Blockade of Wnt-5A/Frizzled 5 signaling inhibits rheumatoid synoviocyte activation

Abstract

Objective It is not understood why cultured fibroblast‐like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) often display a persistently activated phenotype, despite removal from an inflammatory environment. Previously, we found that these FLS expressed high levels of both Wnt‐5A and Frizzled 5 (Fz5), a receptor–ligand pair implicated in both limb bud and bone marrow stem cell development. The objective of the present experiments was to determine whether Wnt‐5A/Fz5 signaling contributes to FLS activation. Methods Wnt‐5A expression in FLS was inhibited by transfection with both antisense and dominant negative (dn) vectors. Fz5 signaling was blocked with an antibody to the extracellular domain of the receptor. The effects of these treatments on the expression of the proinflammatory cytokines interleukin‐6 (IL‐6) and IL‐15 and on the expression of receptor activator of nuclear factor κB ligand (RANKL) were assessed by reverse transcriptase–polymerase chain reaction and immunoblotting. Results Both antisense Wnt‐5A and dnWnt‐5A vectors, but not empty vector, diminished IL‐6 and IL‐15 expression in RA FLS. Anti‐Fz5 antibody exerted similar effects and also reduced RANKL expression. Conclusion Wnt‐5A/Fz5 signaling may contribute to the activated state of FLS in RA. Receptor antagonists of Fz5 should be considered for the treatment of refractory synovitis.

Regulation of fibronectin and metalloproteinase expression by Wnt signaling in rheumatoid arthritis synoviocytes

“…Total RNA was extracted from tissues and cultured FLS by using TRIzol (Gibco BRL, Gaithersburg, MD) as described previously (4,5). For reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analyses, the following primer pairs were used: for G3PDH, 5Ј-ACCACAGTCCAT-GCCCATCAC-3Ј (forward) and 5Ј-TCCACCACCCTGTTG-CTGTA-3Ј (reverse); for ␤-actin, 5Ј-CAGGTGATCACGAT-AGGTAACGA-3Ј (forward) and 5Ј-CATCCACATTTGTTG-GAACGT-3Ј (reverse); for Wnt-1, 5Ј-CACGACCTCGTCT-ACTTCGAC-3Ј (forward) and 5Ј-ACAGACACTCGTGCA-GTACGC-3Ј (reverse); for luciferase, 5Ј-CATAGTTGACCG-CTTGAAGTCTT-3Ј (forward) and 5Ј-TTGGACTTTC-CGCCCTTCTTGG-3Ј (reverse); and for ␤-galactosidase, 5Ј-GATCTGCCATTGTCAGACATGTA-3Ј (forward) and 5Ј-ACACCAGACCAACTGGTAATGG-3Ј (reverse).…”

“…The primer pairs 5Ј-AGGTGAATTCATGCCCCAACTTTCCG-3Ј (forward) and 5Ј-TGGAGATATCGATGTAGGCAGCTGTC-3Ј (reverse) were used to amplify the human LEF-1 DNA fragment from a fetal complementary DNA library (Clontech) by PCR. The PCR product with Eco RI and Eco RV sites at the two ends was then introduced into the same sites of a pcDNA-HA vector with the HA tag introduced between the Eco RV and Xho I sites (4,5). 2868 …”

“…Cell lysates were made according to the same procedure described elsewhere (4,5). Approximately 30 g of each lysate was used for Western blotting.…”

“…They produce chemotactic and proinflammatory cytokines that promote leukocyte infiltration in the joints (4)(5)(6)(7)(8)(9)(10). In addition, the FLS synthesize extracellular matrix proteins, such as fibronectin, that facilitate cell attachment to the articular surface and promote cell survival (1,2,11).…”

“…To different degrees, various pairs of wnt/fz genes can mediate either cell activation or cell growth (20)(21)(22)(23)(24)(25)(26). Our earlier experiments showed that Wnt-5A/Fz-5 signaling regulated interleukin-6 (IL-6), IL-8, IL-15, and receptor activator of nuclear factor B ligand (RANKL) production by FLS from RA patients (4,5). Thus, inhibition of Wnt-5A/Fz-5 function by neutralizing antibodies or dominant-negative expression vectors reduced cytokine production.…”