Our results demonstrate the presence of active pathways for the modulation of protein synthesis in the placentas from high-altitude pregnancies, which is likely to be the result of ER stress. In vitro studies further confirmed that hypoxia activates similar pathways in placental cells, resulting in protein synthesis inhibition and a subsequent reduction in cell proliferation. Together, these data suggest that placental hypoxia-induced protein synthesis inhibition could be one of the mechanistic explanations for the pathophysiology of SGA babies (). Although this conclusion is drawn from a small sample size, the remarkable consistency of the increased P-eIF2α and 4E-BP1 total protein levels, the two most important components regulating cap-dependent translation, observed across 4 different regions of each of the high-altitude placentas, suggest these findings are unlikely to be an artifact.
Schematic diagram showing how protein synthesis inhibition may be linked to the growth restriction (SGA) caused by chronic hypoxia in high-altitude pregnancies.
Dilation of the endoplasmic reticulum cisternae has previously been reported in Nepalese placentas, although the full significance of the finding was not realized at that time (41
). Protein synthesis is the most energy-demanding cellular process, and up to 35% of intracellular ATP production is used by the translation machinery in eukaryotic cells (42
). During cell growth and proliferation, one of the primary limiting factors is the rate of protein translation. Total protein translation must reach a critical threshold level before cell replication is allowed to occur. In response to mitogenic signals, a series of signaling cascades may be triggered in order to activate protein translation machinery, while withdrawal of extracellular cues results in a reduction in protein synthesis and exit from the cell cycle (37
The majority of ATP production takes place in mitochondria, where oxygen molecules are used as the final electron acceptor. Therefore, limitation of oxygen availability during chronic hypoxia at high altitude likely affects this process, resulting in a potential energy crisis. Our previously published data showed that although there is a significant reduction of the ATP:ADP ratio in the same high-altitude placentas, the concentrations of ATP and ADP show no significant difference between sea level and high altitude (31
). This may reflect a metabolic reprogramming, with an increase in anaerobic glycolysis and a reduction in mitochondrial oxygen consumption (43
). Interestingly, we have recently found that the protein levels of at least one subunit of mitochondrial complex I and/or complex IV are reduced in both the high-altitude placentas and placental cells maintained at 1% O2
). Mitochondrial respiratory activity can also be inhibited by phosphocreatine, which is used as an energy reserve to maintain ATP levels (45
). Indeed, there is a trend toward higher concentrations of phosphocreatine in these high-altitude placentas (31
), suggesting the placentas are preconditioned to store energy for use during hypoxic stress. Similar observations have been reported in the rat hippocampus, in which phosphocreatine concentrations increase under chronic hypoxia (46
). We did not observe any increase in phosphorylation of AMPK in the high-altitude placentas (Supplemental Fig. S2), indicating the placentas are unlikely to be suffering an energy crisis because of chronic hypoxia. This may reflect successful restoration of energy supply and demand as a result of activation of these homeostatic pathways.
To achieve the maximal efficiency in energy savings, regulatory mechanisms for protein synthesis have been evolutionarily established at the initiation stage and are regulated by a family of eIFs (47
). Phosphorylation of eIF2α at Ser51 directly halts the translation initiation as it acts as a competitive inhibitor of eIF2B, thus blocking recycling of eIF2. Indeed, we observed elevation of P-eIF2α in the high-altitude placentas, as well as in response to hypoxic incubation of JEG-3 and BeWo ( and B
). This mechanism does not block translation initiation for all mRNAs. Those mRNAs containing small upstream open reading frames (uORFs) within their 5′-UTR regions or internal ribosome entry site (IRES) sequences are selectively translated independent of eIF2α regulation (48
). It has been shown that many stress-response and essential genes, including ATF4, ER chaperones (GRP78 and GRP94), N-myc downstream regulated gene 1 (NDRG1) and vascular endothelial growth factor A (VEGF-A) are able to bypass this regulation and increase their expression on ER stress (49
). This phenomenon may explain the increase of total 4E-BP1 protein that we observed (), for ATF4 can induce expression of 4E-BP1
Koritzinsky et al.
) reported a biphasic hypoxic stress response in the protein synthesis inhibition pathways, in which the P-PERK/P-eIF2α pathway is initiated within 1 h of anoxia, and by 16 h, there is a transition to predominant control by the P-AKT/4E-BP1 pathway. Both pathways remain active, but the P-PERK/P-eIF2α pathway activity is reduced. We did observe a dramatic inhibition of P-4E-BP1 phosphorylation and a significant increase of total 4E-BP1 in BeWo cells under hypoxia for 3 d, but not in JEG-3 and placental fibroblasts. This difference is likely due to the differential susceptibility of the cells to the hypoxic environment, possibly reflecting their endocrine activities. This speculation is supported by the cell proliferation assay, in which there was an 18, 40, and 60% suppression of cell proliferation in placental fibroblasts, JEG-3 cells, and BeWo cells, respectively.
It is apparent that after the initial eIF2α response, translation initiation is primarily regulated by AKT-mTOR-4E-BP1 signaling (15
). 4E-BP1 regulates cap-dependent translation initiation by binding to eIF4E, preventing recruitment of mRNA to ribosomes (53
). Phosphorylation of 4E-BP1 by mTORC1 blocks this interaction (54
). In high-altitude placentas, there was a significant decrease in AKT phosphorylation and P-4E-BP1 (), suggesting loss of AKT-mTOR signaling. Interestingly, we also observed a 2.2-fold increase of total 4E-BP1 in high-altitude placenta, suggesting a long-term role of 4E-BP1 in the prolonged inhibition of protein synthesis. High 4E-BP1 levels protect pancreatic β cells from ER stress-induced cell death (21
), suggesting that the increase seen in the high-altitude placenta may perform a similar function.
Increased angiogenesis is an important adaptation under hypoxia, and studies have shown increased vascularity in the high-altitude placenta of indigenous populations (41
). For many years, much emphasis has been placed on the role of HIF1α. However, ER stress has recently been identified as a regulator of angiogenesis through a HIF1α-independent pathway by direct regulation of VEGF mRNA expression (57
). IRE1 is another proximal sensor involved in ER stress pathways. Knockout of IRE1α genes reduces VEGF mRNA expression, as well as protein levels, and compromises placental vascularization in the labyrinthine zone of the mouse placenta (58
). We did not observe any spliced variant of XBP-1
mRNA in the 3100-m placentas, indicating that IRE1α is not activated. This is consistent with the fact that the fractional volume of the villi occupied by the fetal capillaries was the same as in sea-level placentas (30
). Differences in the ER response to hypoxia between indigenous and non-native populations could potentially explain the lack of an angiogenic response in the placentas studied here.
Taken together, ER stress appears on the one hand to conserve energy consumption, and on the other hand to promote angiogenesis for greater oxygen delivery. A recent publication from Bastide et al.
) showed that the VEGF-A 5′UTR contains an uROF within an IRES that controls its translation, allowing VEGF-A to bypass P-eIF2α regulation on ER stress. Therefore, could the mild ER stress we observe at 3100 m be simply considered a homeostatic response, slowing cell proliferation through protein synthesis inhibition and thereby resulting in a lower birth weight that matches the maternal oxygen supply? This view is supported by the findings of Postigo et al.
), who reported that fetal oxygen delivery is constant between altitude levels and ethnic groups when normalized to fetal weight.
Furthermore, we have observed more severe ER stress in placentas associated with intrauterine growth restriction of maternal vascular origin, in particular, when complicated with preeclampsia (39
). In those instances, we saw evidence of activation of the IRE1α and apoptotic pathways, along with greater dilation of the ER cisternae. This differential activation of the ER stress response pathways can be recapitulated in vitro
by applying increasing doses of tunicamycin to JEG-3 cells. Low doses cause only increased phosphorylation of eIF2α, whereas higher doses result in increased expression of the chaperone protein, GRP78, and activation of the IRE1α and the apoptotic pathways (39
). Hence, we speculate that there is a spectrum of placental ER stress in vivo
, stimulating a mild homeostatic response in the chronically hypoxic placenta at altitude and a more severe response with pathological consequences, in cases of deficient maternal spiral arterial conversion.