Sci Rep. 2016 Jun 9;6:27622 

Overexpression of HSPA1A enhances the osteogenic differentiation of bone marrow mesenchymal stem cells via activation of the Wnt/β-catenin signaling pathway

Zhijun Pan


摘要:

HSPA1A, which encodes cognate heat shock protein 70, plays important roles in various cellular metabolic pathways. To investigate its effects on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), its expression level was compared between undifferentiated and differentiated BMSCs. Rat HSPA1A overexpression in BMSCs increased osteoblast-specific gene expression, alkaline phosphatase activity, and mineral deposition in vitro. Moreover, it upregulated β-catenin and downregulated DKK1 and SOST. The enhanced osteogenesis due to HSPA1A overexpression was partly rescued by a Wnt/β-catenin inhibitor. Additionally, using a rat tibial fracture model, a sheet of HSPA1A-overexpressing BMSCs improved bone fracture healing, as determined by imaging and histological analysis. Taken together, these findings suggest that HSPA1A overexpression enhances osteogenic differentiation of BMSCs, partly through Wnt/β-catenin.rBMSCs were purchased from Cyagen Biosciences (Guangzhou, China). These cells can differentiate into osteoblasts, adipoblasts, and chondrocytes under specific inductive conditions. Adherent cells were trypsinized and passaged after reaching 80% confluence. Cells from passages 3–9 were used in subsequent experiments.Recombinant DKK1 was purchased from PeproTech (Rocky Hill, NJ, USA). In accordance with a previous study, the applied concentration of DKK1 was 0.5 μg/mL21.To determine the expression levels of HSPA1A associated with osteogenic differentiation of MSCs, we examined HSPA1A endogenous expression in rBMSCs at days 0, 7, and 14 during the process of osteogenic differentiation. Compared with undifferentiated rBMSCs, the mRNA expression of HSPA1A increased significantly at days 7 and 14 during osteogenic differentiation (Fig. 1A). In addition, the protenin expression of HSPA1A also increased significantly at day 14 during osteogenic differentiation (Fig. 2B,C).We thank Qubo Ni for his expert technical assistance. This work was supported by a grant from the National Natural Science Foundation of China (No.81271973 and No.81201397), the Zhejiang Provincial Natural Science Foundation of China (No. LY15H060001; No. LY15H060002 No. LY16H060003; and No. LY13H060002) and Zhejiang medical and health science and technology plan project (No. 2015KYB182).Author Contributions Z.P. contributed design and funding sources to this study. W.Z. and D.X. drafted the manuscript. H.Y., C.L., S.W., Q.Z. and W.Z. preformed the in vivo experiments. W.Z., D.X., E.C., X.G., D.H. and S.W. did all the in vitro parts of the study. Y.T. and J.Y. carried out statistical work. All authors have contributed significantly. All authors read and approved the final manuscript.
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