Hypoxia regulates the expression of fatty acid-binding proteins in primary term human trophoblasts

Abstract

Objective: Fatty acids (FAs) are essential for fetal development. Cellular FA uptake is modulated by fatty acid-binding proteins (FABPs). We hypothesized that hypoxia regulates the expression of FABPs in human trophoblasts.

Study design: Primary term human trophoblasts were cultured for 72 hours in either standard (O2 = 20%) or hypoxic (O2 < 1%) conditions. FABP expression was interrogated using polymerase chain reaction and Western immunoblotting. Trophoblast lipid droplets were examined using dipyrromethene boron difluoride 493/503 staining.

Results: We detected the expression of FABP1, -3, -4, -5, and pm but not FABP2 or FABP6-9 subtypes in trophoblasts. Exposure to hypoxia markedly increased lipid droplet accumulation in trophoblasts. Consistent with this observation, hypoxia enhanced the expression of FABP1, -3, and -4. Lastly, agonists of peroxisome proliferator-activated receptor-gamma enhanced the expression of FABP1 and -4 in trophoblasts.

Conclusion: Hypoxia enhances the expression of FABP1, -3, and -4 in term human trophoblasts, suggesting that FABPs support fat accumulation in the hypoxic placenta.

Figure 1

The expression of FABP subtypes in PHTs. (A) RT-PCR was used to examine the expression of nine FABP subtypes in intact human placental fragments and in cultured PHTs (48 h in culture, representing cytotrophoblasts and syncytiotrophoblasts). Transcript expression in other human tissues serves as a negative or positive control. (B) Quantitative analysis of FABP transcript expression in cultured term PHTs. Cells were cultured for 24-72 h in standard conditions, and mRNA expression determined in duplicates using RT-qPCR (* denotes p < 0.05, Kruskal-Wallis, n = 12).

Figure 2

The effect of hypoxia (O₂ < 1%) on lipid droplet accumulation in PHTs. Cells were cultured in either O₂ = 20% or O₂ < 1% for 48 h or for the time points indicated. (A) Hypoxia influences trophoblast hCG and transcripts for erythropoietin (Epo), erythropoietin receptor (EpoR) and transferrin receptor (TfR). Medium hCG and mRNA expression were determined as described in Methods. (* denotes p < 0.05, n = 3, Mann-Whitney or Kruskal-Wallis for multiple comparisons). (B) Hypoxia enhances the accumulation of lipid droplets in trophoblasts. Upper panels depict light microscopy, and lower panels depict fluorescence from BODIPY fluorophore 493/503 in PHTs cultured for 48 h in either O₂ = 20% (a;c) or O₂ < 1% (b;d). Shown is a representative experiment (n = 3). Scale bar, 30 μM.

Figure 3

The influence of hypoxia on the expression of FABP1, 3 and 4 in PHTs. Cells were cultured for 48 h (A) or for 24-72 h (B) in either O₂ = 20% or O₂ < 1%. FABP expression was determined using RT-qPCR (* denotes p < 0.05, n = 3-5, Kruskal-Wallis). (C) The influence of the hypoxia-mimetic CoCl on FABP expression in PHTs. Cells were cultured for 48 h in the presence or absence of 0.4 mM CoCl (* denotes p < 0.05, n = 3, Mann-Whitney). (D) The influence of hypoxia (48 h) on FABP proteins. The expression of FABP1 and FABP3 was determined using immunoprecipitation followed by western immunoblotting, and FABP4 or actin using western immunoblotting with a whole cell lysate. The blots represent three experiments.

Figure 4

Trophoblast differentiation does not alter FABP expression. (A) PHTs were cultured in either DME or H/W medium for 72 h in standard conditions, and medium hCG determined daily as described in Methods (* denotes p < 0.05, n = 3, Kruskal-Wallis). (B) The expression of mRNA for FABPs, determined in duplicates using RT-qPCR (p = NS, n = 3, Mann-Whitney).

Figure 5

The influence of PPARγ/RXR agonists on FABP expression in trophoblasts. PHTs were cultured in DME for 48 h in the absence or presence of the PPARγ agonist GW1929 (2 μM), RXR agonist LG268 (0.1 μM) or both. The expression of mRNA for FABPs was determined in duplicates using RT-qPCR (* denotes p < 0.05, n = 3, Kruskal-Wallis).