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Piezo1介导基底硬度调控软骨细胞初级纤毛形态

Piezo1 Mediates the Regulation of Substrate Stiffness on Primary Cilia in Chondrocytes

    摘要
  • 摘要:
    目的 研究基底硬度如何调控软骨细胞初级纤毛的形态，并进一步探究Piezo1如何介导基底硬度调控初级纤毛的形态。
    方法 将聚二甲基硅氧烷（polydimethylsiloxane, PDMS）固化剂和主剂分别以1∶10（硬）、1∶50（中硬度）和1∶70（软）的比例混合，制成厚度为1 mm且具有不同硬度的基底膜：硬基底（2.21±0.12 ） MPa、中硬度基底（54.47±6.06） kPa和软基底（2.13±0.10） kPa。利用3种不同硬度基底培养软骨细胞；再分别用Tubastatin A（Tub A）抑制组蛋白脱乙酰酶6（histone deacetylase 6, HDAC6）、Piezo1激活剂Yoda1和抑制剂GsMTx4处理细胞，基于免疫荧光技术分析HDAC6、Yoda1和GsMTx4对软骨细胞形态和初级纤毛长度的影响。
    结果 硬基底会增加软骨细胞铺展面积，免疫荧光显示与中等基底和软基底相比在硬基底上细胞骨架和细胞核面积显著增加（P<0.05），并且初级纤毛显著伸长（P<0.05），但是并不影响初级纤毛的出现率。软骨细胞HDAC6的活性随着基底硬度变软依次升高，抑制软骨细胞HDAC6的活性后，细胞骨架面积和细胞核面积以及初级纤毛长度在硬基底上增加更为显著（P<0.05）。经测试，加入Piezo1激活剂和抑制剂可以调控软骨细胞HDAC6的活性，并且初级纤毛的长度在加入激活剂Yoda1后显著增加（P<0.05），而在经抑制剂GsMTx4处理后，初级纤毛长度在硬基底上显著缩短（P<0.05）。
    结论 培养皿基底硬度和Piezo1可能都通过调控HDAC6活性进而影响软骨细胞初级纤毛形态。

     

    Abstract:
    Objective To investigate how substrate stiffness regulates the morphology of primary cilia in chondrocytes and to illustrate how Piezo1 mediates the morphology regulation of primary cilia by substrate stiffness.
    Methods Polydimethylsiloxane (PDMS) curing agent and the main agent (Dow Corning, Beijing, China) were mixed at the ratio of 1∶10 (stiff), 1∶50 (medium stiffness), and 1∶70 (soft), respectively, to prepare substrate films with the thickness of 1 mm at different levels of stiffness, including stiff substrate of (2.21±0.12) MPa, medium-stiffness substrate of (54.47±6.06) kPa, and soft substrate of (2.13±0.10) kPa. Chondrocytes were cultured with the substrates of three different levels of stiffness. Then, the cells were treated with Tubastatin A (Tub A) to inhibit histone deacetylase 6 (HDAC6), Piezo1 activator Yoda1, and inhibitor GsMTx4, respectively. The effects of HDAC6, Yoda1, and GsMTx4 on chondrocyte morphology and the length of primary cilia were analyzed through immunofluorescence staining.
    Results The stiff substrate increased the spread area of the chondrocytes. Immunofluorescence assays showed that the cytoskeleton and the nuclear area of the cells on the stiff substrate were significantly increased (P<0.05) and the primary cilia were significantly extended (P<0.05) compared with those on the medium-stiffness and soft substrates. However, the presence rate of primary cilia was not affected. The HDAC6 activity of chondrocytes increased with the decrease in substrate stiffness. When the activity of HDAC6 was inhibited, the cytoskeletal area, the nuclei area, and the primary cilium length were increased more significantly on the stiff substrate (P<0.05). Further testing showed that Piezo1 activator and inhibitor could regulate the activity of HDAC6 in chondrocytes, and that the length of primary cilia was significantly increased after treatment with the activator Yoda1 (P<0.05). On the other hand, the length of primary cilia was significantly shortened on the stiff substrate after treatment with the inhibitor GsMTx4 (P<0.05).
    Conclusion Both substrate stiffness and Piezo1 may affect the morphology of chondrocyte primary cilia by regulating HDAC6 activity.

     

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