CONTROL OF OSTEOCLAST ACTIVATION AND DIFFERENTIATION

破骨细胞骨吸收是骨重建的第一步， 在三个不同的步骤进行调节：破骨细胞分化， 启动骨吸收，并控制吸收速率（或 持续时间）。破骨细胞通过粘附于骨基质蛋白而再吸收骨， 以及在附着部位形成封闭空间， 蛋白水解酶被分泌，质子被主动转运， 一种液泡H+-ATP酶（V-ATP酶），在皱褶的 膜的我们实验室最近的研究表明， V-ATP酶15 kD亚基可能在调节该亚基中起关键作用 在破骨细胞分化模型中的表达依赖于 细胞间相互作用免疫沉淀和免疫印迹将是 进行骨髓培养，以确定15 kD亚单位是否控制 功能性V-ATP酶的总量。15 kD的诱导机制 亚基表达将通过启动子的稳定转导来研究， 报告基因构建体进入鼠单核细胞前体系， 在共培养中分化成破骨细胞样细胞。 在小鼠骨髓培养物中骨吸收起始的研究中， 我们发现间质胶原酶（人MMP-13的同系物）， 由基质细胞产生，在启动骨吸收中起关键作用 这并不涉及清除骨表面的胶原蛋白， 激活骨吸收的胶原蛋白裂解产物。客观 本项目的目的是确定胶原蛋白裂解产物 直接激活分离的破骨细胞;检测酶和细胞 MMP-13启动破骨细胞骨吸收的生物学机制 通过确定胶原蛋白上的位点和结构要求， 使用重组体诱导破骨细胞骨吸收活性 胶原蛋白和体外骨吸收测定，在小鼠和 人类细胞模型。这些研究应加强我们对 调节正常的骨重建，并可能提供新的见解， 代谢性骨病中发生的紊乱。

Osteoclast bone resorption is the first step in bone remodeling, and can be regulated at three distinct steps: osteoclast differentiation, initiation of bone resorption, and control of the resorption rate (or duration). Osteoclasts resorb bone by adhering to bone matrix proteins, and forming an enclosed space at the attachment site into which proteolytic enzymes are secreted, and protons are actively transported by a vacuolar H+-ATPase (V-ATPase) that is highly amplified in the ruffled membrane. Recent studies from our laboratory suggest that expression of the V-ATPase 15 kD subunit may have a key role in regulating this subunit expression in a model for osteoclast differentiation that is dependent on cell-cell interactions. Immunoprecipitation and immunoblotting will be performed marrow cultured to determine if the 15 kD subunit controls the overall amount of functional V-ATPase. The mechanism of induction of 15 kD subunit expression will be studied by stable transduction of promoter- reporter constructs into a murine monocytic precursor line that differentiates into osteoclast-like cells in co-culture. In studies on the initiation of bone resorption in mouse marrow cultures, we discovered that interstitial collagenase (the homolog of human MMP-13), produce by stromal cells, has a key role in initiating bone resorption that does not involve clearing the bone surface of collagen, by production of collagen cleavage products that activate bone resorption. An objective of this project will be to determine if collagen cleavage products activate isolated osteoclasts directly; to examine the enzymatic and cell biologic mechanisms by which MMP-13 initiates osteoclast bone resorption by determining the site(s) on collagen and structural requirements for induction of osteoclast bone resorptive activity using recombinant collagen proteins and in vitro bone-resorption assay, in both murine and human cell models. These studies should enhance our understanding of the regulation of normal bone remodeling, and may provide new insights on derangements that occur in metabolic bone diseases.