Background/Purpose:   Fibroblast-Like Synoviocytes (FLS) play important roles in RA progression. Previous studies have revealed importance of FLS in osteoclast activation, however, the roles of FLS in osteoblast differentiation are not clear. In osteoblast differentiation, Wnt family proteins regulate transcriptions for osteoblast differentiation via stabilization of ß-catenin and TCF activation. It is now known that the Wnt pathway is blocked by several soluble proteins such as Dickkopf1 (DKK1) or Sclerostin (SOST). In this study, we aimed to determine if FLS secrete DKK1 to inhibit Wnt pathway.

Methods:   The expressions of DKK1 in synovial tissue were examined by immunohistochemistry. The mRNA and protein expression in cultured FLS were detected by PCR and immuno-precipitation assay, respectively. The concentrations of DKK1 in culture media were measured by ELISA. mRNA expression was quantified by SYBR Green real-time PCR. Activity of Wnt pathway was analyzed by luciferase assay using TCF reporter plasmid that was transfected to U2OS, an osteosarcoma cell line. Anti DKK1 antibodies were used to neutralize DKK1 in FLS culture medium. siRNA was used to knock down the expressions of DKK1 in FLS.

Results:  DKK1 is observed in RA synovial tissue. The mRNA and protein expressions of DKK1 in cultured FLS are comparable to mesenchymal stem cell (MSC), which is a major source of DKK1 in human. TNF clearly induced the DKK1 secretion form FLS, however, IL-1 and IL-6 did not. FLS supernatant inhibited luciferase activities of TCF reporter induced by addition of recombinant human Wnt3a (200ng/mL). The luciferase activities were blocked by addition of anti DKK1 neutralizing antibodies. By using siRNA, we achieved 90% reduction of DKK1 secretion from FLS. As we expected, the supernatant from these siRNA treated FLS exhibited less inhibitory effect on luciferase activities of TCF reporter. Consistent with these data, FLS supernatant inhibited the RUNX2, COL1A1 mRNA expression in MSC.

Conclusion:  FLS produce considerable amount of DKK1. Importantly, DKK1 secreted form FLS effectively blocks TCF activity induced by recombinant Wnt3a. Therefore, FLS may inhibit osteoblast differentiation by secreting DKK1, especially in the inflamed joint of RA, because TNF promote DKK1 production. Thus, it is possible that DKK1 inhibition allows Wnt to bind to its receptor for activation of osteoblast to repair bone erosion in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-dickkopf1-dampens-anti-osteogenic-effect-of-fibroblast-like-synoviocytes/