Cyclooxygenases catalyze a rate-limiting step in the synthesis of vascular endothelial
prostaglandins. Expression of the inducible
cyclooxygenase-2 (COX-2) gene is increased by
hypoxia in human vascular endothelial cells via the nuclear factor (
NF)-kappaB p65 transcription factor, which is necessary but not sufficient to fully induce COX-2 transcription in response to
hypoxia. After finding that cytoplasmic
NF-kappaB p65 and
IkappaBalpha (an inhibitory
protein that binds
NF-kappaB p65 precursors) levels are not changed by
hypoxia, we hypothesized that other factors might play a role in regulating the COX-2 promoter, like the high-mobility-group (HMG) I(Y) family of
proteins, which features multiple A.T hooks and is associated with
NF-kappaB-mediated transactivation.
Nuclear protein obtained from human umbilical vein endothelial cells (HUVECs) was supplemented with HMG I(Y) during electrophoretic mobility shift assays using an NF-kappaB-3'
element probe. These data suggested that HMG I(Y)
proteins interact with
NF-kappaB p65 to induce COX-2 promoter activity. We also found that TATA-box
DNA demonstrated increased electrophoretic shifting indicative of
DNA binding after incubation with either hypoxic HUVEC
nuclear protein or normoxic
nuclear protein supplemented with HMG I(Y). Transfection of HUVECs with an expression vector containing the COX-2 promoter ligated to HMG I(Y)
cDNA demonstrated positive feedback on COX-2 promoter activity in
hypoxia. We confirmed that COX-2 is transcriptionally regulated by
hypoxia using a nuclear runoff assay.
Hypoxia increased steady-state cellular levels of HMG I(Y)
mRNA as an early event, corresponding with increases in
HMG I(Y) protein. Overexpression of HMG I(Y) was associated in a dose-response relationship with increasing prevalence of the COX-2
protein in hypoxic HUVECs. Furthermore, sense (and antisense) HMG I(Y) overexpression caused stimulation (or inhibition) of COX-2 promoter activity as measured by
luciferase reporter gene expression. The physiological significance of these findings was demonstrated by
cyclooxygenase-dependent release of
prostaglandin E2 by HUVECs in
hypoxia. We concluded that
hypoxia increases expression of HMG I(Y)
proteins while facilitating transactivation of the COX-2 promoter. The HMG I(Y) family of
proteins may therefore function as part of a
hypoxia-induced enhanceosome that helps to promote transcription of COX-2.