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: 9981286
• 负责人: Amjad Javed
• 金额: $ 35.41万
• 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 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9981286
• 关键词: Address; Affinity; Anatomy; Animals; BMP2 gene; Biochemical; Biological; Biological Availability; Biological Process; Biology; Bone Diseases; Bone Matrix; Bone Morphogenetic Proteins; Bone Regeneration; Calvaria; Cartilage; Cartilage Diseases; Cell Differentiation process; Cell Maturation; Cell model; Cells; Chondrocytes; Chromatin; Cleidocranial Dysplasia; Competence; Complex; Cues; DNA Binding; DNA Binding Domain; Data; Development; Differentiation and Growth; Embryo; Erinaceidae; Event; Failure; Family; Functional disorder; Gene Deletion; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Goals; Growth; Human; In Situ Hybridization; Inherited; Innovative Therapy; Intervention; Knockout Mice; Knowledge; Link; MAP Kinase Gene; Maintenance; Mammals; Mediating; Mesenchymal; Messenger RNA; Metabolic Bone Diseases; Modeling; Modification; Molecular; Molecular Biology; Monitor; Mus; Mutagenesis; Mutation; Null Lymphocytes; Osteoblasts; Osteogenesis; Osteogenesis Imperfecta; Pathway interactions; Pattern; Phenotype; Physiologic Ossification; Post-Translational Protein Processing; Process; Protein Biochemistry; Proteins; RBX1 gene; RNA; Regulation; Research; Role; Runx2 protein; Signal Transduction; Skeleton; Specific qualifier value; Specificity; Structure; Subgroup; Testing; Therapeutic Intervention; Tissues; To specify; Tooth eruption; Vertebrates; Zinc Fingers; base; bone; bone cell; embryo tissue; experimental study; improved; in vivo; insight; interdisciplinary approach; intramembranous bone formation; mineralization; morphogens; mutant; novel; osteoblast differentiation; osteogenic; osteoprogenitor cell; prevent; protein function; reconstitution; recruit; repaired; skeletal; skeletal disorder; skeletal tissue; skeletogenesis; stable cell line; therapy development; transcription factor; ubiquitin ligase; ubiquitin mediated proteasome degradation

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.

摘要：哺乳动物骨骼的发育是一个极其复杂的过程。两者都完成 软骨内和膜内骨化需要一个高度复杂但协调良好的过程 模式、细胞命运决定、分化、生长和重塑。这些事件由一个指定 协调基因表达的时间和空间模式。起初，分泌形态发生素，例如 刺猬蛋白、骨形态发生蛋白、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必需的转录因子 差异化。 Runx2基因缺失导致胚胎因骨骼完全衰竭而死亡 形成。在人类中，Runx2 基因突变会导致锁骨颅骨发育不良，这是一种显性遗传的疾病 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 (Sp7)。 Sp7属于 属于以三个锌指 DNA 为特征的 Krüppel 样转录因子家族的 Sp 亚组 结合域。 Sp7基因的缺失会导致成骨细胞和骨形成的失败。在人类中， Sp7 基因突变与隐性成骨不全、骨骼脆弱和 牙齿萌出延迟。然而，人们对于其潜在的分子机制知之甚少。 这两种看似无关的蛋白质的表型惊人地相似。需要 Runx2 Sp7 的表达，因为靶向破坏 Runx2 基因的小鼠完全缺乏 Sp7 的表达。 与此形成鲜明对比的是，Runx2 表达在 Sp7 缺失动物的骨骼细胞中是正常的。功能性的 Sp7 缺失小鼠中 Runx2 的无能表明 Sp7 的存在对于完成 Runx2 成骨活性。需要注意的是，Sp7 缺失小鼠中 Runx2 表达的观察 仅限于RNA，通过胚胎组织的原位杂交确定。我们的数据表明，尽管 尽管 Runx2 mRNA 水平正常，但 Sp7 缺失小鼠的骨骼组织中 Runx2 蛋白高度不稳定。我们 进一步证明Runx2和Sp7蛋白形成分子复合物及其转录活性 受独特的翻译后修饰调节。我们的研究结果强烈表明，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。 我们的实验将评估 Sp7 无效背景中 Runx2 蛋白的内源水平，并通过调节 和骨祖细胞中的选择性基因重建。该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) 支持稳定复合物形成的机制 和保留骨骼基因表达的能力。从这项研究中获得的知识将提供 对骨调节复合体成分的分子洞察，可用于创新 改善软骨和骨形成和修复的疗法。