Sp7 Mediated Control of Runx2 Function for Osteoblast Differentiation

摘要：哺乳动物骨骼的发育是一个极其复杂的过程。完成这两项任务 软骨内和膜内成骨需要一个高度复杂但协调良好的过程 图案化、细胞命运承诺、分化、生长和重塑。这些事件由 基因表达的时空协调模式。一开始，分泌的形态致病菌，如 Hedgehog、骨形态发生蛋白、无翼蛋白等向关键转录因子发出信号 来明确基因的表达。Runx2是软骨细胞和成骨细胞必需的转录因子 差异化。Runx2基因缺失导致骨骼完全衰竭导致胚胎死亡 队形。在人类中，Runx2基因的突变导致锁骨颅骨发育不良，这是一种显性遗传性 骨科疾病。骨骼形成的另一个主要调节因子是特异性蛋白-7(SP7)。SP7属于 Krüppel样转录因子家族的Sp亚群，其特征是三个锌指DNA- 结合域。SP7基因缺失会导致成骨细胞功能衰竭和骨形成。在人类身上， SP7基因突变与隐性形式的成骨不全、骨骼脆性和 延迟的牙萌出。然而，人们对其潜在的分子机制知之甚少。 这两个看似不相关的蛋白质的表型惊人地相似。需要运行Runx2才能 SP7的表达，因为靶向干扰Runx2基因的小鼠完全缺乏SP7的表达。 与之形成鲜明对比的是，在SP7缺失动物的骨骼细胞中，Runx2的表达是正常的。功能界别 SP7缺失小鼠的Runx2功能不全提示SP7的存在是完成 Runx2成骨活性。值得注意的是，在SP7缺失小鼠中观察到Runx2的表达 仅限于RNA，由胚胎组织的原位杂交确定。我们的数据显示，尽管 正常水平的Runx2基因、Runx2蛋白在SP7基因缺失小鼠的骨骼组织中高度不稳定。我们 进一步证明Runx2和SP7蛋白形成分子复合体及其转录活性 由独特的翻译后修饰来调节。我们的发现有力地表明，在骨骼细胞中， SP7起着分子变阻器的作用，对Runx2蛋白的功能稳定性和周转是必需的。 我们的实验将评估在SP7缺失的背景下和通过调节的 以及在骨祖细胞中的选择性基因重建。此应用程序的目标是识别和 定义a)Runx2和SP7调控复合体的空间和时间组织和组装 成骨细胞的形成和/或维持以及b)支持稳定复合体形成的机制 以及保持骨骼基因表达的能力。通过这项研究获得的知识将提供 对可作为创新靶点的骨骼调节复合体成分的分子洞察力 改善软骨和骨骼的形成和修复的治疗。

ABSTRACT: Development of skeleton in mammals is an exceedingly complex process. Completion of both endochondral and intramembranous ossification entails a highly intricate but well-coordinated process of patterning, cell fate commitment, differentiation, growth, and remodeling. These events are specified by a coordinated temporal and spatial pattern of gene expression. At first, secreted morphogens such as hedgehog, bone morphogenetic proteins, wingless proteins, and others, signal to key transcription factors to specify gene expression. Runx2 is an essential transcription factor for both chondrocyte and osteoblast differentiation. Runx2 gene deletion results in embryonic lethality due to a complete failure of bone formation. In humans, mutation of the Runx2 gene causes cleidocranial dysplasia, a dominantly inherited skeletal disorder. Another master regulator of skeletogenesis is the Specificity protein-7 (Sp7). Sp7 belongs to the Sp subgroup of the Krüppel-like family of transcription factors characterized by three zinc-finger DNA- binding domains. Deletion of Sp7 gene results in failure of osteoblasts, and bone formation. In humans, mutation of the Sp7 gene is linked with the recessive form of osteogenesis imperfecta, skeletal fragility and delayed tooth eruption. However, very little is known about the underlying molecular mechanism for the surprisingly similar phenotype from the two seemingly unrelated proteins. Runx2 is required for the expression of Sp7, as mice with targeted disruption of the Runx2 gene completely lack expression of Sp7. In sharp contrast, the Runx2 expression is normal in the skeletal cells of Sp7 null animals. The functional incompetency of Runx2 in the Sp7 null mice suggests that Sp7 presence is obligatory for completion of the Runx2 osteogenic activity. It is important to note that the observation of Runx2 expression in Sp7 null mice is limited to only RNA, determined by in situ hybridization of embryonic tissues. Our data show that despite normal levels of Runx2 mRNA, Runx2 protein is highly unstable in skeletal tissues of Sp7 null mice. We further demonstrate that Runx2 and Sp7 proteins form a molecular complex and their transcriptional activity is regulated by unique posttranslational modifications. Our findings strongly suggest that in skeletal cells, Sp7 acts as a molecular rheostat and is necessary for functional stability and turnover of Runx2 protein. Our experiment will assess endogenous levels of Runx2 protein in Sp7 null background and by a regulated and selective gene reconstitution in osteoprogenitor cells. The goal of this application is to identify and define a) spatial and temporal organization and assembly of Runx2 and Sp7 regulatory complexes for the formation and/or maintenance of osteoblasts and b) mechanisms supporting the stable complex formation and retention of competency for skeletal gene expression. Knowledge obtained from this study will provide molecular insights into components of a bone regulatory complex that can be targeted for innovative therapy to improve cartilage and bone formation and repair.