Hypoxic upregulation of glucose transporters in BeWo choriocarcinoma cells is mediated by hypoxia-inducible factor-1

Abstract

Placental hypoxia has been implicated in pregnancy pathologies, including fetal growth restriction and preeclampsia; however, the mechanism by which the trophoblast cell responds to hypoxia has not been adequately explored. Glucose transport, a process crucial to fetoplacental growth, is upregulated by hypoxia in a number of cell types. We investigated the effects of hypoxia on the regulation of trophoblast glucose transporter (GLUT) expression and activity in BeWo choriocarcinoma cells, a trophoblast cell model, and human placental villous tissue explants. GLUT1 expression in BeWo cells was upregulated by the hypoxia-inducing chemical agents desferroxamine and cobalt chloride. Reductions in oxygen tension resulted in dose-dependent increases in GLUT1 and GLUT3 expression. Exposure of cells to hypoxic conditions also resulted in an increase in transepithelial glucose transport. A role for hypoxia-inducible factor (HIF)-1 was suggested by the increase in HIF-1alpha as a result of hypoxia and by the increase in GLUT1 expression following treatment of BeWo with MG-132, a proteasomal inhibitor that increases HIF-1 levels. The function of HIF-1 was confirmed in experiments where the hypoxic upregulation of GLUT1 and GLUT3 was inhibited by antisense HIF-1alpha. In contrast to BeWo cells, hypoxia produced minimal increases in GLUT1 expression in explants; however, treatment with MG-132 did upregulate syncytial basal membrane GLUT1. Our results show that GLUTs are upregulated by hypoxia via a HIF-1-mediated pathway in trophoblast cells and suggest that the GLUT response to hypoxia in vivo will be determined not only by low oxygen tension but also by other factors that modulate HIF-1 levels.

Fig. 1

Time course of the effects of hypoxic agents on BeWo glucose transporter 1 (GLUT1). Serum-starved BeWo cells were incubated in 0.2 mM desferroxamine (DFO; A) or 0.2 mM cobalt chloride (B) for up to 72 h (n = 6). At the time points noted, samples were extracted, analyzed for GLUT1 expression by slot immunoblotting, and compared with timed, untreated control incubations. *P < 0.05 vs. control.

Fig. 2

Effect of hypoxic agents on BeWo markers of hypoxia. A: serum-starved BeWo cells were incubated in 0.2 mM DFO or 0.2 mM cobalt chloride for up to 24 h (n = 4). At the time points noted, samples were extracted and analyzed for transferrin receptor (TfR) expression compared with untreated control samples. B: serum-starved BeWo cells were incubated in 0.2 mM DFO or 0.2 mM cobalt chloride for 24 h. Samples were extracted, analyzed for GLUT1 (n = 6), TfR (n = 4), and β-actin (n = 3) expression by slot immunoblotting, and compared with untreated controls. *P < 0.05 vs. control.

Fig. 3

Effects of reduced oxygen tension on BeWo GLUT1 expression. A: time course of GLUT1 expression for serum-starved BeWo cells incubated in 1% oxygen for up to 72 h (n = 6). B: oxygen dependence of GLUT1 expression in BeWo cells incubated for 24 h in 1%, 3%, 5%, or 20% oxygen (n = 6). *P < 0.05 vs. samples incubated in 20% oxygen.

Fig. 4

Time course of the effects of reduced oxygen tension on BeWo GLUT3 expression. Serum-starved BeWo cells were incubated in 1% or 20% oxygen for up to 72 h. At the time points noted, samples were extracted and analyzed for GLUT3 expression (n = 4). *P < 0.05 vs. samples incubated in 20% oxygen.

Fig. 5

Localization of GLUT1 and GLUT3 in BeWo cells. BeWo apical (AP) and basal (BL) membrane fractions were isolated using magnesium precipitation, extracted, and then separated by SDS-PAGE. After transfer, membranes were blotted for GLUT1 and GLUT3. Adjacent AP and BL samples were obtained from the same cell samples.

Fig. 6

Effects of sense (S) and antisense (AS) oligonucleotides on GLUT1 expression. A: serum-starved BeWo cells were incubated in sense or antisense oligonucleotides for 6 h, at which time cells were divided into groups and treated with 0.2 mM cobalt chloride or vehicle (n = 4). B: serum-starved BeWo cells were incubated in sense or antisense oligonucleotides for 6 h, at which time cells were divided into groups and incubated in 3% or 20% oxygen for a further 18 h (n = 4). *P < 0.05 vs. sense oligonucleotide-treated control; ** P < 0.05, sense oligonucleotide-treated control compared with sense oligonucleotide-treated experimental sample (3%).

Fig. 7

Effects of sense and antisense oligonucleotides on GLUT3 expression stimulated by reduced oxygen tension. Serum-starved BeWo cells were incubated in sense or antisense oligonucleotides for 6 h, at which time cells were divided into groups and incubated in 3% or 20% oxygen for a further 18 h (n = 4). *P < 0.05 vs. sense-treated control.