title: Amiodarone induces angiotensinogen gene expression in lung alveolar epithelial cells through activation protein-1.
abstract: Previous work from this laboratory has shown that amiodarone induces alveolar epithelial cell apoptosis that was abrogated by antagonists of angiotensin II. In this study, amiodarone up-regulated angiotensinogen mRNA and protein in primary cultures of rat type II pneumocytes and in the human A549 cell line. The mechanism of amiodarone-induced angiotensinogen expression was studied in A549 cells with a human angiotensinogen promoter-luciferase reporter (angiotensinogen/luciferase). Amiodarone (3 microg/ml) induced both angiotensinogen/luciferase and endogenous angiotensinogen mRNA; the latter was completely blocked by actinomycin-D. Amiodarone did not affect the half-life of endogenous angiotensinogen mRNA. Deletion analyses of angiotensinogen/luciferase identified at least two amiodarone-responsive domains in the angiotensinogen promoter between -350 to -260 bp and -203 to -46 bp. DNA/Protein array and electrophoretic mobility shift assays showed that amiodarone increases DNA binding of both activation protein-1 and STAT-5 transcription factors. Site-directed mutagenesis of three IL-6-responsive signal transducer activator of transcription (STAT) binding sites within the amiodarone-response domains had no effect on amiodarone-induced angiotensinogen/luciferase expression. In contrast, amiodarone-induced angiotensinogen/luciferase expression was abrogated by a dominant-negative fos construct and was stimulated over five times by c-fos and c-jun expressed together but not separately. Mutagenesis of the activation protein-1 binding site at -15 to -12 bp completely eliminated the response to amiodarone. These data show that activation protein-1 family transcription factors mediate amiodarone-induced angiotensinogen expression in human alveolar epithelial cells and identify an activation protein-1 site, located between the TATA (DNA sequence) box and the transcription initiation site, that is required for the response.
authors.affiliation: Department of Physiology, Michigan State University, East Lansing, MI 48824, USA. uhal@msu.edu
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Uhal
authors.forename: Huiying
authors.initials: H
authors.lastname: Zhang
authors.forename: Amal
authors.initials: A
authors.lastname: Abdul-Hafez
authors.forename: Ruijie
authors.initials: R
authors.lastname: Shu
authors.forename: Xiaopeng
authors.initials: X
authors.lastname: Li
mesh_terms: D000230:Adenocarcinoma; D000638:Amiodarone / Q000494:pharmacology*; D000809:Angiotensins / Q000235:genetics* / Q000378:metabolism; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D008175:Lung Neoplasms; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020411:Oligonucleotide Array Sequence Analysis; D040081:Protein Array Analysis; D011650:Pulmonary Alveoli / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D000809:Angiotensins; D000889:Anti-Arrhythmia Agents; D012333:RNA, Messenger; D050799:STAT5 Transcription Factor; D018808:Transcription Factor AP-1; D000638:Amiodarone