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

OBJECTIVE: To clarify the mechanism of impaired bone formation during low dose 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 line (MC3T3-E1) with the capacity to differentiate into osteoblastic or osteocytes, (2) a human osteoblastic osteosarcoma cell line (SaOS-2) with a mature osteoblastic phenotype, and (3) mouse bone marrow stromal cells containing osteoblast precursors. Osteoblast function was assessed by measuring the cellular activity of alkaline phosphatase (ALP) and the mineralization capacity of cultures. RESULTS: MTX suppressed ALP activity dose-dependently in growing MC3T3-E1 cells, but proliferation of these cells was only inhibited by a high concentration 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 proliferation 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 concentrations between l0(-10) and 10(-7) M. CONCLUSION: These results suggest that MTX suppresses bone formation by inhibiting the differentiation of early osteoblastic cells.