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

in Reproduction
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  • 1 Department of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria

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.

Supplementary Materials

    • Supplementary figure 1. Effect of low oxygen tension, insulin and their interaction on PERK 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 nM, 1 nM and 10 nM) for 24h (A-C) and 48h (D-F). Phospho-PERK (p-PERK) and total PERK protein levels were quantified by Western blotting (A). PERK phosphorylation was calculated as p-PERK/Total PERK ratio (B). Total PERK protein levels were normalized to &#x03B1;-tubulin, used as loading control (C). Results were calculated relative to REF, arbitrarily set to 1. Data are presented as median &#x00B1; 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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