Modulation of annexin-A1 promoter by inflammatory stimuli in human monocytes

Phuong A. Vo, Roderick J. Flower & Fulvio D’Acquisto, Biochemical Pharmacology, William Harvey Research Institute, Charterhouse Square, London, EC1M 6BQ, UK.

Annexin-A1 (Anx-A1) is a multifunctional protein that mediates several beneficial effects of glucocorticoids (GCs), including inhibition of leukocyte migration, acute and chronic inflammation, ischaemic damage, pain and fever (Perretti et al., 2003). A growing body of evidence has suggested that Anx-A1 might play a role in the modulation of rheumatoid arthritis (RA). In fact, Anx-A1 is found to be expressed highly in human peripheral blood leukocytes and RA synovial tissues (Goulding et al., 1995). Furthermore, administration of bioactive Anx-A1 peptides significantly inhibits experimental arthritis, whereas anti-Anx-A1 antibody exacerbates arthritis severity and prevents the anti-inflammatory effect of the dexamethasone in different animal models of RA (Yang et al., 1998).

In light of these findings, we investigated the regulation of Anx-A1 promoter by the main cytokines involved in RA, namely tumor necrosis factor-α (TNF-α ), interleukin-1β(IL-1 β ) and interferon-γ (IFN-γ ). To this aim, we cloned a region 1400 base pair (bp) upstream of the TATA box (–1400 to +4) of the Anx-A1 gene into a luciferase reporter vector pGL3-Enhancer, and measured luciferase activity as an index of promoter activation. U937 monocytes were transfected with constructs containing the promoter sequence (or mutants, see below) or the empty vector as negative control, using Fugene® 6. Transfection of the full-length insert (-1400 bp) led to a 10-fold increase in luciferase activity compared with pGL3-Enhancer alone. Treatment of transfected cells with a cytokine mixture (CM) of TNF-α (5 ng/ml), IL-1β (1 ng/ml) and IFN-γ (10 ng/ml) for 4 h further increased the relative luciferase activity to 18-fold. The same trends were observed from two to three independent experiments. In addition, analysis of Anx-A1 mRNA expression by quantitative real-time PCR analysis revealed that CM produced a 2.5-fold increase in Anx-A1 expression. In contrast, TNF-α , IL-1β or IFN-γ alone was ineffective in modulating both luciferase activity and Anx-A1 expression. Together these results suggest that Anx-A1 gene is specifically switched on by the concerted action of multiple cytokines that are involved in the development of RA.

To study the molecular mechanism responsible for these effects, we next investigated transcriptional factors (TFs) for Anx-A1 promoter. Interestingly, bioinformatic analysis of a region 1400 bp upstream of the TATA box (–1400 to +4) of the Anx-A1 gene by PROMO and TRANSFAC5.4 databases revealed putative consensus binding sites for previously described TFs such as CREB, AP-1, NF-1 and C/EBP and some new TFs such as GATA-3. Preliminary studies in our laboratory observed that TFs involved in Th2 cells differentiation, such as GATA-3, suppressed Anx-A1 gene expression. Therefore, we hypothesized that activation of GATA-3 might contribute to the down-regulation of Anx-A1 promoter. To this aim we tested different constructs containing deletion and site-specific mutations of four GATA-3 elements (positions: -643, -784, -914 and -1291) found within the -1400 bp fragment and we identified two GATA-3 sites (-643, -784) that possess suppressor effects. Taken together, these data identify a novel role of GATA-3 as a suppressor of Anx-A1 expression and suggest that the down regulation of this TF during the development of Th1 diseases, such as RA, might be responsible for the increased expression of Anx-A1 observed in other autoimmune pathologies.

Goulding et al. (1995) Ann Rheum Dis, 54, 841-5.
Perretti et al. (2003) News Physiol Sci, 18, 60-4.
Yang et al. (1998) Clin Exp Immunol, 111, 117-22.

This project is funded by the Wellcome Trust. FDA is funded by the Medical Research Council.