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. 2007 Oct;28(10):1012-9.
doi: 10.1016/j.placenta.2007.04.005. Epub 2007 Jun 13.

Role of hypoxia-inducible transcription factors 1alpha and 2alpha in the regulation of plasminogen activator inhibitor-1 expression in a human trophoblast cell line

E S Meade  1 , Y Y Ma, S Guller
Affiliations

Role of hypoxia-inducible transcription factors 1alpha and 2alpha in the regulation of plasminogen activator inhibitor-1 expression in a human trophoblast cell line

E S Meade et al. Placenta. 2007 Oct.

Abstract

The plasminogen activator inhibitors (PAIs) play critical roles in regulating hemostatic and invasive functions of trophoblasts through suppression of plasmin-dependent fibrinolysis and extracellular matrix degradation. The expression of PAI-1 is increased under hypoxic conditions, although the mechanism remains incompletely understood. In the current study we used HTR-8/SVneo cells, a first trimester extravillous trophoblast cell line, and siRNA technology to examine the role of hypoxia-inducible transcription factors (HIFs)-1alpha and -2alpha in the regulation of PAI-1 expression. Using serum-containing and serum-free media culture media it was initially noted that levels of PAI-1, but not PAI-2 protein, were markedly induced by hypoxic (2-3% oxygen) treatment. Under hypoxic conditions, Western blotting revealed that the presence of siRNAs to HIF-1alpha and HIF-2alpha suppressed expression of their respective proteins, whereas treatment with non-targeting and cyclophilin B siRNAs did not. Importantly, incubation with siRNA to HIF-1alpha or HIF-2alpha alone reduced PAI-1 protein levels to a similar extent, with the combined treatment inducing a more profound effect. The presence of HIF siRNAs reduced levels of PAI-1 mRNA as measured by quantitative real-time PCR, indicating that HIF-1alpha and HIF-2 alpha regulate PAI-1 expression at a transcriptional level. These results indicate that both HIF-1alpha and HIF-2alpha play important and similar roles in hypoxia-mediated stimulation of PAI-1 expression in HTR-8/SVneo cells. Our findings provide insight into the physiological regulation of trophoblast PAI-1 expression in early pregnancy when placental oxygen levels are low, as well as a mechanism for over-expression of placental PAI-1 noted in pregnancies with preeclampsia.

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Figures

Figure 1
Figure 1
Hypoxic treatment enhances PAI-1 expression in HTR-8/SVneo cells. HTR-8/SVneo cells were cultured in either serum-containing (Panel A) or serum-free (Panel B) media under normoxic (unfilled bars) or hypoxic (filled bars) conditions. At the indicated time points, the concentration of PAI-1 in culture media were analyzed by ELISA and normalized to total cell protein. Results are expressed as a mean + SE from 5 to 8 independent experiments for each time point studied. *P < 0.05 vs normoxic control by Student's t test **P < 0.01 vs normoxic group by Student's t test ***P < 0.001 vs normoxic group by Student's t test
Figure 2
Figure 2
Effect of hypoxic treatment on PAI-2 levels in HTR-8/SVneo cells. Cells were incubated in serum-containing (A) or serum-free (B) medium under normoxic (unfilled bars) or hypoxic (filled bars) conditions, and at the indicated time level of PAI-2 in culture media was determined by ELISA and normalized to cell protein. Results are presented as a mean + SE from 3 or 4 independent experiments at each time point studied.
Figure 3
Figure 3
Hypoxia-dependent induction of HIF-1α and HIF-2α in HTR-8/SVneo cells. Cells were maintained for the indicated time under normoxic (N) or hypoxic (H) conditions and levels of HIF-1α (Top Panel), HIF-2α (Middle Panel) and cyclophilin B Bottom Panel) were analyzed by Western blotting. The predominant hypoxia-responsive species for both HIFs was detected at a molecular weight of approximately 120 kD and is indicated by an arrow. A lower molecular weight, hypoxia-unresponsive species was also detected by the HIF-2α antibody at approximately 110 kDA. These blots are from a single experiment representing 3 identically conducted ones.
Figure 4
Figure 4
Western blot of siRNA-mediated suppression of HIF-1α and HIF-2α expression. HTR8/SVneo cells were maintained for 48 h under normoxic (N) or hypoxic (H) conditions alone, or transfected under hypoxic conditions with the following siRNAs: 1, HIF-1α; 2, HIF-2α; 1+2, HIF-1α and HIF-2α; NT, non-targeting; Cy, cyclophilin B; M, mock transfection. Levels of HIF-1α (Top Panel), HIF-2α (Middle Panel), and cyclophilin B (Bottom Panel) were detected following Western blotting and immunodetection. These results were obtained in a single experiment representing 4 independently conducted ones.
Figure 5
Figure 5
Effect of HIF siRNA treatment on PAI-1 levels in HTR-8/SVneo cells. HTR8/SVneo cells were incubated for 48 h under normoxic (N) or hypoxic (H) conditions alone, or were maintained under hypoxic conditions with the following siRNAs: NT, non-targeting; Cyc, cyclophilin B; M, mock transfection; 1, HIF-1α; 2, HIF-2α; 1+2, HIF-1α and HIF-2α. Levels of PAI-1 in culture media were determined by ELISA and normalized to total cellular protein. Results are expressed as a mean + SE obtained in 4 to 8 independent experiments. *P < 0.02 vs hypoxic treatment alone group +P < 0.001 vs non-targeting and mock transfection groups
Figure 6
Figure 6
Effect of HIF siRNAs on PAI-1 mRNA levels in HTR-8/SVneo cells. Cells were incubated for 48 h under normoxic (N) or hypoxic (H) conditions alone, or under hypoxia with non targeting (NT), HIF-1α (1), HIF-2α (2), or HIF-1α and HIF-2α (1+2) siRNAs. Levels of PAI-1 mRNA were determined by quantitative real-time PCR and were normalized to that of 18S RNA. Results are expressed as a mean + SE from duplicate determinations in a single experiment representing 3 identically conducted ones.
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