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.
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.
ABSTRACT
BACKGROUND: Fractalkine (CX3CL1) is a chemokine with a unique CX3C motif and is produced by endothelial cells stimulated with lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, interleukin (IL)-1, and interferon-γ. There have been several reports that the caspase/calpain system is activated in endotoxemia, which leads to cellular apoptosis and acute inflammatory processes. We aimed to determine the role of the caspase/calpain system in cell viability and regulation of fractalkine production in LPS-treated endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were stimulated with 0.01–100 μg/mL of LPS to determine cell viability. The changes of CX3CL1 expression were compared in control, LPS (1 μg/mL)-, IL-1α (1 μg/mL)-, and IL-1β (1 μg/mL)-treated HUVECs. Cell viability and CX3CL1 production were compared with 50 μM of inhibitors of caspase-1, caspase-3, caspase-9, and calpain in LPS-treated HUVECs. RESULTS: Cell viability was significantly decreased from 1 to 100 μg/mL of LPS. Cell viability was significantly restored with inhibitors of caspase-1, caspase-3, caspase-9, and calpain in LPS-treated HUVECs. The expression of CX3CL1 was highest in IL-1β-treated HUVECs. CX3CL1 production was highly inhibited with a calpain inhibitor and significantly decreased with the individual inhibitors of caspase-1, caspase-3, and caspase-9. CONCLUSION: The caspase/calpain system is an important modulator of cell viability and CX3CL1 production in LPS-treated endothelial cells.