Identification of the transcriptional regulators of chondrocyte hypertrophy

描述(申请人提供)：Lassar实验室的工作已经确定，在体节强制表达Runx2可以激活软骨细胞肥大标记物(即IHH和X胶原)的表达，前提是这些细胞首先被各种处理诱导成为软骨细胞，这表明软骨形成辅助因素允许Runx2激活软骨细胞肥大标记物的表达。与这一概念一致，Lassar实验室已经证实，异位Runx2与BMP信号转导因子Smad 1共转染只能在软骨细胞而不是成纤维细胞中诱导X胶原蛋白调控序列驱动的荧光素酶的表达。通过对鸡胶原X调节序列进行系统的凝胶迁移率改变分析(EMSA)，Lassar实验室已经确定了肥大软骨细胞中的DNA结合活性(称为Fast Mobility Complex)，该活性包含转录因子FOXA2，并与驱动连接报告基因的软骨细胞特异性表达所必需的序列相互作用。Lassar实验室最近的工作已经证实：(1)FoxA因子在分化的软骨细胞中特异表达，(2)FoxA结合位点在禽类和哺乳动物的X型胶原增强子中都是保守的，(3)禽类X型胶原增强子中FoxA结合位点的突变严重降低了Runx2/Smad1介导的鸡胸骨软骨细胞中该增强子的激活，(4)虽然FoxA1和FOXA2是在鸡肢芽间充质细胞的微团培养中诱导软骨形成的，但FOXA2和FoxA3在小鼠成软骨分化过程中特异地表达，(5)迫使FoxA1、FoxA2和FoxA3的表达FOXA2或FoxA3能有效地激活软骨细胞或成纤维细胞中X-型胶原报告基因的表达，(6)shRNA介导的FoxA因子在鸡胸骨软骨细胞中的敲除既能抑制Runx2/Smad1激活X-荧光素酶报告基因的表达，又能抑制内源性X-荧光素酶、IHH和MMP13的表达。根据这两个新发现，以及其他人发表的研究结果表明FOXA2/3在小鼠生长板软骨细胞中表达，我推测FOXA2和FoxA3可能在促进小鼠软骨细胞成熟方面发挥重要作用。在这项建议中，我建议确定FOXA2和FoxA3是否在调节小鼠软骨细胞成熟方面发挥作用。这项建议的目的是阐明控制软骨细胞肥大的因素，希望这一知识将导致开发出能够阻止关节软骨中这一程序的试剂，从而减缓骨关节炎的进展。 公共卫生相关性：骨关节炎(OA)的软骨退化过程涉及到受影响软骨中软骨细胞肥大程序的激活，这表明软骨细胞成熟程序的激活可能在骨关节炎软骨破坏的进展中发挥关键作用。这项建议的目的是阐明控制软骨细胞肥大的因素，希望这一知识将导致开发出能够阻止关节软骨中这一程序的试剂，从而减缓骨关节炎的进展。

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.