Different regulation of IRE1α and eIF2α pathways by oxygen and insulin in ACH-3P trophoblast model

in Reproduction
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Veronika TandlDepartment of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria

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Denise HochDepartment of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria

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Julia Bandres-MerizDepartment of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria

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Sanela NikodijevicDepartment of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria

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Gernot DesoyeDepartment of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria

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Alejandro Majali-MartinezDepartment of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria

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https://orcid.org/0000-0002-9009-5810

Correspondence should be addressed to G Desoye; Email: gernot.desoye@medunigraz.at
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Endoplasmic reticulum (ER)-stress activates the unfolded protein response (UPR), which plays a (patho)physiological role in the placenta. Oxygen and hyperinsulinemia are major regulators of placental development. Thus, we hypothesized that oxygen, insulin and their interplay modulate ER-stress in early pregnancy. Using the human first-trimester trophoblast cell line ACH-3P, we quantified mRNA and protein of several members of UPR by RT-qPCR and Western blotting, respectively. ER-stress induction using tunicamycin and brefeldin A resulted in increased CHOP (4.6-fold change; P ≤ 0.001), XBP1 expression (1.7- and 1.3-fold change, respectively; P ≤ 0.001 and P < 0.05) and XBP1 splicing (7.9- and 12.8-fold change, respectively; P ≤ 0.001). We subsequently analyzed the effect of oxygen (6.5%, 2.5%), insulin (0.1–10 nM) and their interaction using ANCOVA adjusted for cell passage as co-variate. Although GRP78 protein remained unaffected, low oxygen (2.5% O2) increased IRE1α phosphorylation (+52%; P < 0.05) and XBP1 splicing (1.8-fold change; P ≤ 0.001) after 24 h, while eIF2α protein and CHOP expression were downregulated (−28%; P < 0.05 and −24%; P ≤ 0.001; respectively). eIF2α phosphorylation was also reduced after 48 h by low oxygen (−61%; P < 0.05) but increased in the presence of insulin (+46%; P ≤ 0.01). These changes were not PERK-mediated, since PERK phosphorylation and total protein were not altered. Overall, our results suggest that IRE1α and eIF2α UPR-pathways are differentially regulated by oxygen and insulin in early pregnancy.

Abstract

Endoplasmic reticulum (ER)-stress activates the unfolded protein response (UPR), which plays a (patho)physiological role in the placenta. Oxygen and hyperinsulinemia are major regulators of placental development. Thus, we hypothesized that oxygen, insulin and their interplay modulate ER-stress in early pregnancy. Using the human first-trimester trophoblast cell line ACH-3P, we quantified mRNA and protein of several members of UPR by RT-qPCR and Western blotting, respectively. ER-stress induction using tunicamycin and brefeldin A resulted in increased CHOP (4.6-fold change; P ≤ 0.001), XBP1 expression (1.7- and 1.3-fold change, respectively; P ≤ 0.001 and P < 0.05) and XBP1 splicing (7.9- and 12.8-fold change, respectively; P ≤ 0.001). We subsequently analyzed the effect of oxygen (6.5%, 2.5%), insulin (0.1–10 nM) and their interaction using ANCOVA adjusted for cell passage as co-variate. Although GRP78 protein remained unaffected, low oxygen (2.5% O2) increased IRE1α phosphorylation (+52%; P < 0.05) and XBP1 splicing (1.8-fold change; P ≤ 0.001) after 24 h, while eIF2α protein and CHOP expression were downregulated (−28%; P < 0.05 and −24%; P ≤ 0.001; respectively). eIF2α phosphorylation was also reduced after 48 h by low oxygen (−61%; P < 0.05) but increased in the presence of insulin (+46%; P ≤ 0.01). These changes were not PERK-mediated, since PERK phosphorylation and total protein were not altered. Overall, our results suggest that IRE1α and eIF2α UPR-pathways are differentially regulated by oxygen and insulin in early pregnancy.

Introduction

The placenta is a transient organ that fulfills a wide range of functions to sustain adequate fetal development (Burton & Fowden 2015). Beginning in the first trimester, placental villous cytotrophoblasts (vCTB) proliferate and fuse to form the syncytiotrophoblast (STB), a multinucleated syncytium constituting the feto-maternal barrier that is involved in nutrient and oxygen transport as well as hormone secretion (Maltepe & Fisher 2015). vCTB can also differentiate into extravillous cytotrophoblasts (evCTB), which invade the decidua and anchor the feto-placental unit in the uterus (Knöfler et al. 2019).

Early placental development occurs in a physiological low oxygen environment. During the first weeks of gestation, evCTB reach and plug the decidual spiral arteries, thereby preventing maternal blood from reaching the intervillous space. Toward the end of the first trimester, evCTB remodel the spiral arteries into low resistance and high capacity vessels, leading to the establishment of the utero-placental circulation (Kaufmann et al. 2003). As a result, the oxygen tension in the intervillous space raises steadily from 2.5 to 6.5% along the first trimester of human pregnancy (Jauniaux et al. 2000). Therefore, oxygen is considered a major regulator of placental function (Chang et al. 2018).

Placental development is also highly influenced by the maternal environment (Hoch et al. 2019). Maternal obesity and gestational diabetes mellitus (GDM) are associated with derangements in the intrauterine environment (Desoye & Hauguel-de Mouzon 2007, Bautista-Castaño et al. 2013). This ultimately influences placental development and function, entailing adverse consequences for maternal and fetal health (Catalano & Shankar 2017, Desoye 2018, Hoch et al. 2020). Interestingly, both conditions have been associated with the endoplasmic reticulum (ER)-stress (Westermeier et al. 2014, Yung et al. 2016).

ER-stress occurs when ER-homeostasis is compromised, affecting protein folding, processing and trafficking (Schwarz & Blower 2016). However, ER-stress also plays a physiological role, regulating several biological processes including lipid metabolism, calcium homeostasis and cell differentiation (Li et al. 2019). There are three main ER-transmembrane proteins that act as stress sensors, including protein kinase RNA-like ER kinase (PERK), inositol-requiring transmembrane kinase endoribonuclease-1α (IRE1α) and activating transcription factor 6 (ATF6) (Kadowaki & Nishitoh 2013). Under physiological conditions, the luminal domain of these proteins is bound to glucose-regulated protein 78 kDa (GRP78). GRP78 is an ER chaperone involved in adequate protein refolding (Wang et al. 2009). Thus, upon ER-stress, the misfolded proteins accumulating in the ER-lumen are targeted by GRP78, which is released from IRE1α, PERK and ATF6 resulting in their activation (Frakes & Dillin 2017). Misfolded proteins can directly bind to the luminal domains of IRE1α and PERK, activating them in a GRP78-independent manner (Karagöz et al. 2017, Wang et al. 2018).

After IRE1α, PERK and ATF6 are dissociated from GRP78, they initiate different downstream pathways. IRE1α is activated by dimerization and autophosphorylation (Bertolotti et al. 2000). Through its cytosolic endoribonuclease domain, active IRE1α leads to the excision of a 26-nucleotide intron within the mRNA encoding X-box binding protein 1 (XBP1) (Yoshida et al. 2001, Hetz 2012). PERK activation leads to its oligomerization and autophosphorylation (Bertolotti et al. 2000). This results in the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Active p-eIF2α inhibits guanine nucleotide exchange factor (eIF2B) leading to a decrease in protein synthesis, whereas the expression of C/EBP homologous protein (CHOP) is upregulated (Harding et al. 2000). ATF6 translocates to the Golgi apparatus where it is cleaved. The resulting cytosolic fragment acts as a transcription factor for several genes, including above-mentioned GRP78, XBP1 and CHOP (Wang et al. 2000, Hillary & FitzGerald 2018).

As a result, these conserved signaling pathways, usually referred as the unfolded protein response (UPR), trigger ER size-expansion, chaperone synthesis, attenuation of protein translation and degradation of unfolded proteins, allowing the cell to restore normal ER function (Oslowski & Urano 2011). However, in situations of prolonged ER stress, UPR can lead to apoptosis (Hetz 2012).

ER-stress can be induced by stimuli such as hypoxia, inflammation and nutrient deprivation (Urra et al. 2016). Likewise, insulin can induce ER-stress in human adipocytes and neuroblastoma cells (Inageda 2010, Boden et al. 2014). We have recently described an increase in maternal insulin levels associated with obesity already in the first trimester of human pregnancy (Bandres-Meriz et al. 2020), a time window where placental development is especially susceptible to the intrauterine environment (Desoye 2018). However, the role of oxygen and insulin in the context of placental ER-stress in early pregnancy remains barely investigated.

We hypothesized that low oxygen tension, hyperinsulinemia and their interplay modulate placental ER-stress in the human first-trimester trophoblast. This is important to study because ER-stress may impair placental function in early pregnancy (Burton & Yung 2011, Lee et al. 2019). Since the first-trimester placental tissue availability is limited, studies in early pregnancy most often have to resort to cell lines. Here, we chose the cell line ACH-3P (Hiden et al. 2007), which closely resembles primary human first-trimester trophoblasts (Bilban et al. 2010, Lee et al. 2016). We first demonstrated that ACH-3P is a suitable model for studying ER-stress, and then studied whether low and physiological oxygen tension (2.5% and 6.5% O2, respectively) and insulin concentrations within the (patho)physiological range can regulate the expression and protein levels of several members of the UPR.

Materials and methods

Cell culture

The human first-trimester trophoblast cell line ACH-3P was cultured in Ham’s F-12 medium (Thermo Fisher Scientific ), supplemented with 10% (v/v) fetal calf serum (FCS, Cytiva, Marlborough, MA, USA) and 1% (v/v) penicillin/streptomycin (Thermo Fisher Scientific). ACH-3P cells between passage 7 and 18 were maintained in a hypoxic workstation (Xvivo System Model X3, BioSpherix, Redfield, NY, USA), and incubated at 37°C in a humidified atmosphere containing 5% CO2 and 2.5% or 6.5% O2 for 48 h. Subsequently, cells were cultured under low serum conditions (2% (v/v) FCS) for 24 h. ACH-3P cells were then incubated in the absence (control) or presence of insulin (0.1, 1 or 10 nM, dissolved in 0.02 N HCl, Calbiochem) under 2.5% and 6.5% O2 during 24 h or 48 h for gene expression and protein studies. Cells were also cultured in the presence of the ER-stress inducers tunicamycin (TM, 3 µg/mL, dissolved in DMSO, Sigma–Aldrich) and brefeldin A (BFA, 10 µg/mL, dissolved in ethanol, Sigma–Aldrich) (Nakashima et al. 2019) for 24 h under 6.5% O2.

RNA isolation and RT-qPCR analysis

Total RNA from ACH-3P cells was isolated using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen). Genomic DNA contamination was removed by on-column digestion with DNAse I (Qiagen). RNA was reverse transcribed using the LunaScript RT SuperMix Kit (New England BioLabs, Ipswich, MA, USA) as per the manufacturer’s guidelines. CHOP, XBP1 and spliced XBP1 (sXBP1) mRNA levels were determined by RT-qPCR using 6-carboxyfluorescein (FAM)-labeled TaqMan gene expression assays (CHOP: Hs000358796_g1; XBP1: Hs02856596_m1; sXBP1: Hs03929085_g1; Life Technologies), TaqMan universal PCR master mix (Life Technologies) and the CFX384 RT-qPCR detection system (Bio-Rad Laboratories). Bio-Rad CFX Manager 3.1 software (Bio-Rad Laboratories) automatically generated Ct values. For statistical analysis, ΔCt was calculated using TATA-binding protein (TBP, Hs00427620_m1, 2′-chloro-7′phenyl-1,4-dichloro-6-carboxy-fluorescein (VIC)-labeled; Life Technologies) and peptidylprolyl isomerase A (PPIA, Hs04194521_s1, VIC-labeled; Life Technologies) as housekeeping genes. XBP1 splicing was calculated as ΔCt using sXBP1 and XBP1 (sXBP1 Ct – XBP1 Ct). For graphical representation purposes, results were normalized to the control under 6.5% O2 using the 2−ΔΔCt method (Pfaffl 2001).

Protein isolation and Western blotting

ACH-3P total protein was isolated in RIPA buffer (50 mM Tris–HCl, pH 8.0, with 150 mM sodium chloride, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) sodium dodecyl sulfate; Sigma–Aldrich) containing protease inhibitors (Roche). Protein concentration was determined using the bicinchoninic acid assay (Thermo Fisher Scientific) as per the manufacturer’s guidelines. Equal amounts of protein (5 µg) were mixed with 4× Laemmli buffer (62.5 mM Tris–HCl, pH 6.8, 10% (v/v) glycerol, 1% (w/v) sodium dodecyl sulfate (SDS), 0.005% (w/v) bromophenol blue and 10% (v/v) 2-mercaptoethanol, Bio-Rad Laboratories), denatured for 5 min at 96°C and loaded onto 4–20% precast SDS-PAGE gels (Bio-Rad Laboratories). Electrophoresis was performed at 120 V for 70 min. Thereafter, proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories). Non-specific binding sites were blocked for 1 h with 5% (w/v) non-fat dry milk (Bio-Rad Laboratories) or 1% (w/v) BSA in Tris-EDTA buffer (10 mM Tris–HCl, 1 mM disodium EDTA, pH 8.0) + 0.1% (v/v) Tween 20 (Sigma–Aldrich). The membranes were further incubated overnight at 4°C with primary antibodies against GRP78 (1:5000; BD Biosciences, San Jose, CA, USA), phospho-IRE1α (p(Ser724)-IRE1α, 1:1000, Novus Biologicals, Littleton, CO, USA), total IRE1α (1:1000, Cell Signaling), phospho-eIF2α (p(Ser51)-eIF2α, 1:1000, Cell Signaling), total eIF2α (1:1000, Cell Signaling), phospho-PERK (p(Thr980)-PERK, 1:250, Cell Signaling), total PERK (1:2000, Cell Signaling) or α-tubulin (1:1000, Merck). Subsequently, membranes were washed and incubated with the appropriate HRP-conjugated secondary antibody (1:5000 for GRP78 or 1:2000 for the rest of the primary antibodies, Bio-Rad Laboratories). Immunodetection was performed using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) and the ChemiDoc Touch imaging system (Bio-Rad Laboratories). Band densitometry was assessed using the Image Lab 6.0.1 software (Bio-Rad Laboratories). GRP78, IRE1α and eIF2α protein levels were normalized to α-tubulin, used as a loading control. To determine IRE1α and eIF2α phosphorylation, the ratio of phospho- to total IRE1α and eIF2a was calculated, respectively. To account for inter-membrane variation, data were normalized to an internal calibrator sample (untreated ACH-3P protein lysate) included in every gel. For graphical representation purposes, results were further normalized to the control under 6.5% O2.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8 and SPSS Statistics 25. Outliers were identified using robust regression and outlier removal (ROUT) method (Q = 0.1%). Normal distribution of data was determined by Shapiro–Wilk test. When required, the dependent variables were log-transformed to ensure a normal distribution.

The effect of TM and BFA on XBP1 splicing and expression and CHOP expression was analyzed using t-test. Associations between oxygen tension, insulin concentrations and their interaction (independent variables, exposures) and gene expression and protein levels of UPR members (dependent variables, outcomes) were assessed using analysis of covariance (ANCOVA) adjusted for passage number, which was a priori defined as co-variate. Cells cultured under 6.5% O2 without exogenous insulin supplementation were used as the referent category (REF). P < 0.05 was considered statistically significant. The interaction between oxygen and insulin was deemed significant if P < 0.10.

Results

ACH-3P cells as a model to study ER-stress in early pregnancy

To test whether the ACH-3P cell line is susceptible to ER-stress, cells were treated with the ER-stress inducers TM and BFA for 24 h. The expression of CHOP, XBP1 and its spliced variant (sXBP1) as readouts for ER-stress was determined by RT-qPCR. Both ER-stress inducers significantly increased CHOP mRNA levels to a similar extent (4.6-fold change; P < 0.001; Fig. 1A) when compared to the control. XBP1 expression was also increased in the presence of TM (1.7-fold change; P ≤ 0.001) and BFA (1.3-fold change; P < 0.05; Fig. 1B). Likewise, XBP1 splicing, calculated as sXBP1:XBP1 ratio, was significantly upregulated by TM and BFA (7.9- and 12.8-fold change, respectively; P ≤ 0.001; Fig. 1C).

Figure 1
Figure 1

The ACH-3P cell line is a suitable model to study ER-stress in early pregnancy. ACH-3P cells were cultured in the absence (control) or the presence of the ER-inducers tunicamycin (3 µg/mL) and brefeldin A (10 µg/mL). CHOP and XBP1 expression were determined by RT-qPCR and normalized to the mean expression of TBP and PPIA, used as housekeeper genes (A and B). To assess XBP1 splicing, the mRNA levels of the spliced variant of XBP1 were quantified by RT-qPCR and calculated relative to total XBP1 expression (C). Results were expressed as fold change relative to control arbitrarily set to 1. Data are presented as mean ± s.e.m. and are representative for at least three independent experiments, with each condition assayed in triplicates. Statistical analysis was performed using t-test. *P < 0.05; ***P ≤ 0.001.

Citation: Reproduction 162, 1; 10.1530/REP-20-0668

Effect of oxygen and insulin on GRP78 protein levels

GRP78 is one of the main outputs of the UPR activating downstream pathways. Thus, we quantified GRP78 protein in ACH-3P cells by Western blotting after 24 (Fig. 2A) and 48 h (Fig. 2C). To assess whether GRP78 is regulated by oxygen, insulin or their interaction, we used ANCOVA adjusted for cell passage. Cells cultured under 6.5% O2 in the absence of exogenous insulin served as the referent value (REF). We did not find any differences in GRP78 protein levels between the low (2.5% O2) and the physiological oxygen tension (6.5% O2) regardless of the time point assessed (Fig. 2B and D). Similarly, insulin concentrations spanning the (patho)physiological range (0.1, 1 and 10 nM) did not alter GRP78 protein levels under 2.5 or 6.5% O2 (Fig. 2B and D).

Figure 2
Figure 2

Effect of low oxygen tension, insulin and their interaction on GRP78 protein levels in ACH-3P cells. Cells were cultured in the absence of exogenous insulin supplementation under 6.5% O2 (referent value, REF) or 2.5% (control, CTL), or supplemented with insulin (0.1, 1and 10 nM) for 24 h (A and B) and 48 h (C and D). GRP78 protein levels were quantified by Western blotting (A and C). Results were normalized to α-tubulin, used as a loading control and calculated relative to REF, arbitrarily set to 1 (B and D). Data are presented as median ± IQR and are representative for at least three independent experiments, with each condition assayed in duplicates or triplicates. Statistical analysis was performed using ANCOVA adjusted for cell passage as co-variate.

Citation: Reproduction 162, 1; 10.1530/REP-20-0668

Low oxygen and insulin treatment might compromise cell survival. Thus, we measured lactate dehydrogenase (LDH) activity in cell supernatants as a proxy measure for cell viability. However, we were not able to detect LDH activity (data not shown), suggesting that cell viability was not affected by low oxygen, insulin treatment or their combination.

Effect of oxygen and insulin on the IRE1α pathway

During ER-stress, the autophosphorylation of the activation loop, including phosphorylation at Ser724, activates IRE1α (Ali et al. 2011). This induces XBP1 splicing, which in turn regulates several downstream genes involved in the UPR. Thus, we characterized the IRE1α pathway in ACH-3P cells exposed to low and physiological oxygen tension and insulin. Total IRE1α protein and its phosphorylation at Ser724 (p-IRE1α) were quantified by Western blotting (Fig. 3A and D). After 24 h, IRE1α phosphorylation (p-IRE1α/total IRE1α) was increased under low oxygen tension (+52% vs REF; P < 0.05; Fig. 3B) but remained unaffected by insulin treatment under both low and physiological oxygen tension. No differences in IRE1α phosphorylation were found after 48 h (Fig. 3E). Likewise, we did not observe any oxygen- or insulin-mediated effect on total IRE1α protein levels at the time points studied (Fig. 3C and F).

Figure 3
Figure 3

Effect of low oxygen tension, insulin and their interaction on IRE1α phosphorylation and protein levels in ACH-3P cells. Cells were cultured in the absence of exogenous insulin supplementation under 6.5% O2 (referent value, REF) or 2.5% (control, CTL), or supplemented with insulin (0.1, 1 and 10 nM) for 24 h (A, B and C) and 48 h (D, E and F). Phospho-IRE1α (p-IRE1α) and total IRE1α protein levels were quantified by Western blotting (A and C). IRE1α phosphorylation was calculated as p-IRE1α/Total IRE1α ratio (B and E). Total IRE1α protein levels were normalized to α-tubulin, used as a loading control (C and F). Results were calculated relative to REF, arbitrarily set to 1. Data are presented as median ± IQR and are representative for at least three independent experiments, with each condition assayed in duplicates or triplicates. Statistical analysis was performed using ANCOVA adjusted for cell passage as co-variate. *P < 0.05.

Citation: Reproduction 162, 1; 10.1530/REP-20-0668

We measured total and spliced (s) XBP1 levels by RT-qPCR. Parallel to the increase observed in IRE1α phosphorylation, low oxygen tension also upregulated XBP1 splicing after 24 h (1.8-fold change vs REF; P ≤ 0.001; Fig. 4A), whereas total XBP1 expression remained unaffected (Fig. 4B). Neither insulin or oxygen nor their combination changed XBP1 splicing or expression after 48 h (Fig. 4C and D).

Figure 4
Figure 4

Effect of low oxygen tension, insulin and their interaction on XBP1 splicing and expression in ACH-3P cells. Cells were cultured in the absence of exogenous insulin supplementation under 6.5% O2 (referent value, REF) or 2.5% (control, CTL), or supplemented with insulin (0.1, 1 and 10 nM) for 24 h (A and B) and 48 h (C and D). The mRNA levels of XBP1 and its spliced variant (sXBP1) were determined by RT-qPCR. XBP1 splicing was calculated as ΔCt (sXBP1 Ct − XBP1 Ct) (A and C). Total XBP1 expression was normalized to the mean expression of TBP and PPIA, used as housekeeper genes (B and D). Results were calculated relative to REF, arbitrarily set to 1. Data are presented as mean ± s.e.m. and are representative for three independent experiments, with each condition assayed in duplicates or triplicates. Statistical analysis was performed using ANCOVA adjusted for cell passage as co-variate. ***P ≤ 0.001.

Citation: Reproduction 162, 1; 10.1530/REP-20-0668

Effect of oxygen and insulin on the eIF2α pathway

eIF2α phosphorylation at Ser51 is a key event within the UPR, leading to the inhibition of protein translation. Therefore, we also studied whether eIF2α is modulated by oxygen, insulin or their interaction after 24 and 48 h. Total and phospho (p-) eIF2α protein levels were determined by Western blotting (Fig. 5A and D).

Figure 5
Figure 5

Effect of low oxygen tension, insulin and their interaction on eIF2α phosphorylation and protein levels in ACH-3P cells. Cells were cultured in the absence of exogenous insulin supplementation under 6.5% O2 (referent value, REF) or 2.5% (control, CTL), or supplemented with insulin (0.1, 1 and 10 nM) for 24 h (A, B and C) and 48 h (D, E and F). Phospho-eIF2α (p-eIF2α) and total eIF2α protein levels were quantified by Western blotting (A and C). eIF2α phosphorylation was calculated as p-eIF2α/Total eIF2α ratio (B and E). Total eIF2α protein levels were normalized to α-tubulin, used as a loading control (C and F). Results were calculated relative to REF, arbitrarily set to 1. Data are presented as median ± IQR and are representative for three independent experiments, with each condition assayed in duplicates or triplicates. Statistical analysis was performed using ANCOVA adjusted for cell passage as co-variate. *P < 0.05; **P ≤ 0.01.

Citation: Reproduction 162, 1; 10.1530/REP-20-0668

eIF2α phosphorylation at Ser51 (p-eIF2α/Total eIF2α) was decreased under low oxygen after 24 and 48 h (Fig. 5B and E), the effect reaching statistical significance only after 48 h (−61% vs REF; P < 0.05; Fig. 5E). Total eIF2α protein levels were also downregulated under 2.5% O2 after 24 h (−28% vs REF; P < 0.05; Fig. 5C) but remained unaffected by treatments after 48 h (Fig. 5F).

Although insulin had no effect on eIF2α phosphorylation or protein levels under 6.5% O2 (Fig. 5B, C, E and F), 10 nM insulin induced a significant increase in eIF2α phosphorylation under 2.5% O2 after 48 h (+46% vs REF; P ≤ 0.01; Fig. 5E). When mediated by ER-stress, eIF2α is phosphorylated by PERK. Therefore, we quantified total and phospho (p-) PERK protein by Western blotting (Supplementary Fig. 1A, see section on supplementary materials given at the end of this article). Interestingly, neither oxygen, insulin or their combination altered both PERK phosphorylation at Thr980 (p-PERK/Total PERK) and its protein (Supplementary Fig. 1B and C).

One of the potential consequences of eIF2α activation is CHOP induction, which is also involved in UPR. Thus, we quantified CHOP mRNA by RT-qPCR. Low oxygen tension (2.5% O2) decreased CHOP expression after 24 h relative to 6.5% O2 (−24%; P ≤ 0.001; Fig. 6A), whereas insulin had no effect at both 2.5 and 6.5% O2. Neither insulin or oxygen nor their combination altered CHOP expression after 48 h (Fig. 6B).

Figure 6
Figure 6

Effect of low oxygen tension, insulin and their interaction on CHOP expression in ACH-3P cells. Cells were cultured in the absence of exogenous insulin supplementation under 6.5% O2 (referent value, REF) or 2.5% (control, CTL), or supplemented with insulin (0.1, 1 and 10 nM) for 24 h (A) and 48 h (B). CHOP mRNA levels were quantified by RT-qPCR and normalized to the mean expression of TBP and PPIA, used as housekeeper genes. Results were calculated relative to REF, arbitrarily set to 1. Data are presented as mean ± s.e.m. and are representative for three independent experiments, with each condition assayed in duplicates or triplicates. Statistical analysis was performed using ANCOVA adjusted for cell passage as co-variate. ***P ≤ 0.001.

Citation: Reproduction 162, 1; 10.1530/REP-20-0668

Discussion

ER-stress occurs when misfolded proteins accumulate in the lumen of the ER. This activates the UPR, which in turn regulates several cellular processes including cell proliferation, survival and apoptosis, ultimately deciding cell fate (Hetz 2012, Corazzari et al. 2017). Despite its interest, ER-stress in pregnancy has been mainly studied in pregnancy complications such as preeclampsia (PE) and GDM (Burton et al. 2009, Yung et al. 2016), but its role in early pregnancy has remained elusive. Here, we used the first-trimester trophoblast cell line ACH-3P to assess whether ER-stress can be induced by low oxygen tension or insulin treatment in early pregnancy. Importantly, we used ANCOVA for our statistical analysis, a method that allowed us to control the potential effect of a priori defined co-variate. We specifically adjusted for cell passage number, since it has been shown to affect cell behavior and metabolism (Kwist et al. 2016).

We first focused on GRP78, since its translocation into the ER-lumen is considered one of the initial steps in the UPR (Schwarz & Blower 2016). In pregnancy, GRP78 also regulates several physiological processes including cytotrophoblast fusion (Bastida-Ruiz et al. 2020) and invasion (Arnaudeau et al. 2009). Interestingly, in the present study, neither oxygen nor insulin was able to alter GRP78 protein levels in ACH-3P under the conditions used. These results might initially argue for an absence of ER-stress, that is, lack of activation of the UPR. However, ER-stress can lead to increased GRP78 expression without directly affecting GRP78 protein (Gülow et al. 2002). Thus, based on our results we cannot rule out UPR activation, since GRP78 long protein half-life and its abundant steady-state levels in the ER-lumen might have precluded detection of differences in GRP78 abundance by Western blotting.

The IRE1α-sXBP1 pathway regulates several physiological processes including cell differentiation (Zhang et al. 2005), angiogenesis (Ghosh et al. 2010) and apoptosis (Shemorry et al. 2019) among others. This pathway is also crucial for embryonic viability and adequate placental development in mice (Iwawaki et al. 2009, Oikawa et al. 2010). In human, it is upregulated in pregnancy complications such as PE and fetal growth restriction (FGR) (Lian et al. 2011). Outside pregnancy, oxygen is a regulator of this pathway, since both IRE1α and sXBP1 levels are increased by hypoxia in the breast cancer cell lines MDA-MB-231 and HCC1937 (Liang et al. 2018). Concordantly, we also observed increased IRE1α phosphorylation paralleled by an increase in XBP1 splicing in ACH-3P cells cultured under low oxygen tension. Thus, sustained sub-physiological low oxygen tension, potentially as a result of impaired remodeling of the spiral arteries, might compromise cellular processes such as trophoblast proliferation and differentiation, hindering placental development in early pregnancy. Interestingly, after 48 h, low-oxygen did no longer affect IRE1α phosphorylation or XBP1 splicing. This might reflect a time-dependent regulation of this UPR branch since sustained ER-stress can decrease IRE1α activation (Lin et al. 2007).

We were unable to detect any effect of insulin on the IRE1α-sXBP1 pathway. Hyperinsulinemia is one of the hallmarks of maternal obesity and GDM (Desoye & Hauguel-de Mouzon 2007) present already in the first trimester in obese women (Bandres-Meriz et al. 2020). These conditions, in which ER-stress plays a role (Westermeier et al. 2014, Yung et al. 2016), are also associated with hyperglycemia. This has been recently shown to increase XBP1 splicing in BeWo cells (Lorenzon et al. 2020) and might trigger placental ER-stress in early pregnancy. However, we failed to demonstrate hyperglycemia in the early pregnancy of obese women (Bandres-Meriz et al. 2020), which is why hyperglycemia effects were not studied here.

Finally, we studied the UPR branch involving eIF2α and CHOP. The eIF2α-CHOP axis regulates placental protein translation and apoptosis, which has been linked to pregnancy disorders such as PE and FGR (Yung et al. 2008). This pathway is also involved in other trophoblast-specific physiological processes such as syncytialization (Bastida-Ruiz et al. 2020). Previous studies suggested that hypoxia and hypoxia-reperfusion can induce eIF2α phosphorylation in the human first-trimester trophoblast cell lines JEG-3 and BeWo (Yung et al. 2012, 2014). In contrast, in ACH-3P cells used in our study, low oxygen tension induced downregulation of eIF2α phosphorylation. It is important to highlight that both studies were conducted not only with different cell lines but also under different oxygen tensions. Here, cells were maintained under low and physiological oxygen tensions (2.5 and 6.5% O2, respectively), whereas Yung et al. used hypoxia and hyperoxia (1% vs 21% O2). Altogether, this suggests that eIF2α phosphorylation is regulated in an oxygen- and cell type-specific manner.

eIF2α phosphorylation is mediated by four different upstream kinases, with PERK being the one predominantly involved in ER-stress (Donnelly et al. 2013). In our study, PERK activation remained unaffected by oxygen suggesting that the eIF2α regulation observed might not be ER-stress related.

Severe ER-stress increases CHOP expression and induces apoptosis in JEG-3 cells (Yung et al. 2008). Upregulation of CHOP mRNA was also found in our study after TM or BFA treatment of ACH-3P cells, whereas low oxygen tension decreased CHOP expression. This contrasts to results obtained in other cell types such as endothelial and neuronal cells (Tajiri et al. 2004, Moszyńska et al. 2020), in which ischemia, that is, low oxygen, induces CHOP expression and cell death. Thus, CHOP downregulation in early pregnancy might ensure trophoblast cell survival allowing placental development under adverse environmental conditions in utero.

In our study, eIF2α was the only UPR member regulated by insulin. The insulin-induced increase in eIF2α phosphorylation has been previously reported in human adipose tissue (Boden et al. 2014). Interestingly, the insulin effect was only significant under low oxygen tension, suggesting an interplay between oxygen and insulin in early pregnancy, when spiral arteries are not fully remodeled and oxygen tension in the intervillous space is low, that is, around 2.5% O2. Nevertheless, we suggest this not to be ER-stress mediated since PERK activation was also not altered by insulin treatment.

To the best of our knowledge, this is the first study assessing the effect of insulin on ER-stress in early pregnancy. Although oxygen is one of the major factors regulating trophoblast biology, experiments in the first trimester of pregnancy are usually performed under extreme hypoxic (1% O2) or hyperoxic (21% O2) conditions. Thus, one of the major strengths of this study is the use of (patho)physiological concentrations of oxygen and insulin, which mimic more closely the situation in vivo. The main limitation of this study is the lack of an in-depth analysis of the cellular consequences of ER-stress in ACH-3P cells, and also not having included ATF6, which itself can regulate CHOP expression (Yang et al. 2020). Likewise, the results obtained are based exclusively on a first-trimester trophoblast cell line and future studies should determine whether similar changes in the UPR are also observed in human first-trimester placental explants. Finally, the interplay between insulin and other metabolites that are also dysregulated in obesity such palmitate needs to be addressed since palmitate is capable of regulating ER-stress in cell models of early pregnancy (Lee et al. 2020).

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/REP-20-0668.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

D H received funding from the Doctorate Program MOLIN (FWF, W1241) and the Medical University of Graz. J B-M received funding from the Doctorate Program DP-iDP (FWF, DOC 31-B26) and the Medical University of Graz. A M-M and G D are supported by funds of the Oesterreichische Nationalbank (Anniversary Fund, project number: 17950).

Author contribution statement

V T performed the majority of the experiments and data analysis and participated in the study design. S N performed experiments. D H, J B-M and G D participated in the conception and design of the study, were involved in data discussion and critically reviewed the manuscript. A M-M participated in the conception and design of the study and was involved in data discussion as well as drafting the manuscript.

Acknowledgements

The authors thank Renate Michlmaier and Dr Francisco Algaba Chueca for their technical support and expertise.

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    Figure 1

    The ACH-3P cell line is a suitable model to study ER-stress in early pregnancy. ACH-3P cells were cultured in the absence (control) or the presence of the ER-inducers tunicamycin (3 µg/mL) and brefeldin A (10 µg/mL). CHOP and XBP1 expression were determined by RT-qPCR and normalized to the mean expression of TBP and PPIA, used as housekeeper genes (A and B). To assess XBP1 splicing, the mRNA levels of the spliced variant of XBP1 were quantified by RT-qPCR and calculated relative to total XBP1 expression (C). Results were expressed as fold change relative to control arbitrarily set to 1. Data are presented as mean ± s.e.m. and are representative for at least three independent experiments, with each condition assayed in triplicates. Statistical analysis was performed using t-test. *P < 0.05; ***P ≤ 0.001.

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    Figure 2

    Effect of low oxygen tension, insulin and their interaction on GRP78 protein levels in ACH-3P cells. Cells were cultured in the absence of exogenous insulin supplementation under 6.5% O2 (referent value, REF) or 2.5% (control, CTL), or supplemented with insulin (0.1, 1and 10 nM) for 24 h (A and B) and 48 h (C and D). GRP78 protein levels were quantified by Western blotting (A and C). Results were normalized to α-tubulin, used as a loading control and calculated relative to REF, arbitrarily set to 1 (B and D). Data are presented as median ± IQR and are representative for at least three independent experiments, with each condition assayed in duplicates or triplicates. Statistical analysis was performed using ANCOVA adjusted for cell passage as co-variate.

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    Figure 3

    Effect of low oxygen tension, insulin and their interaction on IRE1α phosphorylation and protein levels in ACH-3P cells. Cells were cultured in the absence of exogenous insulin supplementation under 6.5% O2 (referent value, REF) or 2.5% (control, CTL), or supplemented with insulin (0.1, 1 and 10 nM) for 24 h (A, B and C) and 48 h (D, E and F). Phospho-IRE1α (p-IRE1α) and total IRE1α protein levels were quantified by Western blotting (A and C). IRE1α phosphorylation was calculated as p-IRE1α/Total IRE1α ratio (B and E). Total IRE1α protein levels were normalized to α-tubulin, used as a loading control (C and F). Results were calculated relative to REF, arbitrarily set to 1. Data are presented as median ± IQR and are representative for at least three independent experiments, with each condition assayed in duplicates or triplicates. Statistical analysis was performed using ANCOVA adjusted for cell passage as co-variate. *P < 0.05.

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    Figure 4

    Effect of low oxygen tension, insulin and their interaction on XBP1 splicing and expression in ACH-3P cells. Cells were cultured in the absence of exogenous insulin supplementation under 6.5% O2 (referent value, REF) or 2.5% (control, CTL), or supplemented with insulin (0.1, 1 and 10 nM) for 24 h (A and B) and 48 h (C and D). The mRNA levels of XBP1 and its spliced variant (sXBP1) were determined by RT-qPCR. XBP1 splicing was calculated as ΔCt (sXBP1 Ct − XBP1 Ct) (A and C). Total XBP1 expression was normalized to the mean expression of TBP and PPIA, used as housekeeper genes (B and D). Results were calculated relative to REF, arbitrarily set to 1. Data are presented as mean ± s.e.m. and are representative for three independent experiments, with each condition assayed in duplicates or triplicates. Statistical analysis was performed using ANCOVA adjusted for cell passage as co-variate. ***P ≤ 0.001.

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    Figure 5

    Effect of low oxygen tension, insulin and their interaction on eIF2α phosphorylation and protein levels in ACH-3P cells. Cells were cultured in the absence of exogenous insulin supplementation under 6.5% O2 (referent value, REF) or 2.5% (control, CTL), or supplemented with insulin (0.1, 1 and 10 nM) for 24 h (A, B and C) and 48 h (D, E and F). Phospho-eIF2α (p-eIF2α) and total eIF2α protein levels were quantified by Western blotting (A and C). eIF2α phosphorylation was calculated as p-eIF2α/Total eIF2α ratio (B and E). Total eIF2α protein levels were normalized to α-tubulin, used as a loading control (C and F). Results were calculated relative to REF, arbitrarily set to 1. Data are presented as median ± IQR and are representative for three independent experiments, with each condition assayed in duplicates or triplicates. Statistical analysis was performed using ANCOVA adjusted for cell passage as co-variate. *P < 0.05; **P ≤ 0.01.

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    Figure 6

    Effect of low oxygen tension, insulin and their interaction on CHOP expression in ACH-3P cells. Cells were cultured in the absence of exogenous insulin supplementation under 6.5% O2 (referent value, REF) or 2.5% (control, CTL), or supplemented with insulin (0.1, 1 and 10 nM) for 24 h (A) and 48 h (B). CHOP mRNA levels were quantified by RT-qPCR and normalized to the mean expression of TBP and PPIA, used as housekeeper genes. Results were calculated relative to REF, arbitrarily set to 1. Data are presented as mean ± s.e.m. and are representative for three independent experiments, with each condition assayed in duplicates or triplicates. Statistical analysis was performed using ANCOVA adjusted for cell passage as co-variate. ***P ≤ 0.001.

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