MODULATION OF EXPRESSION AND CELL-SURFACE BINDING OF MEMBERS OF THE TRANSFORMING GROWTH-FACTOR-BETA SUPERFAMILY DURING RETINOIC ACID-INDUCED OSTEOBLASTIC DIFFERENTIATION OF MULTIPOTENTIAL MESENCHYMAL CELLS

We have evaluated the effects of retinoic acid as a differentiating agent on two pluripotential mesenchymal stem cell lines, the mouse cell line C3H-10T1/2 (10T1/2), which has the capacity to differentiate in vitro into myoblasts, adipocytes, chondrocytes, and osteoblasts, and the rat cell line ROB-C26 (C26), which can, in culture, give rise to adipocytes, myoblasts, and osteoblasts. Retinoic acid (10(-6) m) reduces the incidence of myoblast and adipocyte formation and induces or increases alkaline phosphatase expression and responsiveness to PTH, two indicators of the osteoblastic phenotype. Because transforming growth factor-beta (TGFbeta) superfamily members, including the different TGFbeta isoforms and the bone morphogenetic proteins (BMPs), are thought to play a role in regulating bone and cartilage formation, and because exogenous TGFbeta and BMP-2 have already been found to modulate osteoblastic differentiation of C26 and 10T1/2 cells, we evaluated the endogenous expression of these factors in both cell lines cultured in the presence or absence of retinoic acid. Our data show that C26 and 10T1/2 cells constitutively express a broad spectrum of TGFbeta superfamily members. However, this pattern of expression is dramatically altered in response to retinoic acid. Specifically, expression of TGFbeta1 and especially TGFbeta2 is strongly increased, whereas TGFbeta3 expression is down-regulated. These changes are accompanied by a striking decline in TGFbeta receptor expression levels at the cell surface. Furthermore, BMP-2 and -4 expression are decreased after treatment with retinoic acid, whereas vgr-1/BMP-6 expression is induced in C26 cells, but decreased in 10T1/2 cells. These results clearly show a dynamic changing pattern of TGFbeta superfamily expression consequent to the induction of osteogenic differentiation and provide the first indication that TGFbeta receptor down-regulation may be an essential part of this differentiation process. These data also establish the C26 and 10T1/2 cell lines as convenient in vitro model systems for exploring the autoregulation of osteogenic differentiation by members of the TGFbeta superfamily.