Mesenchymal stem cell-secreted KGF ameliorates acute lung injury via the Gab1/ERK/NF-κB signaling axis

Abstract

Background: The epithelial sodium channel (ENaC) situated in the apical membrane of alveolar epithelial type 2 (AT2) cells is beneficial to edematous fluid reabsorption in acute lung injury (ALI). Recently, mesenchymal stem cells (MSCs), particularly their secretome, has emerged as a novel approach for treating pulmonary diseases. Among these secreted factors, keratinocyte growth factor (KGF) plays a critical role in mediating alveolar epithelial repair during ALI by enhancing epithelial cell proliferation, restoring epithelial integrity, and alleviating pulmonary edema, making it a promising candidate for therapeutic strategies. This study primarily focused on investigating the impact of KGF secreted from MSC on ALI, and clarifying its specific mechanism in regulating the expression of ENaC.

Methods: Lipopolysaccharide (LPS)-stimulated primary mouse AT2 cells were treated with KGF in vitro, and western blots along with immunofluorescence assays were performed to investigate the regulatory mechanism of KGF on ENaC protein expression. To further confirm the role of mouse bone marrow MSC-derived KGF, co-culture experiments with AT2 cells and either MSC or MSC with KGF knockdown (MSC-siKGF) were conducted. In vivo, an ALI model was established in mice by LPS-induced lung injury. The therapeutic effects of tail vein-injected MSC or MSC-siKGF were assessed using hematoxylin-eosin staining, lung wet/dry weight ratio, and alveolar fluid clearance.

Results: In primary mouse AT2 cells, KGF stimulation effectively restored the reduction of growth factor receptor-bound protein 2-associated binding protein 1 (Gab1) and α/γ-ENaC protein levels induced by LPS. KGF inhibited the activation of the LPS-induced extracellular regulated protein kinases (ERK) and nuclear factor-kappaB (NF-κB) signaling pathway. Treatment with the ERK pathway inhibitor PD98059 reversed the LPS-induced reduction in ENaC protein levels but had no effect on Gab1 levels. In addition, PD98059 suppressed LPS-induced activation of the NF-κB signaling pathway. Further analysis revealed that LPS stimulation weakened the interaction between the NF-κB p65 subunit and inhibitor kappaB (IκB), while KGF enhanced this interaction and inhibited the nuclear translocation of p65. Both KGF and the NF-κB inhibitor QNZ reversed the LPS-induced downregulation of ENaC protein levels and gene expression. Furthermore, both agents effectively restored the functional activity of ENaC channels. Co-culture with MSCs increased Gab1 protein levels, inhibited ERK/NF-κB signaling activation, and suppressed p65 nuclear translocation in LPS-treated AT2 cells, whereas these effects were attenuated in cells co-cultured with MSC-siKGF. In an ALI mouse model, tail-vein injection of MSCs alleviated lung injury and pulmonary edema, while the therapeutic effects of MSC-siKGF were weaker they were partly restored by the combination of QNZ.

Conclusions: Our study validated that the efficacy of MSCs in the treatment of edematous ALI was significantly associated with KGF, which potentially enhanced the upregulation of ENaC through the Gab1/ERK/NF-κB signaling pathway.

Keywords: Acute lung injury; Epithelial sodium channel; Keratinocyte growth factor; Mesenchymal stem cell; Nuclear factor-kappaB.

Fig. 1

KGF increased Gab1 and ENaC protein expression in AT2 cells. A An analysis of the protein interaction network for the KGFR protein was performed utilizing the string database. B The results of molecular docking were visualized, subsequently followed by a detailed magnification of the interacting amino acid pairs. C Flow-cytometry results showed 90.0% positivity for the AT2 cell surface marker SPC. D Representative graphs showed the effect of LPS or KGF stimulation on α/γ-ENaC and Gab1 protein expression in AT2 cells. E, F Statistical data were obtained from western blots and quantified through gray analysis. **P < 0.01, compared with control group; ^&&P < 0.01, ^&&&P < 0.001, compared with LPS group; n = 3. Comparison among different groups was analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. G, H Immunofluorescence staining of α/γ-ENaC (shown in red) was observed in AT2 cells from different treatment groups. Scale bar: 25 μm

Fig. 2

ERK1/2 interacted with Gab1 protein in AT2 cells. A Protein interaction network analysis of the Gab1 protein was conducted using the string database. B Visualization of molecular docking results, followed by local magnification of the interacting amino acid pairs. C Co-IP assay demonstrated the interaction between Gab1 and ERK1/2

Fig. 3

KGF upregulated ENaC protein expression by inhibiting the ERK phosphorylation. A, B Representative and statistical data of t-ERK1/2 and p-ERK1/2 after PD98059 or KGF treatment. C–F Representative western blot and statistical data for Gab1 and α/γ-ENaC. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group; ^&P < 0.05, ^&&&P < 0.001, compared with LPS group; ^#P < 0.05, compared with LPS + PD group, n = 3. Comparison among different groups was analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test

Fig. 4

LPS-induced activation of the NF-κB was suppressed by KGF or PD98059. A Illustrative western blot of p-p65/t-p65 and p-IκB/t-IκB in whole cell lysates, along with the nuclear protein p65. B Graphical depiction of data derived from western blot assays for nuclear p65 protein. C Graphical illustration of data acquired through western blot analysis of whole cell lysates, showing ratios of phosphorylated to total proteins for p65 and IκB. D Co-IP assay demonstrates the interaction between t-p65 and t-IκB. E, F Representative western blot assay and corresponding graphical representation for Co-IP assay. *P < 0.05, ***P < 0.001, compared with control group; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001, compared with LPS group, n = 3–5. Comparison among the different groups was analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test

Fig. 5

KGF upregulated ENaC protein expression through inhibiting NF-κB phosphorylation. A Illustrative western blot of α/γ-ENaC in whole cell lysates, in addition to the nuclear and pulp protein of p65. B Statistical graph of pulp protein p65. C Nuclear protein of p65. D, E Statistical graph of α/γ-ENaC protein expression in whole cell lysates. F Electrophoretic mobility shift assay. G–I The mRNA expression of ENaC by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001, compared with LPS group, n = 3–4. Comparisons among different groups were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test

Fig. 6

KGF rescues ENaC function. A Schematic representation of Calu-3 cells cultured under air–liquid interface conditions in a Transwell. B Change in TEER of the Calu-3 monolayer over the culture period. ***P < 0.001, compared with day 4. C Isc traces recorded from the Ussing chamber perfusion experiment. D Statistical analysis of ASI. E, F Representative graphs and corresponding graphical representation of ASL height. ***P < 0.001, compared with control group; ^&&P < 0.01, ^&&&P < 0.001, compared with LPS group, n = 3. Comparisons among different groups were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test

Fig. 7

MSC-secreted KGF regulated Gab1 and ENaC protein level in primary mouse AT2 cells. A The co-culture system with AT2 cells and MSCs. B KGF knockdown efficiency in primary MSCs. C–F Representative western blot and corresponding graphical representation for Gab1 and α/γ-ENaC. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001, compared with LPS group; ^#P < 0.05, ^##P < 0.01, compared with LPS + MSC-NC group, n = 3–4. Comparisons among different groups were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test

Fig. 8

MSC-secreted KGF inhibited ERK/NF-κB signaling pathway activation. A Representative western blot of p-ERK1/2/t-ERK1/2, p-p65/t-p65, and p-IκB/t-IκB in whole cell lysates, as well as the nuclear protein fraction of p65. B–E Corresponding graphical representation of data obtained from western blot assays for p-ERK1/2/t-ERK1/2, p-p65/t-p65, and p-IκB/t-IκB in whole cell lysates, and nuclear protein p65, respectively. **P < 0.01, ***P < 0.001, compared with control group; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001, compared with LPS group; ^##P < 0.01, ^###P < 0.001, compared with LPS + MSC-NC group, n = 3–4. Comparisons among different groups were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test

Fig. 9

NF-κB participated in the KGF-mediated improvement of pathological damage and upregulation of ENaC expression in vivo. A HE staining of mouse lung tissues. Scale bar: 50 µm. B ALI scores. C AFC of mice. D, E The lung W/D weight ratio and lung index. F–H Representative western blot and corresponding graphical representation for α/γ-ENaC in lung tissues. ***P < 0.001, compared with control group; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001, compared with LPS group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared with LPS + MSC group; ^$P < 0.05, ^$$P < 0.01, ^$$$P < 0.001, compared with LPS + MSC-siKGF group, n = 3–4. Comparisons among different groups were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test

Fig. 10

The schematic diagram depicts the function of MSCs in enhancing the expression of ENaC in ALI induced by LPS. The accumulation of edema fluid in alveoli was a major characteristic in ALI. MSC-secreted KGF upregulates Gab1 protein expression and inhibits the activation of the ERK/NF-κB signaling pathway, thereby upregulating the ENaC protein level in AT2 cells and alleviating LPS-induced pulmonary edema. ALI, acute lung injury; AT1 cell, alveolar type 1 cell; AT2 cell, alveolar type 2 cell; ENaC, epithelial sodium channel; ERK1/2, extracellular regulated protein kinases1/2; Gab1, growth factor receptor binding 2-associated binding protein 1; IκB, inhibitor κB; KGF, keratinocyte growth factor; MSCs, mesenchymal stem cells; NF-κB, nuclear factor-kappa B