Reconstitution of Runx2/Cbfa1-null cells identifies a requirement for BMP2 signaling through a Runx2 functional domain during osteoblast differentiation
Reconstitution of Runx2/Cbfa1-null cells identifies a requirement for BMP2 signaling through a Runx2 functional domain during osteoblast differentiation
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DOI：
10.1002/jcb.21039
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发表时间：
2007-02-01
影响因子：
4
通讯作者：
Javed, Amjad
中科院分区：
文献类型：
--
作者：
Bae, Jong-Sup;Gutierrez, Soraya;Javed, Amjad
Runx2/Cbfa1转录因子是一种促进成骨细胞分化的支架蛋白；然而，诱导成骨谱系所需的特定 Runx2 功能域仍有待确定。我们使用源自 Runx2 缺失小鼠颅盖的 TERT 永生化细胞系，通过用 Runx2 的野生型和缺失突变体重建成骨活性来解决这个问题。成骨介质（磷酸 β-甘油和抗坏血酸）和/或 BMP2 的存在或不存在不会刺激 Runx2-null 细胞中的成骨细胞基因表达。然而，感染野生型 Runx2 腺病毒的细胞表现出成骨细胞标记基因表达的短暂增加，并且能够对 BMP2 做出反应。早期标记物（即 1 型胶原蛋白、碱性磷酸酶）在培养第 4 天和第 8 天被诱导（四到八倍）。代表成熟成骨细胞的基因（例如 Runx2、骨桥蛋白、骨唾液蛋白、骨钙蛋白）在第 8 天和第 12 天暂时表达并诱导 18 至 36 倍。有趣的是，TGF β 和维生素 D 介导的成骨细胞基因（骨桥蛋白除外）的转录需要 Runx2 的存在。缺少C端96个氨基酸的Runx2（Runx2 Delta 432）显示出与野生型蛋白相似的基因表达模式，证明Groucho相互作用和部分激活结构域对于Runx2成骨活性是可有可无的。进一步删除含有核基质靶向信号和 Smad 相互作用结构域 (Delta 391) 的 Runx2 C 末端后，我们发现没有任何成骨细胞标志物表达。因此，Runx2 391-432 结构域对于 BMP2 成骨信号的执行至关重要。
The Runx2/Cbfa1 transcription factor is a scaffolding protein that promotes osteoblast differentiation; however, the specific Runx2-functional domains required for induction of the osteogenic lineage remain to be identified. We approached this question using a TERT-immortalized cell line derived from calvaria of Runx2-null mice by reconstituting the osteogenic activity with wild-type and deletion mutants of Runx2. The presence or absence of osteogenic media (beta-glycerol phosphate and ascorbic acid) and/or with BMP2 did not stimulate osteoblastic gene expression in the Runx2-null cells. However, cells infected with wild-type Runx2 adenovirus showed a robust temporal increase in the expression of osteoblast marker genes and were competent to respond to BMP2. Early markers (i.e., collagen type-1, alkaline phosphatase) were induced (four- to eightfold) at Days 4 and 8 of culture. Genes representing mature osteoblasts (e.g., Runx2, osteopontin, bone sialoprotein, osteocalcin) were temporally expressed and induced from 18-to 36-fold at Days 8 and 12. Interestingly, TGF beta and Vitamin D-mediated transcription of osteoblast genes (except for osteopontin) required the presence of Runx2. Runx2 lacking the C-terminal 96 amino acids (Runx2 Delta 432) showed a pattern of gene expression similar to wild-type protein, demonstrating the Groucho interaction and part of the activation domain are dispensable for Runx2 osteogenic activity. Upon further deletion of the Runx2 C-terminus containing the nuclear matrix targeting signal and Smad-interacting domain (Delta 391), we find none of the osteoblast markers are expressed. Therefore, the Runx2 391-432 domain is essential for execution of the BMP2 osteogenic signal.