Luppen Cynthia A, Smith Elisheva, Spevak Lyudmila, Boskey Adele L, Frenkel Baruch
Department of Biochemistry, Institute for Genetic Medicine, University of Southern California Keck School of Medicine, Los Angeles, California 90033, USA.
J Bone Miner Res. 2003 Jul;18(7):1186-97. doi: 10.1359/jbmr.2003.18.7.1186.
The anti-glucocorticoid potential of BMP-2 in osteoblasts was tested in MC3T3-E1 cells using dexamethasone (1 microM) and rhBMP-2 (10 or 100 ng/ml). rhBMP-2 restored mineralization but not condensation or collagen accumulation. These results demonstrate the potential and limitations of BMPs in counteracting glucocorticoids.
Pharmacologic glucocorticoids (GCs) inhibit osteoblast function and induce osteoporosis. Bone morphogenetic proteins (BMPs) stimulate osteoblast differentiation and bone formation. Here we tested the anti-glucocorticoid potential of BMP-2 in cultured osteoblasts.
MC3T3-E1 cells were treated with dexamethasone (DEX; 1 microM) and/or recombinant human BMP-2 (rhBMP-2; 10 or 100 ng/ml). Culture progression was characterized by cell cycle profiling, biochemical assays for DNA, alkaline phosphatase (ALP), collagen, and calcium, and by reverse transcriptase-polymerase chain reaction (RT-PCR) of osteoblast phenotypic mRNAs. Mineralization was characterized by Alizarin red and von Kossa staining and by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD).
DEX inhibited differentiation-related cell cycle, nodule formation, collagen accumulation, osteocalcin, and BMP-2 gene expression as well as mineralization. Replenishment of GC-inhibited cultures with 10 or 100 ng/ml rhBMP-2 dramatically rescued mineral deposition. The rhBMP-2-rescued mineral was bone-like apatite nearly identical to the mineral of control cultures. The rhBMP-2 rescue was associated with increased mRNA levels for alpha1(I) collagen, osteocalcin, and Cbfa1 types I and II, as well as ALP activity. In contrast, rhBMP-2 did not rescue the GC-inhibited differentiation-related cell cycle, nodule formation, or collagen accumulation. When administered alone, rhBMP-2 also increased the mRNA levels for alpha1(I) collagen, osteocalcin, and Cbfa1 types I and II, as well as ALP activity. However, treatment with rhBMP-2 alone inhibited cell cycle progression, nodule formation, and collagen accumulation. Surprisingly, in contrast to its rescue of mineralization in DEX-treated cultures, rhBMP-2 inhibited mineralization in the absence of DEX. In parallel to its bimodal effect on mineralization, rhBMP-2 stimulated endogenous BMP-2 mRNA in the presence of DEX, but inhibited endogenous BMP-2 mRNA in the absence of DEX.
Suppression of BMP-2 gene expression plays a pivotal role in GC inhibition of osteoblast differentiation. However, the inability of rhBMP-2 to rescue the entire osteoblast phenotype suggests BMP-2-independent inhibitory effects of CCs. BMP-2 exerts both positive and negative effects on osteoblasts, possibly depending on the differentiation stage and/or the existing BMP signaling.
使用地塞米松（1微摩尔）和重组人骨形态发生蛋白-2（rhBMP-2，10或100纳克/毫升）在MC3T3-E1细胞中测试了BMP-2在成骨细胞中的抗糖皮质激素潜力。rhBMP-2恢复了矿化，但未恢复凝聚或胶原蛋白积累。这些结果证明了骨形态发生蛋白在对抗糖皮质激素方面的潜力和局限性。
药理糖皮质激素（GCs）抑制成骨细胞功能并导致骨质疏松症。骨形态发生蛋白（BMPs）刺激成骨细胞分化和骨形成。在此，我们测试了BMP-2在培养的成骨细胞中的抗糖皮质激素潜力。
用1微摩尔地塞米松（DEX）和/或重组人BMP-2（rhBMP-2，10或100纳克/毫升）处理MC3T3-E1细胞。通过细胞周期分析、DNA、碱性磷酸酶（ALP）、胶原蛋白和钙的生化测定以及成骨细胞表型mRNA的逆转录聚合酶链反应（RT-PCR）来表征培养进程。通过茜素红和冯科萨染色以及傅里叶变换红外光谱（FTIR）和X射线衍射（XRD）来表征矿化。
DEX抑制与分化相关的细胞周期、结节形成、胶原蛋白积累、骨钙素和BMP-2基因表达以及矿化。用10或100纳克/毫升rhBMP-2补充受GC抑制的培养物可显著挽救矿物质沉积。rhBMP-2挽救的矿物质是类似于对照培养物矿物质的类骨磷灰石。rhBMP-2的挽救与α1（I）胶原蛋白、骨钙素以及I型和II型Cbfa1的mRNA水平增加以及ALP活性增加有关。相反，rhBMP-2未能挽救受GC抑制的与分化相关的细胞周期、结节形成或胶原蛋白积累。单独使用时，rhBMP-2也增加了α1（I）胶原蛋白、骨钙素以及I型和II型Cbfa1的mRNA水平以及ALP活性。然而，单独用rhBMP-2处理会抑制细胞周期进程、结节形成和胶原蛋白积累。令人惊讶的是，与其在DEX处理的培养物中挽救矿化相反，rhBMP-2在无DEX时抑制矿化。与其对矿化的双峰效应并行，rhBMP-2在有DEX时刺激内源性BMP-2 mRNA，但在无DEX时抑制内源性BMP-2 mRNA。
BMP-2基因表达的抑制在GC抑制成骨细胞分化中起关键作用。然而，rhBMP-2无法挽救整个成骨细胞表型表明CCs存在不依赖BMP-2的抑制作用。BMP-2对成骨细胞发挥正负两方面的作用，可能取决于分化阶段和/或现有的BMP信号传导。
