Hatakeyama Yuji, Nguyen Jennifer, Wang Xibin, Nuckolls Glen H, Shum Lillian
J Bone Joint Surg Am. 2003;85-A Suppl 3:13-8. doi: 10.2106/00004623-200300003-00004.
Bone morphogenetic proteins (BMPs) are pleiotropic differentiation factors that regulate cell fate determination by orchestrating the activities of downstream signal transducers. Although BMP ligands can elicit signal transduction from heterodimeric combinations of several type-I and type-II receptors, cytoplasmic transducers of the BMP signal include only three known BMP-specific regulatory Smad proteins: Smad1, 5, and 8. In order to determine the combination of signals that regulate chondrogenesis by BMPs, we analyzed the functions of BMP Smad subtypes.
Multipotential mesenchymal C3H10T1/2 cells and monopotential chondroprogenitor MC615 cells were placed in micromass culture in the presence or absence of BMP4. Chondrogenic differentiation was assayed by measuring Sox9 and type-II collagen gene expression and by alcian blue staining. Transactivation of type-II collagen by regulatory Smads singly, or in combination with Smad4, which partners with regulatory Smads, was assayed by luciferase activity.
In the absence of BMP4, mesenchymal cells did not exhibit chondrogenic differentiation, whereas chondroprogenitor cells showed increased cartilage marker expression. In the presence of BMP4, the rate and extent of chondrogenesis increased in a dose-dependent manner for both cell types. We further determined that Smad1 or Smad5, but not Smad8, synergized with Smad4 in the transactivation of the type-II collagen promoter in chondroprogenitor cells. In contrast, Smad8 and Smad4 presented modest synergy in mesenchymal cells.
Taken together, our data suggest that uncommitted mesenchymal cells do not have the cellular competence to respond to the rate-limiting chondroinductive factor BMP. However, in chondroprogenitor cells, BMP stimulates differentiation through mechanisms mediated by Smad1 or Smad5 in combination with Smad4.
骨形态发生蛋白（BMPs）是多效性分化因子，通过协调下游信号转导子的活性来调节细胞命运决定。尽管BMP配体可从几种I型和II型受体的异二聚体组合引发信号转导，但BMP信号的细胞质转导子仅包括三种已知的BMP特异性调节性Smad蛋白：Smad1、5和8。为了确定由BMP调节软骨生成的信号组合，我们分析了BMP Smad亚型的功能。
将多能间充质C3H10T1/2细胞和单能软骨祖细胞MC615细胞在有或无BMP4的情况下进行微团培养。通过测量Sox9和II型胶原蛋白基因表达以及阿尔新蓝染色来检测软骨分化。通过荧光素酶活性检测调节性Smad单独或与与调节性Smad结合的Smad4组合对II型胶原蛋白的反式激活。
在没有BMP4的情况下，间充质细胞未表现出软骨分化，而软骨祖细胞显示软骨标记物表达增加。在有BMP4的情况下，两种细胞类型的软骨生成速率和程度均呈剂量依赖性增加。我们进一步确定，在软骨祖细胞中，Smad1或Smad5而非Smad8与Smad4协同作用于II型胶原蛋白启动子的反式激活。相比之下，Smad8和Smad4在间充质细胞中表现出适度的协同作用。
综上所述，我们的数据表明，未分化的间充质细胞不具备对限速软骨诱导因子BMP作出反应的细胞能力。然而，在软骨祖细胞中，BMP通过Smad1或Smad5与Smad4组合介导的机制刺激分化。
