NR4A2 expression is not altered in placentas from cases of growth restriction or preeclampsia, but is reduced in hypoxic cytotrophoblast

Abstract

Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) transcripts are elevated in the circulation of individuals whose pregnancies are complicated by preterm fetal growth restriction (FGR). In this paper, we show that the cases with preeclampsia (PE) have increased circulating NR4A2 transcripts compared to those with normotensive FGR. We aimed to establish whether the dysfunctional placenta mirrors the increase in NR4A2 transcripts and further, to uncover the function of placental NR4A2. NR4A2 expression was detected in preterm and term placental tissue; expressed higher at term. NR4A2 mRNA expression and protein were not altered in placentas from preterm FGR or PE pregnancies. Hypoxia (1% O₂ compared to 8% O₂) significantly reduced cytotrophoblast NR4A2 mRNA expression, but not placental explant NR4A2 expression. Silencing cytotrophoblast NR4A2 expression under hypoxia (via short interfering (si)RNAs) did not alter angiogenic Placental Growth Factor, nor anti-angiogenic sFlt-1 mRNA expression or protein secretion, but increased expression of cellular antioxidant, oxidative stress, inflammatory, and growth genes. NR4A2 expression was also not altered in a model of tumour necrosis factor-α-induced endothelial dysfunction, or with pravastatin treatment. Further studies are required to identify the origin of the circulating transcripts in pathological pregnancies, and investigate the function of placental NR4A2.

Figure 1

Circulating NR4A2 mRNA in cases of normotensive preterm fetal growth restriction and preeclampsia with growth restriction (< 34 weeks gestation). RNA levels were assessed by qPCR. Cases with preeclampsia and growth restriction had significantly higher circulating NR4A2 mRNA compared to normotensive cases of growth restriction. Data presented as relative change from normotensive levels; mean ± SEM. **p < 0.01. Normotensive, n = 45; preeclampsia, n = 71.

Figure 2

NR4A2 expression in preterm and term placental tissue assessed by qPCR. Term placental tissue had significantly higher NR4A2 mRNA expression compared to preterm tissue. Data presented as relative change from preterm levels; mean ± SEM. **p < 0.01. Preterm n = 30, 24–36 weeks; term n = 29, 37–41 weeks.

Figure 3

NR4A2 expression and protein in preterm control (PT), preeclamptic (PE) and fetal growth restricted (FGR) placental tissue (≤ 34 weeks gestation). (a) mRNA expression assessed by qPCR. (b) Representative western blot. (c) Densitometric analysis of western blot. NR4A2 expression was not altered in the PE and FGR placental tissue compared to respective PT control tissue (PT n = 10, PE n = 49, FGR n = 14). There was no significant difference in NR4A2 protein between the pathological and control tissue (PT n = 15, PE n = 31, FGR n = 17). β-actin acted as the loading control. Data presented as relative change from preterm controls ± SEM.

Figure 4

NR4A2 expression in placental explant tissue and primary cytotrophoblast under normoxic (8% O₂) and hypoxic (1% O₂) conditions. NR4A2 expression was unaltered in placental explant tissue with hypoxia (a). In primary cytotrophoblasts, NR4A2 expression was significantly decreased under hypoxic conditions (b). There was no change in NR4A2 protein production with hypoxia (c). Data presented as fold change from control ± SEM. qPCR: n = 4–5 experimental replicates, each sample from a different patient. Each sample was run in triplicate. Western blot: n = 4 experimental replicates, with each sample from a different patient. Each experiment was run in triplicate and replicate lysates were pooled. β-actin acted as the loading control. ****p < 0.0001.

Figure 5

Primary cytotrophoblast expression and secretion of anti- and pro-angiogenic factors, sFLT-1 and PGF under hypoxic (1% O₂) conditions. Expression assessed by qPCR and secretion by ELISA. There were no significant differences in expression of either sFLT-1 isoform mRNA expression, sFLT-e15a (a) or sFLT-i13 (b), nor sFLT-1 protein secretion (c) or PGF mRNA expression (d) under hypoxia. Data presented as fold change from control ± SEM. n = 3 experimental replicates, with each sample from a different patient. Each experiment was run in triplicate.

Figure 6

Effect of NR4A2 knockdown in primary cytotrophoblasts on expression of genes associated with oxidative stress, inflammation, and placental insufficiency under hypoxic (1% O₂) conditions. The expression of HMOX-1 (a), NOX4 (b), GCLC (c), NLRP3 (d) and SPINT1 (e) were significantly increased with NR4A2 knockdown compared to negative control. Data presented as relative change from control ± SEM. *p < 0.05, **p < 0.01. n = 3 experimental replicates, with each sample from a different patient. Each experiment was run in triplicate.

Figure 7

NR4A2 expression in human umbilical vein endothelial cells with TNF-α induced dysfunction, and pravastatin treatment. There was no significant change in NR4A2 mRNA expression with the addition of TNF-α. Pravastatin treatment did not alter NR4A2 expression from TNF-α only levels. The control group was not exposed to TNF-α. Data presented as fold change from TNF-α treatment ± SEM. n = 3 experimental replicates, with each sample from a different patient. Each experiment was run in duplicate.