly test the feasibility of using Wnt to stimulating bone regeneration. We envision that this information will be of considerable value in therapeutic approaches to treating musculoskeletal conditions.