Nakade O, Baylink D J, Lau K H
Department of Medicine, Loma Linda University, California, USA.
J Bone Miner Res. 1996 Dec;11(12):1880-8. doi: 10.1002/jbmr.5650111208.
We have recently demonstrated that phenytoin, a widely used therapeutic agent for seizure disorders, has osteogenic effects in rats and in humans in vivo, and in human bone cells in vitro. The goal of the present study was to determine the mechanism of the osteogenic action of phenytoin in normal human mandible-derived bone cells. Because many osteogenic agents increased bone cell proliferation through mediation by growth factors, we tested the hypothesis that the osteogenic effects of phenytoin involved the release of a growth factor by measuring the mRNA level of several bone cell growth factors and insulin-like growth factor (IGF) binding proteins with Northern blots using specific cDNA probes. Treatment with 5-50 microM phenytoin reproducibly and markedly increased (up to 6-fold, p < 0.001) the mRNA of transforming growth factor (TGF)-beta 1, but not that of other growth factors (i.e., IGF-II, platelet-derived growth factor-A [PDGF-A], PDGF-B, and TGF-beta 2) and IGF binding proteins (i.e., IGFBP-3, -4, and -5). The stimulation was dose dependent, with an optimal dose of 10-50 microM. Maximal increase was seen after 1 h of phenytoin treatment. The release of biologically active TGF-beta activity in conditioned media was measured with the mink lung cell proliferation inhibition assay. Twenty-four hours of phenytoin treatment significantly increased the production of biologically active TGF-beta (2-fold, p < 0.05) with the optimal dose between 5-50 microM. Comparisons between the in vitro osteogenic effects of phenytoin and those of TGF-beta 1 reveal that these two agents at their respective optimal doses had similar maximal stimulatory effects on [3H]thymidine incorporation, alkaline phosphatase (ALP)-specific activity, and type I alpha-2 collagen mRNA expression in human bone cells. The stimulatory effects of phenytoin on [3H]thymidine incorporation and ALP-specific activity were completely blocked by a neutralizing anti-TGF-beta antibody. In conclusion, these findings demonstrate for the first time that at least some of the osteogenic actions of phenytoin in human bone cells could be in part mediated by TGF-beta 1.
我们最近证实，苯妥英钠作为一种广泛用于治疗癫痫疾病的药物，在大鼠和人类体内以及人类骨细胞体外实验中均具有成骨作用。本研究的目的是确定苯妥英钠在正常人类下颌骨来源的骨细胞中发挥成骨作用的机制。由于许多成骨药物通过生长因子介导来增加骨细胞增殖，我们通过使用特异性cDNA探针的Northern印迹法测量几种骨细胞生长因子和胰岛素样生长因子（IGF）结合蛋白的mRNA水平，来检验苯妥英钠的成骨作用涉及生长因子释放这一假设。用5 - 50微摩尔/升的苯妥英钠处理可重复性地且显著增加（高达6倍，p < 0.001）转化生长因子（TGF）-β1的mRNA水平，但不影响其他生长因子（即IGF-II、血小板衍生生长因子-A [PDGF-A]、PDGF-B和TGF-β2）以及IGF结合蛋白（即IGFBP-3、-4和-5）的mRNA水平。这种刺激是剂量依赖性的，最佳剂量为10 - 50微摩尔/升。苯妥英钠处理1小时后可见最大增加。用貂肺细胞增殖抑制试验测量条件培养基中生物活性TGF-β活性的释放。苯妥英钠处理24小时显著增加了生物活性TGF-β的产生（2倍，p < 0.05），最佳剂量在5 - 50微摩尔/升之间。苯妥英钠与TGF-β1体外成骨作用的比较表明，这两种药物在各自的最佳剂量下对人骨细胞中[3H]胸苷掺入、碱性磷酸酶（ALP）特异性活性和I型α-2胶原mRNA表达具有相似的最大刺激作用。苯妥英钠对[3H]胸苷掺入和ALP特异性活性的刺激作用可被中和性抗TGF-β抗体完全阻断。总之，这些发现首次证明，苯妥英钠在人骨细胞中的至少一些成骨作用可能部分由TGF-β1介导。
