Hypoxia induces cyclooxygenase-2 via the NF-κB p65 transcription factor in human vascular endothelial cells

John F. Schmedtje, Yan Shan Ji, W. L. Liu, Raymond N. DuBois, Marschall S. Runge

    Research output: Contribution to journalArticlepeer-review

    619 Scopus citations

    Abstract

    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-κB p65 (Rel A) to the one out of the two NF-κB 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 μM of the same NF-κB element found to bind NF-κB 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-κB consensus element in the COX-2 upstream promoter region in human vascular endothelial cells.

    Original languageEnglish (US)
    Pages (from-to)601-608
    Number of pages8
    JournalJournal of Biological Chemistry
    Volume272
    Issue number1
    DOIs
    StatePublished - 1997

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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