Studies On The Transcriptional Regulation Mechanisms Of Transcription Factors Required For Skeletal Development

The skeleton is a very complicated and continuously changing tissue. Multiple factors influence skeletal development. Among these factors, transcription factors should play a central regulatory role. Runx2 and Osterix are both key transcription factors involved in osteoblast differentiation and bone formation. However, whether Runx2 positively regulats Osterix is not known.By using RT-PCR, we found that transient expression of Runx2 in a number of non-osteoblastic cell lines, either pluripotent or differentiated, and in the osteoblastic cell lines was able to induce the expression of Osterix. We cloned the 3.2 kb 5'-flanking region of human Osterix gene and analyzed the promoter linked to a firefly luciferase reporter gene. We found that Runx2 could upregulate the promoter activity. The fragment between 1.1 kb and 0.5 kb upstream of the first translation start codon was needed for basal promoter activity. A main functional Runx2 binding site"AGTGGTT"was then identified within the promoter, whereas, another Runx2 binding site"TGTGGT"was canonical, but not conserved. Moreover, the transient transfection and dual-luciferase assay showed Osterix up-regulated the activity of the 2.3kb type I collagen promoter in the non-osteoblastic cells, but Runx2 did not. This defference implies that Osterix, the down stream transcription factor of Runx2 during osteoblast differentiation, is needed to stimulate the osteoblast-specific gene expression of type I collagen.The three Sox transcription factors, L-Sox5, Sox6, and Sox9, are essentially required for chondrocyte differentiation and cartilage development. They are sufficient to induce permanent cartilage. However, their precise mode of action is still poorly understood when they function together in chondrocytes. By using a retroviral system, we generate several stable cell lines overexpressing L-Sox5, Sox6, Sox9, and L-Sox5 and Sox9, respectively. Chondrogenic differentiation was examined by Alcian Blue staining, RT-PCR etc. Specific inhibitors were used to characterize the Stat1-related proliferation or the MAPK-mediated differentiation of the cell lines. The aberrant overexpression of L-Sox5 or Sox6 in ATDC5 dramatically accelerated proliferation and inhibited differentiation during chondrogenesis. In contrast, the aberrant overexpression of Sox9 markedly inhibited proliferation but enhanced the expression of cartilage-specific genes. Exogenous L-Sox5 and Sox9 in the ratio near 1:1 largely secured normal proliferation and differentiation. In addition, the altered expression statuses of the Sox transcription factors differently regulated Stat1 expression. And these alterations also resulted in different responses to the MAPK pathway. The alterations in the starting ratio of the Sox transcription factors modulate proliferation and differentiation during chondrogenesis. Extracellular signals generate different chondrocytes at least in part through establishing varied ratios of the Sox transcription factors in the cartilage.