Regulatory mechanisms in osteoclasts

描述（由申请人提供）：骨骼为身体提供刚性支撑，维持矿物质稳态，并作为造血的主要部位。通过成骨细胞和破骨细胞的平衡和耦合作用来维持骨稳态。骨吸收破骨细胞来源于骨髓系前体细胞，其也产生免疫细胞，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亚基亚型的v-ATP酶以前未知的功能，Atp 6v 0 d2，其表达是高度丰富的破骨细胞。我们发现Atp 6v 0 d2基因敲除小鼠由于骨吸收减少而表现出轻度骨硬化。然而，令我们惊讶的是，Atp 6v 0 d2似乎并不参与v-ATP酶质子泵，这是破骨细胞介导的骨吸收所必需的。相反，Atp 6v 0 d2是多核破骨细胞的最佳生成的关键。此外，Atp 6v 0 d2敲除小鼠表现出骨形成增加，即使在成骨细胞中未检测到Atp 6v 0 d2表达。此外，当直接离体测试时，Atp 6v 0 d2缺陷型成骨细胞在其细胞外基质的分化或矿化中未显示出任何内在缺陷。这些观察结果强烈表明，Atp 6v 0 d2基因敲除小鼠表现出增强的骨形成作为一个未知的成骨细胞的外在机制的结果，可能通过Atp 6v 0 d2缺陷的破骨细胞谱系细胞。最后，通过检查Atp 6v 0 d2的表达，我们发现，NFATc 1可能调节前破骨细胞之间的细胞融合，部分，通过调节诱导Atp 6v 0 d2的表达，这表明信号级联调节前破骨细胞从髓样前体细胞的分化可能是活跃的破骨细胞发育的多个阶段。因此，我们建议通过追求以下特定目标来扩展这些对骨稳态和破骨细胞成熟的调节的研究：（1）确定Atp 6v 0 d2缺失在体内影响骨代谢的程度，（2）确定破骨细胞谱系细胞本身是否可以在体内增加骨形成，（3）通过研究调节Atp 6v 0 d2表达的信号级联和破骨细胞前体的多核化来扩展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交流以交叉调节其功能提供见解，这可能导致用于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。

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.