Altered Cbfa1 expression and biomineralization in an osteosarcoma cell line

Abstract

Osteoblast differentiation and expression are regulated by Cbfa1 transcription factors. Recent evidence suggests that Cbfa1 may also regulate bone mineralization. The purpose of this study was to characterize Cbfa1 expression in relation to mineralization in rat UMR106-01 osteoblastic cell cultures. UMR106-01 BSP cultures consistently form bone-like mineral, whereas the UI subclone mineralize gradually. BSP and UI cultures were grown for 48 h and then treated with β-glycerophosphate. BSP cultures had alizarin red stained calcifications and mineral-like deposits within 24 h of phosphate. Atomic absorption spectroscopy measured significantly ( P<0.0001) more calcium in the phosphate-treated BSP cultures than in the UI. Cbfa1 message was detected in the BSP and UI cultures, but the Cbfa1 N-terminal isoform was deficient in UI and appeared to be up-regulated in the phosphate-treated BSP cultures. Cbfa1 protein levels were also reduced in the UI. DNA sequence from the RT-PCR products was utilized to design Taqman Real-time RT-PCR reagents. Quantitative Real-time RT-PCR analysis showed that Cbfa1 mRNA levels relative to endogenous 18 s rRNA were lower in the slower mineralizing UI cultures. Furthermore, the Cbfa1 N-terminal isoform mRNA levels were significantly ( P<0.001) lower in the slower mineralizing cultures. Transfection with Cbfa1 or isoform antisense caused a significant ( P<0.001) reduction in mineralization. Therefore, Cbfa1 expression may be associated with bone-like mineral formation in rat UMR106-01 osteoblastic cell cultures.