Extracellular matrix stiffness controls osteogenic differentiation of mesenchymal stem cells mediated by integrin α5

Background: Human mesenchymal stem cell (hMSC) differentiation into osteoblasts has important clinical significance in treating bone injury, and the stiffness of the extracellular matrix (ECM) has been shown to be an important regulatory factor for hMSC differentiation. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through integrin alpha 5/ss 1, FAK, and Wnt signaling, subsequently regulating the osteogenic phenotype of hMSCs. Methods: hMSCs were cultured on tunable polyacrylamide hydrogels coated with fibronectin with stiffness corresponding to a Young's modulus of 13-16 kPa and 62-68 kPa. After hMSCs were cultured on gels for 1 week, gene expression of alpha-1 type I collagen, BGLAP, and RUNX2 were evaluated by real-time PCR. After hMSCs were cultured on gels for 24 h, signaling molecules relating to integrin alpha 5 (FAK, ERK, p-ERK, Akt, p-Akt, GSK-3 ss, p-GSK-3 ss, and ss-catenin) were evaluated by western blot analysis. Results: Osteogenic differentiation was increased on 62-68 kPa ECM, as evidenced by alpha-1 type I collagen, BGLAP, and RUNX2 gene expression, calcium deposition, and ALP staining. In the process of differentiation, gene and protein expression of integrin alpha 5/ss 1 increased, together with protein expression of the downstream signaling molecules FAK, p-ERK, p-Akt, GSK-3 ss, p-GSK-3 ss, and ss-catenin, indicating that these molecules can affect the osteogenic differentiation of hMSCs. An antibody blocking integrin alpha 5 suppressed the stiffness-induced expression of all osteoblast markers examined. In particular, alpha-1 type I collagen, RUNX2, and BGLAP were significantly downregulated, indicating that integrin alpha 5 regulates hMSC osteogenic differentiation. Downstream expression of FAK, ERK, p-ERK, and ss-catenin protein was unchanged, whereas Akt, p-Akt, GSK-3 ss, and p-GSK-3 ss were upregulated. Moreover, expression of Akt and p-Akt was upregulated with anti-integrin alpha 5 antibody, but no difference was observed for FAK, ERK, and p-ERK between the with or without anti-integrin alpha 5 antibody groups. At the same time, expression of GSK-3 ss and p-GSK-3 ss was upregulated and ss-catenin levels showed no difference between the groups with or without anti-integrin alpha 5 antibody. Since Akt, p-Akt, GSK-3 ss, and p-GSK-3 ss displayed the same changes between the groups with or without anti-integrin alpha 5 antibody, we then detected the links among them. Expression of p-Akt and p-GSK-3 ss was reduced effectively in the presence of the Akt inhibitor Triciribine. However, Akt, GSK-3 ss, and ss-catenin were unchanged. These results suggested that expression of p-GSK-3 ss was regulated by p-Akt on 62-68 kPa ECM. Conclusions: Taken together, our results provide evidence that matrix stiffness (62-68 kPa) affects the osteogenic outcome of hMSCs through mechanotransduction events that are mediated by integrin alpha 5.