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

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.

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. 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. 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. 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.