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. 2015 Feb 3;20(1):10.
doi: 10.1186/s40001-015-0084-x.

Hypoxia-induced MTA1 promotes MC3T3 osteoblast growth but suppresses MC3T3 osteoblast differentiation

Tielong Liu  1 , Weiwei Zou  2 , Guodong Shi  3 , Jian Xu  4 , Fei Zhang  5 , Jianru Xiao  6 , Yan Wang  7
Affiliations

Hypoxia-induced MTA1 promotes MC3T3 osteoblast growth but suppresses MC3T3 osteoblast differentiation

Tielong Liu et al. Eur J Med Res. .

Abstract

Background: Bone fracture is one of the most common physical injuries in which gene expression and the microenvironment are reprogramed to facilitate the recovery process.

Methods: By specific siRNA transfection and MTT assay, we evaluated the effects of metastasis-associated gene 1 (MTA1) in osteoblast growth. To show the role of MTA1 in osteoblast under hypoxia conditions, by overexpressing and silencing MTA1 expression, we performed mineral deposition and alkaline phosphatase activity assay to observe the differentiation status of osteoblast cells. Real-time PCR and Western blot assays were adopted to detect the expression of certain target genes.

Results: Here, we reported that hypoxia-induced MTA1 expression through hypoxia-induced factor 1 alpha (HIF-1α) and stimulated the growth of osteoblast MC3T3 cells. Silencing of MTA1 through specific siRNA suppressed MC3T3 cell growth and elicited cell differentiation and induced alkaline phosphatase activation and the upregulation of bone morphogenetic protein-2 and osteocalcin.

Conclusions: We found that MTA1 was regulated by HIF-1α in hypoxia circumstance to suppress osteoblast differentiation. These findings provide new insights for bone fracture healing and new strategies to develop potential targets to promote fracture healing.

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Figures

Figure 1
Figure 1
Hypoxia upregulated MTA1 expression. The MC3T3 cells were cultured under 1% oxygen tension (vs. 20% oxygen in the control). The expressions of MTA1 mRNA (A) and protein (B) were evaluated by real-time and Western blot, respectively (n = 3).
Figure 2
Figure 2
Hypoxia upregulated MTA1 expression through HIF-1. (A) Silencing of HIF-1α reversed the hypoxia-induced MTA1 upregulation at mRNA level (P <0.05). (B) Silencing of HIF-1 reversed the hypoxia-induced MTA1 upregulation at protein level (n = 3).
Figure 3
Figure 3
Hypoxia-induced MTA1 promoted the growth of MC3T3 cells. MC3T3 cells were transfected with pCDH-MTA1, MTA1 siRNA, or control siRNA. The growth of MC3T3 cells was evaluated by MTT assay. MTA1 OE, MTA1 overexpression. P <0.05 was considered statistically significant (n = 3).
Figure 4
Figure 4
Silencing of MTA1 slowed the mineralization of osteoblasts. (A) Phase-contrast microscopic images of MC3T3 cells cultured in differentiation medium after overexpression or silencing of MTA1 (×100). MTA1 OE, MTA1 overexpression. (B) The quantitative analysis of bone nodules after MTA1 manipulation (n = 3).
Figure 5
Figure 5
ALP activity assay in MC3T3 cells after MTA1 knockdown or overexpression. The ALP activity was assayed with Alkaline Phosphatase Assay Kit following the instructions. MTA1 OE, MTA1 overexpression (n = 3).
Figure 6
Figure 6
MTA1 regulated the expression of osteocalcin, BMP-2, and ALP. MC3T3 cells were transfected with pCDH-MTA1, MTA1 siRNA, or control siRNA. Null vector and control siRNA were used as controls. The expression of osteocalcin, BMP-2, and ALP was examined by Western blot assay. MTA1 OE, MTA1 overexpression (n = 3).
Figure 7
Figure 7
A schematic presentation showing the signaling pathway from fracture-induced hypoxia to MTA1 upregulation, suppressing osteoblast differentiation.
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