Canonical and Noncanonical Wnt Signaling in Osteoclast Functions

 描述（由申请人提供）：过度的破骨细胞活性会导致骨质流失，并且是一个重大的健康问题，因为它会导致骨质疏松症、肿瘤相关骨病以及其他以骨量和强度降低为特征的病症。 Lrp5/6 拮抗剂硬化蛋白的抗体是一种有前途的新型合成代谢疗法，因为它可以增强骨形成。啮齿动物和人类研究表明，这种疗法还可以减少破骨细胞介导的吸收。 Wnt 活性增强抑制骨吸收的机制尚不清楚。我们的研究表明，破骨细胞前体 (pOC) 中 Lrp5/6 的靶向缺失会降低成骨细胞数量和骨形成率，支持 Wnt/Lrp 在成骨细胞谱系细胞的 pOC 募集中的作用。在 pOC 中靶向删除 β-连环蛋白时，未观察到这种反应，表明 Wnt/Lrp 信号对破骨细胞谱系细胞具有独立于 β-连环蛋白的影响。该项目的重点是研究 Wnt/Lrp 影响 pOC 分化和成骨细胞谱系细胞招募的机制。 Wnt3a 与 Lrp5/6 结合时抑制 pOC 分化。然而，当 Lrp5/6 受体不存在时，Wnt3a 会增强 pOC 分化。我们的初步数据表明，在这些情况下，Wnt3a 的作用更像是 Wnt5a，并通过 Ror2 发出信号来激活非经典 Wnt 信号通路。 pOC 中缺乏 Ror2 的小鼠患有骨硬化症，破骨细胞数量减少。令人惊讶的是，破骨细胞谱系细胞的严重减少对成骨细胞数量没有影响，这表明 Ror2 信号传导抑制了成骨细胞的招募。我们的初步数据表明，Wnt3a 处理 pOC 会抑制 Rank 表达并刺激 SPHK1 酶的表达，从而增加其产物趋化因子 S1P 的产量。相反，Wnt5a 刺激 Rank 表达并抑制 SPHK1/S1P 产生。该提议的中心假设是有两种竞争的 Wnt 通路控制破骨细胞分化。经典臂由 Wnt3a 激活，Wnt3a 通过 Lrp5/6 发出信号，抑制分化并促进 S1P 产生。在正常情况下，非经典臂被 Wnt5a 和 Ror2 激活，以刺激破骨细胞分化并抑制 S1P 水平，但当 Lrp5/6 水平较低时，Ror2 也会被 Wnt 3a 参与。我们检验假设的具体目标是：(1) 确定 Lrp 和 Ror2 对 SPHK1 和 Rank 的调节机制，(2) 检查小鼠和人类破骨细胞谱系细胞中 Ror2 和 SPHK1/S1P 的表达，以及 (3) 评估小鼠和人类衰老模型中成骨细胞谱系细胞的招募。这些研究将显着增强我们对 Wnt 控制骨代谢机制的理解，并提供重要的新途径来增强 Wnt 信号传导对骨合成代谢的影响，同时抑制分解代谢的影响。这些新的治疗方向将改善预防和治疗关节炎和肿瘤引起的骨质溶解等情况下的骨质疏松症和病理性骨质流失的策略。

 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.