Background The gastrin-releasing peptide (GRP) is the mammalian homologue of the bombesin (BN), and its receptor signaling is involved in several functions, including inflammatory response . Both the GRP and its receptor have been found in synovial membrane and fluid of rheumatoid arthritis patients . RC-3095 is an antagonist of the GRP receptor.
Objectives To evaluate the role of gatrin-releasing peptide and the RC-3095, a specific antagonist of the gastrin-releasing peptide receptor, in synovial fibroblast proliferation and invasion.
Methods Mouse DBA/1J fibroblast-like synoviocyte (FLS) were isolated from the tarsus of the hind paws of collagen-induced arthritis . FLS immunocitochemistry was performed to evaluate the presence of GRP-receptor (GRPR) . FLS (2x104 / 96-wells) viability in 24 h treated with RC-3095 (concentration from 0.05 to 10 mM). FLS proliferation stimulated with Lipopolysaccharide (LPS) (1 and 10 µg/mL)  or GRP (0.1, 1 and 10 mM) was performed using the MTT assay in 24 h. The invasion of FLS was assayed in a transwell system using Matrigel-coated inserts from BD (Franklin Lakes, NJ, USA)  and treated with GRP (10 mM), RC-3095 (1 mM) and GRP+RC-3095 (GRP 10 mM and after 30 min RC-3095 1 mM) (n= 3 per group). Differences between experimental groups were compared by ANOVA one-way test.
Results The immunocitochemistry confirmed the presence of GRPR on FLS. RC-3095 concentrations used were not toxic on FLS, maintaining cellular viability. The dose of 1 μM was defined for other experiments because this was the highest dose with lower cell mortality (p<0.05). The GRP 10 mM increased the fibroblast proliferation in 18%, while LPS 10 mM increased 15% compared to FLS unstipulated (p<0.05). Treatment of highly invasive DBA/1J FLS with RC-3095 (1934 ± 941 cells) significantly decreased the number of cells invading Matrigel over 24 h period in 35.3% (p=0.003) compared to GRP (5371 ± 418.1 cells) and non-different to FLS treated with GRP+RC-3095 (3054 ± 794.5 cells).
Conclusions RC-3095 was able to decrease synovial fibroblasts invasion stimulated by GRP. GRP increased FLS proliferation and invasion and could be involved in the development of experimental arthritis through FLS. These findings suggest that interference with the neuropeptide GRP pathway is a potential new strategy for the treatment of arthritis.
– Jensen, R.T. et al. Pharmacol Rev. 2008. 60(1):1-42.
– Grimsholm, O. et al. Cells Tissues Organs. 2008.188(3):299-309.
– Nishioku, T. et al. J Inflam. 2012. 9:44-47.
– Laragione, T. et al. Mol Med. 2010. 16(9-10):352-8
– Miyazawa, S. et al. Rheumatol Int. 2006. 26(8):717-25.
– Tolboom, T.C. et al. Arthritis Rheum. 2005. 52:1999-2002.
Acknowledgements Finnacial support CAPES, CNPq, FIPE (HCPA)
Disclosure of Interest None Declared