Background/Purpose: Wnt family proteins canonically stabilize β-catenin to activate T-cell factor (TCF) for the transcription of several genes, including Runx2, which is important for osteoblastogenesis. Wnt transcripts are highly expressed in RA synovia (Am J Pathol. 2005; 167: 97–105), suggesting the role of Wnt signaling in bone metabolism during articular disease development. Interestingly, fibroblast-like synoviocytes (FLS) also produce remarkable amount of Dickkopf1 (DKK1), a soluble protein that blocks Wnt pathway. Therefore, FLS have both positive and negative effects on Wnt signal activation. It is plausible that a change in WNT and DKK1 expression can affect the strength of Wnt signaling and regulate bone metabolism and joint destruction in articular diseases. In this study, we aimed to determine the cytokines that regulate Wnt signaling in FLS.

Methods: The mRNA expression levels of Wnt1, 2, 3, 3a, 4, 5a, 5b, 7b, 8b, 10A, 10B, 11, DKK1, and Runx2 in FLS were quantified by SYBR green real-time PCR after treatment with cytokines (TNFα, IL-1β, IL-6, IL-17, and IFNγ). Activity of Wnt pathway was assessed by luciferase assay using TCF reporter plasmid, which was transfected to U2OS, an osteosarcoma cell line that lacks DKK1 expression. The concentration of DKK1 in the culture media was measured by ELISA. Anti-DKK1 antibodies were used to neutralize DKK1 in FLS culture medium.

Results: FLS expresses mRNA of Wnt2, 3, 4, 5A, 5B, 7B, 8B 10A, 10B, 11, and DKK1. DKK1 secreted by FLS was functional, as it blocked the activation of TCF luciferase activity induced by recombinant Wnt3a, and was effectively blocked by anti-DKK1 antibody. TNFα and IL-6 induced Wnt2, 4, and Wnt4 mRNA levels in FLS, respectively, whereas, IL-1β increased Wnt2, 5A, 7B, 10A, 10B, and 11 mRNA levels. Importantly, IL-1β dampened DKK1 mRNA expression in FLS, but TNFα and IL-6 did not alter its expression. Consistent with these results, luciferase activity was induced by the supernatant of FLS treated with IL-1β, but not with other cytokines. In addition, Runx2 mRNA expression in FLS was induced by only IL-1β.

Conclusion: Our data indicates that IL-1β has the highest potential to enhance Wnt signaling, among the tested cytokines. It is already known that IL-1β induces phosphorylation of glycogen synthase kinase-3β for the activation of transcriptional factor TCF. Our findings demonstrate that IL-1β also activates the canonical Wnt signaling pathway by changing the expression pattern of Wnt family proteins and DKK1 in FLS. It is suggested that IL-1β, but not the other cytokines, changes the gene expression pattern of Wnt family and DKK1 in FLS to up-regulate Wnt signaling in the joint during aberrant bone formation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/augmentation-of-wnt-signaling-by-il-1%ce%b2-in-fibroblast-like-synoviocytes/