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(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图案化、细胞命运承诺、分化、生长和重塑过程。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，分泌的形态发生蛋白如骨形态发生蛋白、刺猬、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所必需的转录因子。Runx2基因缺失导致骨骼形成完全失败和胚胎死亡。在人类中，Runx2基因的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。骨骼形成的另一个主要调节因子是特异性蛋白-7(SP7)。SP7属于Kruppel类转录因子家族中的Sp亚类，具有三个锌指DNA结合区。靶向破坏SP7/Osterix基因，导致软骨内和膜内骨形成缺失。SP7缺陷的间充质细胞不沉积骨基质，不能分化为成骨细胞。对于这两个看似不相关的蛋白质产生惊人相似表型的潜在分子机制，人们知之甚少。Runx2是SP7表达所必需的，也可能是其功能所必需的，因为靶向干扰Runx2的小鼠不显示SP7的表达。有趣的是，在SP7缺失动物的膜间充质细胞和软骨内骨骼中，Runx2的表达是正常的。在SP7缺陷的细胞中，Runx2的功能不全，提示SP7的存在是完成Runx2成骨活性的必要条件。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作为一个分子变阻器，对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起作用，很有可能Runx2蛋白在SP7缺失细胞中永远不会产生或迅速降解。我们将在实验中通过评估SP7缺失细胞中Runx2蛋白的内源性水平，以及在骨祖细胞中调节和选择性的基因重建/消融来解决这个问题。本应用的目标是识别和定义a)Runx2和SP7调控复合体的时空组织和组装，以形成/维持成骨细胞，以及b)支持稳定的复合体形成和保持骨骼基因表达的能力的机制。从这项研究中获得的知识将为骨调节复合体的成分提供分子洞察力，这些成分可以被靶向创新治疗，以改善软骨和骨的形成和修复。 公共卫生相关性：对参与骨细胞成熟调控的分子机制的关键了解，对于开发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及骨骼疾病的病理生理学。