Abstract. Analysis of MM14 mouse myoblasts demonstrates that terminal differentiation is repressed by pure preparations of both acidic and basic fibroblast growth factor (FGF). Basic FGF is ~30-fold more potent than acidic FGF and it exhibits half maximal activity in clonal assays at 0.03 ng/ml (2 pM). FGF repression occurs only during the G~ phase of the cell cycle by a mechanism that appears to be independent of ongoing cell proliferation. When exponentially growing myoblasts are deprived of FGF, cells become postmitotic within 2-3 h, express muscle-specific proteins within 6-7 h, and commence fusion within 12-14 h. Although expression of these three terminal differentiation phenotypes occurs at different times, all are initiated by a single regulatory "commitment" event in G~. The entire population commits to terminal differentiation within 12.5 h of FGF removal as all cells complete the cell cycle and move into Gt. Differentiation does not require a new round of DNA synthesis. Comparison of MM14 behavior with other myoblast types suggests a general model for skeletal muscle development in which specific growth factors serve the dual role of stimulating myoblast proliferation and directly repressing terminal differentiation. SKE L Ea"A L muscle development involves an initial period of myoblast replication, followed by a phase in which some myoblasts continue to proliferate while others undergo terminal differentiation. The latter process involves the permanent cessation of DNA synthesis, activation of muscle-specific gene expression, and the fusion of single cells into multinucleated muscle fibers. In vitro studies indicate that the onset of terminal differentiation is delayed by media that are rich in serum and/or embryo extract components (3,15,21,22,27,28,33,37,40,42); and there is a general consensus that this is due to the influence of growth factors. Studies from our laboratory with a permanent skeletal muscle line derived from a mouse muscle satellite cell (MM14), indicated that the critical component in embryo extract was probably fibroblast growth factor (FGF) I (27, 28). However, since the FGF preparations available at that time were <1% pure, and since several forms of FGF were thought to exist, the role of FGF remained ambiguous. Subsequent studies of BC3H1 muscle cells have corroborated the role of FGF in repressing muscle differentiation (23,24). However,

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This report describes the first observation of a direct mitogenic effect of androgens on isolated osteoblastic cells in serum-free culture. [3H]thymidine incorporation into DNA and cell counts were used as measures of cell proliferation. The percentage of cells that stained for alkaline phosphatase was used as a measure of differentiation. Dihydrotestosterone (DHT) enhanced mouse osteoblastic cell proliferation in a dose dependent manner over a wide range of doses (10(-8) to 10(-11) molar), and was maximally active at 10(-9) M. DHT also stimulated proliferation in human osteoblast cell cultures and in cultures of the human osteosarcoma cell line, TE89. Testosterone, fluoxymesterone (a synthetic androgenic steroid) and methenolone (an anabolic steroid) were also mitogenic in the mouse bone cell system. The mitogenic effect of DHT on bone cells was inhibited by antiandrogens (hydroxyflutamide and cyproterone acetate) which compete for binding to the androgen receptor. In addition to effects on cell proliferation, DHT increased the percentage of alkaline phosphatase (ALP) positive cells in all three bone cell systems tested, and this effect was inhibited by antiandrogens. We conclude that androgens can stimulate human and murine osteoblastic cell proliferation in vitro, and induce expression of the osteoblast-line differentiation marker ALP, presumably by an androgen receptor mediated mechanism.