Canonical and Noncanonical Wnt Signaling in Osteoclast Functions

 描述(申请人提供)：过多的破骨细胞活动会导致骨质流失，是一个严重的健康问题，因为它会导致骨质疏松症、肿瘤相关性骨病以及其他以骨量和强度降低为特征的疾病。LRP5/6拮抗剂硬化素的抗体是一种很有前途的新的合成代谢疗法，因为它可以促进骨形成。啮齿动物和人类的研究表明，这种疗法还可以减少破骨细胞介导的吸收。增强的Wnt活性抑制骨吸收的机制(S)尚不清楚。我们的研究表明，靶向缺失破骨细胞前体(POC)中的LRP5/6可以减少成骨细胞数量和骨形成速率，支持Wnt/LRP在成骨细胞系细胞POC募集中的作用。在POC中没有发现-连环蛋白的定向缺失，这表明Wnt/Lrp信号对破骨细胞系细胞的影响不依赖于连环蛋白。本课题的重点是研究Wnt/LRP对成骨细胞系细胞POC分化和募集的影响机制。当Wnt3a与LRP5/6结合时，它抑制POC的分化。然而，当LRP5/6受体不存在时，Wnt3a促进POC的分化。我们的初步数据显示，在这些情况下，Wnt3a的作用更像Wnt5a，并通过Ror2发出信号来激活非规范的Wnt信号通路。在POCs中缺乏Ror2的小鼠会出现骨石化，破骨细胞数量减少。令人惊讶的是，破骨细胞系细胞的这种严重减少对成骨细胞的数量没有影响，这表明Ror2信号抑制了成骨细胞的招募。我们的初步数据显示，WNT3a处理POCs抑制Rank的表达，刺激SPHK1酶的表达，增加其产物趋化因子S1P的产生。相反，Wnt5a刺激Rank表达并抑制SPHK1/S1P的产生。这一建议的中心假设是，有两条相互竞争的Wnt通路控制着破骨细胞的分化。Wnt3a激活标准臂，通过LRP5/6发出信号，抑制分化，促进S1P的产生。在正常情况下，Wnt5a和Ror2激活非标准臂以刺激破骨细胞分化并抑制S1P水平，但当LRP5/6水平较低时，Wnt3a也激活Ror2。我们的具体目的是验证我们的假设：(1)确定LRP和Ror2调节SPHK1和Rank的机制；(2)检测Ror2和SPHK1/S1P在小鼠和人破骨细胞系细胞中的表达；(3)评估成骨细胞系细胞在小鼠和人类衰老模型中的募集情况。这些研究将显著提高我们对Wnts调控骨代谢的机制的理解，并为在抑制分解代谢影响的同时增强Wnts信号对骨合成代谢的影响提供重要的新途径。这些新的治疗方向将改进预防和治疗骨质疏松症和关节炎和肿瘤引起的骨溶解等病理性骨丢失的策略。

 DESCRIPTION (provided by applicant): Excessive osteoclast activity causes bone loss and is a significant health issue because it can lead to osteoporosis, tumor-associated bone disease, and other conditions characterized by reduced bone mass and strength. An antibody to the Lrp5/6 antagonist sclerostin is a promising new anabolic therapy because it enhances bone formation. Rodent and human studies indicate that this therapy also reduces osteoclast- mediated resorption. The mechanism(s) by which enhanced Wnt activity suppresses bone resorption is unknown. Our studies reveal that targeted deletion of Lrp5/6 in osteoclast precursors (pOC) reduces osteoblast numbers and bone formation rates, supporting a role for Wnt/Lrp in pOC recruitment of osteoblast lineage cells. This response is not seen with targeted deletion of -catenin in pOCs, indicating -catenin- independent impacts of Wnt/Lrp signaling on osteoclast lineage cells. The focus of this project is to investigate the mechanisms by which Wnt/Lrp influence pOC differentiation and recruitment of osteoblast lineage cells. Wnt3a suppresses pOC differentiation when it binds to Lrp5/6. However, when Lrp5/6 receptors are not present, Wnt3a enhances pOC differentiation. Our preliminary data reveal that in these instances, Wnt3a acts more like Wnt5a and signals through Ror2 to activate noncanonical Wnt signaling pathways. Mice lacking Ror2 in pOCs have osteopetrosis, with reduced osteoclast numbers. Surprisingly, this severe reduction in osteoclast lineage cells had no impact on osteoblast numbers, suggesting that Ror2 signaling suppresses osteoblast recruitment. Our preliminary data reveal that Wnt3a treatment of pOCs represses Rank expression and stimulates expression of the enzyme SPHK1, increasing production of its product, the chemokine S1P. In contrast, Wnt5a stimulates Rank expression and suppresses SPHK1/S1P production. The central hypothesis of this proposal is that there are two competing Wnt pathways that control osteoclast differentiation. The canonical arm is activated by Wnt3a, which signals through Lrp5/6, suppresses differentiation and promotes S1P production. The noncanonical arm is activated by Wnt5a and Ror2 to stimulate osteoclast differentiation and suppress S1P levels under normal conditions, but Ror2 is also engaged by Wnt 3a when Lrp5/6 levels are low. Our Specific Aims to test our hypothesis are to: (1) Determine the mechanisms of Lrp and Ror2 regulation of SPHK1 and Rank, (2) Examine Ror2 and SPHK1/S1P expression in murine and human osteoclast lineage cells, and (3) Evaluate osteoblast lineage cell recruitment in murine and human models of aging. These studies will significantly enhance our understanding of the mechanisms by which Wnts control bone metabolism and provide important new avenues to enhance bone anabolic influences of Wnt signaling while suppressing catabolic influences. These new therapy directions will improve strategies to prevent and treat osteoporosis and pathological bone loss in conditions such as arthritis and tumor-induced osteolysis.