FUNCTION AND REGULATION OF AP-2 IN SKELETAL DEVELOPMENT

描述(申请人提供)：软骨发育在胚胎骨骼形态形成、软骨内骨形成和骨骼关节发育中起着关键作用。在软骨发育过程中，进行性的、协调的软骨发生受到基因表达，尤其是基因转录的精确控制。一些转录因子如Sox9、Sox5、Sox6和Cbfa1被发现通过正向调控成软骨基因的表达而在软骨发育中起关键作用。越来越多的表达和功能研究表明，转录因子AP-2可能通过负调控成软骨基因的表达而在软骨发育中发挥关键作用。多个AP-2在胚胎发育的肢体、出生后的关节软骨和生长板软骨中表达。在体外，AP-2α的表达对成软骨基因(II型胶原和CD-RAP)的表达具有负性调节作用。AP-2α基因敲除会导致颅面和四肢芽发育中的骨骼缺陷(肢体长度缩短和半径丧失)，以及其他胚胎发育缺陷(神经管闭合、体壁形成和眼睛形成)。为了进一步阐明AP-2在软骨发育中的特定作用，我们想要1)通过建立软骨特异的AP-2α基因敲除小鼠和通过显性负的AP-2α突变体的软骨特异性过表达的转基因小鼠来确定靶向消除AP-2表达/活性对软骨发育的影响；2)通过建立在软骨组织中过表达AP-2α的转基因小鼠来确定AP-2的定向过表达对软骨发育的影响；3)通过AP-2α野生型和显性阴性突变体的过度表达，确定AP-2功能的获得和丧失对软骨细胞增殖、分化和存活的影响；以及4)通过全基因组基因芯片分析，确定AP-2调控COL2Alpha1基因表达的分子机制，并通过全基因组基因芯片分析，确定AP-2调控COL2Alpha1基因表达的分子机制，以探讨AP-2Alpha在软骨形成早期调控的分子靶点。研究AP-2基因表达/活性的改变对软骨发育、软骨形成、COL2Alpha1基因转录调控和整体基因表达模式的影响，将为在组织、细胞和基因水平上建立AP-2在软骨发育中的特定作用提供具体证据。这项研究不仅将为正常软骨发育的负调控机制提供至关重要的信息，而且可能有助于揭示由于软骨细胞功能调节不当而导致的软骨疾病，如骨关节炎和出生缺陷。本研究结果也为组织工程和修复中控制软骨细胞的体外功能特性提供了有用的信息和潜在的工具。

DESCRIPTION (provided by applicant): Cartilage development plays a pivotal role in embryonic skeletal pattern formation, endochondral bone formation and skeletal joint development. Progressive, coordinated chondrogenesis during cartilage development is precisely controlled by gene expression, especially by gene transcription. A number of transcription factors such as Sox9, Sox5, Sox6 and Cbfa1 have been found to be critical for cartilage development by positively controlling chondrogenic gene expression. Increasing evidence from expression and function studies has shown that the transcription factor AP-2 may play a critical role in cartilage development through negative regulation of chondrogenic gene expression. Multiple AP- 2s are expressed in embryonic developing limb and in postnatal articular and growth plate cartilage. In vitro expression of AP-2alpha negatively regulates chondrogenic gene expression (type II collagen and CD-RAP). AP-2alpha knockout results in skeletal defects in craniofacial and limb bud development (shortened limb length and loss of radius) as well as other embryonic developmental defects (neural tube closure, body wall formation, and eye formation). To further elucidate specific roles of AP-2 in cartilage development, we would like 1) to determine the effects of targeted elimination of AP-2 expression/activities on cartilage development by generating cartilage-specific AP-2alpha knockout mice and generating transgenic mice with cartilage-specific over-expression of dominant negative AP-2alpha mutants; 2) to determine the effects of targeted over-expression of AP-2 on cartilage development by generating transgenic mice which over-express AP-2alpha in cartilage tissue; 3) to determine the effects of gain and loss of AP-2 function on proliferation, differentiation and survival of chondrogenic cells by over-expression of wild type and dominant negative mutant of AP-2alpha; and 4) to investigate molecular mechanisms underlying AP-2alpha action in chondrogenesis by determining the molecular mechanism of AP-2 regulation of Col2alpha1 gene expression using mutational analysis of Col2alpha1 promoter/enhancer region, and by identifying molecular targets that are controlled by AP-2alpha in early stages of chondrogenesis using genome-wide gene chip analysis. Examining the effects of these alterations in AP-2 gene expression/activities on cartilage development, chondrogenesis, transcriptional regulation of the Col2alpha1 gene and global gene expression pattern will provide concrete evidence to establish specific roles of AP-2 at the levels of tissue, cell and gene in cartilage development. This study will not only provide critically important information on a negative regulatory mechanism underlying normal cartilage development, but also may shed light on cartilage diseases due to improper regulation of chondrogenic cell function such as osteoarthritis and birth defects. The results of this study may also provide useful information and potential tools for controlling functional properties of chondrocytes in vitro for tissue engineering and repair.