Background Rheumatoid arthritis (RA) is an autoimmune disease where the chronic inflammation and subsequent cartilage and bone erosion lead to joint destruction. The fibroblast-like synoviocytes (FLS) have a central role in disease pathogenesis and in vitro FLS invasiveness correlates with articular damage in RA patients. Gastrin-releasing peptide (GRP) plays an important role in the immune and inflammatory response. GRP is found in synovial fluid of RA patients and its receptor (GRPR) is found in synovial membrane of murine arthritis. RC-3095 in an antagonist of GRPR.

Objectives To evaluate the role of GRP and GRPR on invasive behavior of mice FLS and to evaluate the GRP-induced signaling on PI3K/AKT pathway.

Methods FLS were isolated from joints of DBA/1J mice with collagen-induced arthritis. Expression of GRPR in FLS was investigated by immunocytochemistry and western blot (WB). Proliferation of FLS treated with GRP (0.1 μM – 10 μM) (n=3) and RC-3095 (0.05 μM – 10 μM) (n=4) was assessed by Sulforhodamine B assay in 24h, 48h and 72h. Invasion assay with FLS was performed using a Matrigel-coated transwell system over 24h in two different experimental arms: first, FLS were treated with GRP (10 μM), RC-3095 (1 μM) or GRP+RC-3095 (GRP 10 μM and RC-3095 1 μM) (n=6) and after, FLS were treated with GRP (10 μM), Ly294002 (10 μM), or GRP+Ly294002 (GRP 10 μM and Ly294002 10 μM) (n=4). Akt phosphorylation was assessed by WB.

Results GRPR protein was detected in FLS both by immunocytochemistry and WB. GRP and RC-3095 treatments did not affect FLS proliferation. Exposure to GRP increased FLS invasion (5356±1027) by nearly two-fold compared with untreated cells (2845±532) (p<0.02), while the treatment with RC-3095 reduced FLS invasion (1723±271) compared with untreated cells. Treatment with GRP+RC-3095 (2670±499) reversed the invasiveness effect of GRP (p<0.0001). GRP also increased phosphorylated AKT expression in FLS by 30% (p<0.001). When Ly294002 was added with GRP (7772±1200), it prevented the GRP-induced increased cell invasiveness (11036±953.6) (p<0.001) (Figure 1).

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Conclusions Our group demonstrated for the first time GRPR expression in FLS and that GRP are able to activate FLS invasion through AKT pathway. Finally, our results suggest that GRP/GRPR pathway could be relevant in the development of FLS-targeted therapy for RA.

References

1. Scott TL et al. Lancet 376;9746:1094–108.

2. Bottini N, Firestein GS. Nat Rev Rheumatol 2013;9:24–33.

3. Grimsholm O et al. Arthritis Res Ther 2005;7:R416–26.

4. Oliveira PG et al. Arthritis Rheum 2011;63:2956–2965.

References

Acknowledgements FIPE-HCPA, CAPES, CNPq.

Disclosure of Interest None declared