Bone Matrix and Bone Resorption

描述(申请人提供)：这些研究都源于我们之前探索骨基质和骨吸收相互作用的工作，重点关注影响骨骼发育和骨吸收的基因和其他因素。特定目标1建议定位导致大鼠破骨细胞发育和功能失败的两个独特的突变。这一目标将使用经典遗传学结合分子作图和位置克隆技术来识别导致无门牙(Ia)和无牙(Tl)大鼠突变的骨化表型的基因。这些我们已经维持多年的近交系将与一个高度多态的品系进行异交。将使用一系列的聚合酶链式反应、连锁研究以及最终的详细序列分析来识别突变的基因。目前正在对来自TL RAT中新确定地区的候选人进行评估。目的2探讨成骨细胞分泌结缔组织生长因子(CTGF)与破骨细胞募集和激活之间的功能关系。我们已经在体内显示了成骨细胞在三个大鼠和一个小鼠的成骨突变中上调CTGF的mRNA和蛋白，所有这些突变都在不同的点上截断了破骨细胞的分化和/或功能。CTGF过度表达的共性表明，骨吸收是降低CTGF至正常水平的必要条件。我们将检验这一假设：(1)通过治疗突变动物并在体内跟踪对CTGF水平的影响；(2)通过使用破骨细胞和骨器官培养系统，CTGF/破骨细胞的结合将被抗CTGF抗体和饱和破骨细胞α(V)β整合素阻止，并且将检测其对吸收的影响；(3)将检测来自吸收培养物的上清是否能够下调成骨细胞CTGF的表达。具体目标3将确定胶原基因转换失调是否是颅面联合软骨和缝合生长异常的共同特征。我们已经在tl大鼠的两个关键生长中心显示了胶原基因切换(I型和I11型以及II型和X型)的失败。我们将把这些研究扩展到颅底的合并症，并使用这种方法来筛选一系列具有颅面部和/或骨性突变的小鼠，以寻找类似的失败。这将确定胶原蛋白表达异常伴随颅面缺陷的频率，它是否构成颅面生长缺陷的潜在“读出”，从而确定是否有必要进行机械性未来的研究。假说/特定目的4.对骨质疏松性突变中异常基因表达的扩展研究将发现更多参与调节正常骨骼代谢的基因。我们以前已经使用mRNA的差异显示来发现几个重要的骨骼基因。我们将使用高密度基因阵列技术来扩展这些研究，以比较正常、骨质疏松症和治愈的骨质疏松症骨RNA，以确定更多在骨质疏松症突变中上调或下调的基因和途径，以及体内支持破骨细胞分化的基因和途径。这种方法也将允许我们跟踪异常表达的持续或在细胞因子治疗恢复吸收后其恢复正常。

DESCRIPTION (provided by applicant): These studies, all derived from our previous work exploring the interactions of bone matrix and bone resorption, focus on genes and other factors impacting skeletal development and resorption. Specific Aim 1 proposes mapping two unique mutations responsible for the failure of osteoclast development and function in the rat. This aim will be pursued using classical genetics combined with molecular mapping and positional cloning techniques to identify the genes responsible for the osteopetrotic phenotypes of the incisor absent (ia) and toothless (tl) rat mutations. These inbred strains that we have maintained for many years will be outcrossed with a highly polymorphic strain. A series of PCR reactions, linkage studies, and ultimately detailed sequence analyses will be used to identify the mutated genes. Candidates from a newly identified region in the tl rat are currently being evaluated. Specific Aim 2 explores the functional relationship between connective tissue growth factor (CTGF) secretion by osteoblasts and osteoclast recruitment and activation. We have shown up-regulation of CTGF mRNA and protein in vivo by osteoblasts in three osteopetrotic mutations in the rat and one in the mouse, all of which intercept osteoclast differentiation and/or function at distinct points. The commonality of CTGF over-expression implies that bone resorption is required to reduce CTGF to normal levels. We will test this hypothesis: (1) by curing mutant animals and following the impact on CTGF levels in vivo; (2) using osteoclast and bone organ culture systems, CTGF/osteoclast binding will be prevented by anti-CTGF antibodies and by saturating osteoclast alpha(v)beta(3) integrins with recombinant CTGF, and the effect on resorption will measured; (3) supernatants from resorbing cultures will be tested for their ability to down-regulate CTGF expression by osteoblasts. Specific Aim 3 will establish whether collagen gene switching dis-regulation is a common feature of growth abnormalities at craniofacial synchondroses and sutures. We have shown failures of collagen gene switching (types I and Ill and types II and X) at two key growth centers in the tl rat. We will extend these studies to the synchondroses of the skull base, and use this approach to screen a series of mice with craniofacial and/or osteopetrotic mutations for similar failures. This will establish how frequently collagen expression abnormalities accompany craniofacial defects, whether it constitutes a potential "read-out" for craniofacial growth defects, and therefore whether mechanistic future investigations are warranted. Hypothesis/Specific Aim 4. Extending studies of abnormal gene expression in osteopetrotic mutations will uncover more genes involved in regulating normal skeletal metabolism. We have previously used differential display of mRNA to discover several important skeletal genes. We will extend these studies using high-density gene array technology to compare normal, osteopetrotic, and cured osteopetrotic bone RNA to identify more genes and pathways that are up- or down-regulated in osteopetrotic mutations and that underlie osteoclast differentiation in vivo. This method will also permit us to follow the persistence of abnormal expression or its return to normal following restoration of resorption by cytokine treatments.