Identification of the transcriptional regulators of chondrocyte hypertrophy

软骨细胞肥大转录调节因子的鉴定

• 批准号: 8040942
• 负责人: Andrew Bruce Lassar
• 金额: $ 36.61万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8040942
• 关键词: Affect; Alleles; Antibodies; Binding; Binding Sites; Birds; Cartilage; Cells; Chick Embryo; Chickens; Chondrocytes; Chondrogenesis; Collagen; Competence; Complex; DNA Binding; Degenerative polyarthritis; Development; Electrophoretic Mobility Shift Assay; Enhancers; Environment; Epiphysial cartilage; Family member; Fibroblasts; Gene Expression; Goals; Hypertrophy; Knowledge; Lead; Light; Limb Bud; Link; Luciferases; Mediating; Mesenchymal; Mesoderm; Mus; Mutation; Nucleic Acid Regulatory Sequences; Play; Publishing; Reagent; Reporter; Reporter Genes; Role; Signal Transduction; Somites; Techniques; Transfection; Transgenes; Transgenic Mice; Work; articular cartilage; cell type; embryo tissue; in vivo; programs; public health relevance; small hairpin RNA; transcription factor; transgene expression

DESCRIPTION (provided by applicant): Work in the Lassar lab has established that forced expression of Runx2 in somites can activate expression of chondrocyte hypertrophy markers (i.e., Ihh and Collagen X) only if these cells are first induced to become chondrocytes by a variety of treatments, suggesting that a chondrogenic co-factor allows Runx2 to activate expression of markers of chondrocyte hypertrophy. Consistent with this notion, the Lassar lab has established that co-transfection of ectopic Runx2 together with the BMP signal tranducer, Smad 1, can induce expression of a luciferase construct driven by Collagen X regulatory sequences only in chondrocytes and not in fibroblasts. By employing a systematic electrophoretic mobility shift assay (EMSA) with the chicken collagen X regulatory sequences, the Lassar lab has identified a DNA binding activity (termed Fast Mobility Complex) specifically in hypertrophic chondrocytes that contains the transcription factor FoxA2 and interacts with a sequence that is necessary to drive chondrocyte-specific expression of linked reporter genes. Recent work in the Lassar lab has established: (1) that FoxA factors are specifically expressed in differentiated chondrocytes, (2) that FoxA binding sites are conserved in both avian and mammalian collagen X enhancers, (3) that mutation of FoxA binding sites in the avian collagen X enhancer severely decreases Runx2/Smad1-mediated activation of this enhancer in chicken sternal chondrocytes, (4) that while FoxA1 and FoxA2 are induced during chondrogenesis in micromass cultures of chicken limb bud mesenchymal cells, FoxA2 and FoxA3 are specifically expressed during murine chondrogenic differentiation, (5) that forced expression of either FoxA1, FoxA2 or FoxA3 robustly activates the expression of collagen X-reporter constructs in either chondrocytes or fibroblasts, and (6) that shRNA-mediated knockdown of FoxA factors in chicken sternal chondrocytes both inhibits the ability of Runx2/Smad1 to activate the expression of a collagen X-luciferase reporter and inhibits the expression of endogenous collagen X, Ihh, and MMP13. In light of both these new findings, and published findings of others indicating that FoxA2/3 are expressed in murine growth plate chondrocytes, I hypothesize that both FoxA2 and FoxA3 may play an important role in promoting chondrocyte maturation in mice. In this proposal I propose to determine whether FoxA2 and FoxA3 play a role in regulating chondrocyte maturation in mice. The goal of this proposal is to elucidate the factors that control chondrocyte hypertrophy in the hope that this knowledge will lead to the development of reagents that could block this program in articular cartilage and thereby slow the progression of osteoarthritis. PUBLIC HEALTH RELEVANCE: The course of cartilage degradation in osteoarthritis (OA) involves an activation of the chondrocyte hypertrophy program in the affected cartilage, suggesting that activation of the chondrocyte maturation program could play a key role in progression of cartilage destruction in OA. The goal of this proposal is to elucidate the factors that control chondrocyte hypertrophy in the hope that this knowledge will lead to the development of reagents that could block this program in articular cartilage and thereby slow the progression of OA.

描述（由申请人提供）：Lassar实验室的工作已经证实，只有当这些细胞首先通过各种治疗诱导成为软骨细胞时，在某些细胞中强制表达Runx2才能激活软骨细胞肥大标志物（即Ihh和胶原X）的表达，这表明一种成软骨辅助因子允许Runx2激活软骨细胞肥大标志物的表达。与这一观点一致的是，lasar实验室已经证实，异位Runx2与BMP信号转导Smad 1共转染可以诱导由胶原X调节序列驱动的荧光素酶构建体的表达，仅在软骨细胞中，而不是在成纤维细胞中。通过对鸡胶原X调节序列进行系统的电泳迁移迁移测定（EMSA）， lasar实验室已经确定了一种DNA结合活性（称为快速迁移复合物），特别是在肥厚性软骨细胞中，该细胞含有转录因子FoxA2，并与驱动相关报告基因的软骨细胞特异性表达所必需的序列相互作用。拉萨尔实验室最近的工作已经确定：(1) FoxA因子在分化的软骨细胞中特异性表达；(2)FoxA结合位点在禽类和哺乳动物的胶原X增强子中都是保守的；(3)禽类胶原X增强子中FoxA结合位点的突变严重降低了Runx2/ smad1介导的该增强子在鸡胸骨软骨细胞中的激活；(4)在鸡肢芽间充质细胞微块培养中，FoxA1和FoxA2在软骨形成过程中被诱导。FoxA2和FoxA3在小鼠软骨分化过程中特异性表达，(5)FoxA1、FoxA2或FoxA3的强制表达均可强烈激活软骨细胞或成纤维细胞中构建的胶原X报告蛋白的表达，(6)鸡胸骨软骨细胞中shrna介导的FoxA因子的敲低既抑制Runx2/Smad1激活胶原X荧光素酶报告蛋白表达的能力，又抑制内源性胶原X的表达。嗯，还有MMP13。鉴于这两个新发现，以及其他已发表的研究结果表明FoxA2/3在小鼠生长板软骨细胞中表达，我假设FoxA2和FoxA3可能在促进小鼠软骨细胞成熟中发挥重要作用。在本提案中，我提议确定FoxA2和FoxA3是否在调节小鼠软骨细胞成熟中发挥作用。本提案的目标是阐明控制软骨细胞肥大的因素，希望这些知识将导致能够阻断关节软骨中这一程序的试剂的开发，从而减缓骨关节炎的进展。