Osteoclast Regulation of Bone Formation

 描述（由申请人提供）：使用小鼠模型，我们已经表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。 因为破骨细胞从骨基质释放并激活TGF-β，并且是TGF-β反应性细胞，我们询问破骨细胞TGF-β反应是否有助于TGF-β与骨细胞的偶联。 骨吸收到随后的骨形成。在小鼠中，破骨细胞中显性负性TGF-β受体（Tgfbr 2 OclKO）的表达在对破骨细胞数量没有任何影响的情况下引起显著的骨质减少。重要的是，成骨细胞数量和骨形成减少，支持破骨细胞TGF-β反应有助于成骨细胞募集和/或分化。Wnt 1是正常骨形成的重要调节因子。我们发现TGF-β处理野生型破骨细胞或在骨上培养破骨细胞导致Wnt 1 mRNA和蛋白质的显著增加。这种反应在Tgfbr 2 OclKO破骨细胞中减弱。有趣的是，破骨细胞Wnt 1蛋白表达在Tgfbr 2 OclKO骨中低得多，提示Wnt 1是候选的TGF-β诱导的破骨细胞偶联因子.这导致了我们的中心假设，即局部释放的TGF-β作用于破骨细胞以产生包括Wnt 1的偶联因子，其通过增强成骨细胞分化来刺激骨形成。我们已经设计了研究，以测试这一假设在小鼠模型（目标1）和人类受试者（目标2）。我们检验假设的具体目的是：1。在小鼠模型中，研究功能性破骨细胞TGF-β受体信号转导的丧失对成骨细胞的影响以及 破骨细胞系Wnt 1在骨代谢中的作用。我们将（A）评价Tgfbr 2 OclKO小鼠破骨细胞谱系细胞偶联因子表达和成骨细胞谱系细胞在改变的Tgfbr 2 OclKO微环境中的反应，（B）确定破骨细胞Wnt 1破骨细胞产生在破骨细胞-成骨细胞偶联中的作用，和（C）确定基质结合的TGF-β调节偶联因子产生和成骨细胞反应的机制。2.确定绝经后妇女体内破骨细胞减少对偶联因子产生和成骨细胞祖细胞的影响。我们将（A）检查骨微环境中潜在偶联因子的水平和（B）评价破骨细胞谱系细胞偶联因子基因表达及其在成骨细胞中的靶基因表达水平。我们希望这些研究将揭示破骨细胞源性TGF-β诱导的偶联因子，包括Wnt 1，在骨形成和转换中的作用。为了将偶联的概念推向可行的治疗靶点以供进一步开发，需要记录其在人类中的重要性。这是目标2的目标。该项目的创新之处在于：（1）将在小鼠和人类中研究破骨细胞调节成骨细胞的机制，（2）将确定破骨细胞衍生的Wnt 1在骨骼发育和破骨细胞-成骨细胞偶联中的作用，以及（3）将采用一种新的方法来评估体内成骨细胞/骨细胞/破骨细胞谱系细胞中的基因表达，而无需体外操作。

 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.