Piezo1 Regulates the Migration and Adhesion of Osteoblasts during the Fracture Healing Process while Mediating Signal Transduction through the PI3K/AKT/mTOR/Cyclin D1 Pathway

Abstract

Background: Osteoblasts play a pivotal role during fracture healing. Piezo-Type Mechanosensitive Ion Channel Component 1 (Piezo1) is crucial in regulating bone homeostasis. Hence, the objective of this investigation was to explore the molecular mechanisms associated with Piezo1 in the fracture healing process and to clarify its influence on the physiological activities of osteoblasts. Methods: At the outset, the MC3T3-E1 cells were categorized into three sets: the control group, the Yoda1-treated group, and the Grammostola spatulata Mechanotoxin 4 (GsMTx4)-treated group. Subsequently, the cells underwent transfection with Scramble siRNA (Sc siRNA), Piezo1-specific siRNA, pcMV-FLAG Expression (pcMV-FLAG) vector, or pcMV-FLAG-Piezo1. The quantification of gene and protein expression levels was carried out utilizing quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Following that, the effects of manipulating Piezo1 expression, along with the use of the Piezo1 agonist (Yoda1) and inhibitor (GsMTx4), on cell proliferation, adhesion, and migration were assessed through the Cell Counting Kit-8 (CCK-8) assay, cell adhesion assay, scratch assay, and Transwell assay. Ultimately, the influence of Yoda1 and GsMTx4 on the Piezo1/Phosphoinositide 3-kinase (PI3K)/Protein kinase B (AKT)/Mammalian target of rapamycin (mTOR)/Cyclin D1 signaling pathway was examined utilizing Western blot analysis. Results: The overexpression of Piezo1 and the presence of Yoda1 significantly enhanced the viability, adhesion, and metastatic capabilities of MC3T3-E1 osteoblasts (p < 0.01 and p < 0.001). Conversely, Piezo1 knockdown and the application of GsMTx4 significantly suppressed the viability, adhesion, and metastasis of MC3T3-E1 cells (p < 0.01 and p < 0.001). Moreover, Yoda1 induced an elevation in the protein levels of Piezo1, Cyclin D1, p-PI3K, p-AKT, and p-mTOR (p < 0.01 and p < 0.001). In contrast, GsMTx4 resulted in a marked decrease in their expression levels (p < 0.05 and p < 0.01). Conclusion: Piezo1 modulates the migration and adhesion of osteoblasts during the bone healing process, acting as a mediator in the signal transduction through the PI3K/AKT/mTOR/Cyclin D1 pathway.