Regulation of Fracture Healing by Macrophage-Derived Wnt Ligands

项目摘要 骨折愈合是一个复杂的骨愈合过程，受各种细胞和信号的调节 机制等其中，Wnt信号传导长期以来被认为在指导分化中至关重要。 骨髓间充质干细胞（MSC）在骨愈合过程中转化为成骨细胞。然而，这些Wnt的来源 配体是未知的。最近，出现了一系列文献，发现 巨噬细胞衍生的Wnt配体对其他组织（肝、肾、心脏和肠）再生的影响，通常 通过调节祖细胞的行为。由于巨噬细胞已经被很好地描述为 因此，他们使用Wnt信号来调节组织再生并不令人惊讶。然而，没有研究表明 还鉴定了巨噬细胞在骨折愈合期间对Wnt信号传导的贡献。有趣的是，最近 未发表的数据表明巨噬细胞是骨愈合过程中Wnt配体的主要来源， 强调了巨噬细胞在驱动这一过程中的重要性。在这里提出的工作中，表达式 在骨折过程中，将首先测量巨噬细胞对Wnt配体的释放，以及Wnt配体靶向的细胞 治愈然后，编码必需的Wnt配体运输蛋白的基因Wls将在Csf1r中缺失。 表达细胞（单核细胞和巨噬细胞），抑制所有Wnt配体的分泌， 用他莫昔芬诱导Cre重组酶。删除巨噬细胞Wls后， 巨噬细胞衍生的Wnt配体的特征将在炎症、软愈伤组织和硬愈伤组织中进行。 治愈的阶段。通过这些阶段的适当过渡，并最终成功愈合，将是 使用qPCR、流式细胞术、微计算机断层扫描和组织学进行评价。最后， Wnt向MSC的运输，以及它们对MSC行为的影响的后果，将通过在 体外共培养实验。本提案中定义的研究将阐明巨噬细胞的重要性 Wnt配体对骨愈合的影响，并可能将Wnt信号传导确定为增强骨愈合成功的靶向途径。 骨愈合

PROJECT SUMMARY Fracture healing is a complex bone healing process regulated by various classes of cells and signaling mechanisms. Of these, Wnt signaling has long been known to be critically important in directing the differentiation of mesenchymal stem cells (MSCs) into osteoblasts during bone healing. However, the source of these Wnt ligands is not known. Recently, a body of literature has arisen finding both beneficial and detrimental effects of macrophage-derived Wnt ligands on the regeneration of other tissues (liver, kidney, heart, and intestine), often by their modulation of progenitor cell behavior. As macrophages have been well-characterized as orchestrators of healing, their use of Wnt signaling to regulate tissue regeneration is unsurprising. However, no studies have yet identified the contribution of macrophages to Wnt signaling during fracture healing. Interestingly, recent unpublished data suggests that macrophages represent a major source of Wnt ligands during bone healing and underscore the importance of macrophages in driving this process. In the work proposed here, the expression of Wnt ligands by macrophages will first be measured, as well as the cells targeted by Wnt ligands, during fracture healing. Then, Wls, the gene encoding a necessary Wnt ligand trafficking protein, will be deleted in Csf1r- expressing cells (monocytes and macrophages), inhibiting the secretion of all Wnt ligands by these cells after induction of Cre recombinase with tamoxifen. Following deletion of macrophage Wls, the consequences of loss of macrophage-derived Wnt ligands will be characterized during the inflammatory, soft callus, and hard callus phases of healing. Appropriate transition through these phases, and ultimately successful healing, will be evaluated using qPCR, flow cytometry, micro-computed tomography, and histology. Finally, the mechanism of Wnt trafficking to MSCs, and the consequences of their effects on MSC behavior, will be evaluated through in vitro co-culture experiments. The studies defined in this proposal will elucidate the importance of macrophage Wnt ligands on bone healing and potentially identify Wnt signaling as a targetable pathway to enhance successful osseous healing.