Effects of bone morphogenetic protein-2 and transforming growth factor beta1 on gene expression of transcription factors, AJ18 and Runx2 in cultured osteoblastic cells.

Osteoblast differentiation is controlled by multiple transcription factors, Runx2, AJ18, Osterix, Dlx5 and Msx2. The mechanisms of regulation of AJ18 mRNA expression by the transforming growth factor beta (TGF-beta) superfamily remain poorly understood. However, it is known that BMP-2 induces differentiation of C26 cells into more mature osteoblastic cells. The present study, using Northern blot and real-time reverse transcription polymerase chain reaction analyses, investigated the effects of bone morphogenetic protein-2 (BMP-2) and TGF-beta1 on mRNA expression of AJ18 and Runx2 in a clonal osteoblast precursor cell line ROB-C26 (C26) cultured for 3, 6 or 9 days in the presence or absence of BMP-2. Although mRNA expression of Osterix and bone sialoprotein (BSP) was undetectable in the C26 culture, BMP-2 induced Osterix expression on days 3-9, but not BSP expression. BMP-2 also stimulated significantly Dlx5 expression on days 3-9, Msx2 and matrix Gla protein expressions on days 3 and 6, Runx2, alkaline phosphatase and osteocalcin expressions on days 6 and 9 in the culture. Furthermore, BMP-2 increased significantly Smad5 mRNA in the culture on day 3, indicating BMP-2 involvement in the regulation of Smad5 mRNA expression. In contrast, the inhibitory effects of BMP-2 on AJ18 mRNA expression were significant on days 3-9, indicating that a decrease in AJ18 mRNA expression is essential for the increased osteoblastic differentiation. Furthermore, TGF-beta1 (0, 0.1, 1.0 and 5.0 ng/ml) treatment of C26 cells cultured for 6 days in the presence or absence of BMP-2 for 24h stimulated mRNA levels of AJ18 and Runx2, maximal stimulation occurring principally at 1.0 ng/ml. These observations indicate that the expression of AJ18 and Runx2 mRNAs in C26 cells is under the control of BMP-2 and TGF-beta1, which exert different effects on AJ18 mRNA expression, but are potent stimulators of Runx2 mRNA expression during osteoblast differentiation.