Korean J Physiol Pharmacol.  2021 Jul;25(4):307-319. 10.4196/kjpp.2021.25.4.307.

Wnt-C59 inhibits proinflammatory cytokine expression by reducing the interaction between β-catenin and NF-κB in LPS-stimulated epithelial and macrophage cells

• Jang J
• Song J
• Sim I
• Yoon Y

• KMID: 2516870
• DOI: http://doi.org/10.4196/kjpp.2021.25.4.307

Abstract

Dysregulation of the Wnt pathway causes various diseases including cancer, Parkinson’s disease, Alzheimer’s disease, schizophrenia, osteoporosis, obesity and chronic kidney diseases. The modulation of dysregulated Wnt pathway is absolutely necessary. In the present study, we evaluated the anti-inflammatory effect and the mechanism of action of Wnt-C59, a Wnt signaling inhibitor, in lipopolysaccharide (LPS)-stimulated epithelial cells and macrophage cells. Wnt-C59 showed a dose-dependent anti-inflammatory effect by suppressing the expression of proinflammatory cytokines including IL6, CCL2, IL1A, IL1B, and TNF in LPS-stimulated cells. The dysregulation of the Wnt/β-catenin pathway in LPS stimulated cells was suppressed by WntC59 treatment. The level of β-catenin, the executor protein of Wnt/β-catenin pathway, was elevated by LPS and suppressed by Wnt-C59. Overexpression of β-catenin rescued the suppressive effect of Wnt-C59 on proinflammatory cytokine expression and nuclear factor-kappa B (NF-κB) activity. We found that the interaction between β-catenin and NF-κB, measured by co-immunoprecipitation assay, was elevated by LPS and suppressed by Wnt-C59 treatment. Both NF-κB activity for its target DNA binding and the reporter activity of NF-κB-responsive promoter showed identical patterns with the interaction between β-catenin and NF-κB. Altogether, our findings suggest that the anti-inflammatory effect of Wnt-C59 is mediated by the reduction of the cellular level of β-catenin and the interaction between β-catenin and NF-κB, which results in the suppressions of the NF-κB activity and proinflammatory cytokine expression.

Keyword

Catenins; Cytokines; Inflammation; NF-kappa B

Figure

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  Fig. 1 Suppressive effect of Wnt-C59 on lipopolysaccharide (LPS)-stimulated proinflammatory cytokine expressions in BEAS-2B human bronchial epithelial cells. (A–F) Cells were treated with 0 or 50 μM of Wnt-C59, followed by LPS stimulation at 0.1 μg/ml for various time periods of 0.5 to 4 h. (A–E) Messenger RNA levels of proinflammatory cytokines were measured by RT-qPCR. (F) Cell viability was measured. Cells treated with 0 or 50 μM of Wnt-C59 with the same time period of LPS stimulation were compared. *p < 0.05, **p < 0.01, ***p < 0.001. (G–L) Cells were treated with 0 to 50 μM of Wnt-C59, followed by LPS stimulation at 0.1 μg/ml for 2 h. (G–K) Messenger RNA levels of proinflammatory cytokines were measured by RT-qPCR. (L) Cell viability was measured. **p < 0.01, ***p < 0.001 compared with cells stimulated with LPS with 0 μM of Wnt-C59. ###p < 0.001 compared with unstimulated cells. Experiments were conducted in triplicate. Data are shown as mean ± standard deviation, and statistical significance was measured by unpaired t-test.

• 

  Fig. 2 Suppressive effect of Wnt-C59 on lipopolysaccharide (LPS)-induced proinflammatory cytokine expression in RAW264.7 murine macrophage cells. (A–F) Cells were treated with 0 or 50 μM of Wnt-C59, followed by LPS stimulation at 0.1 μg/ml for various time periods of 0.5 to 4 h. (A–E) Messenger RNA levels of proinflammatory cytokines were measured by RT-qPCR. (F) Cell viability was measured. Cells treated with 0 or 50 μM of Wnt-C59 with the same time period of LPS stimulation were compared. *p < 0.05, **p < 0.01, ***p < 0.001. (G–L) Cells were treated with 0 to 50 μM of Wnt-C59, followed by LPS stimulation at 0.1 μg/ml for 4 h. (G–K) Messenger RNA levels of proinflammatory cytokines were measured by RT-qPCR. (L) Cell viability was measured. *p < 0.05, **p < 0.01, ***p < 0.001 compared with cells stimulated with LPS with 0 μM of Wnt-C59. ###p < 0.001 compared with unstimulated cells. Experiments were conducted in triplicate. Data are shown as mean ± standard deviation, and statistical significance was measured by unpaired t-test.

• 

  Fig. 3 Suppressive effect of Wnt-C59 on lipopolysaccharide (LPS)-induced activation of the Wnt/β-catenin pathway in BEAS-2B human bronchial epithelial cells. Cells were treated with 0, 30, or 50 μM of Wnt-C59, followed by LPS stimulation at 0.1 μg/ml for 2 h. (A) Cellular protein levels were measured by Western blotting. Original uncut Western blot images were shown in Supplementary Data 2. β-Actin was used an equal loading control. (B–F) Protein band intensities were quantified using ImageJ. *p < 0.05, **p < 0.01, ***p < 0.001 compared with cells stimulated with LPS with 0 μM of Wnt-C59. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with unstimulated cells. Experiments were conducted in triplicate. Data are shown as mean ± standard deviation, and statistical significance was measured by unpaired t-test. p-LRP6, anti-phospho LRP6; p-GSK-3β, anti-phospho GSK-3β; p-β-catenin, anti-phospho β-catenin.

• 

  Fig. 4 Suppressive effect of Wnt-C59 on lipopolysaccharide (LPS)-induced activation of the Wnt/β-catenin pathway in RAW264.7 murine macrophage cells. Cells were treated with 0, 30, or 50 μM of Wnt-C59, followed by LPS stimulation at 0.1 μg/ml for 4 h. (A) Cellular protein levels were measured by Western blotting. Original uncut Western blot images were shown in Supplementary Data 2. β-Actin was used an equal loading control. (B–F) Protein band intensities were quantified using ImageJ. *p < 0.05, **p < 0.01, ***p < 0.001 compared with cells stimulated with LPS with 0 μM of Wnt-C59. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with unstimulated cells. Experiments were conducted in triplicate. Data are shown as mean ± standard deviation, and statistical significance was measured by unpaired t-test. p-LRP6, anti-phospho LRP6; p-GSK-3β, anti-phospho GSK-3β; p-β-catenin, anti-phospho β-catenin.

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  Fig. 5 Rescuing effects of β-catenin overexpression on anti-inflammatory effects of Wnt-C59 in BEAS-2B human bronchial epithelial cells. Cells grown in 12 well plate were transfected with control vector plasmid (1.5 μg/well) or β-catenin overexpressing plasmid (1.0 or 1.5 μg/well). After one day, cells were treated with 50 μM of Wnt-C59, followed by lipopolysaccharide (LPS) stimulation at 0.1 μg/ml for 2 h. (A) Messenger RNA level of β-catenin were measured by RT-qPCR. (B) Nuclear factor-kappa B (NF-κB) activity for target DNA binding was measured by ELISA. (C, D) Messenger RNA levels of proinflammatory cytokines were measured by RT-qPCR. *p < 0.05, **p < 0.01, ***p < 0.001 compared with cells transfected with control plasmid. ##p < 0.01, ###p < 0.001 compared with unstimulated cells. Experiments were conducted in triplicate. Data are shown as mean ± standard deviation, and statistical significance was measured by unpaired t-test.

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  Fig. 6 Rescuing effects of β-catenin overexpression on anti-inflammatory effects of Wnt-C59 in RAW264.7 murine macrophage cells. Cells grown in 12 well plate were transfected with control vector plasmid (1.5 μg/well) or β-catenin overexpressing plasmid (1.0 or 1.5 μg/well). After one day, cells were treated with 50 μM of Wnt-C59, followed by lipopolysaccharide (LPS) stimulation at 0.1 μg/ml for 4 h. (A) Messenger RNA level of β-catenin were measured by RT-qPCR. (B) Nuclear factor-kappa B (NF-κB) activity for target DNA binding was measured by ELISA. (C, D) Messenger RNA levels of proinflammatory cytokines were measured by RT-qPCR. **p < 0.01, ***p < 0.001 compared with cells transfected with control plasmid. ###p < 0.001 compared with unstimulated cells. Experiments were conducted in triplicate. Data are shown as mean ± standard deviation, and statistical significance was measured by unpaired t-test.

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  Fig. 7 Suppressive effect of Wnt-C59 on the interaction between β-catenin and NF-κB along with the activities of Nuclear factor-kappa B (NF-κB) and its responsive promoter in BEAS-2B human bronchial epithelial cells. Cells were treated with 0, 30, or 50 μM of Wnt-C59, followed by lipopolysaccharide (LPS) stimulation at 0.1 μg/ml for 2 h. (A, C) The protein-protein interaction between β-catenin and NF-κB p65 was analyzed by co-immunoprecipitation assays followed by Western blotting. Original uncut Western blot images were shown in Supplementary Data 2. Input samples of co-immunoprecipitation assays are shown in Supplementary Data 3. (B, D) The amount of co-immunoprecipitated β-catenin or NF-κB p65 was measured by ImageJ. (E) NF-κB activity for its target DNA binding was measured by ELISA. (F) Transcriptional activity of NF-κB-responsive promoter was measured by luciferase reporter assay. *p < 0.05, **p < 0.01, ***p < 0.001 compared with cells stimulated with LPS with 0 μM of Wnt-C59. ##p < 0.01, ###p < 0.001 compared with unstimulated cells. Experiments were conducted in triplicate. Data are shown as mean ± standard deviation, and statistical significance was measured by unpaired t-test. IP, immunoprecipitation; WB, Western blot.

• 

  Fig. 8 Suppressive effect of Wnt-C59 on the interaction between β-catenin and Nuclear factor-kappa B (NF-κB) along with the activities of NF-κB and its responsive promoter in RAW264.7 murine macrophage cells. Cells were treated with 0, 30, or 50 μM of Wnt-C59, followed by lipopolysaccharide (LPS) stimulation at 0.1 μg/ml for 4 h. (A, C) The protein-protein interaction between β-catenin and NF-κB p65 was analyzed by co-immunoprecipitation assays followed by Western blotting. Original uncut western blot images were shown in Supplementary Data 2. Input samples of co-immunoprecipitation assays are shown in Supplementary Data 3. (B, D) The amount of co-immunoprecipitated β-catenin or NF-κB p65 was measured by ImageJ. (E) NF-κB activity for its target DNA binding was measured by ELISA. (F) Transcriptional activity of NF-κB-responsive promoter was measured by luciferase reporter assay. *p < 0.05, **p < 0.01, ***p < 0.001 compared with cells stimulated with LPS with 0 μM of Wnt-C59. ##p < 0.01, ###p < 0.001 compared with unstimulated cells. Experiments were conducted in triplicate. Data are shown as mean ± standard deviation, and statistical significance was measured by unpaired t-test. IP, immunoprecipitation; WB, Western blot.

• 

  Fig. 9 Summary of the findings of this study. The lipopolysaccharide (LPS)-induced activations of Wnt signaling and β-catenin-NF-κB interaction were shown in left panel. Wnt-C59 was reported to inhibit Wnt signaling by suppressing the secretion of Wnt. The inhibitory effects of Wnt-C59 on Wnt signaling and β-catenin-NF-κB interaction were shown in right panel. NF-κB, nuclear factor-kappa B.

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