Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Aug 15;316(14):2291-300.
doi: 10.1016/j.yexcr.2010.05.011. Epub 2010 May 17.

Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

Dun Hong  1 , Hai-Xiao Chen, Hai-Qiang Yu, Yong Liang, Carrie Wang, Qing-Quan Lian, Hai-Teng Deng, Ren-Shan Ge
Affiliations

Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

Dun Hong et al. Exp Cell Res. .

Abstract

Bone remodeling 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 a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the 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 the cytoskeleton using a 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 the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.

PubMed Disclaimer

Figures

Fig 1
Fig 1. Methods for morphological measurement of MC3T3-E1 cells
A, B, C, and D: the measurement of the width (W), length (L), and area of the fusiform MC3T3-E1 cells. E: MC3T3-E1 cell stained with rhodamine phalloidin; F: Merge images show the number of nucleus; G: Selection the area of actin stress fibers; H: Selection for measurement of the cell area;
Fig 2
Fig 2. Functional and morphological analysis of osteoblast differentiation in MC3T3-E1 cells
A: The mineral nodule formation by Alizarin red staining, B: Alkaline phosphatase (ALP) activity of the MC3T3-E1 cells in 21-day- culture; C: Cell proliferation assay. D: The width and length of the MC3T3-E1 cells on day 3; E: The cell area of the MC3T3-E1 cells on day 3 and 7; F: The actin stress fiber density of MC3T3-E1 cells on day 3 and 7. *: P < 0.5, **: P < 0.01, comparison between CON and OS; ##: P < 0.01, value comparison of day 7, 14, and 21 to day 3.
Fig 3
Fig 3. Comparison of the number of matched proteins according to classification of PIGOK in OS and CON group
A: Celluar components classification; B: The molecular function classification; C: The KEGG signaling pathway.
Fig 4
Fig 4. The regulation of proteins in the signaling pathway of regulation of actin cytoskeleton and focal adhesion on day 7
A: Western Blot analysis of proteins. Molecular weight: FLNA (280 K), FLNB (280 K), IQGAP1 (195 K), Vinculin (117 K), Gelsolin (90 K), and β-actin (45 k); B, C, D: The MS/MS spectra of the peptides.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sampath TK, Reddi AH. Homology of bone-inductive proteins from human, monkey, bovine, and rat extracellular matrix. Proc Natl Acad Sci U S A. 1983;80:6591–6595. - PMC - PubMed
    1. Sampath TK, Reddi AH. Importance of geometry of the extracellular matrix in endochondral bone differentiation. J Cell Biol. 1984;98:2192–2197. - PMC - PubMed
    1. Komori T. Regulation of osteoblast differentiation by transcription factors. J Cell Biochem. 2006;99:1233–1239. - PubMed
    1. Day TF, Guo X, Garrett-Beal L, Yang Y. Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell. 2005;8:739–750. - PubMed
    1. Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res. 1992;7:683–692. - PubMed

Publication types