Evidence for the involvement of bone morphogenetic protein-2 in phenytoin-stimulated osteocalcin secretion in human bone cells.

Recent work has shown that the actions of phenytoin on bone cell proliferation and differentiation are, in part, mediated through the upregulation of transforming growth factor-beta1 (TGF-beta(1)). The present study was undertaken to examine the effect of phenytoin on bone morphogenetic proteins (BMP)-2 and -4, which are well-recognized osteoinductive proteins of the TGF-beta superfamily, in osteoblastic cells. Treatment with 5-50 microM of phenytoin increased the amount of mRNA for BMP-2 after a 0.5-24 h incubation in normal human mandible-derived bone cells (HOB-M cells), but failed to affect the mRNA for BMP-4. Phenytoin treatment for 48 h significantly increased the secretion of BMP-2 by approx. four-fold, at an optimal concentration of 10 microM. While TGF-beta(1) inhibited osteocalcin secretion from HOB-M cells, both phenytoin and BMP-2 significantly stimulated it. Importantly, the stimulatory effects of phenytoin on osteocalcin release were completely blocked by the neutralizing antihuman BMP-2 monoclonal antibody. These results indicate that the stimulatory action of phenytoin on osteocalcin secretion in normal human bone cells is mediated, at least partly, through the upregulation of BMP-2, rather than that of TGF-beta(1).