Hypoxia Induces Cyclooxygenase-2 via the NF-κB p65 Transcription Factor in Human Vascular Endothelial Cells

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-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.

Understanding complexity in the HIF signaling pathway using systems biology and mathematical modeling

“…In addition, besides HIF responsive elements, NF-B consensus sequences are also present in the miR-155 promoter indicating the capacity of NF-B-mediated stimuli to influence the HIF pathway via miR-155 [49]. Indeed, although the detailed mechanism is still not fully understood, it is clear that basal NF-B is moderately activated in hypoxia [87][88][89].…”

Molecular typing of epithelial ovarian carcinomas using inflammatory markers

“…It has been shown that the increase in COX-2 expression seen under hypoxic conditions is under the transcriptional control of NF-jB. 35 In addition, hypoxia inducible transcription factor 1 (HIF-1) alpha subunit activates NF-jB, and that NF-jB controls HIF-1alpha transcription, 36 which in turn, drives the de novo synthesis of Glut-1 in chronic hypoxia. 37 Current limitations to this study include the retrospective design, and the finding that use of IHC to evaluate protein levels is only semiquantitative and does not represent the structure or functionality of the protein.…”

NFκB and HIF display synergistic behaviour during hypoxic inflammation

“…This creates a situation whereby both the hypoxic signalling pathway, through the Hypoxia Inducible factor (HIF), and the inflammatory signalling pathway, through Nuclear factor-!B (NF!B), are activated. Recent studies have shown that hypoxia influences the NF!B pathway and that HIF may play an important role in inflammation [4][5][6][7][8]. However, the relative contributions of HIF and NF!B into creating transcriptional activation profiles leading to a coordinated regulation of hypoxia-induced inflammatory gene expression remain unclear.…”