Luppen Cynthia A, Leclerc Nathalie, Noh Tommy, Barski Artem, Khokhar Arvinder, Boskey Adele L, Smith Elisheva, Frenkel Baruch
Department of Biochemistry, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.
J Biol Chem. 2003 Nov 7;278(45):44995-5003. doi: 10.1074/jbc.M306730200. Epub 2003 Aug 20.
Glucocorticoids (GCs) inhibit bone formation in vivo. In MC3T3-E1 osteoblasts, chronic administration of 1 microm dexamethasone (DEX) starting at confluency results in >98% inhibition of bone morphogenetic protein 2 (BMP-2) expression and apatite mineral deposition. Here, it is shown that brief exposure to recombinant human BMP-2 (rhBMP-2), as short as 6 h, is sufficient to induce irreversible commitment to mineralization in DEX-treated cultures. RhBMP-2 dose dependently rescued mineralization but not collagen accumulation in DEX-inhibited cultures. The selective restoration of mineralization was evident in the higher mineral to matrix ratios of DEX/rhBMP-2 co-treated cultures compared with control. We tested the involvement of the runt-related transcription factor 2 (Runx2) in the DEX inhibition and rhBMP-2 rescue of mineralization. Surprisingly, DEX did not decrease Runx2 DNA binding activity, transactivation, or association with the endogenous osteocalcin gene promoter. Furthermore, the rhBMP-2 rescue did not involve Runx2 stimulation, suggesting an important role for factors other than Runx2 in BMP-2 action. Finally, we studied the differentiation-related cell cycle, which persists during commitment to mineralization in untreated cultures, but is inhibited along with mineralization in DEX-treated cultures. Although both rhBMP-2 alone and DEX alone were antimitogenic, rhBMP-2 stimulated this cell cycle in DEX-inhibited cultures. In conclusion, brief rhBMP-2 treatment restores mineralization in DEX-inhibited MC3T3-E1 osteoblasts via a mechanism different from Runx2 stimulation. This restoration may be functionally related to the accompanying rescue of the differentiation-related cell cycle. The efficacy of short term BMP-2 treatment supports the potential of short-lived BMP vectors for skeletal therapy in both traditional and gene therapeutic approaches.
糖皮质激素（GCs）在体内会抑制骨形成。在MC3T3-E1成骨细胞中，从汇合状态开始长期给予1微摩尔地塞米松（DEX）会导致骨形态发生蛋白2（BMP-2）表达及磷灰石矿物沉积受到>98%的抑制。在此研究中发现，短暂暴露于重组人BMP-2（rhBMP-2），短至6小时，就足以在DEX处理的培养物中诱导不可逆的矿化进程。rhBMP-2能剂量依赖性地挽救矿化作用，但在DEX抑制的培养物中对胶原蛋白积累无作用。与对照组相比，DEX/rhBMP-2共同处理的培养物中较高的矿物质与基质比例表明矿化作用得到了选择性恢复。我们测试了矮小相关转录因子2（Runx2）在DEX对矿化的抑制作用以及rhBMP-2对矿化的挽救作用中的参与情况。令人惊讶的是，DEX并未降低Runx2的DNA结合活性、反式激活作用或与内源性骨钙素基因启动子的结合。此外，rhBMP-2的挽救作用并不涉及Runx2的刺激，这表明在BMP-2作用中，除Runx2外的其他因子发挥着重要作用。最后，我们研究了与分化相关的细胞周期，在未处理的培养物中，该细胞周期在矿化进程中持续存在，但在DEX处理的培养物中会随着矿化作用受到抑制。尽管单独使用rhBMP-2和单独使用DEX都具有抗有丝分裂作用，但rhBMP-2能在DEX抑制的培养物中刺激这个细胞周期。总之，短暂的rhBMP-2处理通过一种不同于刺激Runx2的机制，恢复了DEX抑制的MC3T3-E1成骨细胞中的矿化作用。这种恢复可能在功能上与伴随的分化相关细胞周期的挽救有关。短期BMP-2治疗的有效性支持了在传统和基因治疗方法中，使用短效BMP载体进行骨骼治疗的潜力。
