OSTEOGENIC ACTIONS OF PHENYTOIN IN HUMAN BONE-CELLS ARE MEDIATED IN PART BY TGF-BETA-1

We have recently demonstrated that phenytoin, a widely used therapeuti c agent for seizure disorders, has osteogenic effects in rats and in h umans in vivo, and in human bone cells in vitro, The goal of the prese nt study was to determine the mechanism of the osteogenic action of ph enytoin in normal human mandible-derived bone cells, Because many oste ogenic agents increased bone cell proliferation through mediation by g rowth factors, we tested the hypothesis that the osteogenic effects of phenytoin involved the release of a growth factor by measuring the mR NA level of several bone cell growth factors and insulin-like growth f actor (IGF) binding proteins with Northern blots using specific cDNA p robes, Treatment with 5-50 mu M phenytoin reproducibly and markedly in creased (up to 6-fold, p < 0.001) the mRNA of transforming growth fact or (TGF)-beta 1, but not that of other growth factors (i.e., IGF-II pl atelet-derived growth factor-A [PDGF-A], PDGF-B, and TGF-beta 2) and I GF binding proteins (i.e. IGFBP-3, -4, and -5), The stimulation was do se dependent, with an optimal dose of 10-50 mu M. Minimal increase was seen after 1 h of phenytoin treatment. The release of biologically ac tive TGF-beta activity in conditioned media was measured with the mink lung cell proliferation inhibition assay. Twenty-four hours of phenyt oin treatment significantly increased the production of biologically a ctive TGP-beta (2-fold, p < 0.05) with the optimal dose between 5-50 m u M. Comparisons between the in vitro osteogenic effects of phenytoin and those of TGF-beta 1 reveal that these two agents at their respecti ve optimal doses had similar maximal stimulatory effects on [H-3] thym idine incorporation, alkaline phosphatase (ALP)-specific activity, and type I alpha-2 collagen mRNA expression in human bone cells, The stim ulatory effects of phenytoin on [H-3]thymidine incorporation and ALP-s pecific activity were completely blocked by a neutralizing anti-TGF-be ta antibody. In conclusion, these findings demonstrate for the first t ime that at least some of the osteogenic actions of phenytoin in human bone cells could be in part mediated by TGF-beta 1.