We investigated the regulation of Sox9, a transcription factor known to play a 
role in chondrogenesis, by bone morphogenetic protein-2 (BMP-2) and hedgehog 
proteins in order to better understand their signaling function in endochondral 
bone formation. The mesenchymal progenitor cell line C3H10T1/2 was stimulated 
with BMP-2. Sox9 expression levels were measured by quantitative reverse 
transcriptase-polymerase chain reaction and Northern analysis. We found that 
Sox9 was up-regulated by BMP-2 in a dose-dependent manner. The expression of 
Col2a1, a downstream response gene of Sox9, was also significantly increased 
upon BMP-2 addition. We also monitored Sox9 expression after the addition of 
BMP-2 to osteosarcoma cell lines; BMP-2 treatment increased Sox9 mRNA levels in 
MG63, considered to be early osteoblast-like, but not in human osteogenic 
sarcoma (HOS) cells, which are thought to be more advanced in the osteoblastic 
lineage. This response seems to be influenced by differences in BMP receptor 
expression; MG63 cells express BMP receptor IA (BMPR-IA), whereas HOS cells 
express BMPR-IA and BMPR-IB. We also saw an increase in Sox9 mRNA levels in 
BMP-2-treated primary human bone cells (HBCs) derived from femoral heads. We 
found that in addition to BMP-2, Sonic and Indian hedgehog can increase Sox9 
expression in C3H10T1/2 and primary HBCs. Time course studies with C3H10T1/2 
cells after BMP-2 stimulation showed increasing expression of cartilage markers, 
decrease of collagen I mRNA, and a late induction of osteocalcin expression. 
Moreover, the treatment of C3H10T1/2 cells with Sox9 antisense oligonucleotides 
revealed that Sox9 is a downstream mediator of BMP-2 affecting the expression of 
chondrocyte and osteoblast marker genes. Our data show that Sox9 is an important 
downstream mediator of the BMP-2 and hedgehog signaling pathways in osteogenic 
cells.