WNT5A increases rheumatoid synoviocyte migration through RYK receptor and RHO-ROCK pathway

Abstract

Background: Fibroblast-like synoviocytes (FLS) are pivotal in inflammation and joint damage of rheumatoid arthritis (RA). These cells acquire an aggressive and invasive phenotype and secrete inflammatory mediators, metalloproteases and cathepsins that perpetuate inflammation and lead to cartilage and bone damage. Given the importance of FLS in the pathogenesis of RA, there is a growing interest in developing new therapies aimed to revert their aggressive phenotype. To this end, a better knowledgement of molecular mechanisms involved in FLS behavior is needed. Recently, the role of non-canonical Wnt pathway has been highlighted in several processes as migration, invasion and inflammatory responses. The non-canonical Wnt signaling pathway included the planar cell polarity (PCP), with the activation of Rho and Rac GTPases, and the Wnt/Ca2+ pathways. We have previously demonstrated that Wnt5a contributes to the aggressive phenotype of FLS by increasing their migration and invasion ability, and by stimulating the inflammatory response.

Objectives: To analyse the molecular mechanisms involved in the enhancing effects of non-canonical Wnt5a pathway on the aggressive phenotype of rheumatoid synoviocytes.

Methods: The expression of receptors of non-canonical Wnt5a pathway was assessed in FLS from six RA patients by real-time PCR. Expression of these receptors was suppressed by siRNA transfection (Dharmacon) and the antibody (anti-RYK, Abgent). Pharmacological inhibitors were used to identify the signaling pathways. Thus, an inhibitor of ROCK pathway (Y-27632, Selleckchem) was used and a calcium chelator (BAPTA-AM, Enzo) for the inhibition of the PKC pathway. The expression of MAPKs was analyzed by western blot. Migration after stimulation with recombinant Wnt5a protein was analyzed by the wound healing assay using Ibidi inserts. The occupation area was determined by Image J software.

Results: We analyzed the expression of Wnt5a receptors, FZD1, FZD2, FZD4, FZD5, FZD7, ROR1, ROR2 and RYK of RA FLS. Although all receptors were expressed in theses cells, whereas ROR2 was weakly expressed, RYK showed the highest expression. Migration assays after receptor suppression by siRNA transfection or blocking antibodies showed the involvement of RYK on Wnt5a signaling in RA FLS. In effect, Wnt5a-induced migration was reduced by more than 25% in FLS treated with the anti-RYK antibody. In addition, Wnt5a-induced migration in FLS treated with ROCK inhibitor (Y-27632) was 32% lower than in FLS not treated. No change in spontaneous migration was observed in FLS treated with Y-27632. However, the treatment with the calcium chelator, BAPTA-AM, did not show any differential effect between the spontaneous or the Wnt5a-induced migration. In addition, we found the activation of MAP kinases, ERK and P38, as well as, AKT and GSK3β in FLS treated with rWnt5a. Nevertheless rWnt5a did not activate JNK. These effects were reversed after treatment with the ROCK inhibitor or after blocking the RYK receptor.

Conclusion: These results indicate that Wnt5a contributes to the aggressive phenotype of RA FLS by binding to RYK receptor, through Rho-ROCK pathway and the activation of MAPKs, ERK and P38, as well as the activation of AKT and GSK3β.