Department of Pediatric Dentistry / Special Needs Dentistry, Division of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8549, Japan; Department of Pediatric Dentistry, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, 770-8504, Japan.
Department of Pediatric Dentistry / Special Needs Dentistry, Division of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8549, Japan.
Biochem Biophys Res Commun. 2023 Nov 19;682:39-45. doi: 10.1016/j.bbrc.2023.09.080. Epub 2023 Sep 27.
Cells sense and respond to extracellular mechanical stress through mechanotransduction receptors and ion channels, which regulate cellular behaviors such as cell proliferation and differentiation. Among them, PIEZO1, piezo-type mechanosensitive ion channel component 1, has recently been highlighted as a mechanosensitive ion channel in various cell types including mesenchymal stem cells. We previously reported that PIEZO1 is essential for ERK1/2 phosphorylation and osteoblast differentiation in bone marrow-derived mesenchymal stem cells (BMSCs), induced by hydrostatic pressure loading and treatment with the PIEZO1-specific activator Yoda1. However, the molecular mechanism underlying how PIEZO1 induces mechanotransduction remains unclear. In this study, we investigated that the role of the C-terminus in regulating extracellular Ca influx and activating the ERK1/2 signaling pathway. We observed the activation of Fluo-4 AM in the Yoda1-stimulated human BMSC line UE7T-13, but not in a calcium-depleted cell culture medium. Similarly, Western blotting analysis revealed that Yoda1 treatment induced ERK1/2 phosphorylation, but this induction was not observed in calcium-depleted cell culture medium. To investigate the functional role of the C-terminus of PIEZO1, we generated HEK293 cells stably expressing the full-length mouse PIEZO1 (PIEZO1-FL) and a deletion-type PIEZO1 lacking the C-terminal intracellular region containing the R-Ras-binding domain (PIEZO1-ΔR-Ras). We found that Yoda1 treatment predominantly activated Flou-4 AM and ERK1/2 in PIEZO1-FL-trasfected cells but neither in PIEZO1-ΔR-Ras-transfected cells nor control cells. Our results indicate that the C-terminus of PIEZO1, which contains the R-Ras binding domain, plays an essential role in Ca influx and activation of the ERK1/2 signaling pathway, suggesting that this domain is crucial for the mechanotransduction of osteoblastic differentiation in BMSCs.
细胞通过机械转导受体和离子通道感知和响应细胞外机械应激，这些受体和离子通道调节细胞增殖和分化等细胞行为。其中，PIEZO1，即压电型机械敏感离子通道成分 1，最近在各种细胞类型（包括间充质干细胞）中被强调为机械敏感离子通道。我们之前报道过，PIEZO1 在骨髓间充质干细胞（BMSCs）中对于由静压加载和 PIEZO1 特异性激活剂 Yoda1 处理诱导的 ERK1/2 磷酸化和成骨细胞分化是必不可少的。然而，PIEZO1 如何诱导机械转导的分子机制尚不清楚。在这项研究中，我们研究了 C 末端在调节细胞外 Ca 流入和激活 ERK1/2 信号通路中的作用。我们观察到 Yoda1 刺激的人 BMSC 系 UE7T-13 中 Fluo-4 AM 的激活，但在钙耗尽的细胞培养基中则没有。同样，Western blot 分析表明，Yoda1 处理诱导了 ERK1/2 的磷酸化，但在钙耗尽的细胞培养基中则没有观察到这种诱导。为了研究 PIEZO1 的 C 末端的功能作用，我们生成了稳定表达全长小鼠 PIEZO1（PIEZO1-FL）和缺乏包含 R-Ras 结合域的细胞内 C 末端区的缺失型 PIEZO1（PIEZO1-ΔR-Ras）的 HEK293 细胞。我们发现，Yoda1 处理主要在 PIEZO1-FL 转染的细胞中激活 Flou-4 AM 和 ERK1/2，但在 PIEZO1-ΔR-Ras 转染的细胞和对照细胞中则没有。我们的结果表明，PIEZO1 的 C 末端包含 R-Ras 结合域，在 Ca 流入和 ERK1/2 信号通路的激活中发挥着重要作用，这表明该结构域对于 BMSCs 中成骨细胞分化的机械转导至关重要。
