Sp7 Mediated Control of Runx2 Function for Osteoblast Differentiation

Sp7介导的Runx2功能对成骨细胞分化的控制

基本信息

批准号：
8220442
负责人：
Amjad Javed
金额：
$ 32.19万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-01 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8220442
关键词：
Ablation Address Affinity Anatomy Animals BMP2 gene Biochemical Biological Biological Process Biology Bone Diseases Bone Matrix Bone Morphogenetic Proteins Bone Regeneration Bone Tissue Cartilage Cartilage Diseases Cell Differentiation process Cell Maturation Cell model Cells Chondrocytes Cleidocranial Dysplasia Complex Cues DNA Binding DNA Binding Domain Data Deposition Development Differentiation and Growth Embryo Erinaceidae Event Failure Family Functional disorder Gene Deletion Gene Expression Gene Expression Profile Gene Targeting Genes Genetic Genetic Transcription Goals Growth Human In Situ Hybridization Inherited Innovative Therapy Intervention Knockout Mice Knowledge MAP Kinase Gene Maintenance Mammals Mediating Mesenchymal Metabolic Bone Diseases Modeling Modification Molecular Molecular Biology Monitor Mus Mutagenesis Mutation Null Lymphocytes Osteoblasts Osteogenesis Pathway interactions Pattern Phenotype Process Protein Biochemistry Proteins RNA Regulation Research Role Runx2 protein Signal Transduction Skeleton Specific qualifier value Specificity Subgroup Testing Tissues To specify Ubiquitin Ubiquitination Vertebrates Work Zinc Fingers base bone bone cell improved in vivo insight interdisciplinary approach intramembranous bone formation mineralization morphogens multicatalytic endopeptidase complex mutant novel osteoblast differentiation osteogenic osteoprogenitor cell prevent protein function reconstitution repaired skeletal skeletal disorder skeletal tissue skeletogenesis stable cell line transcription factor ubiquitin ligase

项目摘要

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.

描述（由申请人提供）：哺乳动物骨骼的发育是一个极其复杂的过程，涉及膜内骨化和软骨内骨化。任一类骨化的完成都意味着高度复杂但协调良好的模式化、细胞命运决定、分化、生长和重塑过程。这些事件由基因表达的协调时间和空间模式指定。首先，分泌的形态发生素，如骨形态发生蛋白、刺猬蛋白、无翼蛋白等，向关键转录因子发出信号以指定基因表达。 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 基因突变会导致锁骨颅骨发育不良，这是一种显性遗传性骨骼疾病。骨骼发生的其他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 亚组，其特征在于三个锌指 DNA 结合域。 Sp7/Osterix 基因的定向破坏导致软骨内和膜内骨形成的缺失。 Sp7缺陷的间充质细胞不沉积骨基质并且不能分化成成骨细胞。对于这两种看似无关的蛋白质惊人相似的表型的潜在分子机制知之甚少。 Runx2 是 Sp7 表达及其功能所必需的，因为靶向破坏 Runx2 的小鼠不会表现出 Sp7 的表达。有趣的是，Runx2 表达在 Sp7 缺失动物的膜间充质细胞和软骨内骨骼中是正常的。 Runx2 在 Sp7 缺陷细胞中的功能不全表明 Sp7 的存在对于 Runx2 成骨活性的完成是必需的。值得注意的是，在 Sp7 缺失小鼠中观察到 Runx2 的存在仅限于 RNA，这是通过 Sp7 缺失胚胎组织切片的原位杂交确定的。我们最近的数据表明，在骨骼细胞中，Sp7 充当分子变阻器，对于 Runx2 蛋白的功能稳定性和周转是必需的。鉴于复杂的转录后调控网络在骨骼细胞中发挥作用，Runx2 蛋白很可能在 Sp7 缺失细胞中从未产生或快速降解。我们将通过评估 Sp7 无效细胞中 Runx2 蛋白的内源水平以及通过骨祖细胞中受调节和选择性的基因重建/消融来实验性地解决这个问题。该应用的目标是确定和定义a）用于成骨细胞形成/维持的Runx2和Sp7调节复合物的空间和时间组织和组装，以及b）支持稳定复合物形成和保留骨骼基因表达能力的机制。从这项研究中获得的知识将为骨调节复合物的成分提供分子见解，这些成分可以作为创新疗法的目标，以改善软骨和骨的形成和修复。公共卫生相关性：对骨细胞成熟调节所涉及的分子机制的重要了解对于开发生长异常和代谢性骨疾病的介入疗法具有巨大潜力。这项研究的结果将帮助我们了解骨骼组织以及软骨和骨骼疾病的病理生理学。