Calcium, copper, iron, oxygen, and carbon dioxide are essential factors in fetal growth. These molecules are transferred by specific receptors located on the cell membrane or cytoplasm in placenta. Calcium, copper, and iron transfer genes are regulated by oestrogen, placental lactogen, and vitamin D. During pregnancy, expression of these receptors is controlled by the nutritional status of the maternal and fetal environment. Some synthetic plastics contain endocrine-disrupting chemicals (EDC), which have similar structures to steroid hormones or endogenous hormones related to reproduction. These substances disturb action of hormones (e.g. increasing oestrogen or progesterone) by interacting with their receptors or affecting the expression of transporting genes for cations. We used a BeWo cell line (human trophoblast cell line) to test the effect of EDC during pregnancy. The cells were cultured in phenol red-free DMEM supplemented with 5% charcoal dextran-stripped fetal bovine serum for 48 h to ensure the depletion of steroid hormones in the cells. Ethinyl oestradiol (EE), which activates oestrogen receptors, was used as a positive control. Then, EE (10–9, 10–8, and 10–7 M), octylpehnol (OP; 10–7, 10–6, and 10–5 M), nonylphenol (NP; 10–8, 10–7, and 10–6 M), and bisphenol A (BPA; 10–7, 10–6, and 10–5 M) were treated in BeWo cells for 48 h, and the cells were harvested. The mRNA and protein levels for calcium transporting genes (PMCA1 and TRPV6), copper transporting genes (CTR1 and ATP7A), and iron transporting genes (IREG1 and HEPH) were quantified by RT-qPCR, and Western blotting, respectively. Experiments were carried 3 times, and results were statistically analysed by GraphPad Prism6 (GraphPad Software, San Diego, CA, USA). We observed dose-dependent decreases in mRNA levels of PMCA1, TRPV6, ATP7A, and IREG1 compared with control group in OP-, NP-, or BPA-treated groups. Protein levels showed a similar pattern to mRNA levels. Based on our data, we confirmed that these EDC affect metal ion channels such as calcium, copper, and iron transporters during pregnancy.
Endoplasmic reticulum (ER) regulates calcium ion concentration as a reservoir in the cell. ER stress is a cellular stress response related to the endoplasmic reticulum. At the initial stage of ER stress, ER tries to restore normal function by halting protein translation, degrading misfolded proteins, and increasing production of chaperones involved in protein folding. If ER fails to restore ER stress, ER stress can lead cells to apoptosis. To study the signaling between ER stress and calcium channels under ER-stressed circumstances, we designed a hypoxia-induced diabetic model. Nine-week-old male mice were chosen, maintained under hypoxic condition under 10% O2, 5% CO2 for 10 days, and the expression of ER stress markers and calcium channel gene expression were examined by real-time PCR. By maintaining hypoxic condition, the mice showed high glucose levels. Under this diabetic condition, in pancreatic beta cells, ER stress markers were elevated. This tendency showed an increase in calbindin-D9k KO mice. Chaperones such as calreticulin and calnexin were decreased, but in calbindin-D9k KO mice chaperone calnexin was not decreased. Interestingly, the calbindin-D9k KO normoxia mice showed increased glucose level compared with wild-type normoxia mice. Also, calnexin expression of pancreas was decreased in calbindin-D9k KO normoxia mice. This result indicates that pancreas cells were under endoplasmic reticulum stress. Taken together, calbindin may play an important role in endoplasmic reticulum stress in pancreas. This work was supported by the National Research Foundation of Korea (NRF) grant of Korean government (MEST) (No. 2013-010514).
Preeclampsia (PE) is a disorder of pregnancy characterized by high blood pressure and large amounts of protein in the urine. Preeclampsia is thought in many cases to be caused by a shallowly implanted placenta that becomes hypoxic. The hypoxic condition during the pregnancy can result from a failure at any stage in the delivery of oxygen to the cells. In peripheral tissues, oxygen diffuses down a pressure gradient into cells and moves into their mitochondria, where it is used to produce energy. As an expression of beta-oxidation-related genes, ACADVL was detected by gene-fishing technology using the placenta of human. We conducted in vitro and in vivo experiments to confirm preliminary study by inducing hypoxic stress in the BeWo cells and mice placenta. BeWo cells were cultured at 37°C under 1% O2, 5% CO2, and balanced with N2. Pregnant mice were maintained from GD 6.5 to 17.5 under 11% O2, 5% CO2, and balanced with N2. The expression of beta-oxidation related genes (ACADVL, EHHADH, HADH, ACAA1) were observed under hypoxic condition at mRNA and protein levels. The expression of genes known as biomarkers for hypoxia, HIF-1α, was increased in BeWo cells and mouse placenta, which induced PE. The beta-oxidation-related genes ACADVL expression was significantly increased by hypoxic stress both BeWo cells and mouse placenta. The elevated level of HIF-1α indicates that our experimental conditions closely mimicked PE. These results indicate that changes of beta-oxidation-related genes are correlated with PE induced hypoxic condition.
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