Characterization of SHOX functions in bone formation: Identification of target genes and interacting proteins

生长是一个复杂的过程，受到各种不同的环境和遗传因素的影响。在遗传因子中，转录因子SHOX在生长和骨发育中起重要作用。SHOX突变是Leri-Weill、Langer和Turner Syndrome相关的身材矮小和骨骼异常的基础。SHOX基因内的突变也已在约3 - 5%的先前描述为特发性的矮小个体中被鉴定。最近我们发现SHOX在骨肉瘤细胞和原代软骨细胞中的异位表达可导致细胞周期阻滞和凋亡。我们还发现内源性SHOX在生长板软骨细胞中表达。总之，这些结果表明SHOX参与软骨细胞分化，但SHOX在软骨形成中的确切作用尚未阐明。本课题的目的是研究SHOX在骨形成中的作用，并鉴定其调控基因以及指导骨细胞和软骨分化的下游靶基因。为了实现我们的目标，我们将集中在两个不同的方法：识别SHOX靶基因。我们将采用我们最近开发的体内系统，该系统基于间充质干细胞向软骨细胞的特异性分化。使用该系统的先前结果表明，SHOX诱导与这些细胞的软骨细胞分化相一致。SHOX基因在软骨细胞分化的某个阶段的特异性激活提供了使用该模型进行旨在鉴定SHOX靶基因的基因表达研究的机会。SHOX相互作用蛋白的鉴定。我们将用全长SHOX作为诱饵，对自制的软骨细胞原代cDNA文库进行酵母双杂交筛选。通过鉴定SHOX伴侣和靶基因来了解SHOX在骨形成中的功能，将为骨发育过程提供新的见解。

Growth is a complex process that is influenced by a variety of different environmental and genetic factors. Among the genetic factors, an important role of growth and bone development can be attributed to the transcription factor SHOX. SHOX mutations underlie the short stature and the skeletal abnormalities associated with Leri-Weill, Langer and Turner Syndromes. Mutations within the SHOX gene have also been identified in approximately 3 - 5 % of short individuals previously described as idiopathic. Recently we have found that ectopic SHOX expression in osteosarcoma cells and primary chondrocytes leads to cell cycle arrest and apoptosis. We have also shown that endogenous SHOX is expressed in growth plate chondrocytes. Together, these results suggest that SHOX is involved in chondrocyte differentiation but the exact role of SHOX in chondrogenesis has not been elucidated yet. The goat of our proposed project is to characterize the role of SHOX in bone formation and to identify the genes that represent the regulators as well as the downstream target genes instructing bone cell and cartilage differentiation. To accomplish our aim we will focus on two different approaches: Identification of SHOX target genes. We will employ our recently developed in vivo system which is based on the specific differentiation of mesenchymal stem cells into chondrocytes. Previous results using this system suggest that SHOX induction coincides with the chondrocytic differentiation of these cells. The specific activation of the SHOX gene at a certain stage of chondrocyte differentiation provides the opportunity of using this model for gene expression studies aimed at identifying SHOX target genes. Identification of SHOX interacting proteins. We will perform a yeast two hybrid screen of a self-made primary chondrocyte cDNA library using full length SHOX as a bait. The understanding of SHOX functions in bone formation through the identification of SHOX partners and target genes will provide new insights on the processes which take place during bone development.