Molecular Immunology 46 (2009) 3345–3357 Contents lists available at ScienceDirect Molecular Immunology journal homepage: www.elsevier.com/locate/molimm Identification of NR4A2 as a transcriptional activator of IL-8 expression in human inflammatory arthritis  Carol M. Aherne a,c , Jason McMorrow a,c , David Kane b , Oliver FitzGerald b , Kimberlee S. Mix a,c , Evelyn P. Murphy a,c,∗ a College of Life Sciences, UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland b St. Vincent’s University Hospital, Elm Park, University College Dublin, Belfield, Dublin 4, Ireland c UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland article info Article history: Received 2 June 2009 Accepted 26 July 2009 Available online 3 September 2009 Keywords: Orphan nuclear receptors Cytokines Chemokines Arthritis Promoter activity Transactivation Gene expression abstract Expression of the orphan nuclear receptor NR4A2 is controlled by pro-inflammatory mediators, suggest- ing that NR4A2 may contribute to pathological processes in the inflammatory lesion. This study identifies the chemoattractant protein, interleukin 8 (IL-8/CXCL8), as a molecular target of NR4A2 in human inflam- matory arthritis and examines the mechanism through which NR4A2 modulates IL-8 expression. In TNF--activated human synoviocyte cells, enhanced expression of IL-8 mRNA and protein correspond to temporal changes in NR4A2 transcription and nuclear distribution. Ectopic expression of NR4A2 leads to robust changes in endogenous IL-8 mRNA levels and co-treatment with TNF- results in significant (p < 0.001) secretion of IL-8 protein. Transcriptional effects of NR4A2 on the human IL-8 promoter are enhanced in the presence of TNF-, suggesting molecular crosstalk between TNF- signalling and NR4A2. A dominant negative IB kinase antagonizes the combined effects of NR4A2 and TNF- on IL-8 promoter activity. Co-expression of NR4A2 and the p65 subunit of NF-B enhances IL-8 transcription and functional studies indicate that transactivation occurs independently of NR4A2 binding to DNA or heterodimeriza- tion with additional nuclear receptors. The IL-8 minimal promoter region is sufficient to support NR4A2 and NF-B/p65 co-operative activity and NR4A2 can interact with NF-B/p65 on a 39 bp sequence within this region. In patients treated with methotrexate for active inflammatory arthritis, a reduction in NR4A2 synovial tissue levels correlate significantly (n = 10, r = 0.73, p = 0.002) with changes in IL-8 expression. Collectively, these data delineate an important role for NR4A2 in modulating IL-8 expression and reveal novel transcriptional responses to TNF- in human inflammatory joint disease. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction Chronic inflammatory arthropathies including rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are complex systemic diseases, characterized by hyperplasia of synovial tissue that trans- forms into an invasive tongue of destructive tissue (pannus) at the interface with cartilage and bone, ultimately leading to pro- gressive joint destruction (Muller-Ladner et al., 2005). The pannus consists of invasive synoviocyte and macrophage cells which dis- play features suggesting a high level of activation (Firestein, 1994; Tak and Bresnihan, 2000). Pro-inflammatory mediators including tumor necrosis factor alpha (TNF-) and interleukin-1 (IL-1) are released by macrophage cells (Tak and Bresnihan, 2000) to promote  Supported by grants from the Health Research Board of Ireland and Science Foundation Ireland to EPM. ∗ Corresponding author at: UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland. Tel.: +353 1 7166215; fax: +353 1 7166219. E-mail address: evelyn.murphy@ucd.ie (E.P. Murphy). synoviocyte cell proliferation; the release of matrix-degrading enzymes; the induction of pro-angiogenic factors; and secretion of cytokines and chemokines including interleukin-8 (IL-8/CXCL8) (Alvaro-Gracia et al., 1990; Ben-Av et al., 1995; Sakurada et al., 1996; Shi et al., 2004). While it has been established that the co- operation of activated synoviocyte cells with other cellular partners is pivotal to the pathogenesis of joint destruction (Muller-Ladner et al., 2005), the molecular mechanisms and transcriptional events that control this inflammatory transformation are not well under- stood. Increased IL-8 expression is associated with the development and clinical symptoms of human inflammatory arthritis (Kraan et al., 2001). In early RA synovitis, enhanced IL-8 expression is observed by synoviocyte cells of the lining layer (Takahashi et al., 1999) while, in established disease, both synoviocyte and macrophage cells at the cartilage–pannus junction demon- strate significantly increased expression of IL-8 (Deleuran et al., 1994). In an animal model of arthritis, neutralizing IL-8 anti- bodies protect against leukocyte infiltration and tissue damage in the early phase of joint inflammation (Akahoshi et al., 1994). 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.07.019