MECHANISMS OF MINERALIZED MATRIX ACCUMULATION

在揭示许多分子事件的重大进展， 关于控制骨形成的研究大大增加了我们 了解基质矿化。 已充分证实 不溶性胶原蛋白的积累和交联对于 功能性矿化细胞外基质的发育。 是什么 尚不清楚骨胶原成熟是如何在 发展和细胞因子是否通过相同的调节这一过程 机制等前胶原蛋白C-蛋白酶和赖氨酰氧化酶已被 被认为是细胞外翻译后 胶原蛋白的改性。 除了处理前胶原外， 前胶原C蛋白酶将前赖氨酰氧化酶切割成32 kDa 活性酶和18 kDa前肽。 赖氨酰氧化酶催化 胶原蛋白交联的必要步骤。 最近我们 一个实验室发现了18 kDa前肽的生物学作用 基质矿化 在目的1中，调节前胶原C蛋白酶和赖氨酰的作用， 氧化酶在矿化细胞外基质形成中的作用将是 在发育中的胎鼠颅骨成骨细胞培养中研究。 分析将包括各种分子参数，包括mRNA 水平，蛋白质水平和酶活性，以及评估 通过测量不溶性胶原蛋白形成细胞外基质， 无机钙积累 基因敲除小鼠的研究进展 缺乏编码前胶原蛋白C-蛋白酶的BPM-1基因， 一种补充方法，以确定前胶原C的作用- 蛋白酶和赖氨酰氧化酶处理对体外骨形成的影响。 在 目的2，TGF-β和TNF-α对前胶原C- 蛋白酶和赖氨酰氧化酶、胶原蛋白合成和胶原蛋白 将在发育中的成骨细胞培养物中测定积累。 在目的3中，18 kDa前肽的成骨活性将是 表征了 为此目的，重组赖氨酰氧化酶前肽 将在真核表达系统中制备并用于实施 功能研究和探索受体介导的机制。 从这些研究中获得的结果将大大增加 了解细胞外事件如何导致基质成熟， 成矿 一旦分子参数解决了这个问题， 建议明确定义，可以利用 为发展治疗方法而获得的信息， 影响骨完整性的各种疾病。

Significant progress in uncovering many of the molecular events with respect to control of bone formation has added considerably to our understanding of matrix mineralization. It is well established that insoluble collagen accumulation and cross-linking are essential for the development of a functional mineralized extracellular matrix. What is not known is how bone collagen maturation is regulated during development and whether cytokines modulate this process via the same mechanisms. Procollagen C-proteinase and lysyl oxidase have been identified as key players in the extracellular post-translational modification of collagen. In addition to processing procollagen, procollagen C-proteinase cleaves pro-lysyl oxidase into the 32 kDa active enzyme and the 18 kDa propeptide. Lysyl oxidase catalyzes the essential step necessary for collagen cross-linking. Recently, our laboratory has discovered a biological role for the 18 kDa propeptide in matrix mineralization. In Aim 1, the role of regulation of procollagen C-proteinase and lysyl oxidase in the formation of a mineralized extracellular matrix will be investigated in developing fetal rat calvaria osteoblastic cultures. Analyses will include a variety of molecular parameters including mRNA levels, protein levels, and enzyme activity, as well as assessment of extracellular matrix formation by measuring insoluble collagen and inorganic calcium accumulation. The recent development of knockout mice that lack the BPM-1 gene that encodes procollagen C-proteinase provides a supplementary approach to establish the role of procollagen C- proteinase and lysyl oxidase processing on in vitro bone formation. In Aim 2, the cytokine effects of TGF-beta and TNF-alpha on procollagen C- proteinase and lysyl oxidase, collagen synthesis, and collagen accumulation will be determined in developing osteoblastic cultures. In Aim 3, the osteogenic activity of the 18 kDa propeptide will be characterized. For this purpose recombinant lysyl oxidase propeptide will be prepared in a eukaryotic expression system and used to carry out functional studies and to explore receptor mediated mechanisms. The results obtained from these studies will add significantly to the understanding of how extracellular events lead to matrix maturation and mineralization. Once the molecular parameters addressed in this proposal are clearly defined, it may be possible to exploit the information gained for the development of therapeutic approaches in a variety of diseases affecting bone integrity.