Transcriptional control of adrenomedullin induction by phorbol ester in human monocytic leukemia cells

Adrenomedullin is a potent vasodilator peptide that was originally identified from human Pheochromocytoma. In this study, we investigated the induction of adrenomedullin gene expression in THP-1 acute monocytic leukemia cells during differentiation into macrophage-like cells by 12-O- tetradecanoylphorbol-13-acetate (TPA), and identified a cis-regulatory region of the human adrenomedullin gene responsible for TPA-induced adrenomedullin expression. Upon treatment with TPA (100 ng·mL^-1) for 24 h, immunoreactive adrenomedullin concentrations in the culture medium and adrenomedullin mRNA levels were increased more than 10-fold, concomitant with the differentiation of THP-1 cells into macrophage-like cells. Actinomycin D abolished the TPA- induced adrenomedullin expression, indicating that the induction of ADM gene expression by TPA was regulated at the transcriptional level. Transient transfection assay revealed that a cis-acting region (positions -70 to -30) of human adrenomedullin gene was necessary for TPA-induced reporter gene expression. This region contains multiple copies of activator protein 2 (AP- 2) binding sites, which are bound by purified AP-2 protein, as judged by electrophoretic mobility shift assay. The binding activity to this region was undetectable in nuclear extracts prepared from untreated THP-1 cells, but was increased in extracts prepared from TPA-treated cells. The protein binding was abolished by unlabeled oligonucleotides containing the AP-2 consensus sequence. These results indicate that the region (-70 to -30) of the human ADM gene containing multiple AP-2 binding sites is responsible for TPA- induced adrenomedullin expression in THP-1 cells.