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

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.

Glucose, Insulin and Oxygen Modulate Expression of Serotonin-Regulating Genes in Human First-Trimester Trophoblast Cell Line ACH-3P

“…We chose to use first-trimester trophoblast-derived cell line ACH-3P, which is particularly suitable for in vitro experiments because it closely resembles primary human first-trimester trophoblasts, but compared to the primary trophoblasts, has proliferative properties [ 26 ]. It also expresses insulin receptors, essential for the present study [ 27 ].…”

“…This approach aimed to provide more controlled and standardized experimental conditions by mitigating potential interference from insulin, glucose, and serotonin present in serum. The time points for treatments were selected based on published studies on the effects of glucose and insulin in trophoblast cellular models [ 27 , 28 ] and the results of our preliminary experiments.…”

“…For the insulin treatment experiments, the ACH-3P cells were cultured for 2 days before treatment under low-serum conditions (2% FBS) in Ham’s F-12 medium, and subsequently incubated in the absence (control) or presence of 10 nM insulin (Calbiochem, EMD Chemicals, San Diego, CA, USA) at 2.5% and 6.5% O 2 for 24 and 48 h. Insulin concentration was chosen based on our previous dose-response experiments on ACH-3P cells, showing that the respective concentration mimicked hyperinsulinemia in our experimental setup [ 27 ]. This concentration also corresponds to the physiologically relevant postprandial concentration of insulin [ 30 ].…”