The transcription factor Sox9 is involved in BMP-2 signaling

We investigated the regulation of Sox9, a transcription factor known to pla y a role in chondrogenesis, by bone morphogenetic protein-2 (BMP-2) and hed gehog proteins in order to better understand their signaling function in en dochondral bone formation. The mesenchymal progenitor cell line C3H10T1/2 w as stimulated with BMP-2. Sox9 expression levels were measured by quantitat ive reverse transcriptase-polymerase chain reaction and Northern analysis. We found that Sox9 was up-regulated by BMP-2 in a dose-dependent manner. Th e expression of Col2a1, a downstream response gene of Sox9, was also signif icantly increased upon BMP-2 addition, We also monitored Sox9 expression af ter the addition of BMP-2 to osteosarcoma cell lines; BMP-2 treatment incre ased 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 a dvanced in the osteoblastic lineage. This response seems to be influenced b y differences in BMP receptor expression; MG63 cells express BMP receptor I A (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 (HBC s) derived from femoral heads. We found that in addition to BMP-2, Sonic an d Indian hedgehog can increase Sox9 expression in C3H10T1/2 and primary HBC s. Time course studies with C3H10T1/2 cells after BMP-2 stimulation showed increasing expression of cartilage markers, decrease of collagen I mRNA, an d a late induction of osteocalcin expression. Moreover, the treatment of C3 H10T1/2 cells with Sox9 antisense oligonucleotides revealed that Sox9 is a downstream mediator of BMP-2 affecting the expression of chondrocyte and os teoblast marker genes. Our data show that Sox9 is an important downstream m ediator of the BMP-2 and hedgehog signaling pathways in osteogenic cells.