Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells.
The inducible cyclooxygenase, COX-2, has been associated with vascular inflammation and cellular proliferation. We have discovered that hypoxia increases expression of the COX-2 gene in human vascular endothelial cells in culture independent of other stimuli. Western analysis of human umbilical vein endothelial cells (HUVEC) revealed a greater than 4-fold induction of protein by hypoxia (1% O2). The steady-state level of COX-2 mRNA was correspondingly elevated by both Northern blot and reverse transcriptase-polymerase chain reaction analysis. Using electrophoretic mobility shift assays with antibody supershifting, we also found that hypoxia causes increased binding of NF-kappaB p65 (Rel A) to the one out of the two NF-kappaB consensus elements in the COX-2 promoter which is closest to the transcription start site of the COX-2 gene. Transfection of an immortalized human microvascular endothelial cell line (HMEC-1) with mutation reporter gene constructs and HUVEC with both mutation and deletion reporter gene constructs suggested that transcription of the COX-2 gene was enhanced by hypoxia. In transcription factor decoy experiments, hypoxic HUVEC were exposed in culture to 20 microM of the same NF-kappaB element found to bind NF-kappaB protein. The wild type transcription factor decoy prevented hypoxic induction of COX-2, presumably by binding with cytoplasmic p65; however, mutated or scrambled oligonucleotides did not prevent the increase in COX-2 protein expression by hypoxia. Thus, the intracellular signaling mechanism that leads to induction of COX-2 by hypoxia includes binding of p65 to the relatively 3' NF-kappaB consensus element in the COX-2 upstream promoter region in human vascular endothelial cells.
Schmedtje
J F
JF
Sealy Center for Molecular Cardiology, Department of Medicine, The University of Texas Medical Branch, Galveston 77555-1064, USA. john@schmedtje.utmb.edu
Ji
Y S
YS
Liu
W L
WL
DuBois
R N
RN
Runge
M S
MS
D001483:Base Sequence; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D004268:DNA-Binding Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D000860:Hypoxia / Q000201:enzymology*; D007527:Isoenzymes / Q000096:biosynthesis*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000502:physiology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis*; D051996:Transcription Factor RelA
D004268:DNA-Binding Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D051996:Transcription Factor RelA; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases