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标题:Extracellular matrix stiffness controls osteogenic differentiation of mesenchymal stem cells mediated by integrin α5.
时间:2020-02-14 23:08:09
DOI:10.1186/s13287-018-0798-0
PMID:29490668
作者:Meiyu Sun;Guangfan Chi;Juanjuan Xu
关键词:Matrix stiffness;Mesenchymal stem cells;Differentiation;Integrin α5
出版源: 《Stem Cell Research & Therapy》 ,2018 ,9 (1) :52
摘要: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 α5/β1, FAK, and Wnt signaling, subsequently regulating the osteogenic phenotype of hMSCs. 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 ofalpha-1typeIcollagen,BGLAP, andRUNX2were evaluated by real-time PCR. After hMSCs were cultured on gels for 24 h, signaling molecules relating to integrin α5 (FAK, ERK, p-ERK, Akt, p-Akt, GSK-3β, p-GSK-3β, and β-catenin) were evaluated by western blot analysis. Osteogenic differentiation was increased on 62–68 kPa ECM, as evidenced byalpha-1 type I collagen,BGLAP, andRUNX2gene expression, calcium deposition, and ALP staining. In the process of differentiation, gene and protein expression of integrin α5/β1 increased, together with protein expression of the downstream signaling molecules FAK, p-ERK, p-Akt, GSK-3β, p-GSK-3β, and β-catenin, indicating that these molecules can affect the osteogenic differentiation of hMSCs. An antibody blocking integrin α5 suppressed the stiffness-induced expression of all osteoblast markers examined. In particular,alpha-1 type I collagen,RUNX2, andBGLAPwere significantly downregulated, indicating that integrin α5 regulates hMSC osteogenic differentiation. Downstream expression of FAK, ERK, p-ERK, and β-catenin protein was unchanged, whereas Akt, p-Akt, GSK-3β, and p-GSK-3β were upregulated. Moreover, expression of Akt and p-Akt was upregulated with anti-integrin α5 antibody, but no difference was observed for FAK, ERK, and p-ERK between the with or without anti-integrin α5 antibody groups. At the same time, expression of GSK-3β and p-GSK-3β was upregulated and β-catenin levels showed no difference between the groups with or without anti-integrin α5 antibody. Since Akt, p-Akt, GSK-3β, and p-GSK-3β displayed the same changes between the groups with or without anti-integrin α5 antibody, we then detected the links among them. Expression of p-Akt and p-GSK-3β was reduced effectively in the presence of the Akt inhibitor Triciribine. However, Akt, GSK-3β, and β-catenin were unchanged. These results suggested that expression of p-GSK-3β was regulated by p-Akt on 62–68 kPa ECM. 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 α5. The online version of this article (10.1186/s13287-018-0798-0) contains supplementary material, which is available to authorized users.
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目录:
  • Abstract
    • Background
    • Methods
    • Results
    • Conclusions
  • Background
  • Methods
    • Cell culture and characterization
    • Growth of cells
    • Oil Red O and Alizarin Red S staining
    • Cell karyotype analysis
    • ECM fabrication
    • CCK8 cytotoxicity assays
    • Scanning electron microscopy
    • Alkaline phosphatase staining
    • Confocal microscopy and flow cytometry
    • FITC-Phalloidin staining of F-actin
    • Gene expression analysis
    • Integrin blocking experiments
    • Western blot analysis
    • Statistical analysis
  • Results
    • The characteristics of hMSCs
    • ECM preparation and analysis
    • ECM stiffness induced the osteogenic differentiation of hMSCs
    • Altered distribution of integrin α5/β1 during stiffness induced osteogenic differentiation of hMSCs
    • Increased expression of integrin α5 on 62–68 kPa ECM
    • Increased protein expression of molecules downstream of integrin α5/β1 and Wnt/β-catenin on 62–68 kPa ECM
    • Integrin α5-mediated osteogenic differentiation induced by matrix and expression of signaling proteins has changed after blocking integrin α5
  • Discussion
  • Conclusions
  • Additional file
  • Abbreviations
  • Funding
  • Availability of data and materials
  • Authors’ contributions
  • Ethics approval and consent to participate
  • Consent for publication
  • Competing interests
  • Publisher’s Note
  • References

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