FRI0018 Sp1 Interference Prevents Joint Destruction of Ra through Inhibitory Effects of Gliostatin and Matrix Metalloproteinase-3

Background Gliostatin (GLS) has angiogenic and arthritogenic activities, which plays an important role in joint destruction as well as matrix metalloproteinase (MMP)- 3 in rheumatoid arthritis (RA). We previously reported that the expression of GLS in serum from RA patients was significantly correlated with disease activity, and that the expression of MMP-3 was induced by GLS in RA fibroblast-like synoviocytes (FLSs). The human GLS and MMP-3 gene promoter contains consensus binding sites for the DNA binding protein Sp1. Here we examined whether Sp1 was necessary for GLS and MMP-3 productions in RA. Objectives The purpose of this study was to investigate whether the effect of Sp1 interference using the Sp1 inhibitor mithramycin and RNA interference on GLS and MMP-3 productions in FLSs derived from patients with RA. Methods FLSs were cultured from synovial specimens obtained from 10 RA patients during total knee arthroplasty, and were stimulated by tumour necrosis factor (TNF)-α with or without treatment of mithramycin. The gene and protein expressions of GLS and MMP-3 were studied using the quantitative reverse-transcription polymerase chain reaction (RT-PCR) and an enzyme immunoassay. Intracellular signalling pathway activation was determined by a small interfering RNA (siRNA) transfection. Results GLS and MMP-3 productions were suppressed in FLSs by siRNA against Sp1 transfection. TNF-α (1 ng/ml) significantly increased GLS and MMP-3 expressions in RA FLSs; these effects were reduced by pre-treatment with mithramycin (300 nM). Conclusions Our results showed that pretreatment of mithramycin and Sp1 silencing resulted in a significant suppression of GLS and MMP-3 productions in TNF-α-stimulated FLSs compared to controls. The present study indicates that expression of the GLS and MMP-3 gene is mediated, in part, through the transcription factor Sp1.As physiological concentrations of mithramycin can regulate GLS and MMP-3 productions in RA, mithramycin is a promising candidate for anti-rheumatic therapy. References Takeuchi, et al.: Aberrant production of gliostatin/platelet-derived endothelial cell growth factor in rheumatoid arthritis. Arthritis Rheum 1994; 37: 662–72. Muro H, et al. Autocrine induction of gliostatin/platelet-derived endothelial cell growth factor (GLS/PD-ECGF) and GLS-induced expression of matrix metalloproteinases in rheumatoid arthritis synoviocytes. Rheumatol 1999; 38: 1195 -1202. Ikuta K, et al. The Sp1 transcription factor is essential for the expression of gliostatin/thymidine phosphorylase in rheumatoid fibroblast-like synoviocytes. Arthritis Res Ther. 2012; 14:R87 Acknowledgement This research was supported by a Grant-in-Aid for Scientific Research (C) (26462309) from the Japan Society for the Promotion of Science. Disclosure of Interest K. Ikuta: None declared, Y. Waguri-Nagaya Grant/research support from: Biomet Japan, Inc, Chugai Pharmaceutical Co., Ltd., Astellas Pharma Inc., Merck Sharp & Dohme Co., AbbVie Japan Co., Ltd., Ono Pharmaceutical Co., Ltd., N. Tatematsu: None declared, Y. Kawaguchi: None declared, T. Terazawa: None declared, M. Kobayashi: None declared, M. Aoyama: None declared, K. Asai: None declared, T. Otsuka: None declared