Osteoclast Regulation of Bone Formation

 描述(申请人提供)：使用小鼠模型，我们已经证明破骨细胞衍生因子促进成骨细胞谱系细胞的募集和分化。 由于破骨细胞从骨基质中释放并激活转化生长因子-，并且是转化生长因子-反应细胞，我们询问破骨细胞转化生长因子-反应是否有助于偶联 骨吸收到随后的骨形成。在小鼠中，在破骨细胞中表达显性负向转化生长因子-受体(Tgfbr2OclKO)导致明显的骨量减少，而对破骨细胞数量没有任何影响。重要的是，成骨细胞数量和骨形成减少，支持破骨细胞转化生长因子-反应有助于成骨细胞招募和/或分化。WNT1是正常骨形成的重要调节因子。我们发现，转化生长因子-处理野生型破骨细胞或在骨上培养破骨细胞可显著增加WNT1mRNA和蛋白的表达。这种反应在Tgfbr2OclKO破骨细胞中被钝化。有趣的是，破骨细胞WNT1蛋白在Tgfbr2OclKO骨中的表达明显降低，提示WNT1可能是转化生长因子诱导的破骨细胞偶联因子。这导致了我们的中心假设，局部释放的转化生长因子-作用于破骨细胞，产生包括WNT1在内的偶联因子，通过促进成骨细胞分化来刺激骨形成。我们设计了在小鼠模型(目标1)和人类受试者(目标2)中测试这一假设的研究。我们的具体目的是验证我们的假设：1.在小鼠模型中，研究功能性破骨细胞转化生长因子-受体信号的丢失对成骨细胞的影响以及 破骨细胞系WNT1在骨代谢中的作用我们将(A)评估Tgfbr2OclKO小鼠破骨细胞系细胞偶联因子在改变的Tgfbr2OclKO微环境中的表达和成骨细胞系细胞反应；(B)确定破骨细胞WNT1在破骨细胞-成骨细胞偶联中的作用；(C)确定基质结合的转化生长因子-调节成骨细胞偶联因子产生和反应的机制。2.测定破骨细胞减量对绝经后妇女体内偶联因子产生和成骨祖细胞的影响。我们将(A)检测骨微环境中潜在偶联因子的水平，(B)评估破骨细胞系细胞偶联因子基因在成骨细胞中的表达及其靶基因表达水平。我们期望这些研究将揭示破骨细胞衍生的转化生长因子-诱导的偶联因子，包括WNT1，在骨形成和转换中的作用。为了将偶联的概念推向进一步发展的可行的治疗目标，需要对其在人类中的重要性进行记录。这就是目标2的目标。该项目的创新方面是：(1)破骨细胞调节成骨细胞的机制将在小鼠和人类中得到检验，(2)破骨细胞衍生的WNT1在骨骼发育和破骨细胞-成骨细胞耦合中的作用将被确定，以及(3)将采用一种新的方法来评估体内成骨细胞/破骨细胞/破骨细胞系细胞中的基因表达，而不需要体外操作。

 DESCRIPTION (provided by applicant): Using murine models, we have shown that osteoclast-derived factors promote the recruitment and differentiation of osteoblast lineage cells. Because osteoclasts release and activate TGF- from the bone matrix and are TGF-responsive cells, we asked whether osteoclast TGF- responses contribute to the coupling of bone resorption to subsequent bone formation. In mice, expression of a dominant negative TGF- receptor in osteoclasts (Tgfbr2OclKO) caused marked osteopenia in the absence of any impact on osteoclast numbers. Importantly, osteoblast numbers and bone formation were reduced, supporting that osteoclast TGF- responses contribute to osteoblast recruitment and/or differentiation. Wnt1 is a crucial regulator of normal bone formation. We found that TGF- treatment of wildtype osteoclasts or culturing osteoclasts on bone resulted in marked increases in Wnt1 mRNA and protein. This response was blunted in Tgfbr2OclKO osteoclasts. Interestingly, osteoclast Wnt1 protein expression was much lower in Tgfbr2OclKO bones, suggesting that Wnt1 is a candidate TGF--induced osteoclast coupling factor. This leads to our central hypothesis that locally released TGF- acts on osteoclasts to produce coupling factors including Wnt1, which stimulate bone formation by enhancing osteoblast differentiation. We have designed studies to test this hypothesis in murine models (Aim 1) and human subjects (Aim 2). Our Specific Aims to test our hypothesis are: 1. In murine models, investigate the impacts of loss of functional osteoclast TGF- receptor signaling on osteoblasts and the roles of osteoclast lineage Wnt1 in bone metabolism. We will (A) Evaluate Tgfbr2OclKO mouse osteoclast lineage cell coupling factor expression and osteoblast lineage cell responses in the altered Tgfbr2OclKO microenvironment, (B) Determine the role of osteoclast Wnt1 osteoclast production in osteoclast-osteoblast coupling and (C) Define the mechanism by which matrix bound TGF- regulates coupling factor production and responses by osteoblasts. 2. Determine the effect of osteoclast reduction on coupling factor production and osteoblast progenitors in vivo in postmenopausal women. We will (A) Examine levels of potential coupling factors in the bone microenvironment and (B) Evaluate osteoclast lineage cell coupling factor gene expression and their target gene expression levels in osteoblasts. We expect that these studies will reveal roles for osteoclast-derived TGF--induced coupling factors, including Wnt1, in bone formation and turnover. In order to move the concept of coupling towards a viable therapeutic target for further development, documentation of its importance in humans is required. This is the goal of Aim 2. The innovative aspects of this project are: (1) the mechanisms by which osteoclasts modulate osteoblasts will be examined in both mice and humans, (2) the role of osteoclast-derived Wnt1 in skeletal development and osteoclast-osteoblast coupling will be determined, and (3) a new approach to evaluate gene expression in osteoblast/osteocyte/osteoclast lineage cells in vivo without in vitro manipulation will be employed.