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. 2019 Sep;14(3):225-233.
doi: 10.1016/j.jds.2019.03.001. Epub 2019 Mar 20.

Fak silencing impairs osteogenic differentiation of bone mesenchymal stem cells induced by uniaxial mechanical stretch

Pei Hu  1   2 , Xiaowen Zhu  1 , Chuang Zhao  1   2 , Jing Hu  1   2 , En Luo  1   2 , Bin Ye  1   2
Affiliations

Fak silencing impairs osteogenic differentiation of bone mesenchymal stem cells induced by uniaxial mechanical stretch

Pei Hu et al. J Dent Sci. 2019 Sep.

Abstract

Background/purpose: Mechanical stretch plays a key role in promoting proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) in distraction osteogenesis (DO). A better understanding of how the extracellular biomechanical stimulation is transferred to intracellular signal expression will benefit DO. Focal adhesion kinase (FAK) is a key factor in integrin signaling pathway. However, little is known about the effect of integrin-FAK signaling during the process of stretch induced osteogenic differentiation of BMSCs.

Materials and methods: A specific short hairpin RNAs (shRNAs) lentiviral expression vector was used to silence Fak gene and a well-established in vitro uniaxial dynamic stretching device was applied to stimulate DO. Fak silencing was confirmed by fluorescence microscopy and the detection of Fak mRNA and FAK, p-FAK protein expression. Alkaline phosphatase (ALP) activity, expression of osteogenic differentiation markers - runt-related transcription factor 2 (RUNX2/Runx2) and alkaline phosphatase (Alp) together with integrin upstream signal transduction molecules integrin beta-1 (ITGB1/Itgb1) and downstream signal transduction molecules integrin-linked kinase (ILK) were detected after the stretch.

Results: The results showed that mechanical stretch in control groups significantly induced the osteogenic differentiation of BMSCs with increased ALP activity, expression of RUNX2/Runx2 and Alp, together with upregulated ITGB1/Itgb1 and ILK, which all vanished in Fak silencing group.

Conclusion: Silencing of the Fak gene inhibited the osteogenic differentiation of rat BMSCs induced by in vitro mechanical stretch through integrin signaling pathway.

Keywords: Distraction osteogenesis; Focal adhesion kinase; Integrin signaling pathway; Mechanical stretch; Mesenchymal stem cells.

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Figures

Figure 1
Figure 1
Uniaxial dynamic stretching device. A. Control system; B. Stretching units; and C. Bone during distraction osteogenesis in vivo; D. Cells during biomechanical stretch in stretching unit; E. Primary rat bone mesenchymal stem cells. Scale bars, 100 μm; F. Cells after mechanical stretch. Scale bars, 100 μm.
Figure 2
Figure 2
Antigens expression on cell surface. Positive for CD29 and CD90, negative for CD34 and CD45.
Figure 3
Figure 3
Representative fluorescence images showing GFP expression in BMSCs of the three groups after transfection: A. Control group; B. shRNA-Neg group and C. shRNA-Fak group. Scale bar, 100 μm.
Figure 4
Figure 4
A. Representative images of Western blots showing FAK, p-FAK and GAPDH protein expression in control group of BMSCs after exposed to mechanical stretch simulating distraction. B. Fak mRNA expression in three groups of BMSCs. C. FAK and p-FAK protein expression in three groups of BMSCs (*p < 0.05).
Figure 5
Figure 5
mRNA expression level of Itgb1, Alp, and Runx2 in three groups of BMSCs after exposed to mechanical stretch simulating distraction (*p < 0.05).
Figure 6
Figure 6
A. Representative images and B. Quantitative analysis of Western blots showing ITGB1, ILK, RUNX2, and GAPDH protein expression in three groups of BMSCs after exposed to mechanical stretch simulating distraction (*p < 0.05).
See this image and copyright information in PMC

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