Inflammatory stimuli modulate the Annexin‐A1 promoter by in human monocytes

Annexin‐A1 (AnxA1) mediates the beneficial effects of glucocorticoids, in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA). In this study we investigate the transcriptional regulation of the AnxA1 promoter following stimulation with inflammatory cytokines. A genomic sequence encompassing a region (−1400 to +1) upstream of the AnxA1 gene was cloned into a luciferase reporter vector pGL3‐Enhancer, and used for transfection in U937 monocytes. Stimulation of transfected cells with a cytokine mixture (CM) of tumor necrosis factor‐α (5 ng/ml), interleukin‐1β (1 ng/ml) and interferon‐γ (10 ng/ml) for 4h increased luciferase activity to 18‐fold compared to 10‐fold in untreated cells (both relative to control empty vector). In addition, CM induced a 2.5‐fold increase in AnxA1 mRNA, measured by quantitative real‐time PCR. Interestingly, analysis of promotor constructs containing deletion and site‐specific mutations for GATA‐3 elements within the −1400 bp fragment revealed suppressor effects on AnxA1 promoter activity by motifs −643 and −784. Consistent with these results, chromatin immunoprecipitation analysis demonstrated constitutive GATA‐3 binding in unstimulated cells, and detachment from the promoter sequence upon CM stimulation. Taken together, these data identify a novel role for GATA‐3 as a suppressor of AnxA1 expression. Down regulation of this transcription factor during the development of Th1 diseases, such as RA, may be responsible for the increased expression of AnxA1 observed in other autoimmune pathologies. This project is funded by the Wellcome Trust. FDA is funded by the Medical Research Council.