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Altered Cbfal expression and biomineralization in an osteosarcoma cell line
Author(s) -
Perinpanayagam H.,
Schneider G.,
Holtman K.,
Zaharias R.,
Stanford C.
Publication year - 2004
Publication title -
journal of orthopaedic research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.041
H-Index - 155
eISSN - 1554-527X
pISSN - 0736-0266
DOI - 10.1016/s0736-0266(03)00196-7
Subject(s) - osteoblast , cell culture , microbiology and biotechnology , mineralization (soil science) , chemistry , gene isoform , runx2 , transfection , alkaline phosphatase , biology , biochemistry , in vitro , enzyme , gene , genetics , organic chemistry , nitrogen
Osteoblast differentiation and expression are regulated by Cbfal transcription factors. Recent evidence suggests that Cbfal may also regulate bone mineralization. The purpose of this study was to characterize Cbfal 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. Cbfal message was detected in the BSP and UI cultures, but the Cbfal N‐terminal isoform was deficient in UI and appeared to be up‐regulated in the phosphate‐treated BSP cultures. Cbfal 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 Cbfal mRNA levels relative to endogenous 18 s rRNA were lower in the slower mineralizing UI cultures. Furthermore, the Cbfal N‐terminal isoform mRNA levels were significantly ( P < 0.001) lower in the slower mineralizing cultures. Transfection with Cbfal or isoform antisense caused a significant ( P < 0.001) reduction in mineralization. Therefore, Cbfal expression may be associated with bone‐like mineral formation in rat UMR106‐01 osteoblastic cell cultures. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.