Evidence of placental translation inhibition and endoplasmic reticulum stress in the etiology of human intrauterine growth restriction

Abstract

Unexplained intrauterine growth restriction of the fetus (IUGR) results from impaired placental development, frequently associated with maternal malperfusion. Some cases are complicated further by preeclampsia (PE+IUGR). Here, we provide the first evidence that placental protein synthesis inhibition and endoplasmic reticulum (ER) stress play key roles in IUGR pathophysiology. Increased phosphorylation of eukaryotic initiation factor 2alpha suggests suppression of translation initiation in IUGR placentas, with a further increase in PE+IUGR cases. Consequently, AKT levels were reduced at the protein, but not mRNA, level. Additionally, levels of other proteins in the AKT-mammalian target of rapamycin pathway were decreased, and there was associated dephosphorylation of 4E-binding protein 1 and activation of glycogen synthase kinase 3beta. Cyclin D1 and the eukaryotic initiation factor 2B epsilon subunit were also down-regulated, providing additional evidence for this placental phenotype. The central role of AKT signaling in placental growth regulation was confirmed in Akt1 null mice, which display IUGR. In addition, we demonstrated ultrastructural and molecular evidence of ER stress in human IUGR and PE+IUGR placentas, providing a potential mechanism for eukaryotic initiation factor 2alpha phosphorylation. In confirmation, induction of low-grade ER stress in trophoblast-like cell lines reduced cellular proliferation. PE+IUGR placentas showed elevated ER stress with the additional expression of the pro-apoptotic protein C/EBP-homologous protein/growth arrest and DNA damage 153. These findings may account for the increased microparticulate placental debris in the maternal circulation of these cases, leading to endothelial cell activation and impairing placental development.

Figure 1

Increased P-eIF2α at Ser51 inhibits protein synthesis and reduces cell proliferation in pathological placentas. A: Western blot showing increased P-eIF2α (Ser51) with constant levels of eIF2α, and reduced cyclin D1 in both IUGR and PE+IUGR placentas. B: Densitometry of bands expressed relative to normal controls (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein, both normalized to β-actin. Data are mean ± SEM for six placentas per group. C: Salubrinal treatment slows cell proliferation in JEG-3 cells. Cell numbers were compared to the untreated control (100%). D: Protein translation is halted under eIF2α phosphorylation. Cells were exposed for 1 hour to [³⁵S]methionine before protein extraction. Whole cell lysates were used to show an equal input of protein. Data are mean ± SEM for three independent experiments. **P < 0.01.

Figure 2

Protein synthesis inhibition induces loss of AKT-GSK-3β signaling in IUGR placentas. A: Western blotting revealed reduced AKT isoforms, P-GSK-3β, GSK-3β eIF2B ε subunit and P-eIF2Bε in IUGR placentas. B: Densitometry of band intensity in IUGR expressed relative to normal controls (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein, both normalized to β-actin. C: Quantitative real-time-PCR revealed no difference in transcript levels for the three AKT isoforms in IUGR. Data are mean ± SEM for six placentas per group. *P < 0.05, **P < 0.01, n.s indicates non-significant change.

Figure 3

Reduction of mTOR signaling in IUGR placentas. A: Western blotting demonstrated reduced phosphorylation of mTOR, TSC2 and 4E-BP1, but not of S6 and eEF2K. Levels of total mTOR, TSC2, raptor, eEF2K but not 4E-BP1 and S6, were lower in IUGR placentas. B: Densitometry of band intensity in IUGR expressed relative to normal controls (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein (P/T), both normalized to β-actin. Data are mean ± SEM for six placentas per group. *P < 0.05 and **P < 0.01.

Figure 4

mTOR signaling is reduced in Akt1−/− placentas. A: Placental weight of Akt1−/− mice. B: Western blot showing reduced phosphorylation of Akt, Tsc2 and 4E-BP1 and increased Akt2 and Akt3 total protein. C: Densitometry of band intensity in Akt1−/− relative to WT (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein, both normalized to β-actin. Data are mean ± SEM for three placentas per group within a single litter. *P < 0.05. D: A positive correlation exists between placental mass and the phosphorylation status of Akt (normalized with β-actin), but not with total Akt. n = 12 placentas with different Akt1 genotypes.

Figure 5

Increased ER stress in IUGR placentas. A: Left panel, tunicamycin induced a dose-dependent increase of P-eIF2α (Ser51), CHOP, GRP78, GRP 94, and apoptotic cell death. RT-PCR also revealed increased splicing of Xbp-1 mRNA. Right graph, densitometric quantification of all bands plotted on a log scale. B: Electron micrographs showing regular cis-ternae of RER (arrowed) in control placenta and dilated cis ternae in IUGR and PE+IUGR placentas. Magnification = 14K. C: Increased ER stress biomarkers, CHOP and GRP94 in PE+IUGR placentas while increased spliced variant of Xbp-1 mRNA in both IUGR and PE+IUGR placentas. D: Densitometry of band intensity in those placentas expressed relative to normal controls (100%). E: IHC showed the nuclear localization of CHOP in both syncytiotrophoblast and endothelial cells in PE+IUGR placentas.

Figure 6

Low-grade ER stress induced by either sublethal dosage of tunicamycin or repetitive H/I-R reduces cell proliferation in JEG-3 cells. A and B: Sublethal dosage (0.3125 μg/ml) with tunicamycin for 48 hours. C and D: Repetitive H/I-R in a 3-hour cyclical pattern at 0.5% and 21% O₂ in the presence of 1.1 mmol/L glucose for 48 hours. A and B: Western blot showing increased eIF2α phosphorylation, decreased Akt protein, and no induction in CHOP. B and D: Cell numbers were compared to the untreated control (100%). Data are mean ± SEM for three independent experiments. **P < 0.01.

Figure 7

Schematic diagram showing the three molecular mechanisms of protein synthesis inhibition and interactions with apoptotic pathways in the etiology of IUGR and PE+IUGR.