1a, 25-Dihydroxyvitamin D3 Inhibits Transforming Growth Factor β1-Induced Epithelial-Mesenchymal Transition via β-Catenin Pathway
Introduction
The epithelial-mesenchymal transition (EMT) is a biological process where epithelial cells lose their characteristic features and acquire a mesenchymal phenotype. This transformation is characterized by the loss of epithelial markers such as E-cadherin and the gain of mesenchymal markers like α-smooth muscle actin (α-SMA) and fibronectin (FN). EMT is a critical mechanism in various pathological conditions, including airway remodeling in asthma. Airway remodeling involves structural changes in the airway walls, such as thickening of the basement membrane, increased smooth muscle mass, and fibrosis, which contribute to the chronicity and severity of asthma.
Transforming growth factor β1 (TGF-β1) is a multifunctional cytokine that plays a pivotal role in inducing EMT. TGF-β1 activates several signaling pathways, including the Smad, non-Smad, and β-catenin pathways, to promote EMT. Among these, the Wnt/β-catenin pathway is particularly significant in the context of airway remodeling in asthma. The Wnt/β-catenin pathway regulates cell proliferation, migration, and differentiation, and its dysregulation is implicated in various diseases, including cancer and fibrosis.
Vitamin D, specifically its active metabolite 1α, 25-dihydroxyvitamin D3 (1α, 25(OH)2D3), has been shown to have anti-fibrotic and immunomodulatory effects. Recent studies suggest that 1α, 25(OH)2D3 can inhibit TGF-β1-induced EMT, although the exact mechanisms remain unclear. This study aims to explore the role of 1α, 25(OH)2D3 in regulating TGF-β1-induced EMT in alveolar epithelial cells, with a focus on the Wnt/β-catenin signaling pathway.
Methods
Cell Culture and Treatment
Alveolar epithelial cells were isolated from rats and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, penicillin, and streptomycin. The cells were maintained in a humidified atmosphere of 5% CO2 at 37°C. Once the cells reached 80% confluency, they were serum-starved overnight and then treated with 1α, 25(OH)2D3, ICG-001 (a selective inhibitor of β-catenin transcriptional activity), or a combination of both, followed by stimulation with TGF-β1. The morphological changes in the cells were observed using inverted fluorescence microscopy.
Western Blotting
Protein expression levels of E-cadherin, α-SMA, fibronectin, and β-catenin were analyzed using western blotting. Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer, and the protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit. Equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were probed with primary antibodies against E-cadherin, α-SMA, fibronectin, and β-catenin, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using a chemiluminescence detection system.
Immunofluorescence Staining
Alveolar epithelial cells were plated on coverslips and treated as described above. After treatment, the cells were fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and blocked with bovine serum albumin. The cells were then incubated with primary antibodies against β-catenin, E-cadherin, α-SMA, and fibronectin, followed by staining with fluorescently labeled secondary antibodies. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and the fluorescence was observed using a fluorescence microscope.
Real-Time Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNA was extracted from the cells using TRIzol reagent, and cDNA was synthesized using a reverse transcription kit. RT-qPCR was performed using specific primers for Snail and β-actin (as an internal control). The reaction conditions included an initial denaturation step at 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 35 seconds, annealing at 60°C for 1 minute, and extension at 72°C for 30 seconds. The relative mRNA expression levels were calculated using the 2-ΔΔCt method.
Gelatin Zymography
Matrix metalloproteinase-9 (MMP-9) activity was assessed using gelatin zymography. Protein samples were mixed with gelatin and subjected to SDS-PAGE. After electrophoresis, the gel was incubated with substrate buffer at 37°C for 36 hours, stained with Coomassie Brilliant Blue, and destained. MMP-9 activity was visualized as clear bands against a blue background.
Top/Fop Flash Assay
The transcriptional activity of β-catenin was measured using the Top/Fop Flash reporter assay. Cells were transfected with Top/Fop Flash plasmids or a control plasmid using Lipofectamine 2000. After 48 hours, the cells were lysed, and luciferase activity was measured using a dual-luciferase reporter assay system. The relative luciferase activity was calculated as the ratio of Firefly luciferase activity to Renilla luciferase activity.
Statistical Analysis
Data were analyzed using SPSS software (version 22.0). Differences between groups were assessed using one-way analysis of variance (ANOVA) followed by the Least Significant Difference (LSD) post-hoc test. A p-value of less than 0.05 was considered statistically significant.
Results
1α, 25(OH)2D3 and ICG-001 Inhibit TGF-β1-Induced EMT
TGF-β1 treatment induced a morphological change in alveolar epithelial cells from a cobblestone-like appearance to a spindle-like mesenchymal phenotype. This change was reversed by treatment with 1α, 25(OH)2D3 or ICG-001, either alone or in combination. Western blot analysis showed that TGF-β1 significantly reduced the expression of E-cadherin and increased the expression of α-SMA and fibronectin. Treatment with 1α, 25(OH)2D3 or ICG-001 reversed these effects, with the combination treatment showing the most significant inhibition of EMT markers.
Immunofluorescence staining confirmed these findings, showing that 1α, 25(OH)2D3 and ICG-001 inhibited the expression of mesenchymal markers and promoted the expression of epithelial markers. The combination of 1α, 25(OH)2D3 and ICG-001 had a synergistic effect, further enhancing the inhibition of EMT.
1α, 25(OH)2D3 and ICG-001 Act as Negative Regulators of the Wnt/β-Catenin Signaling Pathway
The Top/Fop Flash reporter assay showed that TGF-β1 significantly increased the transcriptional activity of β-catenin. This increase was inhibited by treatment with 1α, 25(OH)2D3 or ICG-001, with the combination treatment showing the most significant reduction in β-catenin activity. Western blot analysis confirmed that TGF-β1 increased the expression of β-catenin, and this effect was reversed by 1α, 25(OH)2D3 and ICG-001.
1α, 25(OH)2D3 and ICG-001 Inhibit Downstream Transcription Factors MMP-9 and Snail in the β-Catenin Signaling Pathway
RT-qPCR analysis showed that TGF-β1 significantly increased the expression of Snail, a downstream transcription factor in the Wnt/β-catenin pathway. This increase was inhibited by treatment with 1α, 25(OH)2D3 or ICG-001, with the combination treatment showing the most significant reduction in Snail expression. Gelatin zymography showed that TGF-β1 increased MMP-9 activity, and this effect was also inhibited by 1α, 25(OH)2D3 and ICG-001.
Discussion
The findings of this study demonstrate that 1α, 25(OH)2D3 inhibits TGF-β1-induced EMT in alveolar epithelial cells by negatively regulating the Wnt/β-catenin signaling pathway. The study also shows that 1α, 25(OH)2D3 has a synergistic effect with ICG-001, a selective inhibitor of β-catenin transcriptional activity, in inhibiting EMT.
The Wnt/β-catenin pathway plays a crucial role in EMT by regulating the expression of downstream transcription factors such as Snail and MMP-9. Snail is a zinc-finger transcription factor that represses epithelial genes and activates mesenchymal genes, while MMP-9 is involved in the degradation and remodeling of the extracellular matrix. By inhibiting the Wnt/β-catenin pathway, 1α, 25(OH)2D3 reduces the expression of Snail and MMP-9, thereby inhibiting EMT.
These findings have important implications for the treatment of airway remodeling in asthma. Vitamin D supplementation could potentially be used as a therapeutic strategy to inhibit EMT and prevent airway remodeling in asthma patients. However, further studies are needed to confirm these findings in vivo and to explore the potential clinical applications of vitamin D in the treatment of asthma.
Conclusion
In conclusion, this study provides evidence that 1α, 25(OH)2D3 inhibits TGF-β1-induced EMT in alveolar epithelial cells by negatively regulating the Wnt/β-catenin signaling pathway. The study also demonstrates that 1α, 25(OH)2D3 has a synergistic effect with ICG-001 in inhibiting EMT. These findings suggest that vitamin D could be a potential therapeutic agent for the prevention and treatment of airway remodeling in asthma.
doi.org/10.1097/CM9.0000000000000830
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