Effects of combining transforming growth factor β and 1,25- dihydroxyvitamin D₃ on differentiation of a human osteosarcoma (MG-63)

Transforming growth factor β (TGFβ) and 1,25-dihydroxyvitamin D₃ (1,25D₃), when added simultaneously to a human osteosarcoma cell line, MG- 63, induce alkaline phosphatase activity 40-70-fold over basal levels, 6-7- fold over 1,25D₃ treatment alone, and 15-20-fold over TGFβ treatment alone. TGFβ and 1,25D₃ synergistically increased alkaline phosphatase specific activity in both matrix vesicles and plasma membrane isolated from the cultures, but the specific activity was greater in and targeted to the matrix vesicle fraction. Inhibitor and cleavage studies proved that the enzymatic activity was liver/bone/kidney alkaline phosphatase. Preincubation of MG-63 cells with TGFβ for 30 min before addition of 1,25D₃ was sufficient for maximal induction of enzyme activity. Messenger RNA for liver/bone/kidney alkaline phosphatase was increased 2.1-fold with TGFβ, 1.7-fold with 1,25D₃, and 4.8-fold with the combination at 72 h. Human alkaline phosphatase protein as detected by radioimmunoassay was stimulated only 6.3- fold over control levels with the combination. This combination of factors was tested for their effect on production of three other osteoblast cell proteins: collagen type I, osteocalcin, and fibronectin. TGFβ inhibited 1,25D₃-induced osteocalcin production, whereas both factors were additive for fibronectin and collagen type I production. TGFβ appears to modulate the differentiation effects of 1,25D₃ on this human osteoblast-like cell and thereby retain the cell in a non-fully differentiated state.