Abstract
BackgroundRheumatoid arthritis (RA) is characterized by excessive synovial inflammation. Cyclooxygenase-2 (COX-2) is an enzyme that catalyzes the conversion of arachidonic acid (AA) into prostaglandins. Epithelium-specific Ets transcription factor-1 (ESE-1) was previously demonstrated to upregulate COX-2 in co-operation with nuclear factor kappa B (NFκB) in macrophages and chondrocytes. However, the role of ESE-1 in RA pathology has remained unclear. In this study, we aimed to elucidate the relationship between ESE-1 and COX-2 in RA synovial fibroblasts (RASFs) using a HD-Ad-mediated knockdown approach.ResultsESE-1 and COX-2 were induced by IL-1β in RASFs that corresponded with an increase in PGE2. Endogenous levels of ESE-1 and COX-2 in human RASFs were analyzed by RT-qPCR and Western blot, and PGE2 was quantified using competitive ELISA. Interestingly, knockdown of ESE-1 using helper-dependent adenovirus (HD-Ad) led to a significant upregulation of COX-2 at a later phase of IL-1β stimulation. Examination of ESE-1 intracellular localization by nuclear fractionation revealed that ESE-1 was localized in the nucleus, occupying disparate cellular compartments to NFκB when COX-2 was increased. To confirm the ESE-1-COX-2 relationship in other cellular systems, COX-2 was also measured in SW982 synovial sarcoma cell line and ESE-1 knockout (KO) murine macrophages. Similarly, knockdown of ESE-1 transcriptionally upregulated COX-2 in SW982 and ESE-1 KO murine macrophages, suggesting that ESE-1 may be involved in the resolution of inflammation.ConclusionESE-1 acts as a negative regulator of COX-2 in human RASFs and its effect on COX-2 is NFκB-independent.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-016-0105-7) contains supplementary material, which is available to authorized users.
Highlights
Rheumatoid arthritis (RA) is characterized by excessive synovial inflammation
ESE‐1 and COX‐2 are induced by IL‐1β in human rheumatoid arthritis synovial fibro‐ blast (RASF) E26 transformation-specific (Ets) transcription factor-1 (ESE-1) was previously shown to be rapidly upregulated by proinflammatory stimuli in human RASFs and maintained up to 24 h [15, 16]
To investigate its relationship to COX-2 expression, we stimulated primary RASFs with 10 ng/mL human IL-1β and quantified the amount of ESE-1 and COX-2 messenger ribonucleic acid (RNA) (mRNA) by quantitative reverse transcription polymerase chain reaction (RT-qPCR) (Fig. 1a)
Summary
Rheumatoid arthritis (RA) is characterized by excessive synovial inflammation. Cyclooxygenase-2 (COX2) is an enzyme that catalyzes the conversion of arachidonic acid (AA) into prostaglandins. ESE-1, the prototype of ESE subfamily, is highly sensitive to inflammatory stimulation [15], where it was found to be expressed in the human RA synovial tissue [16] It was transcriptionally upregulated by proinflammatory stimuli such as IL-1β, TNF-α, or LPS in the resident cell types including synovial fibroblasts, chondrocytes, osteoblasts, and macrophages, typically displaying a peak expression between 2–6 h and dissipation by 24 h in most cells [16]. As RASFs and synovial macrophages are prominent cell types present in the terminal layer of the hyperplastic synovial tissue which secrete inflammatory cytokines and matrix-degrading enzymes [18, 19], elucidation of the role of ESE-1 in COX-2 regulation is important to gain better understanding of the molecular events that occur in RA synovial tissues
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