Molecular basis of estrogen-induced cyclooxygenase type 1 upregulation in endothelial cells.
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.
Gibson
Linda L
LL
Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, TX 75390, USA.
Hahner
Lisa
L
Osborne-Lawrence
Sherri
S
German
Zohre
Z
Wu
Kenneth K
KK
Chambliss
Ken L
KL
Shaul
Philip W
PW
D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D016384:Consensus Sequence; D051545:Cyclooxygenase 1; D004274:DNA, Recombinant / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D042783:Endothelial Cells / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects / Q000502:physiology; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D047629:Estrogen Receptor beta / Q000235:genetics / Q000502:physiology; D000077267:Fulvestrant; D017930:Genes, Reporter; D006801:Humans; D008565:Membrane Proteins; D051379:Mice; D046912:Multiprotein Complexes; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics*; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000502:physiology; D017384:Sequence Deletion; D012756:Sheep; D016329:Sp1 Transcription Factor / Q000378:metabolism; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology*; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; C493673:TFAP2A protein, human; C493668:Tfap2a protein, mouse; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D000077267:Fulvestrant; D004958:Estradiol; D051545:Cyclooxygenase 1; C496511:PTGS1 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; C496512:Ptgs1 protein, mouse