OSTEOBLAST MEDIATED TURNOVER OF COLLAGENASE IN BONE

描述（改编自申请人的摘要）：在一些遗传性的 骨骼疾病，包括石骨症和骨质疏松症， 过量的骨积聚，而在骨质疏松症中， 超过了骨增量。 从历史上看，骨形成与 成骨细胞和破骨细胞的吸收。 然而，数据显示， 积累，这表明成骨细胞在 再吸收过程。 这个细胞被证明是 靶向多种吸收促进剂，并能够 产生中性蛋白酶，如纤溶酶原激活剂， 胶原酶（MMP-1）。 申请人已经证明PTH刺激 大鼠成骨细胞系UMR分泌后一种酶， MMP 1的最大浓度出现在细胞外 培养基中添加激素后12-24 h。 这些随后 下降，到96 h时几乎检测不到。 细胞介导的结合 其机制是通过快速和饱和的去除外源性 纯化的大鼠MMP-1在37 ℃。 结合研究，在4 ℃下使用125 I-MMP-1， 表明一个单一类别的饱和受体和12000 受体/细胞。 一个时间进程显示特定的受体介导的 10 min内结合，60 min达到平衡， 实验证明了可逆性。 该受体被证明是 由于许多相关和不相关的蛋白质， 无法竞争约束力。 内化研究显示， 30 min时细胞内积累，125 I-完全降解 MMP-1 90分钟，表明该受体在UMR中起作用 成骨细胞清除细胞外MMP-1。 UMR的治疗 具有PTH的细胞在24小时后引起结合活性的下调， h，随后反弹至对照结合水平的两倍。 这是 随着内化率的协调变化， PTH处理的细胞中配体的降解。 因此， PTH处理细胞中受体丰度和配体降解 与我们观察到的细胞外 MMP-1的浓度表明受体决定了 和酶在细胞外环境中的功能。 因此， 这一修订后的竞争性延续提案将进一步 表征和识别受体。 这将通过以下方式实现： 1)通过125 I-测定受体的生物化学性质， 标记受体并在亲和柱上纯化; 2）使用 该纯化的材料以获得肽序列数据; 3）克隆 受体; 4）使用克隆表达和检查调节， 受体;和5）受体的突变，以确定其功能 域. 这项工作的结果应该有助于解剖细胞- 调节成骨细胞中加工的MMP-1的介导途径， 其他细胞。 在这样做的时候，数据也应该提供一些见解 骨重建（异常的MMP 1 表达/摄取）已经出错。

DESCRIPTION (Adapted from the Applicant's Abstract): In some inherited disorders of the skeleton, including osteopetrosis and osteosclerosis, excess bone accumulates whereas in osteoporosis bone loss significantly exceeds bone gain. Historically, bone formation was associated with the osteoblast and resorption with the osteoclast. However, data has accumulated which indicates that the osteoblast takes an active role in the resorption process. This cell has been shown to be the primary target for a variety of resorption promoting agents, and to be capable of producing neutral proteinases, such as plasminogen activator and collagenase (MMP-1). The applicant has shown that PTH stimulates secretion of the latter enzyme by the rat osteoblastic cell line, UMR, with maximal concentrations of MMP1 appearing in the extracellular medium 12-24 h after addition of the hormone. These subsequently decline, becoming almost undetectable by 96 h. A cell-mediated binding mechanism was suggested by the rapid and saturable removal of exogenous purified rat MMP-l at 37 C. Binding studies, using 125I-MMP-l at 4 C, indicated a saturable receptor of a single class and 12000 receptors/cell. A time course revealed specific receptor-mediated binding within 10 min, and equilibrium by 60 min, while dissociation experiments demonstrated reversibility. The receptor was shown to be specific for rat MMP-l since a host of related and unrelated proteins failed to compete for binding. Internalization studies revealed maximal intracellular accumulation at 30 min and complete degradation of 125I- MMP-l by 90 min, suggesting this receptor functions in the UMR osteoblastic cells to eliminate extracellular MMP-l. Treatment of UMR cells with PTH causes a downregulation of the binding activity after 24 h, with a later rebound to twice the control binding levels. This is paralleled by coordinate changes in the rate of internalization and degradation of the ligand in PTH-treated cells. Thus, the pattern of receptor abundance and ligand degradation in PTH-treated cells correlates inversely with our observations of the extracellular concentrations of MMP-l suggesting the receptor dictates the abundance and function of the enzyme in the extracellular milieu. Thus, the aims of this revised competing continuation proposal are to further characterize and identify the receptor. This will be accomplished by: 1) determining the biochemical properties of the receptor, by 125I- labelling the receptor and purifying it on a affinity column; 2) using this purified material to obtain peptide sequence data; 3) cloning the receptor; 4) using the clone to express and examine regulation of the receptor; and 5) mutation of the receptor to determine its functional domains. The results of this work should aid in dissection of a cell- mediated pathway for regulation of elaborated MMP-l in osteoblasts and other cells. In so doing, the data should also provide some insight into those many skeletal disorders where bone remodelling (aberrant MMP1 expression/uptake) has gone awry.