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Open Access Research article

The Sp1 transcription factor is essential for the expression of gliostatin/thymidine phosphorylase in rheumatoid fibroblast-like synoviocytes

Kenji Ikuta1, Yuko Waguri-Nagaya1*, Kae Kikuchi1, Takaya Yamagami1, Masahiro Nozaki1, Mineyoshi Aoyama2, Kiyofumi Asai2 and Takanobu Otsuka1

Author Affiliations

1 Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, MuzuhoMizuho-ku, Nagoya, 467-8601, Japan

2 Department of Molecular Neurobiology, Nagoya City University Graduate School of Medical Sciences, Mizuho-Ku, 1 Kawasumi, Mizuho-cho, MuzuhoMizuho-ku, Nagoya, 467-8601, Japan

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Arthritis Research & Therapy 2012, 14:R87  doi:10.1186/ar3811

Published: 25 April 2012

Abstract

Introduction

Gliostatin/thymidine phosphorylase (GLS/TP) has angiogenic and arthritogenic activities, and aberrant GLS production has been observed in the active synovial membranes of rheumatoid arthritis (RA) patients. The human GLS gene promoter contains at least seven consensus binding sites for the DNA binding protein Sp1. Here we examined whether Sp1 is necessary for GLS production in RA. We also studied the effects of the Sp1 inhibitor mithramycin on GLS production in RA fibroblast-like synoviocytes (FLSs).

Methods

FLSs from RA patients were treated with specific inhibitors. The gene and protein expression of GLS were studied using the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and an enzyme immunoassay. Intracellular signalling pathway activation was determined by western blotting analysis, a luciferase assay, a chromatin immunoprecipitation (ChIP) assay and a small interfering RNA (siRNA) transfection.

Results

The luciferase and ChIP assays showed that Sp1 binding sites in the GLS promoter were essential for GLS messenger RNA (mRNA) expression. GLS production was suppressed in FLSs by siRNA against Sp1 transfection. Mithramycin decreased GLS promoter activity, mRNA and protein expression in FLSs. Tumour necrosis factor-α (TNF-α) significantly increased GLS expression in RA FLSs; this effect was reduced by pre-treatment with cycloheximide and mithramycin.

Conclusions

Pretreatment of mithramycin and Sp1 silencing resulted in a significant suppression of GLS production in TNF-α-stimulated FLSs compared to controls. GLS gene expression enhanced by TNF-α was partly mediated through Sp1. As physiological concentrations of mithramycin can regulate GLS production in RA, mithramycin is a promising candidate for anti-rheumatic therapy.