Molecular Basis of Estrogen-Induced Cyclooxygenase Type 1 Upregulation in Endothelial Cells

• Published in: Circulation research 2005-03, Vol.96 (5), p.518-525
• Main Authors: Gibson, Linda L, Hahner, Lisa, Osborne-Lawrence, Sherri, German, Zohre, Wu, Kenneth K, Chambliss, Ken L, Shaul, Philip W
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Biological and medical sciences; Cell Nucleus - metabolism; Cells, Cultured - drug effects; Cells, Cultured - metabolism; Consensus Sequence; Cyclooxygenase 1; DNA, Recombinant - genetics; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - physiology; Endothelial Cells - metabolism; Enzyme Induction - drug effects; Enzyme Induction - physiology; Estradiol - analogs & derivatives; Estradiol - pharmacology; Estrogen Receptor alpha - chemistry; Estrogen Receptor alpha - drug effects; Estrogen Receptor alpha - genetics; Estrogen Receptor alpha - physiology; Estrogen Receptor beta - genetics; Estrogen Receptor beta - physiology; Fundamental and applied biological sciences. Psychology; Genes, Reporter; Humans; Membrane Proteins; Mice; Multiprotein Complexes; Promoter Regions, Genetic - genetics; Prostaglandin-Endoperoxide Synthases - biosynthesis; Prostaglandin-Endoperoxide Synthases - genetics; Protein Interaction Mapping; Protein Structure, Tertiary; Protein Transport; Recombinant Fusion Proteins - physiology; Sequence Deletion; Sheep; Sp1 Transcription Factor - metabolism; Transcription Factor AP-2; Transcription Factors - chemistry; Transcription Factors - physiology; Transcriptional Activation - drug effects; Transfection; Vertebrates: cardiovascular system
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/01.RES.0000158967.96231.88

Estrogen upregulates cyclooxygenase-1 (COX-1) expression in endothelial cells. To determine the basis of this process, studies were performed in ovine endothelial cells transfected with the human COX-1 promoter fused to luciferase. Estradiol (E2) caused activation of the COX-1 promoter with maximal stimulation at 10 mol/L E2, and the response was mediated by either ERα or ERβ. Mutagenesis revealed a primary role for a putative Sp1 binding motif at −89 (relative to the ATG codon) and lesser involvement of a consensus Sp1 site at −111. Electrophoretic mobility shift assays yielded a single complex with the site at −89, and supershift analyses implicated AP-2α and ERα, and not Sp1, in protein-DNA complex formation. In endothelial cells with minimal endogenous ER, the transfection of ERα mutants lacking the DNA binding domain or primary nuclear localization signals caused 4-fold greater stimulation of promoter activity with E2 than wild-type ERα. In contrast, mutant ERα lacking the A-B domains was inactive. Thus, estrogen-mediated upregulation of COX-1 in endothelium is uniquely independent of direct ERα-DNA binding and instead entails protein-DNA interaction involving AP-2α and ERα at a proximal regulatory element. In addition, the process may be initiated by cytoplasmic ERα, and critical receptor elements reside within the amino terminus.