PIEZO1 Ion Channel Mediates Ionizing Radiation-Induced Pulmonary Endothelial Cell Ferroptosis via Ca2+/Calpain/VE-Cadherin Signaling

Abstract

Pulmonary endothelial cell dysfunction plays an important role in ionizing radiation (IR)-induced lung injury. Whether pulmonary endothelial cell ferroptosis occurs after IR and what are the underlying mechanisms remain elusive. Here, we demonstrate that 15-Gy IR induced ferroptosis characterized by lethal accumulation of reactive oxygen species (ROS), lipid peroxidation, mitochondria shrinkage, and decreased glutathione peroxidase 4 (GPX4) and SLC7A11 expression in pulmonary endothelial cells. The phenomena could be mimicked by Yoda1, a specific activator of mechanosensitive calcium channel PIEZO1. PIEZO1 protein expression was upregulated by IR in vivo and in vitro. The increased PIEZO1 expression after IR was accompanied with increased calcium influx and increased calpain activity. The effects of radiation on lung endothelial cell ferroptosis was partly reversed by inhibition of PIEZO1 activity using the selective inhibitor GsMTx4 or inhibition of downstreaming Ca²⁺/calpain signaling using PD151746. Both IR and activation of PIEZO1 led to increased degradation of VE-cadherin, while PD151746 blocked these effects. VE-cadherin knockdown by specific siRNA causes ferroptosis-like phenomena with increased ROS and lipid peroxidation in the lung endothelial cells. Overexpression of VE-cadherin partly recused the ferroptosis caused by IR or PIEZO1 activation as supported by decreased ROS production, lipid peroxidation and mitochondria shrinkage compared to IR or PIEZO1 activation alone. In summary, our study reveals a previously unrecognized role of PIEZO1 in modulating ferroptosis, providing a new target for future mitigation of radiation-induced lung injury.

Keywords: Piezo1; VE-cadherin; calpain; ferroptosis; ionizing radiation.

FIGURE 1

Ionizing radiation induced induces pulmonary endothelial cell ferroptosis and increases expression of PIEZO1 protein. (A) Lipid peroxidation assessment in HULEC-5a cells, 24 h after exposure to 15-Gy of ionizing radiation (IR). Bar graphs showed relative levels of lipid peroxidation by C11-BODIPY staining in indicated cells. (B) ROS measurement in HULEC-5a cells, 24 h after IR. ROS levels were determined using H₂DCFDA and analyzed using flow cytometry. (C) Western blotting analysis of ACSL4, SLC7A11, DMT1, and GPX4 protein expression in HULEC-5a cells, 6, 12, and 24 h after IR. (D) Representative transmission electron microscopy images of HULEC-5a cells after IR. Yellow arrows show mitochondria. (E) Expression of GPX4 and SLC7A11 detected by immunohistochemistry assay in lungs of mice after IR. Scale bars: 100 µm (left) and 50 µm (right). (F) Western blot analysis of PIEZO1 protein expression in HULEC-5a cells after IR. (G) Expression of PIEZO1 protein detected by immunohistochemistry assay. Scale bars: 100 µm (left) and 50 µm (right). Data were plotted as means ± SEM. n = 3 independent repeats. **, p < 0.01 vs control.

FIGURE 2

PIEZO1 mediates pulmonary endothelial cell ferroptosis induced by ionizing radiation. (A) Lipid peroxidation assessment in HULEC-5a cells pre-treated with Yoda1 (2.5 µM) or GsMTx4 (5 µM) before ionizing radiation (IR). (B) ROS measurement in HULEC-5a cell pre-treated with Yoda1 (2.5 µM) or GsMTx4 (5 µM) 30 min before IR until the end of the experiment (24 h post IR). (C) Western blotting analysis of ACSL4, SLC7A11, DMT1, and GPX4 protein expression in HULEC-5a cell pre-treated with Yoda1 (2.5 µM) or GsMTx4 (5 µM) 30 min before IR. (D) Transmission electron microscopy images of HULEC-5a cells without radiation (control) or at 24 h after Yoda1 treatment. Yellow arrows show mitochondria. Data were plotted as means ± SEM. n = 3 independent repeats. **, p < 0.01 vs control; ^##, p < 0.01 vs GsMTx4; ^$$, p < 0.01 vs Yoda1; ^%%, p < 0.01 vs IR.

FIGURE 3

PIEZO1 mediates ionizing radiation-induced pulmonary endothelial cell ferroptosis by increasing intracellular calcium concentration and calpain activity. (A) Intracellular Ca²⁺ measured using Fluo4-AM (5 mM) across groups. Histograms showing differences in Ca²⁺ concentrations across groups. (B) Calpain activity assay in HULEC-5a cell pre-treated with Yoda1 (2.5 µM) or GsMTx4 (5 µM) for 24 h before ionizing radiation (IR). (C) Lipid peroxidation assessment in HULEC-5a cells pre-treated with PD151746 (20 µM) for 24 h before IR. (D) ROS measurement in HULEC-5a cell pre-treated with PD151746 (20 µM) for 24 h before IR or without radiation. (E) Lipid peroxidation assessment in HULEC-5a cells pre-treated with PD151746 (20 µM) and/or Yoda1 for 24 h. The Lipid peroxidation level of the control group is the same result shown in Figure 3C. The grouping experiment belongs to single batch of experiment. (F) ROS measurement in HULEC-5a cells pre-treated with PD151746 (20 µM) and/or Yoda1 for 24 h. (G) Western blotting analysis of ACSL4, SLC7A11, DMT1, and GPX4 expression in HULEC-5a cell pretreated with PD151746 (20 µM) for 24 h followed by IR and/or treatment with Yoda1. Data were plotted as means ± SEM. n = 3 independent repeats. **, p < 0.01 vs control. ^&&, p < 0.01 vs PD151746. ^##, p < 0.01 vs GsMTx4. ^$$, p < 0.01 vs Yoda1; ^%%, p < 0.01 vs IR.

FIGURE 4

VE-cadherin contributes to the ferroptosis-inducing effect of PIEZO1 (A) Expression of VE-cadherin detected by immunohistochemistry assay in lungs of mice 24 h after exposure to 15-Gy of ionizing radiation (IR) or no radiation. Scale bars: 100 µm (left) and 50 µm (right) (B) Western blotting analysis of VE-cadherin and VE-cadherin fragment in HULEC-5a cell at 30 min, 1, 2 and 4 h after treatment with Yoda1. (C) Western blotting analysis of VE-cadherin and VE-cadherin fragment expression in HULEC-5a cell at 6, 12, and 24 h after exposure to IR or no radiation. (D) Western blot and RT-PCR analyses showing successful knockdown of VE-cadherin in HULEC-5a cells. (E) ROS measurement in HULEC-5a cells with knockdown of VE-cadherin. (F) Lipid peroxidation assessment in HULEC-5a cells with knockdown of VE-cadherin. (G) Transmission electron microscopy images of HULEC-5a cells with or without knockdown of VE-cadherin. (H) ROS measurement in HULEC-5a cells with knockdown of VE-cadherin subjected to IR or no radiation. The results are from the same batch of experiment shown in Figure 4E (I) Lipid peroxidation assessment in HULEC-5a cells with knockdown of VE-cadherin subjected to IR or no radiation. The results are from the same batch of experiment shown in Figure 4F (J) Western blot and RT-PCR analyses showing successful overexpression of VE-cadherin in HULEC-5a cells. (K) ROS measurement in HULEC-5a cells with overexpression of VE-cadherin with Yoda1 treatment. (L) Lipid peroxidation assessment in HULEC-5a cells with overexpression of VE-cadherin with Yoda1 treatment. (M) ROS measurement in HULEC-5a cells with overexpression of VE-cadherin with IR. The results are from the same batch of experiment shown in Figure 4K (N) Lipid peroxidation assessment in HULEC-5a cells with overexpression of VE-cadherin with IR. The results are from the same batch of experiment shown in Figure 4L (O) Transmission electron microscopy images of HULEC-5a cells with overexpression of VE-cadherin with Yoda1 treatment. (P) Transmission electron microscopy images of HULEC-5a cells with overexpression of VE-cadherin before IR. Data were plotted as means ± SEM. n = 3 independent repeats. **, p < 0.01 vs control; ^##, p < 0.01 vs NC; ^%%, p < 0.01 vs IR; ^$$, p < 0.01 vs IR + NC (negative control; ^{^^}, p < 0.01 vs Yoda1; ⁺⁺, p < 0.01 vs Yoda1+NC).

FIGURE 5

A schematic diagram summarizing the mechanism of PIEZO1 in regulating ionizing radiation-induced ferroptosis in lung endothelial cells. Ionizing radiation leads to increased PIEZO1 expression in lung endothelial cells. Increased expression of PIEZO1 increases intracellular Ca²⁺ concentration, which further increases calpain activity. Calpain increases degradation of VE-cadherin and promotes the development of ferroptosis possibly by YAP signaling.