Role of Dlx-5 in Chondrocyte Differentiation

描述(申请人提供)：软骨内骨化的过程，即四肢的骨骼在软骨模型产生后形成，依赖于一个仔细调节的软骨细胞成熟程序。识别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控相互作用对于了解骨骼的生长发育至关重要。本研究的目的是探讨同源结构域转录因子Dlx5在软骨内成骨过程中是软骨细胞成熟的关键正向调节因子的假说，并揭示Dlx5与其他参与控制该过程的因素之间的关系。Dlx5在未成熟的增殖期软骨细胞向有丝分裂后肥大前期软骨细胞的转化过程中表达，这是成熟的关键步骤。体内鸡肢体骨骼元素分化过程中，逆转录病毒Dlx5的错误表达导致骨骼元素严重缩短，肥大的软骨细胞数量增多，骨桥蛋白、X型胶原等肥大分化分子标志物的表达区域扩大和上调，矿化软骨基质扩张。在促进细胞肥大成熟的同时，Dlx5的错误表达也显著地抑制了细胞的增殖。这些结果表明，Dlx5至少部分地通过促进未成熟的增殖性软骨细胞向肥大的软骨细胞转化而积极地调节成熟度。Dlx5在软骨细胞成熟中的作用将通过检测Dlx5错误表达对其他肥大成熟标志物表达的影响以及当Dlx5错误表达特异性靶向转基因小鼠四肢软骨模型时是否促进软骨细胞成熟来进一步研究。也将确定Dlx6是否促进成熟，Dlx6是DLX家族中一个假定的功能冗余成员。在肢体间充质细胞和软骨细胞模型培养系统中，Dlx5调节成熟的机制将通过获得和失去功能的方法来研究。Dlx5与其他软骨细胞成熟的正性调节因子包括RMPs、Cbfal/Runx2和(-catenin)介导的Wnt信号之间的可能的调控关系将被研究。各种分子激动剂和拮抗剂将被用来开始解开Dlx5和其他因子如何在调节肥大成熟中相互作用和合作。还将确定Dlx5是否参与了软骨细胞成熟过程中骨桥蛋白、骨涎蛋白和X型胶原基因的转录调控。Dlx5与BMP信号通路和/或Cbfal/Runx2相互作用和合作的可能性将被研究，以调控这些候选靶基因中的一个或多个的启动子。

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.