FUNCTION AND REGULATION OF AP-2 IN SKELETAL DEVELOPMENT

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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.