Molecular basis of estrogen-induced cyclooxygenase type 1 upregulation in endothelial cells

Circ Res. 2005 Mar 18;96(5):518-25. doi: 10.1161/01.RES.0000158967.96231.88. Epub 2005 Feb 10.

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

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • 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
  • Fulvestrant
  • 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

Substances

  • DNA, Recombinant
  • DNA-Binding Proteins
  • Estrogen Receptor alpha
  • Estrogen Receptor beta
  • Membrane Proteins
  • Multiprotein Complexes
  • Recombinant Fusion Proteins
  • Sp1 Transcription Factor
  • TFAP2A protein, human
  • Tfap2a protein, mouse
  • Transcription Factor AP-2
  • Transcription Factors
  • Fulvestrant
  • Estradiol
  • Cyclooxygenase 1
  • PTGS1 protein, human
  • Prostaglandin-Endoperoxide Synthases
  • Ptgs1 protein, mouse