Cyclic adenosine monophosphate-responsive elements are involved in the transcriptional activation of the human IL-10 gene in monocytic cells

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Abstract
IL‐10 plays an important role in the regulation of immune responses. We and others have demonstrated recently that cyclic adenosine monophosphate (cAMP)‐elevating substances up‐regulate monocytic IL‐10 expression in vitro and in vivo. Computer analysis of the IL‐10 promoter/enhancer region localized four putative cAMP‐responsive elements (CRE1– 4) with homology to the CRE consensus motif. In electrophoretic mobility shift assays CRE1 and CRE4 bound protein complexes consisting of transcription factors CREB‐1 and ATF‐1, while CRE3 bound only marginal amounts of CREB‐1/ATF‐1 in combination with unknown protein(s). CRE2 showed no protein binding activity. In vitro mutation of CRE1 and CRE4 reduced the level of cAMP‐stimulated transactivation in reporter gene assays in comparison to the wild‐type promoter by 20 % and 50 %, respectively, while mutation of CRE3 had no effect. The main action of CRE4 on cAMP‐dependent stimulation is probably based on its adjacent localization to the TATA box and its sequence comprising a perfect half site. Experiments with double and triple mutants and with deleted promoter fragments indicated the participation of additional elements beside the CRE motifs in the cAMP‐dependent stimulation. Our data suggest that intracellular cAMP may directly affect expression of the immunoregulatory cytokine IL‐10 in monocytic cells via activation of the eukaryotic transcription factors CREB‐1 and ATF‐1 and their binding to CRE1 and CRE4 in the upstream enhancer of the IL‐10 promoter.