Sp7 Mediated Control of Runx2 Function for Osteoblast Differentiation

描述（申请人提供）：哺乳动物骨骼的发育是一个极其复杂的过程，涉及膜内和软骨内成骨。任何一类骨化的完成都意味着一个高度复杂但协调良好的过程，包括模式、细胞命运承诺、分化、生长和重塑。这些事件是由基因表达的协调的时间和空间模式指定的。首先，分泌的形态因子如骨形态发生蛋白、刺猬蛋白、无翅蛋白等向关键转录因子发出信号，指定基因表达。Runx2是软骨细胞和成骨细胞分化的重要转录因子。Runx2基因缺失导致骨骼形成完全失败和胚胎致死。在人类中，Runx2基因突变导致锁骨颅骨发育不良，这是一种显性遗传性骨骼疾病。另一个骨骼形成的主要调节因子是特异性蛋白-7 （Sp7）。Sp7属于kruppel样转录因子家族的Sp亚群，以三个锌指dna结合结构域为特征。靶向破坏Sp7/Osterix基因，导致软骨内和膜内骨形成缺失。Sp7缺失的间充质细胞不能沉积骨基质，不能向成骨细胞分化。对于这两种看似不相关的蛋白质令人惊讶的相似表型的潜在分子机制知之甚少。Runx2是Sp7的表达所必需的，也可能是其功能所必需的，因为靶向破坏Runx2的小鼠不显示Sp7的表达。有趣的是，Runx2在Sp7阴性动物的膜质间充质细胞和软骨内骨骼中表达正常。Runx2在Sp7缺陷细胞中的功能缺失表明，Sp7的存在是Runx2成骨活性完成的必要条件。值得注意的是，在Sp7基因缺失的小鼠中观察到Runx2的存在仅限于RNA，这是通过对Sp7基因缺失的胚胎组织切片进行原位杂交来确定的。我们最近的数据表明，在骨骼细胞中，Sp7作为一种分子变阻器，是Runx2蛋白功能稳定和周转所必需的。考虑到一个复杂的转录后调控网络在骨骼细胞中起作用，很有可能在Sp7缺失的细胞中，Runx2蛋白从未产生或迅速降解。我们将通过实验来解决这个问题，通过评估Sp7缺失细胞中内源性Runx2蛋白的水平，以及在骨祖细胞中进行调节和选择性的基因重建/消融。本应用程序的目标是确定和定义a) Runx2和Sp7调控复合物形成/维持成骨细胞的时空组织和组装，b)支持稳定复合物形成和骨骼基因表达能力保持的机制。从这项研究中获得的知识将为骨调节复合体的组成部分提供分子见解，可以针对创新疗法来改善软骨和骨的形成和修复。

DESCRIPTION (provided by applicant): Development of skeleton in mammals is an exceedingly complex process and involves both intramembranous and endochondral ossification. Completion of either class of ossification implies 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 bone morphogenetic proteins, hedgehog, wingless proteins and others, signal to key transcription factors to specify gene expression. Runx2 is an essential transcription factor for chondrocyte and osteoblast differentiation. Runx2 gene deletion results in complete failure of skeleton formation and embryonic lethality. In humans, mutation of Runx2 gene causes cleidocranial dysplasia, a dominantly inherited skeletal disorder. Other master regulator of skeletogenesis is the Specificity protein-7 (Sp7). Sp7 belongs to the Sp subgroup of the Kruppel-like family of transcription factors characterized by three zinc-finger DNA-binding domains. Targeted disruption of Sp7/Osterix gene, results in absence of endochondral and intramembranous bone formation. The Sp7 deficient mesenchymal cells do not deposit bone matrix and cannot differentiate into osteoblasts. 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 and possibly for its function as mice with targeted disruption of Runx2 do not show expression of Sp7. Interestingly, Runx2 expression is normal in the mesenchymal cells of membranous and the endochondral skeleton of Sp7 null animals. The functional incompetency of Runx2 in Sp7 deficient cells, suggest that Sp7 presence is obligatory for completion of Runx2 osteogenic activity. It is important to note that the observation of Runx2 presence in Sp7 null mice is limited to only RNA, determined by in situ hybridization of tissue section from Sp7 null embryos. Our recent data demonstrate that in skeletal cells, Sp7 acts as a molecular rheostat and is necessary for functional stability and turnover of Runx2 protein. Given that a complex post-transcription regulatory network is operative in skeletal cells, a strong possibility exist that Runx2 protein is never made or rapidly degraded in Sp7 null cells. We will experimentally address this by assessing endogenous levels of Runx2 protein in Sp7 null cells and by a regulated and selective gene reconstitution/ ablation 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 formation/maintenance of osteoblasts and b) mechanisms supporting stable complex formation and retention of competency for skeletal gene expression. Knowledge obtained from this study will provide molecular insights into components of bone regulatory complex that can be targeted for innovative therapy to improve cartilage and bone formation and repair. PUBLIC HEALTH RELEVANCE: Crucial understanding of molecular mechanism involved in the regulation of bone cell maturation has significant potential for developing interventional therapies in growth anomalies and metabolic bone disorders. Findings from this study will help us in understanding the pathophysiology of skeletal tissues and cartilage and bone disorders.