Atrial Natriuretic Peptide (ANP)-mediated Maturation and Cytokine Phenotype of Human Dendritic Cells (DCs) Involve Demethylation of the SOCS3 Promoter

Abstract

RATIONALE: ANP modulates the maturation and cytokine phenotype of DCs, particularly by altering their IL-10 production, but the mechanism is unclear. Since, the suppressor of cytokine signaling-3, SOCS3, inhibits the activity of DCs, the action of SOCS3 on ANP-activated DCs was studied by demethylation of the SOCS3 promoter. METHODS: Transfected DCs were treated with the demethylating reagent 5′-aza-2′-deoxycytidine (5′-aza-2′dC), SOCS3 expression was determined by immunoblot or RT-PCR, and supernatant cytokines were measured by Bio-Plex array. To assess the effect of methylation on SOCS3 expression, a SOCS3-P-luciferase reporter plasmid was methylated and co-transfected into DCs with pANP. Luciferase activity was measured 48 hours after transfection. The maturation of DCs was assessed using the FITC-dextran phagocytosis assay. RESULTS: ANP downregulated SOCS3 expression compared to control, but SOCS3 was up-regulated by 5′-aza-2′dC demethylation. Also, treatment of pANP-transfected DCs with siRNA to SOCS3 leads to the upregulation of IL-10 and IFN-γ. Moreover, demethylation of CpG islands in the SOCS3 promoter elevates the expression of SOCS3 and enhances maturation of human DCs. CONCLUSIONS: These results indicate that ANP mediated maturation of DCs involves demethylation of the SOCS3 promoter.