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: 8640073
• 负责人: Andrew Bruce Lassar
• 金额: $ 35.88万
• 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/8640073
• 关键词: Affect; 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; LoxP-flanked allele; 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.

描述（由申请人提供）：Lassar实验室的工作已经确定，在体节中Runx 2的强制表达可以激活软骨细胞肥大标志物的表达（即，Ihh和胶原蛋白X），这表明软骨形成辅因子允许Runx 2激活软骨细胞肥大标志物的表达。与这一观点一致，Lassar实验室已经确定，异位Runx 2与BMP信号转导子Smad 1一起共转染可以诱导仅在软骨细胞而不是成纤维细胞中由胶原X调控序列驱动的荧光素酶构建体的表达。通过对鸡X胶原蛋白调节序列进行系统性电泳迁移率变动分析（EMSA），Lassar实验室已经鉴定了肥大软骨细胞中特异性的DNA结合活性（称为快速迁移复合物），该细胞含有转录因子FoxA 2并与驱动连接报告基因的软骨细胞特异性表达所需的序列相互作用。Lassar实验室最近的工作已经建立了：（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胶原X增强子中是保守的，（3）禽类胶原X增强子中FoxA结合位点的突变严重降低了鸡胸骨软骨细胞中Runx 2/Smad 1介导的该增强子的活化，（4）虽然FoxA 1和FoxA 2在鸡肢芽间充质细胞的微团培养物中的软骨形成过程中被诱导，但FoxA 2和FoxA 3在鼠软骨形成分化过程中特异性表达，（5）FoxA 1、FoxA 2或FoxA 3的强制表达强烈激活软骨细胞或成纤维细胞中胶原蛋白X-报道构建体的表达，和（6）鸡胸骨软骨细胞中FoxA因子的shRNA介导的敲低既抑制Runx 2/Smad 1激活胶原X-荧光素酶报告基因表达的能力，又抑制内源性胶原X、Ihh和MMP 13的表达。根据这些新的发现，以及其他人发表的研究结果表明FoxA 2/3在小鼠生长板软骨细胞中表达，我推测FoxA 2和FoxA 3可能在促进小鼠软骨细胞成熟中发挥重要作用。在这个提议中，我建议确定FoxA 2和FoxA 3是否在调节小鼠软骨细胞成熟中发挥作用。该提案的目标是阐明控制软骨细胞肥大的因素，希望这一知识将导致试剂的开发，可以阻止关节软骨中的这一程序，从而减缓骨关节炎的进展。

项目成果

Fibroblast growth factor maintains chondrogenic potential of limb bud mesenchymal cells by modulating DNMT3A recruitment.

• DOI: 10.1016/j.celrep.2014.07.038
• 发表时间: 2014-09-11
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Kumar D;Lassar AB
• 通讯作者: Lassar AB

Colchicine protects against cartilage degeneration by inhibiting MMP13 expression via PLC-γ1 phosphorylation.

• DOI: 10.1016/j.joca.2021.08.001
• 发表时间: 2021-11
• 期刊: Osteoarthritis and cartilage
• 影响因子: 7
• 作者: Takeuchi K;Ogawa H;Kuramitsu N;Akaike K;Goto A;Aoki H;Lassar A;Suehara Y;Hara A;Matsumoto K;Akiyama H
• 通讯作者: Akiyama H

Mechanical motion promotes expression of Prg4 in articular cartilage via multiple CREB-dependent, fluid flow shear stress-induced signaling pathways.

• DOI: 10.1101/gad.231969.113
• 发表时间: 2014-01-15
• 期刊: Genes & development
• 影响因子: 10.5
• 作者: Ogawa H;Kozhemyakina E;Hung HH;Grodzinsky AJ;Lassar AB
• 通讯作者: Lassar AB

GATA6 is a crucial regulator of Shh in the limb bud.

• DOI: 10.1371/journal.pgen.1004072
• 发表时间: 2014-01
• 期刊: PLoS genetics
• 影响因子: 4.5
• 作者: Kozhemyakina E;Ionescu A;Lassar AB
• 通讯作者: Lassar AB