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Cited by 112High Altitude and HypoxiaPregnancy and preeclampsia studiesEndoplasmic Reticulum Stress and Disease

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Evidence of endoplasmic reticulum stress and protein synthesis inhibition in the placenta of non‐native women at high altitude

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Yung, Hong Wa, Cox, Mathew, van Patot, Martha Tissot, Burton, Graham J.·2012·The FASEB Journal
DOI 10.1096/fj.11-190082PMC PMC3336782
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Abstract

Pregnancy at high altitude is associated with a reduction in birth weight of ~100 g/1000 m of ascent. The underlying mechanisms are unclear but may involve alteration in energy‐demanding activities, such as protein synthesis. To test this hypothesis, both in vivo and in vitro approaches were used. Placental tissues from pregnant women residing at 3100 m were studied, and placental cells were incubated under hypoxia. In the 3100‐m placentas, we observed dilation of endoplasmic reticulum (ER) cisternae, increased phosphorylation of eukaryotic initiation factor 2 subunit α (P‐eIF2α), reduced AKT phosphorylation, and reduced P‐4E‐BP1 but increased 4E‐BP1 protein compared to sea level controls. These findings suggest the presence of ER stress and protein synthesis inhibition. Hypoxia (1% O 2 ) reduced proliferation of trophoblast‐like JEG‐3 cells, BeWo cells, and placental fibroblasts by ~40, ~60, and ~18%, respectively. Sublethal dosage of salubrinal, an eIF2α phosphatase inhibitor, increased P‐eIF2α and reduced BeWo cell and placental fibroblast proliferation by ~50%. Administration of the PI‐3K inhibitor LY294002 also reduced JEG‐3 proliferation. Our results demonstrate that exposure to chronic hypobaric hypoxia causes mild placental ER stress, which, in turn, modulates protein synthesis and slows proliferation. These effects may account for the reduced placental villous volume, and contribute to the low birth weight that typifies high‐altitude populations.—Yung, H. W., Cox, M., Tissot van Patot, M., Burton, G. J. Evidence of endoplasmic reticulum stress and protein synthesis inhibition in the placenta of non‐native women at high altitude. FASEB J. 26, 1970‐1981 (2012). www.fasebj.org

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Figure 1. Evidence of ER stress in placentas from high altitude. A) Phosphorylation of PERK and eIF2α and total protein level of PERK, eIF2α, and GRP78 were measured by Western blot analysis. β-Actin was used as loading control. B) Densitometric quantification of band intensity. Levels of phosphorylated and total protein are presented as a ratio to the sea-level control (100%). Data are means ± se from all 24 samples. **P < 0.01. C) Splicing of Xbp-1 mRNA was analyzed by RT-PCR; positive and negative controls were set up by treatment of the JEG-3 cells in the presence or absence of 5 μg/ml tunicamycin for 24 h. D) Electron microscopy showed dilation of ER cisternae at high altitude, but not at sea level. Arrows indicate ER cisternae. Scale bars = 500 nm.
Figure 1. Evidence of ER stress in placentas from high altitude. A) Phosphorylation of PERK and eIF2α and total protein level of PERK, eIF2α, and GRP78 were measured by Western blot analysis. β-Actin was used as loading control. B) Densitometric quantification of band intensity. Levels of phosphorylated and total protein are presented as a ratio to the sea-level control (100%). Data are means ± se from all 24 samples. **P < 0.01. C) Splicing of Xbp-1 mRNA was analyzed by RT-PCR; positive and negative controls were set up by treatment of the JEG-3 cells in the presence or absence of 5 μg/ml tunicamycin for 24 h. D) Electron microscopy showed dilation of ER cisternae at high altitude, but not at sea level. Arrows indicate ER cisternae. Scale bars = 500 nm.CC BY-NC · PubMed Central
Figure 2. Increase of oxidative stress in high-altitude placentas. A) Placental sections were immunohistochemically stained with anti-4HNE antibody, an indicator of lipid peroxidation. The majority of staining was in the syncytiotrophoblast. Scale bar = 300 μm. B) Phosphorylation of p38 kinase and HSP27 and total protein level of p38 kinase and HSP27 were measured by Western blot analysis. β-Actin was used as loading control. C) Densitometric quantification of band intensity. Levels of phosphorylated and total protein are presented as a ratio to the sea-level control (100%). Data are means ± se from all 24 samples. *P < 0.05; **P < 0.01.
Figure 2. Increase of oxidative stress in high-altitude placentas. A) Placental sections were immunohistochemically stained with anti-4HNE antibody, an indicator of lipid peroxidation. The majority of staining was in the syncytiotrophoblast. Scale bar = 300 μm. B) Phosphorylation of p38 kinase and HSP27 and total protein level of p38 kinase and HSP27 were measured by Western blot analysis. β-Actin was used as loading control. C) Densitometric quantification of band intensity. Levels of phosphorylated and total protein are presented as a ratio to the sea-level control (100%). Data are means ± se from all 24 samples. *P < 0.05; **P < 0.01.CC BY-NC · PubMed Central
Figure 3. Reduction of AKT-mTOR signaling in high-altitude placentas. A) Proteins were extracted from nonlabored elective Caesarean placentas at both sea level and 3100 m. Western blot analysis was used to measure phosphorylated and total protein levels of P-AKT (Ser473), P-AKT (Thr308), AKT, P-TSC2 (Thr1462), TSC2, P-4E-BP1 (Ser65), P-4E-BP1 (Thr37/46), and 4E-BP-1. β-Actin was used as a loading control. B) Densitometric quantification of band intensity. Levels of phosphorylated and total protein are presented as a ratio to the sea-level control (100%). Data are means ± se from all 24 samples. *P < 0.05; **P < 0.01. C) No change in 4E-BP1 transcript level. qRT-PCR was used to measure the 4E-BP1 mRNA level by TaqMan; 18S was used as a house-keeping gene.
Figure 3. Reduction of AKT-mTOR signaling in high-altitude placentas. A) Proteins were extracted from nonlabored elective Caesarean placentas at both sea level and 3100 m. Western blot analysis was used to measure phosphorylated and total protein levels of P-AKT (Ser473), P-AKT (Thr308), AKT, P-TSC2 (Thr1462), TSC2, P-4E-BP1 (Ser65), P-4E-BP1 (Thr37/46), and 4E-BP-1. β-Actin was used as a loading control. B) Densitometric quantification of band intensity. Levels of phosphorylated and total protein are presented as a ratio to the sea-level control (100%). Data are means ± se from all 24 samples. *P < 0.05; **P < 0.01. C) No change in 4E-BP1 transcript level. qRT-PCR was used to measure the 4E-BP1 mRNA level by TaqMan; 18S was used as a house-keeping gene.CC BY-NC · PubMed Central
Figure 4. Hypoxia reduces trophoblast cell proliferation and induces eIF2α phosphorylation. A) Both JEG-3 and BeWo cells were cultured at either 20 or 1% O2 in normal culture medium for 3 d. Number of cells was counted using a hemocytometer. Experiments were done in duplicate and repeated 3 times. Data presented are means ± sd; the value of 20% O2 is used as a control and expressed as 100%. B) Top panel: proteins were isolated from both JEG-3 cells and BeWo cells after 3 d incubation and subjected to Western blot analysis. Anti-P-eIF2α, anti-eIF2α, and anti-GRP78 antibodies were used. β-Actin was used as a loading control. Bottom panel: densitometric quantification of band intensity. Levels of phosphorylated and total protein are presented as a ratio to the untreated control (100%). Data are means ± sd from 3 independent experiments. **P < 0.01.
Figure 4. Hypoxia reduces trophoblast cell proliferation and induces eIF2α phosphorylation. A) Both JEG-3 and BeWo cells were cultured at either 20 or 1% O2 in normal culture medium for 3 d. Number of cells was counted using a hemocytometer. Experiments were done in duplicate and repeated 3 times. Data presented are means ± sd; the value of 20% O2 is used as a control and expressed as 100%. B) Top panel: proteins were isolated from both JEG-3 cells and BeWo cells after 3 d incubation and subjected to Western blot analysis. Anti-P-eIF2α, anti-eIF2α, and anti-GRP78 antibodies were used. β-Actin was used as a loading control. Bottom panel: densitometric quantification of band intensity. Levels of phosphorylated and total protein are presented as a ratio to the untreated control (100%). Data are means ± sd from 3 independent experiments. **P < 0.01.CC BY-NC · PubMed Central
Figure 5. Differential effect of hypoxia on AKT and 4E-BP1 phosphorylation in JEG-3 and BeWo cells. JEG-3 cells and BeWo cells were incubated either at 20 or 1% O2 for 3 d. A) Proteins were isolated and subjected to Western blot analysis to measure P-AKT (Thr308), P-AKT (Ser473), AKT, P-4E-BP1 (Ser65) and 4E-BP1. β-Actin was used as loading control. B) Densitometric quantification of band intensity. Levels of phosphorylated and total protein are presented as a ratio to the untreated control (100%). Data are means ± sd from 3 independent experiments. *P < 0.05; **P < 0.01.
Figure 5. Differential effect of hypoxia on AKT and 4E-BP1 phosphorylation in JEG-3 and BeWo cells. JEG-3 cells and BeWo cells were incubated either at 20 or 1% O2 for 3 d. A) Proteins were isolated and subjected to Western blot analysis to measure P-AKT (Thr308), P-AKT (Ser473), AKT, P-4E-BP1 (Ser65) and 4E-BP1. β-Actin was used as loading control. B) Densitometric quantification of band intensity. Levels of phosphorylated and total protein are presented as a ratio to the untreated control (100%). Data are means ± sd from 3 independent experiments. *P < 0.05; **P < 0.01.CC BY-NC · PubMed Central
Figure 6. Inhibition of AKT activity and increased phosphorylation of eIF2α reduce proliferation in placental cells. A) Left panel: BeWo cells were incubated with the eIF2α phosphatase inhibitor salubrinal (17.5 μM) for 3 d. Western blot analysis was used to measure eIF2α phosphorylation. No cell death was detected after incubation. Right panel: proliferation rate of BeWo cells was reduced under salubrinal treatment. B) Left panel: JEG-3 cells were incubated with sublethal dosage (10 μM) of the PI-3K inhibitor LY294002 for 2 d. Western blot analysis was used to show the inhibitory effect of LY294002 on AKT phosphorylation; GSK-3 phosphorylation was used as a readout of AKT activity. Right panel: proliferation rate of JEG-3 cells was reduced under LY294002 incubation. For cell proliferation assay, the number of cells was counted using a hemocytometer. Experiments were done in duplicate and repeated 3 times. Data presented are means ± sd, with the value of the untreated cells used as a control and expressed as 100%. **P < 0.01.
Figure 6. Inhibition of AKT activity and increased phosphorylation of eIF2α reduce proliferation in placental cells. A) Left panel: BeWo cells were incubated with the eIF2α phosphatase inhibitor salubrinal (17.5 μM) for 3 d. Western blot analysis was used to measure eIF2α phosphorylation. No cell death was detected after incubation. Right panel: proliferation rate of BeWo cells was reduced under salubrinal treatment. B) Left panel: JEG-3 cells were incubated with sublethal dosage (10 μM) of the PI-3K inhibitor LY294002 for 2 d. Western blot analysis was used to show the inhibitory effect of LY294002 on AKT phosphorylation; GSK-3 phosphorylation was used as a readout of AKT activity. Right panel: proliferation rate of JEG-3 cells was reduced under LY294002 incubation. For cell proliferation assay, the number of cells was counted using a hemocytometer. Experiments were done in duplicate and repeated 3 times. Data presented are means ± sd, with the value of the untreated cells used as a control and expressed as 100%. **P < 0.01.CC BY-NC · PubMed Central
Figure 7. Schematic diagram showing how protein synthesis inhibition may be linked to the growth restriction (SGA) caused by chronic hypoxia in high-altitude pregnancies.
Figure 7. Schematic diagram showing how protein synthesis inhibition may be linked to the growth restriction (SGA) caused by chronic hypoxia in high-altitude pregnancies.CC BY-NC · PubMed Central

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Metadata

Authors
Yung, H. W., Cox, M., van Patot, M. T., & Burton, G. J.
Affiliations
  • University of Cambridge (GB)
  • Centre for Trophoblast Research University of Cambridge Cambridge UK
Corresponding author
Burton, Graham J.
ORCID
Venue
The FASEB Journal
Publisher
Wiley
Published
2012-01-20
Volume
26
Issue
5
Pages
1970-1981
ISSN
  • 0892-6638 (ISSN-L)
  • 1530-6860
DOI
10.1096/fj.11-190082
PMC
PMC3336782
OpenAlex
W2115115666
Cited by
112
References
62
Citations by year
2026: 2 · 2025: 9 · 2024: 5 · 2023: 6 · 2022: 8 · 2021: 9 · 2020: 8 · 2019: 10 · 2018: 9 · 2017: 7 · 2016: 8 · 2015: 12 · 2014: 5 · 2013: 10 · 2012: 4
FWCI
5.97
Type
journal article
Language
en
Open access
Licence
Locations
  • The FASEB Journal (publishedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
Wellcome Trust (084804/2/08/Z)
Topics
  • Health Sciences › Medicine › Physiology (medical) › High Altitude and Hypoxia
  • Health Sciences › Medicine › Obstetrics and Gynecology › Pregnancy and preeclampsia studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Endoplasmic Reticulum Stress and Disease
Keywords
endoplasmic reticulum stress, hypoxia, high altitude, placenta, protein synthesis inhibition, eIF2α phosphorylation, 4E-BP1, Akt phosphorylation, PI3K signaling, salubrinal, cell proliferation, low birth weight, trophoblast
MeSH terms
Altitude, Endoplasmic Reticulum, Female, Humans, Phosphoproteins, Phosphorylation, Placenta, Pregnancy, Protein Biosynthesis, Blotting, Western, Eukaryotic Initiation Factor-2, Oxidative Stress, Cell Cycle Proteins, Cell Line, Tumor, Adaptor Proteins, Signal Transducing, Proto-Oncogene Proteins c-akt, Real-Time Polymerase Chain Reaction

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