Differentiation of normal human bone cells by transforming growth factor-β and 1,25(OH) 2 vitamin D 3

Abstract

To investigate the role of transforming growth factor-β 1 (TGFβ) in bone metabolism, the effects of this agent on the differentiation characteristics of human bone cells were studied in vitro. Human bone cells were isolated from femoral head samples by collagenase digestion. Differentiation characteristics included alkaline phosphatase activity, osteocalcin production, and mRNA levels for alkaline phosphatase, type I α 2-procollagen, and osteocalcin. The effect of TGFβ on alkaline phosphatase was not constant, but varied with the incubation conditions. At high cell density and in the presence of serum, TGFβ decreased alkaline phosphatase activity. However, at low cell density and under serum-free conditions, TGFβ stimulated alkaline phosphatase activity. The addition of 1,25(OH) 2 vitamin D 3 also stimulated alkaline phosphatase. The combination of the two agents gave a greater increase in activity than the sum of the activities when the two agents were given alone. The percentage of cells that stain positively for alkaline phosphatase changed in parallel with the change in specific activity. The percentage of positive cells increased from 17% to 64%, while the specific activity increased from 22 to 169 mU/mg protein. To investigate the mechanism of this stimulation, mRNA levels were measured at 24 hours. Individually, TGFβ and 1,25(OH) 2D 3 increased message levels for alkaline phosphatase and type I procollagen, but the greatest effect was produced by the combination of the two factors. 1,25(OH) 2D 3 increased osteocalcin mRNA levels, but TGFβ markedly inhibited this stimulation. TGFβ also inhibited production of osteocalcin by the human bone cells. TGFβ appears to modulate differentiation of human bone cells in combination with 1,25(OH) 2D 3 and other factors.