Role of Dlx-5 in Chondrocyte Differentiation

描述（由申请人提供）：软骨模型生成后肢体骨骼形成的软骨内成骨过程依赖于精心调节的软骨细胞成熟程序。鉴定控制肥大软骨细胞成熟的基因和信号分子以及它们之间的调节相互作用对于理解骨生长和发育至关重要。本研究的目的是探讨同源结构域转录因子Dlx5是软骨内成骨过程中软骨细胞成熟的关键正调节因子的假设，并揭示Dlx5与其他参与控制这一过程的因素之间的关系。Dlx5在未成熟增殖软骨细胞向有丝分裂后肥大前软骨细胞的转化过程中表达，这是成熟的关键步骤。在鸡肢体骨骼细胞分化过程中，逆转录病毒错误表达Dlx5会导致严重缩短的骨骼细胞的形成，这些骨骼细胞含有过多的肥大软骨细胞，一些肥厚分化分子标志物（包括骨桥蛋白和X型胶原）的表达域扩大和上调，以及矿化软骨基质的扩张。Dlx5错误表达也显著降低细胞增殖，同时促进肥厚性成熟。这些结果表明Dlx5至少在一定程度上通过促进未成熟增殖软骨细胞向肥大软骨细胞的转化来积极调节成熟。为了进一步研究Dlx5在软骨细胞成熟中的作用，我们将检测鸡肢体中Dlx5错表达对其他肥厚成熟标志物表达的影响，并确定特异性靶向转基因小鼠肢体软骨模型的Dlx5错表达是否促进软骨细胞成熟。还将确定Dlx6是否促进成熟，Dlx6是Dlx家族中一个假定的功能冗余的成员。Dlx5调控成熟的机制将在肢体间充质细胞和软骨细胞模型培养系统中使用功能增益和功能丧失的方法进行研究。Dlx5与其他软骨细胞成熟的正调节因子(包括RMPs、Cbfal/Runx2和（-catenin-介导的Wnt信号）之间可能的调节关系将被研究。多种分子激动剂和拮抗剂将被用于揭示Dlx5和其他因子如何在调节肥厚成熟中相互作用和合作。还将确定Dlx5是否参与软骨细胞成熟过程中骨桥蛋白、骨唾液蛋白和X型胶原蛋白基因的转录调控。Dlx5与BMP信号通路和/或Cbfal/Runx2在一个或多个候选靶基因启动子的转录调控中相互作用和合作的可能性将被研究。

DESCRIPTION (provided by applicant): The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a carefully regulated program of chondrocyte maturation. Identification of the genes and signaling molecules that control hypertrophic chondrocyte maturation and the regulatory interactions among them is crucial to understanding bone growth and development. The goal of this research is to investigate the hypothesis that the homeodomain transcription factor Dlx5 is a key positive regulator of chondrocyte maturation during endochondral ossification, and to unravel the relationships between Dlx5 and other factors involved in controlling the process. Dlx5 is expressed during the conversion of immature proliferating chondrocytes to post-mitotic prehypertrophic chondrocytes, a critical step in maturation. Retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in formation of severely shortened skeletal elements that contain excess numbers of hypertrophying chondrocytes, expanded and upregulated domains of expression of some molecular markers of hypertrophic differentiation including osteopontin and type X collagen, and expansion of mineralized cartilage matrix. Dlx5 misexpression also markedly reduces cell proliferation concomitant with promoting hypertrophic maturation. These results suggest Dlx5 positively regulates maturation at least in part by promoting conversion of immature proliferating chondrocytes to hypertrophying chondrocytes. The role of Dlx5 in chondrocyte maturation will be further investigated by examining effects of Dlx5 misexpression in the chick limb on expression of additional markers of hypertrophic maturation, and determining if chondrocyte maturation is promoted when Dlx5 misexpression is specifically targeted to the cartilage models of the limbs of transgenic mouse embryos. It will also be determined if maturation is promoted by Dlx6, a putative functionally redundant member of the Dlx family. The mechanism of Dlx5 regulation of maturation will be investigated using gain- and loss-of-function approaches in limb mesenchymal cell and chondrocyte model culture systems. The possible regulatory relationships between Dlx5 and other positive regulators of chondrocyte maturation including RMPs, Cbfal/Runx2, and (-catenin-mediated Wnt signaling will be investigated. A variety of molecular agonists and antagonists will be used to begin to unravel how Dlx5 and the other factors interact and cooperate in regulating hypertrophic maturation. It will also be determined if Dlx5 participates in transcriptional regulation of the osteopontin, bone sialoprotein, and type X collagen genes during chondrocyte maturation. The possibility that Dlx5 interacts and cooperates with the BMP signaling pathway and/or Cbfal/Runx2 in the transcriptional regulation of the promoters of one or more of these candidate target genes will be investigated.