FUNCTION AND REGULATION OF AP-2 IN SKELETAL DEVELOPMENT

AP-2 在骨骼发育中的功能和调节

• 批准号: 7117640
• 负责人: ZHENGMIN HUANG
• 金额: $ 26.3万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7117640
• 关键词: cartilage development; cell differentiation; cell growth regulation; cell proliferation; electron microscopy; functional /structural genomics; gene expression; genetically modified animals; immunocytochemistry; in situ hybridization; laboratory mouse; microarray technology; polymerase chain reaction; protein structure function; terminal nick end labeling; tissue /cell culture; transcription factor; vertebrate embryology

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.

描述（由申请人提供）：软骨发育在胚胎骨骼模式形成、软骨内骨形成和骨骼关节发育中起关键作用。软骨发育过程中渐进、协调的软骨形成受基因表达，特别是基因转录的精确控制。已经发现许多转录因子如Sox 9、Sox 5、Sox 6和Cbfa 1通过正向控制软骨形成基因的表达对软骨发育至关重要。越来越多的表达和功能研究表明，转录因子AP-2可能通过负调控软骨形成基因的表达在软骨发育中发挥关键作用。多个AP-2在胚胎发育的肢体和出生后的关节和生长板软骨中表达。AP-2 α的体外表达负调节软骨形成基因表达（II型胶原和CD-RAP）。AP-2 α基因敲除会导致颅面和肢芽发育中的骨骼缺陷（肢长缩短和桡骨缺失）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，我们希望1）通过产生软骨特异性AP-2 α敲除小鼠和产生具有软骨特异性过表达显性失活AP-2 α突变体的转基因小鼠来确定AP-2表达/活性的靶向消除对软骨发育的影响; 2）通过产生在软骨组织中过表达AP-2 α的转基因小鼠来确定AP-2的靶向过表达对软骨发育的影响; 3）通过过量表达AP-2 α的野生型和显性失活突变体，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;和4）通过使用Col 2 α 1启动子/增强子区域的突变分析确定AP-2调节Col 2 α 1基因表达的分子机制，并通过使用全基因组基因芯片分析鉴定在软骨形成的早期阶段由AP-2 α控制的分子靶标，来研究AP-2 α在软骨形成中作用的分子机制。研究AP-2基因表达/活性的这些改变对软骨发育、软骨形成、Col 2alpha 1基因的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。这项研究不仅将提供关于正常软骨发育的负调控机制的重要信息，而且还可能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。本研究的结果也可能提供有用的信息和潜在的工具，在体外控制软骨细胞的功能特性的组织工程和修复。