Slug suppression induces apoptosis via Puma transactivation in rheumatoid arthritis fibroblast-like synoviocytes treated with hydrogen peroxide.

Hoon-Suk Cha,Jaejoon Lee,Joong Kyong Ahn,Eun-Kyung Bae,Kwang-Sung Ahn,Eun-Mi Koh

doi:10.3858/emm.2010.42.6.044

Abstract

Inadequate apoptosis contributes to synovial hyperplasia in rheumatoid arthritis (RA). Recent study shows that low expression of Puma might be partially responsible for the decreased apoptosis of fibroblast-like synoviocytes (FLS). Slug, a highly conserved zinc finger transcriptional repressor, is known to antagonize apoptosis of hematopoietic progenitor cells by repressing Puma transactivation. In this study, we examined the expression and function of Slug in RA FLS. Slug mRNA expression was measured in the synovial tissue (ST) and FLS obtained from RA and osteoarthritis patients. Slug and Puma mRNA expression in FLS by apoptotic stimuli were measured by real-time PCR analysis. FLS were transfected with control siRNA or Slug siRNA. Apoptosis was quantified by trypan blue exclusion, DNA fragmentation and caspase-3 assay. RA ST expressed higher level of Slug mRNA compared with osteoarthritis ST. Slug was significantly induced by hydrogen peroxide (H2O2) but not by exogenous p53 in RA FLS. Puma induction by H2O2 stimulation was significantly higher in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. After H2O2 stimulation, viable cell number was significantly lower in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. Apoptosis enhancing effect of Slug siRNA was further confirmed by ELISA that detects cytoplasmic histone-associated DNA fragments and caspase-3 assay. These data demonstrate that Slug is overexpressed in RA ST and that suppression of Slug gene facilitates apoptosis of FLS by increasing Puma transactivation. Slug may therefore represent a potential therapeutic target in RA.

Highlights

Rheumatoid arthritis (RA) is a chronic joint disorder characterized by inflammation and proliferation of synovial tissue (ST) resulting in bone and cartilage destruction
Slug mRNA was detected in fibroblast-like synoviocytes (FLS) with a tendency of increased expression in RA FLS compared to osteoarthritis FLS (n = 3 each), but did not reach statistical significance (P > 0.10) (Figure 1B)
Tumor-like proliferation of synoviocytes plays a key role in the progression of RA, and inadequate apoptosis of FLS contributes to this process (Firestein, 2003)

Summary

Introduction

Rheumatoid arthritis (RA) is a chronic joint disorder characterized by inflammation and proliferation of synovial tissue (ST) resulting in bone and cartilage destruction. One explanation for the synovial proliferation is an imbalance between cell proliferation and apoptosis or programmed cell death. The amount of DNA fragmentation is significantly increased in rheumatoid synovium (Firestein et al, 1995; Nakajima et al, 1995), which is presumably due to the oxidative stress generated by chronic inflammation (Tak et al, 2000). Only low numbers of apoptotic cells are present in rheumatoid synovium (Nakajima et al, 1995; Matsumoto et al, 1996; Sugiyama et al, 1996; Ceponis et al, 1999), which suggests the discrepancy between apoptotic stimuli and real apoptotic cell death occurring in RA synovium. Impaired apoptosis may play an important role in the pathogenesis of RA

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Conclusion