Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells.
Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells.
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DOI：
10.1016/j.yexcr.2010.05.011
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发表时间：
2010-08-15
影响因子：
3.7
通讯作者：
Ge RS
中科院分区：
文献类型：
--
作者：
Hong D;Chen HX;Yu HQ;Liang Y;Wang C;Lian QQ;Deng HT;Ge RS
骨重塑依赖于骨形成和骨吸收之间的动态平衡，分别由成骨细胞和破骨细胞介导。在一定的刺激下，成骨细胞可分化为早期成骨细胞，并进一步分化为成熟成骨细胞。这一过程的特点是碱性磷酸酶（ALP）活性增加和矿化结核的形成。在本研究中，我们对小鼠成骨祖细胞MC3T3-E1进行了成骨细胞分化，并将此过程分为三个阶段。在第一阶段（第3天），成骨细胞分化的MC3T3-E1细胞不表达ALP，也不沉积矿化结节。第二阶段，MC3T3-E1细胞表达ALP，但不形成矿化结节。第三阶段，MC3T3-E1细胞具有ALP活性，形成矿化结节。在本研究中，我们重点研究了MC3T3-E1细胞在成骨细胞分化早期（即过早成骨细胞向成熟成骨细胞转变的阶段）的形态学和蛋白质组学变化。我们发现，在这一阶段，由于细胞扩散增强，细胞增殖减少，平均细胞面积和平均应力纤维密度增加。我们进一步利用蛋白质组学方法分析细胞骨架调控信号通路中的蛋白，发现IQGAP1、gelsolin、moesin、radixin和Cfl1上调。通过对黏附信号通路的分析，我们发现FLNA、LAMA1、LAMA5、COL1A1、COL3A1、COL4A6、COL5A2表达上调，COL4A1、COL4A2、COL4A4表达下调。综上所述，细胞骨架和局灶黏附调控信号通路在成骨细胞分化早期调控细胞扩散和肌动蛋白骨架形成中起关键作用。
The bone remodelling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodule. In present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation - a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of cytoskeleton using proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.