Methotrexate (MTX) inhibits osteoblastic differentiation in vitro: Possible mechanism of MTX osteopathy

Objective. To clarify the mechanism of impaired bone formation during low d ose methotrexate (MTX) therapy. Methods. The in vitro effects of MTX on the function and differentiation of osteoblastic cells were investigated using (1) a mouse osteogenic cell lin e (MC3T3-E1) with the capacity to differentiate into osteoblastic or osteoc ytes, (2) a human osteoblastic osteosarcoma cell line (SaOS-2) with a matur e osteoblastic phenotype, and (3) mouse bone marrow stromal cells containin g osteoblast precursors. Osteoblast function was assessed by measuring the cellular activity of alkaline phosphatase (ALP) and the mineralization capa city of cultures. Results. MTX suppressed ALP activity dose-dependently in growing MC3T3-E1 c ells, but proliferation of these cells was only inhibited by a high concent ration of MTX. In contrast, inhibition of ALP activity in MC3T3-E1 cells of mature osteoblastic phenotype was only observed with 10(-8) M and 10(-7) M MTX, and proliferation was not influenced. ALP activity and the proliferat ion of SaOS-2 cells were not inhibited by MTX, even when growing cells were treated. However, both ALP activity and formation of calcified nodules in bone marrow stromal cell cultures were significantly suppressed by MTX at c oncentrations between 10(-10) and 10(-7) M. Conclusion. These results suggest that MTX suppresses bone formation by inh ibiting the differentiation of early osteoblastic cells.