Bone morphogenetic protein-2 induces the differentiation of a mesenchymal progenitor cell line, ROB-C26, into mature osteoblasts and adipocytes.

AIMS: Osteoblasts and adipocytes originate from common precursor cells. We examined the effects of bone morphogenetic protein-2 (BMP-2) on the molecular mechanisms governing the diametric actions of BMP-2 on simultaneous mature osteoblast and adipocyte differentiation in a clonal mesenchymal progenitor cell line, ROB-C26 (C26). MAIN METHODS: The present study using RT-PCR, Western blotting and ELISA investigated the effects of BMP-2 on transcription factors for osteoblasts (Runx2, Dlx5, Osterix, Msx2 and AJ18) and adipocytes (PPARgamma2), osteoblastic markers, alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC), and adipocyte differentiation-dependent protein, aP2 in C26 cells. KEY FINDINGS: BMP-2 increased not only mRNA and protein expressions of Dlx5 followed by inducing Osterix and BSP levels in a dose- and time-dependent manner with an increase in Runx2, Msx2, AJ18, ALP and OC levels, but also induced PPARgamma2 and aP2 levels. At the light microscopic level, BMP-2 induced mature osteoblastic and adipogenic differentiation in the C26 cultures not only by an increase in the number of ALP-positive osteoblasts, in their staining intensity and in the number of mature adipocytes with Oil Red O-positive lipid droplets, but also mineralized matrix formation of the cultures assessed by detecting an increase in calcium concentration and the formation of Alizarin Red S-positive mineralization nodules. SIGNIFICANCE: These results indicate that BMP-2 induces the differentiation of C26 mesenchymal progenitors into mature osteoblasts and adipocytes and the usefulness of this cell line for studying the regulatory mechanism of osteoblast and adipocyte differentiation from common mesenchymal progenitors.

The effect of retinoic acid on a zinc finger transcription factor, AJ18, during differentiation of a rat clonal preosteoblastic cell line, ROB-C20, into osteoblasts.

OBJECTIVE: A zinc finger type transcription factor, AJ18, is thought to be a negative regulator of osteoblast differentiation, but its expression mechanism is not fully understood. Retinoic acid (RA) is a metabolite of vitamin A and involves the proliferation and differentiation in a variety of cells. To verify the effect of RA on osteoblast differentiation, AJ18 expression level was examined using a rat clonal preosteoblastic cell line, ROB-C20 (C20). DESIGN: Confluent C20 cells were treated with or without RA (10(-6)M) for several days. Northern, real time RT-PCR and Western blotting analyses were performed to examine AJ18 expression pattern in gene and protein levels. To identify the active promoter sequence of AJ18 gene, luciferase assay was designed. Furthermore, the effect of overexpressed AJ18 in C20 cells on alkaline phosphatase (ALP) mRNA expression and its activity was compared with that of RA-treated cells. RESULTS: RA increased the expression of AJ18 mRNA from 2 to 13 days as well as its protein production. However, no significant changes of Runx2 mRNA expression and undetectable osterix mRNA expression were observed in C20 cells treated with or without RA. Luciferase assay showed increases in promoter activities in some constructs of 5'-flanking region of AJ18 gene in RA-treated C20 cells. On the other hand, RA decreases enzymatic activity and mRNA expression level of ALP, but mRNA expression levels of bone sialoprotein and osteocalcin were not altered. Interestingly, reduced ALP activity and its mRNA expression level were detected in exogenous AJ18-overexpressing C20 cells. CONCLUSIONS: This study supports the hypothesis that RA may restrict to the differentiation of C20 cells into mature osteoblasts via inductive AJ18 expression with activation of multiple signal pathways.

Changes in extracellular activin A:follistatin ratio during differentiation of a mesenchymal progenitor cell line, ROB-C26 into osteoblasts and adipocytes.

We investigated the effects of BMP-2 and dexamethasone (Dex) on follistatin (FS) and activin A expressions in a mesenchymal progenitor cell line, ROB-C26 (C26). C26 cells stimulated to differentiate into osteoblastic cells by blocking myogenic differentiation after BMP-2 treatment and into adipocytes with Dex treatment. Alkaline phosphatase (ALP) mRNA expression and its activity in the confluent C26 cells were dose- and time-dependently stimulated by BMP-2, but inhibited by Dex. The stimulatory effect on FS and activin A mRNA expressions by BMP-2 and Dex were dose-dependent. Cycloheximide pre-treatment indicated that FS and activin A expressions appear to be the direct target of BMP-2 and Dex signaling. BMP-2 time-dependently increased FS and activin A levels. Dex also increased FS level, but induced a time-dependent biphasic effect on activin A level, a decrease (2-6 h) followed by an increase (12-72 h). The data of the ratio of activin A concentration in the culture media to that of FS (activin A:FS ratio) measured by ELISA showed that BMP-2-induced osteoblastic differentiation involved an activin-dominant microenvironment, whereas Dex-induced adipocyte differentiation involved a FS-dominant microenvironment. Excess FS suppressed the stimulatory ALP activity of BMP-2, whereas activin A prevented not only Dex-induced inhibitory ALP activity, but also adipogenesis via suppression of the adipocyte transcriptional factor cascade. These results indicate that BMP-2-induced activin-dominant microenvironment may be critical for osteoblastic differentiation by restricting the antagonistic effects of FS on BMP activity, while Dex-induced FS-dominant microenvironment may be critical for adipocyte differentiation by restricting the inhibitory action of activin A on adipocyte differentiation.

Autoregulatory mechanism of Runx2 through the expression of transcription factors and bone matrix proteins in multipotential mesenchymal cell line, ROB-C26.

Runx2 is essential for osteoblast differentiation and gene expression of bone matrix proteins, however, little is known about the mechanism regulating its activity. In this study, the role of Runx2 on gene expression of transcription factors, AJ18, Msx2, and Dlx5, was examined in vitro. It is known that AJ18 and Msx2 act as repressors to inhibit activity of Runx2, whereas Dlx5 promotes its activity. An expression vector inserted Runx2 cDNA was transiently overexpressed in a rat multipotential mesenchymal cell line, ROB-C26 (C26). Real time reverse transcription-PCR analysis showed that, in exogenous Runx2-overexpressing C26 cells (C26-Rx), AJ18 expression increased 1.8-fold, Msx2 expression increased 3.0-fold, and Dlx5 expression increased 2.7-fold compared to the cells transfected with vector alone (C26-Co). Luciferase assay also showed that, in C26-Rx, AJ18 promoter activity increased 2.1-fold compared to C26-Co. Furthermore, gene expression of alkaline phosphatase (ALP) and bone matrix proteins including type I collagen (Col1), osteocalcin (OC), osteopontin (OPN), and matrix Gla protein (MGP) was examined. In C26-Rx, MGP expression increased 1.8-fold, and OPN expression increased 1.4-fold compared to C26-Co. However, no significant difference in Col1, ALP, and OC expressions was detected between C26-Rx and C26-Co. These results suggest that the existence of autoregulatory feed back loops, which inhibit Runx2 activity through the interaction of AJ18, Dlx5, and Msx2 cooperating with that of MGP and OPN, interferes with the differentiation of C26 cells toward mature osteoblasts.