Piezo1 mediates electrotaxis of alveolar epithelial cells via calcium-dependent PI3K/Akt signaling

Alveolar type II (AT2) epithelial cells play a vital role in lung injury repair, where their directed migration toward damaged regions is essential for epithelial regeneration. However, the underlying regulatory mechanisms remain poorly understood. This study aimed to determine whether direct-current electric fields (EFs) act as directional cues for AT2 cell migration and to identify the key molecular mediators and intracellular signaling pathways involved.

RNA-seq data from acute respiratory distress syndrome patient-derived AT2 cells were integrated with charged membrane protein signatures to identify candidate genes associated with electrotaxis. Cell migration and calcium dynamics were assessed using time-lapse imaging and live-cell calcium imaging, while pharmacological inhibition, quantitative polymerase chain reaction, and Western blot analyses were employed to investigate molecular mechanisms.

Integrative transcriptomic analysis identified Piezo1 as a candidate regulator associated with AT2 cell electrotaxis. Functional assays in A549 cells demonstrated that exposure to a direct-current EF (100 mV/mm) significantly promoted cathode-directed migration and increased Piezo1 expression at both mRNA and protein levels. Inhibition of Piezo1 using the specific antagonist GSMTx4, or chelation of intracellular calcium with BAPTA-AM, markedly disrupted EF-induced directional migration, as evidenced by reduced migration velocity and loss of directionality. Live-cell calcium imaging confirmed that Piezo1 is essential for EF-induced calcium influx. Moreover, Western blot analysis revealed that EF stimulation elevated the phospho-phosphatidylinositol 3-kinase and protein kinase B, which was significantly attenuated upon Piezo1 inhibition.

Piezo1-mediated calcium influx and subsequent phospho-phosphatidylinositol 3-kinase/protein kinase B activation drive the electrotaxis of AT2 cells. These findings identify Piezo1 as a key bioelectrical sensor linking EFs to intracellular calcium signaling and directional migration, and suggest its potential as a therapeutic target for promoting epithelial regeneration in acute lung injury.