Extracellular matrix stiffness controls osteogenic differentiation of mesenchymal stem cells mediated by integrin α5.
Extracellular matrix stiffness controls osteogenic differentiation of mesenchymal stem cells mediated by integrin α5.
复制标题
细胞外基质硬度控制整合素α5介导的间充质干细胞的成骨分化
DOI：
10.1186/s13287-018-0798-0
复制
发表时间：
2018-03-01
影响因子：
7.5
通讯作者：
Li Y
中科院分区：
文献类型：
--
作者：
Sun M;Chi G;Xu J;Tan Y;Xu J;Lv S;Xu Z;Xia Y;Li L;Li Y
人间充质干细胞（hMSC）分化为成骨细胞对于治疗骨损伤具有重要的临床意义，细胞外基质（ECM）的硬度已被证明是hMSC分化的重要调节因素。本研究的目的是进一步阐明基质硬度如何通过整合素 α5/β1、FAK 和 Wnt 信号传导影响细胞内信号传导，从而调节 hMSC 的成骨表型。 hMSC 在涂有纤连蛋白的可调聚丙烯酰胺水凝胶上培养，其硬度对应于 13-16 kPa 和 62-68 kPa 的杨氏模量。 hMSC在凝胶上培养1周后，通过实时PCR评估α-1 I型胶原、BGLAP和RUNX2的基因表达。 hMSC 在凝胶上培养 24 小时后，通过蛋白质印迹分析评估与整合素 α5 相关的信号分子（FAK、ERK、p-ERK、Akt、p-Akt、GSK-3β、p-GSK-3β 和 β-catenin）。 α-1 I 型胶原蛋白、BGLAP 和 RUNX2 基因表达、钙沉积和 ALP 染色证明，62-68 kPa ECM 下成骨分化增加。在分化过程中，整合素α5/β1基因和蛋白表达增加，下游信号分子FAK、p-ERK、p-Akt、GSK-3β、p-GSK-3β和β-catenin蛋白表达增加，表明这些分子可以影响hMSCs的成骨分化。阻断整合素α5的抗体抑制了所检查的所有成骨细胞标志物的僵硬诱导的表达。特别是，α-1 I 型胶原、RUNX2 和 BGLAP 显着下调，表明整合素 α5 调节 hMSC 成骨分化。 FAK、ERK、p-ERK 和 β-catenin 蛋白的下游表达没有变化，而 Akt、p-Akt、GSK-3β 和 p-GSK-3β 则上调。此外，抗整合素α5抗体上调了Akt和p-Akt的表达，但在有或没有抗整合素α5抗体组之间，FAK、ERK和p-ERK没有观察到差异。同时，GSK-3β和p-GSK-3β的表达上调，并且β-连环蛋白水平在有或没有抗整合素α5抗体的组之间没有显示差异。由于 Akt、p-Akt、GSK-3β 和 p-GSK-3β 在有或没有抗整合素 α5 抗体的组之间显示出相同的变化，因此我们检测了它们之间的联系。在 Akt 抑制剂 Triciribine 存在下，p-Akt 和 p-GSK-3β 的表达被有效降低。然而，Akt、GSK-3β 和 β-连环蛋白没有变化。这些结果表明 p-GSK-3β 的表达受 p-Akt 在 62-68 kPa ECM 上的调节。综上所述，我们的结果提供了证据，证明基质硬度 (62-68 kPa) 通过整合素 α5 介导的机械转导事件影响 hMSC 的成骨结果。本文的在线版本 (10.1186/s13287-018-0798-0) 包含补充材料，可供授权用户使用。
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 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 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 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 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, and BGLAP were 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.
登录
查看更多内容
影响因子：
3.7
作者：
Hendesi H;Barbe MF;Safadi FF;Monroy MA;Popoff SN
通讯作者：
Popoff SN
DOI：
10.1016/j.biocel.2015.04.011
发表时间：
2015-07-01
影响因子：
4
作者：
Lambertini, Elisabetta;Penolazzi, Letizia;Piva, Roberta
通讯作者：
Piva, Roberta
影响因子：
5.6
作者：
Chim, Shek Man;Kuek, Vincent;Xu, Jiake
通讯作者：
Xu, Jiake
影响因子：
4.8
作者：
Barbolina, Maria V.;Liu, Yiuying;Stack, M. Sharon
通讯作者：
Stack, M. Sharon
影响因子：
1.2
作者：
Di Benedetto, Adriana;Brunetti, Giacomina;Mori, Giorgio
通讯作者：
Mori, Giorgio