Hypoxia induces transcription of the plasminogen activator inhibitor-1 gene through genistein-sensitive tyrosine kinase pathways in vascular endothelial cells
T. Uchiyama et al., Hypoxia induces transcription of the plasminogen activator inhibitor-1 gene through genistein-sensitive tyrosine kinase pathways in vascular endothelial cells, ART THROM V, 20(4), 2000, pp. 1155-1161
A decline in oxygen concentration perturbs endothelial function, which prom
otes local thrombosis. In this study, we determined whether hypoxia in the
range of that observed in pathophysiological hypoxic states stimulates plas
minogen activator inhibitor-1 (PAI-1) production in bovine aortic endotheli
al cells. PAI-1 production, measured by ELISA, was increased by 4.7-fold (P
<0.05 versus normoxic control, n=4) at 12 hours after hypoxic stimulation.
Northern blot analysis showed the progressive time-dependent increase in th
e steady-state level of PAI-1 mRNA expression by hypoxia, which reached a 7
.5-fold increase: (P<0.05 versus control, n=4) at 12 hours. Deferoxamine, w
hich has been known to bind heme protein and to reproduce the hypoxic respo
nse, induced PAI-1 production at both the mRNA and protein levels. The half
-life of PAI-1 mRNA, as determined by a standard decay assay, was not affec
ted by hypoxia, suggesting that induction of PAI-1 mRNA was regulated mainl
y at the transcriptional level. Transient transfection assays of the human
PAI-1 promoter-luciferase construct indicates that a hypoxia-responsive reg
ion lies between -414 and -107 relative to the transcription start site, wh
ere no putative hypoxia response element is found. The hypoxia-mediated inc
rease in PAI-1 mRNA levels was attenuated by the tyrosine kinase inhibitors
genistein (50 mu mol/L) and herbimycin A (1 mu mol/L), whereas PD98059 (50
mu mol/L, MEK1 inhibitor), SB203580 (10 mu mol/L, p38 mitogen-activated pr
otein kinase inhibitor), and calphostin C (1 mu mol/L, protein kinase C inh
ibitor) had no effect on the induction of PAI-1 expression by hypoxia and d
eferoxamine. Genistein but not daidzein blocked the production of hypoxia-
and deferoxamine-induced PAI-1 protein. Thus, we conclude that hypoxia stim
ulates PAI-1 gene transcription and protein production through a signaling
pathway involving genistein-sensitive tyrosine kinases in vascular endothel
ial cells.