The interaction between A20 and the NR4A subfamily of nuclear receptors in the pathogenesis of rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic, progressive inflammatory disease that affects nearly 1 % of the world’s population. RA is characterized by inflammation of the synovial joints leading to joint damage which can result in deformities and loss of function. Activation of the transcription factor NF-kB is pivotal in the pathogenesis of RA, switching on multiple proinflammatory genes. The zinc finger protein A20 inhibits induction of NF-kB activation by a variety of stimuli including the proinflammatory cytokine TNF-a in a variety of cell types, dampening the immune response. The A20-related genes ABIN-1, ABIN-2 and Cezanne have also been shown to attenuate TNF-a-induced NF-kB activation. The NR4A subfamily of orphan nuclear receptors comprising of NURR1, NUR77 and NOR-1 are ligand-independent transcription factors and evidence suggests that they may have a proinflammatory role in the RA synovium. Previous studies have demonstrated that overexpression of the NR4A subfamily members led to the induction of A20 gene expression, indicating the potential role of this subfamily in the regulation of A20 and A20-interacting proteins. This study investigated the potential interaction between A20 and the NR4A member NURR1 in the context of RA. Bioinformatic analysis of A20, ABIN-1, ABIN-2 and Cezanne identified the NR4A oinding site (NBRE site) in the promoter region of all four genes, indicating that A20 and these A20-interacting genes may be induced by the NR4A subfamily of nuclear receptors. Transient co-transfection experiments and luciferase assays were carried out on cellular models of inflammatory arthritis (fibroblast-like synoviocytes and chondrocytes). The results indicated that A20 overexpression attenuates the induction of NBRE-luciferase activity by endogenous and overexpressed NURR1 in both synoviocyte and chondrocyte cells. Results in synoviocytes revealed a dose-dependent inhibitory effect of A20 on the transcriptional activity of NURR1. Further experiments demonstrate that in synoviocytes, A20 overexpression led to suppression of NR4A driven transactivation of the interleukin (IL)-8 promoter. In chondrocytes, A20 increased NURR1 induction of the IL-8 promoter. These results indicate that A20 may play a role in modulating the transcriptional activity of NURR1. Reverse transcription (RT)-PCR analysis of synoviocytes and chondrocytes stimulated with the proinflammatory cytokine TNF-a indicated that transcripts from A20, ABIN-1 and ABIN-2 are differentially expressed, while Cezanne is not affected by TNF-a treatment in these cellular contexts. qPCR analysis of A20 and ABIN-1 mRNA expression in chondrocytes in response to TNF-a treatment confirmed the end-point PCR results obtained for this cell type.