Abstract
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(-8) mol/L E2, and the response was mediated by either ERalpha or ERbeta. 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-2alpha and ERalpha, and not Sp1, in protein-DNA complex formation. In endothelial cells with minimal endogenous ER, the transfection of ERalpha mutants lacking the DNA binding domain or primary nuclear localization signals caused 4-fold greater stimulation of promoter activity with E2 than wild-type ERalpha. In contrast, mutant ERalpha lacking the A-B domains was inactive. Thus, estrogen-mediated upregulation of COX-1 in endothelium is uniquely independent of direct ERalpha-DNA binding and instead entails protein-DNA interaction involving AP-2alpha and ERalpha at a proximal regulatory element. In addition, the process may be initiated by cytoplasmic ERalpha, and critical receptor elements reside within the amino terminus.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Binding Sites
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Cell Nucleus / metabolism
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Cells, Cultured / drug effects
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Cells, Cultured / metabolism
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Consensus Sequence
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Cyclooxygenase 1
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DNA, Recombinant / genetics
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DNA-Binding Proteins / chemistry
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DNA-Binding Proteins / physiology*
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Endothelial Cells / metabolism
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Enzyme Induction / drug effects
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Enzyme Induction / physiology
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Estradiol / analogs & derivatives
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Estradiol / pharmacology*
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Estrogen Receptor alpha / chemistry
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Estrogen Receptor alpha / drug effects
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Estrogen Receptor alpha / genetics
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Estrogen Receptor alpha / physiology*
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Estrogen Receptor beta / genetics
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Estrogen Receptor beta / physiology
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Fulvestrant
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Genes, Reporter
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Humans
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Membrane Proteins
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Mice
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Multiprotein Complexes
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Promoter Regions, Genetic / genetics*
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Prostaglandin-Endoperoxide Synthases / biosynthesis
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Prostaglandin-Endoperoxide Synthases / genetics*
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Protein Interaction Mapping
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Protein Structure, Tertiary
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Protein Transport
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Recombinant Fusion Proteins / physiology
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Sequence Deletion
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Sheep
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Sp1 Transcription Factor / metabolism
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Transcription Factor AP-2
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Transcription Factors / chemistry
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Transcription Factors / physiology*
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Transcriptional Activation / drug effects*
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Transfection
Substances
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DNA, Recombinant
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DNA-Binding Proteins
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Estrogen Receptor alpha
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Estrogen Receptor beta
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Membrane Proteins
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Multiprotein Complexes
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Recombinant Fusion Proteins
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Sp1 Transcription Factor
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TFAP2A protein, human
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Tfap2a protein, mouse
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Transcription Factor AP-2
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Transcription Factors
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Fulvestrant
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Estradiol
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Cyclooxygenase 1
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PTGS1 protein, human
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Prostaglandin-Endoperoxide Synthases
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Ptgs1 protein, mouse