Elucidation of the Role of Creb5 in Synovial Joint Formation

阐明 Creb5 在滑膜关节形成中的作用

• 批准号: 10228688
• 负责人: Andrew Bruce Lassar
• 金额: $ 67.05万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-18 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10228688
• 关键词: ATAC-seq; Adult; Alleles; Area; Arthritis; Cattle; Cells; Chondrocytes; Competence; Coupled; DNA Binding Domain; Degenerative polyarthritis; Development; Ectopic Expression; Embryo; Engineering; Environment; Epidermal Growth Factor Receptor; Epiphysial cartilage; Event; Exons; Fibroblasts; Friction; Gene Expression; Genes; Genetic Recombination; Genetic Transcription; Goals; Guide RNA; Hallmark Cell; Induced Mutation; Infection; Injury; Joints; Knee joint; Lentivirus; Limb Bud; Maintenance; Mechanics; Mediating; Meniscus structure of joint; Mesenchyme; Mus; Natural regeneration; Newborn Infant; Physiologic Ossification; Play; Protein Binding Domain; Proteins; Proteoglycan; Regulatory Element; Rheumatoid Arthritis; Role; Signal Pathway; Signal Transduction; Synovial joint; Tissues; Transforming Growth Factor Beta 2; Work; articular cartilage; bZIP Domain; insight; joint formation; knock-down; long bone; lubricin; novel; p38 Mitogen Activated Protein Kinase; programs; protein expression; response; small hairpin RNA; stem cell population; tissue regeneration; transcription factor; transcriptome sequencing

Project Summary/Abstract. The broad, long-term goal of this project is to develop a comprehensive understanding of the developmental events that dictate cell fate decisions in formation of the synovial joint. Within this broad area, this proposal focuses on the differentiation of articular cartilage, which plays a central role in maintaining the low-friction environment of the joint space. Indeed, a hallmark of cells comprising the articular cartilage is their expression of proteoglycans, such as the protein lubricin, encoded by the Prg4 gene, that lubricates the joint and protects against the development of arthritis. Prg4 is specifically expressed in the superficial-most layer of the articular cartilage. Findings by both the Lassar lab and others have established that Prg4-expressing cells in the superficial zone of articular cartilage (in embryonic and early post-natal mice) serve as a stem cell population for all deeper regions of the articular cartilage in the adult. Thus, to elucidate how the articular cartilage stem cell population is both generated during development and maintained in the adult, a key objective of this proposal is to identify the factors that regulate the expression of both Prg4 and other genes that are specifically expressed in the superficial zone of the articular cartilage. Recent findings in the Lassar lab indicate that the transcription factor Creb5 is uniquely expressed in superficial zone articular chondrocytes (as opposed to both deeper zone articular chondrocytes and growth plate chondrocytes) and is a crucial regulator of Prg4 expression. Most notably, ectopic expression of Creb5 in deep zone bovine articular chondrocytes (which do not expression Prg4) enabled TGF-b2 and EGFR signals to induce Prg4 expression in these cells, to a level equal to that expressed by superficial zone articular chondrocytes. These findings suggest that Creb5 establishes a competent state in chondrocytes to express Prg4 in response to these signaling pathways. In addition, the Lassar lab has found that mice engineered to lack functional Creb5 fail to form many synovial joints, and that mis-expression of Creb5 throughout the limb bud mesenchyme (with Prx1-Cre) results in a profound loss of growth plate development in long bones. Taken together, these findings indicate that Creb5 plays a critical role in both the formation of synovial joints and is a both a novel and crucial regulator of Prg4/lubricin expression in articular chondrocytes. This project will identify both Creb5-dependent genes and the regulatory elements that drive the expression of these genes in primary bovine superficial zone articular chondrocytes; and mechanistically determine how TGFb, EGFR, and p38 signaling modulate the transcriptional activity of Creb5 in these cells. In addition, this project will determine how Creb5 regulates the formation of synovial joints, and elucidate how Creb5 expression in the epiphyseal perichondrium blocks extension of the growth plate into the epiphyseal ends of the long bones.

项目摘要/摘要。 该项目的广泛、长期目标是全面了解发展 决定滑膜关节形成过程中细胞命运决定的事件。在这个广泛的领域内，本提案 专注于关节软骨的分化，这在维持低摩擦方面发挥着核心作用 关节空间的环境。事实上，构成关节软骨的细胞的一个标志是它们的表达 蛋白多糖，例如由 Prg4 基因编码的蛋白质 lubricin，可以润滑关节并保护关节 防止关节炎的发展。 Prg4具体表达于关节的最浅层 软骨。 Lassar 实验室和其他实验室的研究结果表明，表面的 Prg4 表达细胞 关节软骨区域（在胚胎和产后早期的小鼠中）作为所有更深层次的干细胞群 成人关节软骨区域。因此，阐明关节软骨干细胞群如何 既在发育过程中产生，又在成人中维持，该提案的一个关键目标是确定 调节 Prg4 和其他基因表达的因子 关节软骨的表面区域。 Lassar 实验室的最新发现表明转录因子 Creb5 在浅层关节软骨细胞中独特表达（与深层关节软骨细胞相反） 软骨细胞和生长板软骨细胞），是 Prg4 表达的重要调节因子。最值得注意的是，异位 Creb5 在深区牛关节软骨细胞（不表达 Prg4）中的表达启用 TGF-b2 EGFR 信号诱导这些细胞中 Prg4 的表达，达到与浅层区域表达的水平相同的水平 关节软骨细胞。这些发现表明 Creb5 在软骨细胞中建立了一种有能力的状态 表达 Prg4 来响应这些信号通路。此外，Lassar 实验室还发现小鼠经过基因改造 缺乏功能性的 Creb5 无法形成许多滑膜关节，并且 Creb5 在整个肢体中的错误表达 芽间充质（含有 Prx1-Cre）会导致长骨生长板发育严重丧失。采取 总之，这些发现表明 Creb5 在滑膜关节的形成中发挥着关键作用，并且是 两者都是关节软骨细胞中 Prg4/lubricin 表达的新型且关键的调节因子。该项目将确定 Creb5 依赖性基因和驱动这些基因在原代细胞中表达的调控元件 牛浅层关节软骨细胞；并从机制上确定 TGFb、EGFR 和 p38 信号传导调节这些细胞中 Creb5 的转录活性。此外，该项目将确定 Creb5如何调节滑膜关节的形成，并阐明Creb5如何在骨骺中表达 软骨膜阻止生长板延伸到长骨的骨骺端。