Changes in Runx2/Cbfa1 expression and activity during osteoblastic differentiation of human bone marrow stromal cells
Changes in Runx2/Cbfa1 expression and activity during osteoblastic differentiation of human bone marrow stromal cells
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DOI：
10.1359/jbmr.2003.18.2.213
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发表时间：
2003-02-01
影响因子：
6.2
通讯作者：
Khosla, S
中科院分区：
文献类型：
--
作者：
Shui, CX;Spelsberg, TC;Khosla, S
Runx2/Cbfa1已被确定为控制成骨细胞分化的“主基因”。然而，其在诱导成骨细胞表型中的作用主要在啮齿动物系统中得到表征。因此，我们检测了人骨髓基质 (BMSC) 细胞成骨细胞分化过程中 Runx2/Cbfa1 信使 RNA、蛋白质和活性水平。半定量逆转录聚合酶链反应 (RT-PCR) 分析表明，随着这些细胞 (hMS2-15) 成骨细胞表型的发展，碱性磷酸酶和骨钙素 mRNA 的表达以时间依赖性方式增加。发现 II 型 Runx2/Cbfa1 信使 RNA 在 hMS2-15 细胞中组成型表达，并且在分化过程中没有改变；没有检测到 I 型 Runx2/Cbfa1 转录物的表达。有趣的是，尽管在这些细胞的成骨细胞分化过程中 Runx2/Cbfa1 信使 RNA 水平没有任何变化，但通过与成骨细胞特异性顺式作用元件 2 (OSE2) 结合来评估，Runx2/Cbfa1 的活性在所有检查的时间点均显着增加，在第 7 天看到最高活性水平。在分化程度较低的人类的原代培养物中观察到类似的结果 骨髓来源的间充质干细胞。免疫沉淀和蛋白质印迹分析显示，虽然 hMS2-15 细胞分化时 Runx2/Cbfa1 蛋白水平没有增加，但 Runx2/Cbfa1 磷酸化有所增加。因此，与成骨细胞分化与 Runx2/Cbfa1 合成增加相关的啮齿动物系统相反，我们发现在人 BMSC 中，成骨细胞分化主要与 Runx2/Cbfa1 活性增加相关，而信使 RNA 或蛋白质水平没有变化。我们的研究结果还表明，Runx2/Cbfa1 活性的增加是通过涉及关键残基磷酸化的翻译后机制发生的。
Runx2/Cbfa1 has been identified as a "master gene" controlling osteoblast differentiation. However, its role in inducing the osteoblast phenotype has been characterized primarily in rodent systems. Thus, we examined Runx2/Cbfa1 messenger RNA, protein, and activity levels during osteoblastic differentiation of human bone marrow stromal (BMSC) cells. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that the expression of alkaline phosphatase and osteocalcin mRNAs increased in a time-dependent manner with the development of the osteoblast phenotype by these cells (hMS2-15). Type II Runx2/Cbfa1 messenger RNA was found to be constitutively expressed in hMS2-15 cells and not altered during differentiation; there was no detectable expression of the type I Runx2/Cbfa1 transcript. Interestingly, despite the absence of any change in Runx2/Cbfa1 messenger RNA levels during osteoblastic differentiation of these cells, the activity of Runx2/Cbfa1, as assessed by binding to the osteoblast-specific cis-acting element 2 (OSE2), increased markedly at all time-points examined, with the highest activity level seen at day 7. Similar results were observed in primary cultures of less differentiated human marrow-derived mesenchymal stem cells. Immunoprecipitation and Western blot analysis revealed that whereas there was no increase in Runx2/Cbfa1 protein levels with differentiation in hMS2-15 cells, there was an increase in Runx2/Cbfa1 phosphorylation. Thus, in contrast to rodent systems where osteoblast differentiation is associated with increased synthesis of Runx2/Cbfa1, we find that in human BMSC, osteoblastic differentiation is associated primarily with increases in Runx2/Cbfa1 activity, without a change in messenger RNA or protein levels. Our findings also show that the increase in Runx2/Cbfa1 activity occurs through a posttranslational mechanism involving phosphorylation of key residues.