Hypoxia-Inducible Factor 1alpha (HIF1A) Mediates Distinct Steps of Rat Trophoblast Differentiation in Gradient Oxygen1

Abstract

Defective differentiation of invasive, placental trophoblast cells has been associated with several pregnancy-related disorders. This study examines the molecular, functional, and morphological differentiation of lineage-specific, trophoblast giant cells under a gradient of oxygen concentrations. Low oxygen (3%) inhibited differentiation, but this inhibition was relieved in a stepwise fashion with increasing levels of oxygen. The oxygen-sensitive hypoxia-inducible factor 1alpha (HIF1A) is a major transcriptional regulator of the cellular response to low oxygen, and increased HIF1A protein levels and activity corresponded with the maintenance of the stem cell-like state and inhibition of trophoblast differentiation in low oxygen. Furthermore, constitutive expression of an oxygen-insensitive, active form of HIF1A protein mimicked the effects of low oxygen, inhibiting the differentiation of trophoblast giant cells. This study is the first to delineate the stepwise effects of oxygen on the activation of the trophoblast giant cell differentiation process and establishes a new paradigm from which to investigate trophoblast differentiation. In addition, this is the first reported study to demonstrate that constitutive HIF1A activity mediates oxygen's inhibition of differentiation. These results suggest that a dysregulation of HIF1A could contribute to impaired placental development.

Id2 Mediates Differentiation of Labyrinthine Placental Progenitor Cell Line, SM10

“…Cos7 cells were cultured in DMEM/high glucose, 10% heat-inactivated FBS, and 1% antibiotic-antimycotic. Rcho-1 cells were cultured as previously described [54][55][56]. All cell lines were passaged at 80%-90% confluence.…”

“…Western blotting was performed as previously described using rabbit polyclonal anti-Id2 antibody at 1/500 overnight at 4°C overnight in blocking buffer [5% (w/v) non-fat milk, pH 7.4 in 1· PBS containing 0.05% Tween-20 (PBST)] [54,66]. The membrane was washed with PBST, probed with HRP-conjugated goat anti-rabbit IgG secondary antibody at 1/10,000 for 1 h at room temperature, and visualized using Enhanced Chemi-Luminiscence reagent according to the manufacturer's instructions.…”

AMPK Knockdown in Placental Trophoblast Cells Results in Altered Morphology and Function

“…The cells were rinsed and fixed with a 4% paraformaldehyde solution in 1· PBS and permeabilized with a solution containing 3% goat serum, 250 mM KCl, 20 mM HEPES, 0.1% glycine, and 0.5% Triton X-100 in 1· PBS for 30 min. After incubation with 6.6 mM rhodamine-conjugated phalloidin in 1 · PBS for 1 h, the cells were rinsed in 1· PBS, and incubated with 1 mg/mL of Hoechst dye for 2 min to stain the nuclei [38]. Coverslips were mounted on plates using ClearMount Mounting Solution (Invitrogen; No.…”

“…Glucose is the primary source of energy for the growing baby [38]. The fetus has very little gluconeogenic ability and therefore relies on the mother for most of its energy supply [41].…”

Reproduction and Growth

“…Increased HIF-1alpha protein levels and activity correlated with the inhibition of trophoblast differentiation in low oxygen. In a murine model, constitutive expression of an oxygen-insensitive, active form of HIF-1alpha protein mimicked the effects of hypoxia and inhibited trophoblast differentiation [ 71 ]. HIF-1alpha mRNA and protein expression have been shown to be abnormally elevated in pre-eclamptic placental tissue compared to controls and TGFbeta(3) is overexpressed in preeclamptic pregnancy [ 67 ].…”