Hong Wa Yung

Placental Endoplasmic Reticulum Stress and Oxidative Stress in the Pathophysiology of Unexplained Intrauterine Growth Restriction and Early Onset Preeclampsia

The pregnancy complications of unexplained intrauterine growth restriction and early onset preeclampsia are thought to share a common aetiology in placental malperfusion secondary to deficient maternal spiral artery conversion. A key question is whether the contrasting clinical manifestations reflect different placental pathologies, or whether they are due to altered maternal responses to a common factor derived from the placenta. Recently, molecular evidence of protein synthesis inhibition secondary to endoplasmic reticulum stress has provided an explanation for the small placental phenotype in both conditions. However, other pathways activated by more severe endoplasmic reticulum stress are only observed in placentas from pregnancies associated with early onset preeclampsia. Here, we review the literature and conclude that there is evidence of greater maternal vascular compromise of the placenta in these cases. We speculate that in cases of normotensive intrauterine growth restriction the placental pathology is centred predominantly around endoplasmic reticulum stress, whereas in cases complicated by preeclampsia oxidative stress is further superimposed. This causes the release of a potent mix of pro-inflammatory cytokines, anti-angiogenic factors and trophoblastic aponecrotic debris into the maternal circulation that causes the peripheral syndrome. Maternal and fetal constitutional factors may modulate how the placenta responds to the maternal vascular insult, and how the mother is affected by the placental factors released. However, the principal conclusion is that the difference between these two conditions lies in the severity of the initiating deficit in spiral arterial conversion, and the relative degrees of endoplasmic reticulum stress and oxidative stress induced in the placenta as a result.

Evidence of Placental Translation Inhibition and Endoplasmic Reticulum Stress in the Etiology of Human Intrauterine Growth Restriction

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.

Endoplasmic reticulum stress exacerbates ischemia-reperfusion-induced apoptosis through attenuation of Akt protein synthesis in human choriocarcinoma cells.

Oxidative stress is central to ischemia‐reperfusion injury. The role of the endoplasmic reticulum (ER) in this process is uncertain. In ER signaling, PERK‐Nrf2 and Ire‐CHOP are two pathways that determine cell fate under stress. PERK‐Nrf2 up‐regulates antioxidant enzyme expression whereas Ire‐CHOP promotes apoptosis. We have identified a novel pathway in ER stress‐induced apoptosis after ischemia‐reperfusion in vitro involving translational suppression of the survival kinase PKB/Akt (Akt), and elucidated an alternative protective role of antioxidants in the regulation of Akt activity. Using human choriocarcinoma JEG‐3 cells, we found that sustained activation of ER stress by tunicamycin or thapsigargin exacerbated apoptosis in oxygen‐glucose‐deprived cells during reoxygenation. This was mediated via a reduction in phosphorylated Akt secondary to down‐regulation of protein translation rather than suppression of phosphorylation. Transient overexpression of wild‐type Akt, but not kinase‐dead Akt, in JEG‐3 cells diminished tunicamycin‐OGD reoxy‐genation‐induced apoptosis. The antioxidants Trolox and Edaravone reduced apoptosis, but the protective effect of Trolox was abrogated by the PI3K inhibitor, LY294002. We speculate that sustained ER stress may contribute to the placental dysfunction seen in human pregnancy complications.—Yung, H‐w., Korolchuk, S., Tolkovsky, A. M., Charnock‐Jones, D. S., Burton, G. J. Endoplasmic reticulum stress exacerbates ischemiareperfusion‐induced apoptosis through attenuation of Akt protein synthesis in human choriocarcinoma cells. FASEB J. 21, 872–884 (2007)

Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner.

Endoplasmic reticulum (ER) stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473) confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308). The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling.

Suppression of Mitochondrial Electron Transport Chain Function in the Hypoxic Human Placenta: A Role for miRNA-210 and Protein Synthesis Inhibition

Fetal growth is critically dependent on energy metabolism in the placenta, which drives active exchange of nutrients. Placental oxygen levels are therefore vital, and chronic hypoxia during pregnancy impairs fetal growth. Here we tested the hypothesis that placental hypoxia alters mitochondrial electron transport chain (ETS) function, and sought to identify underlying mechanisms. We cultured human placental cells under different oxygen concentrations. Mitochondrial respiration was measured, alongside levels of ETS complexes. Additionally, we studied placentas from sea-level and high-altitude pregnancies. After 4 d at 1% O2 (1.01 KPa), complex I-supported respiration was 57% and 37% lower, in trophoblast-like JEG3 cells and fibroblasts, respectively, compared with controls cultured at 21% O2 (21.24 KPa); complex IV-supported respiration was 22% and 30% lower. Correspondingly, complex I levels were 45% lower in placentas from high-altitude pregnancies than those from sea-level pregnancies. Expression of HIF-responsive microRNA-210 was increased in hypoxic fibroblasts and high-altitude placentas, whilst expression of its targets, iron-sulfur cluster scaffold (ISCU) and cytochrome c oxidase assembly protein (COX10), decreased. Moreover, protein synthesis inhibition, a feature of the high-altitude placenta, also suppressed ETS complex protein levels. Our results demonstrate that mitochondrial function is altered in hypoxic human placentas, with specific suppression of complexes I and IV compromising energy metabolism and potentially contributing to impaired fetal growth.

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

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% O2) 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

Differential activation of placental unfolded protein response pathways implies heterogeneity in causation of early- and late-onset pre-eclampsia

Based on gestational age at diagnosis and/or delivery, pre‐eclampsia (PE) is commonly divided into early‐onset (<34 weeks) and late‐onset (≥34 weeks) forms. Recently, the distinction between ‘placental’ and ‘maternal’ causation has been proposed, with ‘placental’ cases being more frequently associated with early‐onset and intrauterine growth restriction. To test whether molecular placental pathology varies according to clinical presentation, we investigated stress‐signalling pathways, including unfolded protein response (UPR) pathways, MAPK stress pathways, heat‐shock proteins and AMPKα in placentae delivered by caesarean section for clinical indications at different gestational ages. Controls included second‐trimester, pre‐term and normal‐term placentae. BeWo cells were used to investigate how these pathways react to different severities of hypoxia–reoxygenation (H/R) and pro‐inflammatory cytokines. Activation of placental UPR and stress‐response pathways, including P‐IRE1α, ATF6, XBP‐1, GRP78 and GRP94, P‐p38/p38 and HSP70, was higher in early‐onset PE than in both late‐onset PE and normotensive controls (NTCs), with a clear inflection around 34 weeks. Placentae from ≥ 34 weeks PE and NTC were indistinguishable. Levels of UPR signalling were similar between second‐trimester and term controls, but were significantly higher in pre‐term ‘controls’ delivered vaginally for chorioamnionitis and other conditions. Severe H/R (1/20% O2) induced equivalent activation of UPR pathways, including P‐eIF2α, ATF6, P‐IRE1α, GRP78 and GRP94, in BeWo cells. By contrast, the pro‐inflammatory cytokines TNFα and IL‐1β induced only mild activation of P‐eIF2α and GRP78. AKT, a central regulator of cell proliferation, was reduced in the < 34 weeks PE placentae and severe H/R‐treated cells, but not in other conditions. These findings provide the first molecular evidence that placental stress may contribute to the pathophysiology of early‐onset pre‐eclampsia, whereas that is unlikely to be the case in the late‐onset form of the syndrome.

Endoplasmic reticulum stress in the pathogenesis of early-onset pre-eclampsia

Recent data have provided molecular evidence of high levels of endoplasmic reticulum stress in non-laboured placentas from cases of early-onset pre-eclampsia. Endoplasmic reticulum stress is intricately linked to oxidative stress, and the two often share the same aetiology. In the case of pre-eclampsia this is likely to be placental malperfusion, secondary to deficient conversion of the spiral arteries. Endoplasmic reticulum stress activates a number of signalling pathways aimed at restoring homeostasis, but if these attempts fail then the apoptotic machinery may be activated. The potential consequences for placental development and function are numerous and diverse. Inhibition of protein synthesis results in lower levels of many kinases, growth factors and regulatory proteins involved in cell cycle control, and experiments in vitro reveal that endoplasmic reticulum stress slows cell proliferation. Chronic, low levels of stress during the second and third trimesters may therefore result in a growth restricted phenotype. Higher levels of endoplasmic reticulum stress lead to activation of pro-inflammatory pathways, a feature of pre-eclampsia that may contribute to maternal endothelial cell activation. These findings emphasise the complexity of cellular responses to stress, and the need to approach these in a holistic fashion when considering therapeutic interventions.

Nitric oxide-induced cell death of cerebrocortical murine astrocytes is mediated through p53- and Bax-dependent pathways

We have investigated the mechanism by which nitric oxide (NO) induces the death of mouse astrocytes. We show that NO (from donor diethylenetriamine‐NO adduct) induces death with several features of apoptosis, including chromatin condensation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, Bax translocation to the mitochondria and cytochrome c release, but no caspase activation or nuclear fragmentation is observed. Nitric oxide also elevates p53 expression, causing a concomitant increase in p53 serine 18 phosphorylation and p53 translocation from the cytoplasm to the nucleus. Activation of Bax and p53 is important for NO‐induced apoptosis‐like cell death because Bax‐ or p53‐deficient astrocytes are much more resistant than wild‐type cells to the same NO treatment. We further demonstrate that LY294002‐sensitive kinases are responsible for controlling serine 18 phosphorylation of p53, thereby regulating the pro‐apoptotic activity of p53 in astrocytes. While apoptosis is suppressed in the presence of LY294002, however, death by necrosis is increased, suggesting that LY294002‐sensitive kinases additionally suppress a latent necrotic response to NO. We conclude that NO‐induced death in astrocytes is mediated by p53‐ and Bax‐dependent mechanisms, although full manifestation of apoptosis is aborted by concomitant inhibition of caspase activation. More generally, our data suggest that apoptotic mediators should be evaluated as the cause of cell death even in cases where a full apoptotic phenotype is lacking.

Endoplasmic reticulum stress disrupts placental morphogenesis: implications for human intrauterine growth restriction

We recently reported the first evidence of placental endoplasmic reticulum (ER) stress in the pathophysiology of human intrauterine growth restriction. Here, we used a mouse model to investigate potential underlying mechanisms. Eif2s1tm1RjK mice, in which Ser51 of eukaryotic initiation factor 2 subunit alpha (eIF2α) is mutated, display a 30% increase in basal translation. In Eif2s1tm1RjK placentas, we observed increased ER stress and anomalous accumulation of glycoproteins in the endocrine junctional zone (Jz), but not in the labyrinthine zone where physiological exchange occurs. Placental and fetal weights were reduced by 15% (97 mg to 82 mg, p < 0.001) and 20% (1009 mg to 798 mg, p < 0.001), respectively. To investigate whether ER stress affects bioactivity of secreted proteins, mouse embryonic fibroblasts (MEFs) were derived from Eif2s1tm1RjK mutants. These MEFs exhibited ER stress, grew 50% slower, and showed reduced Akt–mTOR signalling compared to wild‐type cells. Conditioned medium (CM) derived from Eif2s1tm1RjK MEFs failed to maintain trophoblast stem cells in a progenitor state, but the effect could be rescued by exogenous application of FGF4 and heparin. In addition, ER stress promoted accumulation of pro‐Igf2 with altered glycosylation in the CM without affecting cellular levels, indicating that the protein failed to be processed after release. Igf2 is the major growth factor for placental development; indeed, activity in the Pdk1–Akt–mTOR pathways was decreased in Eif2s1tm1RjK placentas, indicating loss of Igf2 signalling. Furthermore, we observed premature differentiation of trophoblast progenitors at E9.5 in mutant placentas, consistent with the in vitro results and with the disproportionate development of the labyrinth and Jz seen in placentas at E18.5. Similar disproportion has been reported in the Igf2‐null mouse. These results demonstrate that ER stress adversely affects placental development, and that modulation of post‐translational processing, and hence bioactivity, of secreted growth factors contributes to this effect. Placental dysmorphogenesis potentially affects fetal growth through reduced exchange capacity. Copyright