Suppression of Skp2 contributes to sepsis-induced acute lung injury by enhancing ferroptosis through the ubiquitination of SLC3A2

Abstract

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The inflammatory cytokine storm causes systemic organ damage, especially acute lung injury in sepsis. In this study, we found that the expression of S-phase kinase-associated protein 2 (Skp2) was significantly decreased in sepsis-induced acute lung injury (ALI). Sepsis activated the MEK/ERK pathway and inhibited Skp2 expression in the pulmonary epithelium, resulting in a reduction of K48 ubiquitination of solute carrier family 3 member 2 (SLC3A2), thereby impairing its membrane localization and cystine/glutamate exchange function. Consequently, the dysregulated intracellular redox reactions induced ferroptosis in pulmonary epithelial cells, leading to lung injury. Finally, we demonstrated that intravenous administration of Skp2 mRNA-encapsulating lipid nanoparticles (LNPs) inhibited ferroptosis in the pulmonary epithelium and alleviated lung injury in septic mice. Taken together, these data provide an innovative understanding of the underlying mechanisms of sepsis-induced ALI and a promising therapeutic strategy for sepsis.

Keywords: Cytokine storm; Ferroptosis; SLC3A2; Sepsis; Skp2.

Fig. 1

Cytokine storm during sepsis inhibited Skp2 expression in lung epithelial cells. A Experimental setup and sample collection (generated by Biorender). BALF samples were collected from septic patients and healthy donors (n = 6). C57BL/6 mice were randomly divided into the sham or CLP sepsis groups (n = 4). The murine monocyte/macrophage line Raw264.7 was stimulated with LPS and the culture medium was collected after 12 h (from Raw264.7 cell-conditioned medium, RCM). MLE-12 murine lung epithelial cells were treated with RCM or LPS for 4 h. B, C The mRNA and protein expression of Skp2 in the BALF of septic patients and healthy donors were measured by qPCR (B) and western blotting (C). D–F Lung tissue of mice was collected 24 h after CLP surgery, and the protein expression of Skp2 in the lungs was measured by western blotting (D) or immunofluorescence staining (E, F). Scale bar = 20 μm. G–I The following four groups were included in the in vitro experiment: control group (Con.), LPS treatment group (LPS), Raw264.7 conditioned medium treatment group (RCM), and RCM plus LPS costimulation group (RCM + LPS). MLE-12 cells were stimulated for 4 h, after which Skp2 protein expression was measured by western blotting (G) and immunofluorescence staining (H, I). Scale bar = 20 μm. J The mouse lung resident macrophage line MH-S was stimulated with LPS and the cell culture supernatant were collected as MH-S-conditioned medium (MCM). MLE-12 cells were then divided into the following groups: control group (Con.), LPS treatment group (LPS), MH-S conditioned medium stimulation group (MCM), and MCM plus LPS costimulation group (MCM + LPS). The data are presented as the means ± standard deviations (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 2

Skp2 deficiency induced ferroptosis of alveolar epithelial cells during sepsis. Wildtype and Skp2^+/− C57BL/6 mice were randomly assigned to the sham or CLP group (n = 4 for Western blotting, n = 6 for other analyses). A Pulmonary mitochondrial ultrastructure was visualized by electron microscopy, scale bar = 500 μm. B The Fe²⁺ levels in 10 mg lung tissues were examined with iron assay kit. C Lipid peroxidation was examined by measuring the MDA content in the lungs. D The GSSG/GSH ratio in the lung was measured with a GSSG/GSH quantification kit. The relative protein expression of GPX4 and Skp2 in the lungs was measured by western blotting (E) and immunofluorescence staining (F). G, H MLE-12 lung epithelial cells were transfected with Skp2 lentivirus (Skp2-3*Flag-PGK-Puro) to overexpress Skp2 or with control lentivirus, and the transfected cells were then selected with puromycin. G Lipid peroxidation was measured by flow cytometry using Bodipy. H The fluorescent dye Bodipy was used to visualize lipid peroxidation (green) and reduction (red) under a fluorescence microscope, scale bar = 10 μm. I, J Skp2 in MLE-12 cells was knocked down by siRNA, and the cells were treated with RCM for 4 h. I Cells transfected with the lipid peroxidation probe Bodipy were analyzed by flow cytometry for the detection of positive cells. J The fluorescent dye Bodipy was used to visualize lipid peroxidation (green) and reduction (red) under a fluorescence microscope. Scale bar = 10 μm. The data are presented as the means ± standard deviations (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 3

RCM-induced activation of the MEK-ERK pathway mediated Skp2 inhibition in alveolar epithelial cells. A MLE-12 cells were treated with RCM, and changes in Skp2 and the MEK/ERK pathway, the p38 pathway, and the JNK pathway were measured by western blotting. B MLE-12 cells were subjected to treatment with RCM and specific inhibitors targeting the MEK, p38, and JNK signaling pathways. Alterations in protein expression levels in MLE-12 cells were assessed using western blot analysis. C MLE-12 cells were treated with RCM and a specific activator targeting MEK/ERK (C16-PAF and (rel)-AR234960), and the protein levels were measured by western blotting. D MLE-12 cells were treated with RCM and/or a MEK/ERK inhibitor (AZD8330/FR180204). The relative protein expression of MEK/ERK kinase pathway and Skp2 was measured by western blotting. E Western blotting was used to measure the expression of Skp2 and GPX4 in mouse lung epithelial cells. F Cells transfected with the lipid peroxidation probe Bodipy were analyzed by flow cytometry for the detection of positive cells. G The fluorescent dye Bodipy was used to visualize lipid peroxidation (green) and reduction (red) under a fluorescence microscope. Scale bar = 10 μm. The data are presented as the means ± standard deviations (ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 4

Skp2 deficiency aggravated sepsis-induced ALI via the induction of ferroptosis. A Experimental setup for C57BL/6 (generated by Biorender). Wildtype and Skp2^+/− C57BL/6 mice were randomly divided into the sham, CLP and CLP + Fer-1 groups, C57BL/6 mice were intravenously injected with Fer-1 (3 mg/kg). n = 24 for survival analysis, n = 6 for other analyses. Survival curve analysis was conducted to evaluate the 7 day survival rate of the mice, while the remaining measurements were carried out at the 24-h time point. B Kaplan–Meier survival analysis of the mice. C The protein concentration in BALF (mg/mL) was determined by BCA Protein Assay Kits. D Total cell numbers in BALF were counted. E Wet/dry weight ratio of lung tissues. F H&E staining of lung tissues, scale bar = 100 μm. G, H TUNEL staining of lung tissues (scale bar = 20 μm) and quantification of TUNEL-positive cells among the total cells in each group. The data are presented as the means ± standard deviations (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 5

Skp2 promoted the ubiquitination of SLC3A2 via a K48-linked nondegradative mechanism. A MLE-12 cells overexpressing Skp2 were subjected to immunoprecipitation-mass spectrometry (IP-MS). The identified proteins were annotated with GO terms. B The expression levels of Skp2, GPX4 and SLC3A2 in the lungs of wildtype/Skp2^+/− Sham and CLP mice were measured by western blotting. C HEK293T cells were transfected with Flag-Skp2, Myc-SLC3A2, or HA-Ub and treated with MG132, and the IP samples and whole cell lysates were analyzed by immunoblotting. D HEK293T cells were transfected with different mutants, and the IP samples and whole cell lysates were analyzed by immunoblotting. E HEK293T cells were transfected with Flag-Skp2, Myc-SLC3A2, or K48/K63/wildtype-Ub and treated with MG132, and the IP samples and whole cell lysates were analyzed by immunoblotting. F IP samples and whole cell lysates of HEK293T cells transfected with Flag-Skp2 and Myc-SLC3A2 without MG132 treatment were analyzed by immunoblotting. G The distribution of Skp2 and SLC3A2 in MLE-12 cells was detected by immunofluorescence staining, scale bar = 10 μm

Fig. 6

Skp2 deficiency triggered ferroptosis by inhibiting SLC3A2 ubiquitination and disrupting cystine/glutamate exchange. A–C MLE-12 mouse lung epithelial cells were transfected with Skp2-3*Flag-PGK-Puro to overexpress Skp2, and Puro Lentivirus transfected MLE-12 cells were used as controls. A EpCAM functions as an indicator of epithelial cells and was used to indicate the interplay between SLC3A2 and the alveolar epithelium. The distributions of Skp2, SLC3A2 and EpCAM were detected by immunofluorescence staining. Scale bar = 20 μm; B, C transfected MLE-12 cells were stimulated with RCM or Skp2 inhibitor SZL P1-41. B The glutamate content in the cell culture supernatant was analyzed with a glutamate assay kit. C Cystine uptake by MLE-12 cells was analyzed with a cystine uptake kit. D–F MLE-12 cells were transfected with Skp2 siRNA to knock down Skp2. Scrambled siRNA was used as a control. D The distributions of Skp2 and SLC3A2 were detected by immunofluorescence staining in MLE-12 cells transfected with Skp2 siRNA, scale bar = 20 μm; E, F transfected cells were stimulated with RCM or control medium (Con.). E The glutamate content in the medium of MLE-12 cells transfected with Skp2 siRNA and stimulated with RCM was analyzed by a glutamate assay kit; F the cystine uptake of MLE-12 cells transfected with Skp2 siRNA and stimulated with RCM was determined with a cystine uptake detection kit. The data are presented as the means ± standard deviations (ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 7

Skp2 overexpression improves sepsis-induced acute lung injury. Mice were assigned to Vehicle-Sham group, Vehicle-CLP group, Skp2-OE-sham (LNP injected) group or Skp2-OE-CLP (LNP injected) group. For LNP mice, lipid nanoparticles (LNPs) carrying Skp2 mRNA were injected via the tail vein. A Schematic diagram of LNP assembly (generated by Biorender). B After administering LNPs via intravenous injection, luciferase intensity in the heart, liver, spleen, lung and kidney was measured. C Survival analysis of each group, n = 24. D, E Histochemical staining (scale bar = 50 μm) and H&E staining (scale bar = 100 μm) of lungs were used to evaluate lung injury. F Cell counts in BALF of each group. G Protein concentration in BALF (mg/mL) of mice in each group. H Wet/dry ratio of lung tissues in each group. I Examination of the Fe²⁺ levels in lungs. J Lung GSSG/GSH ratio. K Lipid peroxidation as measured by the MDA concentration in the lungs. L TUNEL staining of the lungs of mice in each group, scale bar = 20 μm. M Quantification of TUNEL-positive cells among the total cell in each group. N Electron microscopy was used to visualize the ultrastructure of pulmonary mitochondria, scale bar = 500 μm. The data are presented as the means ± standard deviations (ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 8

Skp2 regulates ferroptosis by modulating the distribution of SLC3A2 and affecting cystine and glutamate transport (schematic diagram by Biorender). Skp2 mediates K48-linked ubiquitination but not SLC3A2 degradation, which is essential for the normal transportation of cystine and glutamate antiporters through the cell membrane. In sepsis, inflammatory cytokine storms suppress Skp2 expression, leading to reduced SLC3A2 ubiquitination and hence hindering the localization of SLC3A2 to the cell membrane. The abnormal distribution of SLC3A2 disturbed its exclusion and led to the intracellular accumulation of glutamate. GSH levels were reduced, ultimately increasing ferroptosis in lung epithelial cells. Thus, Skp2 overexpression can reduce mortality and ameliorate lung injury in septic mice by inhibiting ferroptosis (generated by Biorender)