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FUNCTION AND REGULATION OF AP-2 IN SKELETAL DEVELOPMENT

AP-2 在骨骼发育中的功能和调节

基本信息

批准号：
7471897
负责人：
ZHENGMIN HUANG
金额：
$ 24.53万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-08-03 至 2008-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7471897
关键词：
Age Alkaline Phosphatase Apoptosis Apoptotic Biological Assay Biological Markers Bone Marrow Bone and Cartilage Funding Cartilage Cartilage Diseases Cell Count Cell Death Cell Lineage Cell Proliferation Cell physiology Cells Cessation of life Chondrocytes Chondrogenesis Classification Collaborations Collagen Type II Complex Condition Congenital Abnormality DNA biosynthesis DNA chemical synthesis Data Data Analyses Defect Degenerative polyarthritis Development Digit structure Dominant-Negative Mutation Ear Electron Microscope Embryo Enhancers Epiphysial cartilage Exencephalies Exons Extracellular Matrix Face Forelimb Fracture Gene Chips Gene Expression Gene Expression Profiling Genes Genetic Recombination Genetic Transcription Genome Human Immunohistochemistry In Situ Hybridization In Vitro Indium Infection Investigation Joints Knock-out Knockout Mice Label Length Light Limb Bud Limb structure Measures Mediating Mesenchymal Messenger RNA Molecular Molecular Target Mus Mutant Strains Mice Neural Tube Closure Neural Tube Defects Nodule Nose Numbers Osteogenesis Partner in relationship Pathogenesis Pattern Pattern Formation Perinatal Play Production Property Protein Overexpression Proteoglycan Regulation Regulator Genes Reverse Transcriptase Polymerase Chain Reaction Role Site-Directed Mutagenesis Skeletal Development Skeletal system Skeleton Staging TFAP2A gene TdT-Mediated dUTP Nick End Labeling Assay Testing Tissue Engineering Tissues Transcript Transcriptional Regulation Transfection Transgenes Transgenic Mice Undifferentiated Wild Type Mouse annexin A5 base cartilage development craniofacial enhancer-binding protein AP-2 eye formation in vivo mineralization mutant postnatal promoter radius bone structure recombinase repaired tool transcription factor transgene expression

项目摘要

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（申请人提供）：软骨发育在胚胎骨骼模式形成、软骨内骨形成和骨关节发育中起着关键作用。在软骨发育过程中，渐进的、协调的软骨形成是由基因表达，尤其是基因转录精确控制的。许多转录因子如Sox9、Sox5、Sox6和Cbfa1已被发现通过正向控制软骨基因表达对软骨发育至关重要。越来越多来自表达和功能研究的证据表明，转录因子AP-2可能通过负调控软骨基因表达在软骨发育中发挥关键作用。多个AP- 2s在胚胎发育肢体和出生后的关节和生长板软骨中表达。体外表达AP-2alpha负调控软骨基因（II型胶原和CD-RAP）的表达。AP-2alpha敲除导致颅面和肢体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（神经管关闭、体壁形成和眼睛形成）。为了进一步阐明AP-2在软骨发育中的具体作用，我们希望：1)通过培养软骨特异性AP-2alpha敲除小鼠和培养具有软骨特异性过表达显性阴性AP-2alpha突变体的转基因小鼠，确定靶向消除AP-2表达/活性对软骨发育的影响；2)通过培养软骨组织中过表达AP-2 α的转基因小鼠，确定靶向过表达AP-2对软骨发育的影响；3)通过AP-2 α野生型和显性负突变体的过表达，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；4)通过Col2alpha1启动子/增强子区域的突变分析确定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在软骨形成早期阶段由AP-2alpha控制的分子靶点，研究AP-2alpha在软骨形成中作用的分子机制。研究这些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，将为确定AP-2在组织、细胞和基因水平上在软骨发育中的具体作用提供具体证据。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，而且还可能揭示由于软骨细胞功能调节不当导致的软骨疾病，如骨关节炎和出生缺陷。本研究结果也可能为体外组织工程和修复软骨细胞的功能特性控制提供有用的信息和潜在的工具。