Immortalized murine osteoblasts derived from BMP 2-T-antigen expressing transgenic mice.

Abstract

Osteoblast cell lines capable of undergoing bone formation in vitro would provide useful models for understanding gene expression during bone cell differentiation. To that end, transgenic mice were produced using a 2.9-kilobase bone morphogenetic protein 2 (BMP-2) promoter fragment, driving simian virus 40 T antigen as the transgene. The expression of simian virus 40 T antigen driven by the BMP-2 promoter immortalizes the cells. From the calvaria of the transgenic mouse, several osteoblastic cell lines were isolated and cloned. One clonal osteoblast cell line, called 2T3, has been characterized and shown to produce mineralized bone nodules. Recombinant human BMP-2 (rhBMP-2) accelerates the formation of these mineralized bone nodules. 2T3 cells express alkaline phosphatase, collagen type I, osteocalcin, and endogenous BMP-2 messenger RNA (mRNA) in a similar chronological order as normal freshly isolated fetal rat calvarial cells during early nodule formation and subsequent mineralization. The 2T3 cells also exhibit extensive growth and multilayering during differentiation, as demonstrated by growth curves and transmission electron microscopy. As with freshly isolated fetal rat calvarial cells, 1,25-dihydroxyvitamin D3 inhibited alkaline phosphatase activity and alkaline phosphatase mRNA expression, but stimulated osteocalcin mRNA expression, but stimulated osteocalcin mRNA expression. rhBMP-2 also accelerated the expression of alkaline phosphatase activity and mRNA, osteocalcin mRNA, and BMP-2 mRNA in 2T3 cells along with the formation of larger and more mineralized bone nodules. The 2T3 cell exhibits autoregulation at the mRNA and transcriptional levels. The 2T3 osteoblast cell line offers a system for examining autoregulation of the BMP-2 gene and downstream gene expression during osteoblast differentiation. 2T3 cells are reclonable and maintain their differentiation capabilities.

Phosphatidylinositol 3-Kinase Regulates Bone Morphogenetic Protein-2 (BMP-2)-induced Myocyte Enhancer Factor 2A-dependent Transcription of BMP-2 Gene in Cardiomyocyte Precursor Cells

“…We have shown previously that BMP-2 autoregulates its expression during osteogenesis (29). This is one of the mechanisms by which BMP-2 maintains its sustained effect during osteoblast differentiation.…”

“…Transfection and Luciferase Assay-The BMP-2-LUC reporter plasmid, in which the firefly luciferase gene is driven by a 2.7-kb 5Ј-flanking sequence of BMP-2 gene, has been described before (17,28,29). BMP-2-LUC reporter plasmid was cotransfected with different expression plasmids using LipofectAMINE Plus reagent as described (20,21,26,30).…”

“…These data indicate that BMP-2-induced PI 3-kinase activity is necessary for transcriptional activation of MEF-2. (29,30). To investigate the involvement of PI 3-kinase in BMP-2 transcription, we used a reporter construct in which the firefly luciferase gene is driven by the promoter of the BMP-2 gene (BMP-2-LUC) (29).…”

“…(29,30). To investigate the involvement of PI 3-kinase in BMP-2 transcription, we used a reporter construct in which the firefly luciferase gene is driven by the promoter of the BMP-2 gene (BMP-2-LUC) (29). This plasmid was transfected either with vector alone or dominant negative p85 subunit of PI 3-kinase into CL6 cells.…”

Human Serine Protease HTRA1 Positively Regulates Osteogenesis of Human Bone Marrow-derived Mesenchymal Stem Cells and Mineralization of Differentiating Bone-forming Cells Through the Modulation of Extracellular Matrix Protein

“…These results are in stark contrast to the findings presented in a previous report by Hadfield et al [20], where HTRA1 was shown to impart a negative influence over mineralized matrix formation by mouse 2T3 osteoblasts. One possible explanation may lie within the fact that 2T3 cells are derived from transgenic mice overexpressing the simian virus 40 (SV40) T antigen and as such are immortalized [34]. The fact that HTRA1 gene expression is known to be significantly affected in SV40 transformed cells [35] would imply that modifications of the osteogenic status of these cells brought about by changes in HTRA1 expression and activity may not be truly representative of nonimmortalized, primary cells.…”