CONTROL OF OSTEOCLAST ACTIVATION AND DIFFERENTIATION

破骨细胞骨吸收是骨重建的第一步，可以 在三个不同的步骤中被调节：破骨细胞分化， 启动骨吸收，控制骨吸收速度(或 持续时间)。破骨细胞通过粘附骨基质蛋白吸收骨， 并在附着点形成封闭空间，其中 蛋白水解酶被分泌，质子被活跃地通过 一种在皱纹中高度扩增的液泡型H+-ATPase(V-ATPase) 薄膜。我们实验室最近的研究表明， V-ATPase 15kD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 细胞与细胞的相互作用。免疫沉淀和免疫印迹将被 进行骨髓培养以确定15kD亚基是否控制 功能性V-ATPase总量。15kD辐射诱导机制的研究 亚基表达的研究将通过稳定的转导启动子- 记者构建了一种小鼠单核细胞前体细胞系 在共培养中分化为破骨细胞样细胞。 在小鼠骨髓培养中启动骨吸收的研究中， 我们发现间质胶原酶(人基质金属蛋白酶-13的同源物)， 由基质细胞产生，在启动骨吸收中起关键作用 这不包括通过产生清除骨骼表面的胶原 激活骨吸收的胶原蛋白裂解产物。一个目标 这个项目的关键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 基质金属蛋白酶-13启动破骨细胞骨吸收的生物学机制 通过确定部位(S)上的胶原蛋白和结构要求 重组人破骨细胞诱导骨吸收活性的实验研究 胶原蛋白和体外骨吸收试验 人体细胞模型。这些研究应该会加深我们对 对正常骨重建的调节，并可能为 发生在代谢性骨骼疾病中的紊乱。

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.