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

• 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
• 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

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，涉及膜内和软骨内骨化。任何一类骨化的完成都意味着模式化、细胞命运定型、分化、生长和重塑的高度复杂但协调良好的过程。这些事件由基因表达的协调时间和空间模式指定。首先，分泌的形态发生蛋白如骨形态发生蛋白、刺猬蛋白、无翅蛋白等向关键转录因子发出信号以指定基因表达。Runx 2是软骨细胞和成骨细胞分化所必需的转录因子。Runx 2基因缺失导致骨骼形成完全失败和胚胎死亡。在人类中，Runx 2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。骨骼发生的另一个主要调节因子是特异性蛋白-7（Sp 7）。Sp 7属于Kruppel样转录因子家族的Sp亚组，其特征在于具有三个锌指DNA结合结构域。Sp 7/Osterix基因的靶向破坏导致软骨内和膜内骨形成的缺失。Sp 7缺陷的间充质细胞不存款骨基质，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。Runx 2是Sp 7表达所必需的，并且可能是其功能所必需的，因为具有Runx 2靶向破坏的小鼠不显示Sp 7的表达。有趣的是，Runx 2的表达是正常的间充质细胞的膜和内软骨骨架的Sp 7无效的动物。Sp 7缺陷细胞中Runx 2的功能丧失表明Sp 7的存在对于完成Runx 2成骨活性是必需的。重要的是要注意，在Sp 7缺失小鼠中观察到Runx 2的存在仅限于RNA，通过Sp 7缺失胚胎的组织切片的原位杂交来确定。我们最近的数据表明，在骨骼肌细胞中，Sp 7作为一个分子变阻器，是Runx 2蛋白的功能稳定性和营业额所必需的。考虑到复杂的转录后调控网络在骨骼细胞中起作用，Runx 2蛋白在Sp 7无效细胞中从未产生或迅速降解的可能性很大。我们将通过评估Sp 7缺失细胞中Runx 2蛋白的内源性水平和通过骨祖细胞中的调节性和选择性基因重建/消融来实验性地解决这一问题。本申请的目的是鉴定和定义a）用于形成/维持成骨细胞的Runx 2和Sp 7调节复合物的空间和时间组织和组装，和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，这些成分可以用于创新疗法，以改善软骨和骨的形成和修复。 公共卫生关系：对骨细胞成熟调控的分子机制的关键理解对于发展生长异常和代谢性骨疾病的介入治疗具有重要的潜力。这项研究的结果将有助于我们了解骨骼组织和软骨和骨骼疾病的病理生理学。