Regulatory mechanisms in osteoclasts

描述(申请人提供)：骨骼为身体提供坚硬的支撑，维持矿物质动态平衡，是造血的主要部位。骨平衡是通过成骨细胞和破骨细胞的平衡和耦合作用来维持的。骨吸收破骨细胞来源于髓系前体细胞，也产生免疫细胞，如巨噬细胞和树突状细胞。这一应用部分源于我们试图了解破骨细胞在功能和发育上与免疫细胞的不同。在我们的初步数据中，我们描述了一种新的功能未知的v-ATPase亚基亚型Atp6v0d2，其在破骨细胞中的表达高度丰富。我们发现，Atp6v0d2基因敲除小鼠由于骨吸收减少而表现出轻微的骨化病。然而，令我们惊讶的是，Atp6v0d2似乎不参与v-ATPase质子泵，而v-ATPase质子泵是破骨细胞介导的骨吸收所必需的。相反，Atp6v0d2对于多核破骨细胞的最佳生成至关重要。此外，尽管在成骨细胞中没有检测到Atp6v0d2的表达，但Atp6v0d2基因敲除的小鼠表现出更多的骨形成。此外，当直接在体外测试时，Atp6v0d2缺陷的成骨细胞在其细胞外基质的分化或矿化方面没有表现出任何内在的缺陷。这些观察有力地表明，Atp6v0d2基因敲除小鼠表现出增强的骨形成，这是未知的成骨细胞外部机制的结果，可能是通过Atp6v0d2缺陷的破骨细胞谱系细胞。最后，通过检测Atp6v0d2的表达，我们发现NFATc1可能部分地通过调节Atp6v0d2表达的诱导来调节破骨前细胞之间的细胞-细胞融合，这表明调节破骨前细胞与髓系前体分化的信号级联在破骨细胞发育的多个阶段可能是活跃的。因此，我们建议通过以下具体目标扩展这些关于骨稳态和破骨细胞成熟调控的研究：(1)确定Atp6v0d2缺失对体内骨代谢的影响程度；(2)确定破骨细胞系细胞本身是否可以促进体内骨形成；(3)通过研究调节Atp6v0d2表达和前破骨细胞多核的信号级联反应，扩大我们对破骨细胞成熟的理解。从这些研究中获得的知识将为不同分子如何合作诱导破骨细胞分化，以及破骨细胞和成骨细胞如何沟通以交叉调节其功能提供见解，这可能导致治疗和预防骨质疏松症和其他骨骼疾病的新的或改进的治疗策略。公共卫生相关性：破骨细胞是主要的，如果不是唯一的，可以吸收骨的细胞。因此，了解导致破骨细胞分化和激活的分子途径将有助于改善骨质疏松症以及其他涉及骨破坏的疾病的治疗和预防。

DESCRIPTION (provided by applicant): Bones provide rigid support for the body, maintain mineral homeostasis, and serve as the primary site for hematopoiesis. Bone homeostasis is maintained through the balanced and coupled actions of osteoblasts and osteoclasts. Bone-resorbing osteoclasts are derived from myeloid-lineage precursor cells, which also yield immunocytes such as macrophages and dendritic cells. This application stems in part from our attempt to understand how osteoclasts differ functionally and developmentally from immunocytes. In our preliminary data, we describe a novel subunit isoform of v-ATPase of previously unknown function, Atp6v0d2, expression of which is highly enriched in osteoclasts. We show that Atp6v0d2 knockout mice exhibit mild osteopetrosis due to decreased bone resorption. To our surprise, however, Atp6v0d2 does not appear to be involved in the v-ATPase proton pump, which is required for osteoclast-mediated bone resorption. Rather, Atp6v0d2 is critical for the optimal generation of multinucleated osteoclasts. In addition, Atp6v0d2 knockout mice exhibit increased bone formation, even though Atp6v0d2 expression was not detected in osteoblasts. Moreover, when tested directly ex vivo, Atp6v0d2-deficient osteoblasts do not show any intrinsic defects in their differentiation or mineralization of extracellular matrix. These observations strongly suggest that Atp6v0d2 knockout mice show enhanced bone formation as a result of unknown osteoblast-extrinsic mechanisms, possibly via Atp6v0d2-deficient osteoclast lineage cells. Finally, by examining Atp6v0d2 expression, we show that NFATc1 may regulate cell-cell fusion between preosteoclasts, in part, by regulating the induction of Atp6v0d2 expression, suggesting that signaling cascades that regulate the differentiation of preosteoclasts from myeloid precursors may be active during multiple stages of osteoclast development. Therefore, we propose to extend these studies of the regulation of bone homeostasis and osteoclast maturation by pursuing the following specific aims: (1) determining the extent to which Atp6v0d2 deletion affects bone metabolism in vivo, (2) determining whether osteoclast lineage cells per se can increase bone formation in vivo, and (3) extending our understanding of osteoclast maturation by studying signaling cascades that regulate Atp6v0d2 expression and the multinucleation of preosteoclasts. The knowledge gained from these studies will provide insights into how different molecules cooperate to induce osteoclast differentiation, and how osteoclasts and osteoblasts communicate to cross-regulate their functions, which may lead to novel or improved therapeutic strategies for the treatment and prevention of osteoporosis and other bone diseases. PUBLIC HEALTH RELEVANCE: Osteoclasts are the principal, if not the only, cells that can resorb bone. Thus, understanding the molecular pathways leading to the differentiation and activation of osteoclasts will help improve the treatment and prevention of osteoporosis as well as other diseases involving bone destruction.