Article
Rescue of chondrocytic phenotype using small peptides to inhibit canonical WNT/β-catenin signaling
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Published: | September 1, 2015 |
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Introduction: In osteoarthritis (OA), chondrocytes undergo hypertrophic differentiation. During OA, the matrix is remodeled, including down-regulation of genes such as Acan and Col2a1. One of the signaling pathways involved in chondrocyte differentiation is the canonical WNT/β-catenin signaling pathway. SAH-Bcl9 (stabilized alpha-helix of Bcl9) and StAx-35R (stapled axin β-catenin binding domain), two small peptides have been developed to inhibit the oncogenic canonical WNT signaling by directly targeting β-catenin. We hypothesized that SAH-Bcl9 and StAx-35R may inhibit the differentiation of chondrocytes towards hypertrophy.
Methods: In vitro analyses were performed using primary chondrocytes isolated from neonatal mice. Micromass cultures were stained with alcian blue to examine proteoglycan production. Expression of marker genes was measured by quantitative RT-PCR. Effects of the inhibitors on protein level were analyzed using Western blot. Using dual luciferase assays the efficacy of the inhibitors on the TCF/Lef blockade was tested. Intracellular localization of the inhibitors was investigated by confocal laser microscopy.
Results: Confocal laser microscopy demonstrated intracellular localization of FITC-labeled SAH-Bcl9 and StAx-35R peptides. Treatment of hip caps with inhibitors showed their penetration into cartilage independent of previous IL-1 treatment. WNT3A induced TCF/Lef promotor activity was significantly reduced at high concentrations of SAH-Bcl9 and StAx-35R. In contrast to StAx-35R, which did not affect phosphorylation of LRP6 and β-catenin, Western blot results indicated less phosphorylation of LRP6 using SAH-Bcl9 in addition to WNT3A. Quantitative RT-PCR revealed decreased expression of Acan, Col2a1 and Sox9, while expression of ColX, Axin2 and Lef1 are up-regulated upon stimulation with WNT3A. However this effect could not be restored by addition of SAH-Bcl9. Alcian blue staining of micromass cultures showed that WNT3A induced proteoglycan loss could not be reversed by SAH-Bcl9.
Conclusion: Our data indicate that at high concentrations SAH-Bcl9 and StAx-35R inhibit the canonical WNT signaling pathway regarding TCF/Lef reporter activity. In this study, however we could not observe that inhibition of canonical WNT signaling has beneficial effects on WNT3A induced changes on chondrocyte phenotype.