Mono-(2-ethylhexyl) phthalate induces trophoblast hypoxia and mitochondrial dysfunction through HIF-1α-miR-210-3p axis in HTR-8/SVneo cell line
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PROVIDER: S-EPMC11338994 | biostudies-literature | 2024 Jan
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Project description:Preeclampsia is associated with first trimester placental dysfunction. miR-210, a small non-coding RNA, is increased in the preeclamptic placenta. The effects of elevated miR-210 on placental function remain unclear. The objectives of this study were to identify targets of miR-210 in first trimester primary extravillous trophoblasts (EVTs) and to investigate functional pathways altered by elevated placental miR-210 during early pregnancy. EVTs isolated from first trimester placentas were exposed to cobalt chloride (CoCl2), a HIF-1α stabilizer and hypoxia mimetic, and miR-210 expression by qPCR, HIF1α protein levels by western blot and cell invasion were assessed. A custom TruSeq RNA array, including all known/predicted miR-210 targets, was run using miR-210 and miR-negative control transfected EVTs. Mitochondrial function was assessed by high resolution respirometry in transfected EVTs. EVTs exposed to CoCl2 showed a dose and time-dependent increase in miR-210 and HIF1α and reductions in cell invasion. The TruSeq array identified 49 altered genes in miR-210 transfected EVTs with 27 genes repressed and 22 enhanced. Three of the top six significantly repressed genes, NDUFA4, SDHD, and ISCU, are associated with mitochondrial function. miR-210 transfected EVTs had decreased maximal, complex II and complex I+II mitochondrial respiration. This study suggests that miR-210 alters first trimester trophoblast function. miR-210 overexpression alters EVT mitochondrial function in early pregnancy. Mitochondrial dysfunction may lead to increased reactive oxygen species, trophoblast cell damage and likely contributes to the pathogenesis of preeclampsia.
| S-EPMC6590495 | biostudies-literature
Project description:Hepatocyte growth factor (HGF) is reported to be down-regulated in pregnancy complications like intrauterine growth retardation and preeclampsia, which are associated with abnormal trophoblast migration/invasion. In this study, role of HGF and associated signaling pathways has been investigated in HTR-8/SVneo trophoblastic cells migration/invasion under normoxia (20% O2) and hypoxia (2% O2). HTR-8/SVneo cells exposed to hypoxia showed increase in migration and invasion as compared to cells incubated under normoxic conditions. The migration/invasion under both normoxic and hypoxic conditions was further enhanced after treatment with HGF. Subsequent to treatment with HGF, a significant increase in expression of MMP2 & MMP3 under normoxia and MMP1 & MMP9 under hypoxia was observed. Treatment of HTR-8/SVneo cells with HGF under hypoxia also led to decrease in TIMP1. Treatment of the cells with HGF led to activation of mitogen activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. Inhibition of MAPK by U0126 and PI3K by LY294002 led to concomitant decrease in the HGF-mediated migration/invasion of HTR-8/SVneo cells. HGF treatment under hypoxia also led to a significant increase in hypoxia inducible factor (HIF-1α) expression. Additionally, inhibition of HIF-1α by siRNA led to decrease in HGF-mediated migration of HTR-8/SVneo cells under hypoxic conditions. Inhibition of HGF activated MAPK and PI3K signaling led to reduction in HIF-1α expression under hypoxia. In conclusion, HGF facilitates HTR-8/SVneo cell migration/invasion by activation of MAPK/PI3K signaling pathways and increased expression of MMPs. HIF-1α has a role in HGF-mediated increase in migration under hypoxic conditions.
| S-EPMC6946778 | biostudies-literature
Project description:Phthalates are ubiquitous plasticizer chemicals found in consumer products. Exposure to phthalates during pregnancy has been associated with adverse pregnancy and birth outcomes and differences in placental gene expression in human studies. The objective of this research was to evaluate global changes in placental gene expression via RNA sequencing in two placental cell models following exposure to the phthalate metabolite mono(2-ethylhexyl) phthalate (MEHP). HTR-8/SVneo and primary syncytiotrophoblast cells were exposed to three concentrations (1, 90, 180 µM) of MEHP for 24 h with DMSO (0.1%) as a vehicle control. mRNA and lncRNAs were quantified using paired-end RNA sequencing, followed by identification of differentially expressed genes (DEGs), significant KEGG pathways, and enriched transcription factors (TFs). MEHP caused gene expression changes across all concentrations for HTR-8/SVneo and primary syncytiotrophoblast cells. Sex-stratified analysis of primary cells identified different patterns of sensitivity in response to MEHP dose by sex, with male placentas being more responsive to MEHP exposure. Pathway analysis identified 11 KEGG pathways significantly associated with at least one concentration in both cell types. Four ligand-inducible nuclear hormone TFs (PPARG, PPARD, ESR1, AR) were enriched in at least three treatment groups. Overall, we demonstrated that MEHP differentially affects placental gene expression based on concentration, fetal sex, and trophoblast cell type. This study confirms prior studies, as enrichment of nuclear hormone receptor TFs were concordant with previously published mechanisms of phthalate disruption, and generates new hypotheses, as we identified many pathways and genes not previously linked to phthalate exposure.
| S-EPMC9968694 | biostudies-literature
Project description:Gordonia sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The K(m) and V(max) values for mono-2-ethylhexyl phthalate were 26.9 +/- 4.3 microM and 18.1 +/- 0.9 micromol/min . mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of meta-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.
| S-EPMC1449075 | biostudies-literature
Project description:RationaleThe most frequently occurring phthalate, di(2-ethylhexyl) phthalate (DEHP), causes adverse effects on glucose homeostasis and insulin sensitivity in several cell models and epidemiological studies. However, thus far, there is no information available on the molecular interaction of phthalates and one of the key regulators of the metabolism, the peroxisome proliferator-activated receptor gamma (PPARγ). Since the endogenous ligand of PPARγ, 15-deoxy-delta-12,14-prostaglandin J2 (15Δ-PGJ2 ), features structural similarity to DEHP and its main metabolites produced in human hepatic metabolism, mono(2-ethylhexyl) phthalate (MEHP) and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), we tested the hypothesis of direct interactions between PPARγ and DEHP or its transformation products.MethodsHydrogen/deuterium exchange mass spectrometry (HDX-MS) and docking were conducted to obtain structural insights into the interactions and surface plasmon resonance (SPR) analysis to reveal information about binding levels. To confirm the activation of PPARγ upon ligand binding on the cellular level, the GeneBLAzer® bioassay was performed.ResultsHDX-MS and SPR analyses demonstrated that the metabolites MEHP and MEOHP, but not DEHP itself, bind to the ligand binding pocket of PPARγ. This binding leads to typical activation-associated conformational changes, as observed with its endogenous ligand 15Δ-PGJ2 . Furthermore, the reporter gene assay confirmed productive interaction. DEHP was inactive up to a concentration of 14 μM, while the metabolites MEHP and MEOHP were active at low micromolar concentrations.ConclusionsIn summary, this study gives structural insights into the direct interaction of PPARγ with MEHP and MEOHP and shows that the DEHP transformation products may modulate the lipid metabolism through PPARγ pathways.
| S-EPMC6367069 | biostudies-literature
Project description:BackgroundBone morphogenetic protein 9 (BMP9) has been shown to regulate processes such as angiogenesis, endothelial dysfunction, and tumorigenesis. However, the role of BMP9 in preeclampsia (PE) is unclear. The purpose of this study was to investigate the role and mechanism of BMP9 in PE.MethodsThe effects of BMP9 on the viability, migration and invasion of HTR-8/Svneo cells were investigated by CCK-8 assay, wound healing assay and Transwell invasion assay. The effect of BMP9 on apoptosis of HTR-8/Svneo cells was detected by flow cytometry. Plasma levels of BMP9, SDF1 and CXCR4 were detected by ELISA kit. qRT-PCR and Western blot were used to detect the expression levels of each gene in the cells.ResultsOverexpression of BMP9 promoted the proliferation and migration of trophoblast cells and inhibited apoptosis. Knockdown of BMP9 had the opposite effect. The levels of BMP9, SDF1 and CXCR4 in the plasma of PE patients were down-regulated, and BMP9 was positively correlated with the levels of SDF1 and CXCR4. BMP9 also significantly upregulated the mRNA and protein levels of SDF1 and CXCR4 in HTR-8/SVneo cells. Further mechanistic studies found that BMP9 promoted the migration and invasion of HTR-8/SVneo cells and inhibited apoptosis by activating the SDF1/CXCR4 pathway.ConclusionWe demonstrate for the first time that BMP9 promoted the migration and invasion of HTR-8/SVneo cells and inhibits apoptosis by activating the SDF1/CXCR4 pathway. This suggests that BMP9 may be a biomarker molecule for PE.
| S-EPMC10405378 | biostudies-literature
Project description:BackgroundHow nutrition and growth factor restriction due to serum depletion affect trophoblast function remains poorly understood. We performed a proteomic differential study of the effects of serum depletion on a first trimester human immortalized trophoblast cell line.MethodsThe viability of HTR-8/SVneo trophoblast cells in culture with 0, 0.5 and 10 % fetal bovine serum (FBS) were assayed via MTT at 24, 48 and 64 h. A comparative proteomic analysis of the cells grown with those FBS levels for 24 h was performed using two-dimensional electrophoresis (2DE), followed by mass spectrometry for protein spot identification, and a database search and bioinformatics analysis of the expressed proteins. Differential spots were identified using the Kolmogorov-Smirnov test (n = 3, significance level 0.10, D > 0.642) and/or ANOVA (n = 3, p < 0.05).ResultsThe results showed that low serum doses or serum depletion differentially affect cell growth and protein expression. Differential expression was seen in 25 % of the protein spots grown with 0.5 % FBS and in 84 % of those grown with 0 % FBS, using 10 % serum as the physiological control. In 0.5 % FBS, this difference was related with biological processes typically affected by the serum, such as cell cycle, regulation of apoptosis and proliferation. In addition to these changes, in the serum-depleted proteome we observed downregulation of keratin 8, and upregulation of vimentin, the glycolytic enzymes enolase and pyruvate kinase (PKM2) and tumor progression-related inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) enzyme. The proteins regulated by total serum depletion, but not affected by growth in 0.5 % serum, are members of the glycolytic and nucleotide metabolic pathways and the epithelial-to-mesenchymal transition (EMT), suggesting an adaptive switch characteristic of malignant cells.ConclusionsThis comparative proteomic analysis and the identified proteins are the first evidence of a protein expression response to serum depletion in a trophoblast cell model. Our results show that serum depletion induces specific changes in protein expression concordant with main cell metabolic adaptations and EMT, resembling the progression to a malignant phenotype.
| S-EPMC5415790 | biostudies-literature
Project description:Phthalate exposure is widespread and has a global impact. Growing evidence shows that mono-2-ethylhexyl phthalate (MEHP) exposure has a negative impact on human health. However, whether MEHP exposure is associated with mortality and other adverse outcomes in hemodialysis patients remains unknown. This study prospectively enrolled 217 patients on maintenance hemodialysis from June 30, 2021, to August 16, 2022. Baseline serum MEHP, di-2-ethylhexyl phthalate (DEHP), and indoxyl sulfate (IS) concentrations were measured. Primary endpoints were all-cause mortality or composite adverse outcomes, including all-cause death plus hospitalization due to cardiovascular disease, heart failure, stroke, infection, or cancer. Serum MEHP concentrations were positively associated with DEHP but not indoxyl sulfate concentrations in hemodialysis patients. Additionally, serum MEHP concentrations were significantly and independently associated with all-cause mortality and composite adverse outcomes (adjusted hazard ratios [HRs], 1.04 and 1.03 per ng/mL, 95% confidence intervals [CIs], 1.01-1.07 and 1.00-1.05; p = 0.016 and 0.015, respectively). We found a cutoff value of MEHP for predicting both endpoints. Patients with serum MEHP concentrations of ≥ 41.8 ng/mL had much higher risks for all-cause mortality and composite adverse outcomes (adjusted HRs, 39.2 and 13; 95% CIs, 2.44-65.7 and 2.74-61.4; p = 0.011 and 0.001, respectively). MEHP exposure is significantly associated with higher risks for all-cause mortality and composite adverse outcomes. Hemodialysis patients with serum MEHP concentrations above 41.8 ng/mL had much poorer prognoses regarding both outcomes.
| S-EPMC10697867 | biostudies-literature
Project description:Recently, numerous studies have identified that immature cell populations including stem cells and progenitor cells can be found among "side-population" (SP) cells. Although SP cells isolated from some adult tissues have been reported elsewhere, isolation and characterization of human trophoblast SP remained to be reported. In this study, HTR-8/SVneo cells and human primary villous cytotrophoblasts (vCTBs) were stained with Hoechst 33342 and SP and non-SP (NSP) fractions were isolated using a cell sorter. A small population of SP cells was identified in HTR-8/SVneo cells and in vCTBs. SP cells expressed several vCTB-specific markers and failed to express syncytiotrophoblast (STB) or extravillous cytotrophopblast (EVT)-specific differentiation markers. SP cells formed colonies and proliferated on mouse embryonic fibroblast (MEF) feeder cells or in MEF conditioned medium supplemented with heparin/FGF2, and they also showed long-term repopulating property. SP cells could differentiate into both STB and EVT cell lineages and expressed several differentiation markers. Microarray analysis revealed that IL7R and IL1R2 were exclusively expressed in SP cells and not in NSP cells. vCTB cells sorted as positive for both IL7R and IL1R2 failed to express trophoblast differentiation markers and spontaneously differentiated into both STB and EVT in basal medium. These features shown by the SP cells suggested that IL7R and IL1R2 are available as markers to detect the SP cells and that vCTB progenitor cells and trophoblast stem cells were involved in the SP cell population.
| S-EPMC3131303 | biostudies-literature
Project description:Phthalates are extensively used as plasticizers in a variety of daily-life products, resulting in widespread distribution in aquatic environments. However, limited information is available on the endocrine disrupting effects of phthalates in aquatic organisms. The aim of the present study was to examine whether exposure to mono-(2-ethylhexyl) phthalate (MEHP), the hydrolytic metabolite of di-(2-ethylhexyl) phthalate (DEHP) disrupts thyroid endocrine system in fish. In this study, zebrafish (Danio rerio) embryos were exposed to different concentrations of MEHP (1.6, 8, 40, and 200 μg/L) from 2 h post-fertilization (hpf) to 168 hpf. The whole-body content of thyroid hormone and transcription of genes involved in the hypothalamic-pituitary-thyroid (HPT) axis were examined. Treatment with MEHP significantly decreased whole-body T4 contents and increased whole-body T3 contents, indicating thyroid endocrine disruption. The upregulation of genes related to thyroid hormone metabolism (Dio2 and UGT1ab) might be responsible for decreased T4 contents. Elevated gene transcription of Dio1 was also observed in this study, which might assist to degrade increased T3 contents. Exposure to MEHP also significantly induced transcription of genes involved in thyroid development (Nkx2.1 and Pax8) and thyroid hormone synthesis (TSHβ, NIS and TG). However, the genes encoding proteins involved in TH transport (transthyretin, TTR) was transcriptionally significantly down-regulated after exposure to MEHP. Overall, these results demonstrate that acute exposure to MEHP alters whole-body contents of thyroid hormones in zebrafish embryos/larvae and changes the transcription of genes involved in the HPT axis, thus exerting thyroid endocrine toxicity.
| S-EPMC3962405 | biostudies-literature