Effects of methotrexate on human osteoblasts in vitro: Modulation by 1,25‐dihydroxyvitamin D₃

This study was designed to investigate whether methotrexate (MTX), used in the treatment of rheumatoid arthritis (RA), affects proliferation and differentiation of human osteoblasts in culture. The effects of MTX were assessed by analyzing markers of proliferation and differentiation of human trabecular bone–derived osteoblast‐like cells cultured in the presence or absence of 1,25‐dihydroxyvitamin D₃ (1,25[OH]₂D₃). Treatment of the osteoblastic cells with MTX resulted in a strong dose‐dependent inhibition of cell proliferation with half maximal response at a dose of 30 nM. MTX did not interfere with cellular alkaline phosphatase (AP) activity, the number of cells expressing cytochemical AP, or basal osteocalcin production. Addition of 1,25(OH)₂D₃ to the cultures caused an enhanced AP expression and osteocalcin production coinciding with a decreased osteoblast proliferation. Coincubation of 1,25(OH)₂D₃ with MTX in doses ≥100 nM further inhibited osteoblast growth and induced a significant stimulation of AP expression and activity, and production of osteocalcin above the values reached in the 1,25(OH)₂D₃ cultures. In conclusion, MTX proved to be a potent inhibitor of osteoblast proliferation but did not affect basal osteoblastic phenotypic expression. In the presence of the osteoblast differentiation‐promotor, 1,25(OH)₂D₃, MTX further inhibited cell growth which was associated with enhanced AP activity and osteocalcin production. Thus, MTX may have profound effects on bone metabolism and remodeling by interfering with bone cell turnover.