Open AccessTranscriptional control of adrenomedullin induction by phorbol ester in human monocytic leukemia cells
Author(s) -
Nakayama Masaharu,
Takahashi Kazuhiro,
Kitamuro Tomomi,
Murakami Osamu,
Shirato Kunio,
Shibahara Shigeki
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2000.01384.x
Subject(s) - adrenomedullin , microbiology and biotechnology , monocytic leukemia , transfection , electrophoretic mobility shift assay , gene expression , activator (genetics) , reporter gene , phorbol , cell culture , messenger rna , chemistry , gene , biology , protein kinase c , biochemistry , peptide , signal transduction , genetics
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.