Human Placental Cell Line Research Articles

Synthesis and Anticancer Activity of 5-(1,2-Diselenolan-3-Yl)pentanoic Acid and its Derivatives

A green and high-yielding synthetic route for the preparation of 5-(1,2-diselenolan-3-yl)pentanoic acid (SeA) was reported. Some SeA derivatives, N′-substituted benzylidene-5-(1,2-diselenolan-3-yl)pentanehydrazide, were prepared, and they were screened for their anticancer activity against human breast MCF-7, leukemia HL-60, cervixuterus Hela, and placental villus Bewo cancer cell lines. The minimum inhibitory concentrations (MICs) of the synthetic compounds showed moderate anticancer activity at low concentrations (0.5–5 μg/mL).

Bioinformatic Analysis of Benzo-α-Pyrene-Induced Damage to the Human Placental Insulin-Like Growth Factor-1 Gene

Intrauterine growth restriction (IUGR) has been associated with exposure to polyaromatic hydrocarbons (PAHs) which are released in the combustion of oil, fuel, gas, garbage, and tobacco. Pregnant women exposed to PAHs are at risk of the effects of these environmental toxins; for example, benzo-α-pyrene (BαP) is able to enter the blood stream and could contribute to IUGR or other developmental abnormalities via effects on the placental cells. Since IUGR has been associated with decreased cord blood concentrations of immunoreactive insulin-like growth factor 1 (ir-IGF-1) and IUGR has been associated with disordered development and fetal programming, we tested the effects of BαP on human placental trophoblast cells in culture. IGF-1 expression and activation was studied using an immortalized human placental trophoblast cell line (HTR-8). The cells were treated with vehicle control or 1 µmol/L BαP, or 5 µmol/L BαP for 12 hours. RNA was extracted and the exons of IGF-1 were amplified using reverse transcriptase-polymerase chain reaction (RT-PCR). The ir-IGF-1 expression levels were compared using gel electrophoresis. The PCR products were sequenced, and levels of mutation were measured with comparative sequence analysis. A computational protein analysis (computer simulation) was performed in order to assess the potential impact of BαP-associated mutation on IGF-1 protein function. The IGF-1 expression decreased considerably in BαP-treated cells relative to untreated controls (P < .05), also in a dose-dependent manner. Comparative sequence analysis indicated that the level of BαP exposure correlated with the percentage of base pair mutations in IGF-1 nucleotide sequences for both treatment groups (P < .05). Shifts were observed in the open reading frame, indicating a possible change in the IGF-1 start codon. Protein folding simulation analysis indicated that the base pair changes induced by BαP weakened IGF-1-IGF binding protein (IGFBP) interaction. In concordance with the previous findings, exposure of human placental trophoblast cells to BαP exposure results in reduction of IGF-1 expression and base pair mutations. The direct action of BαP on the placenta indicates that it may not be necessary for BαP to access other maternal tissues in order for gene abnormalities to occur. Given that PAHs are known to work through aryl hydrocarbon hydrolase (AHH), these results are likely due to the presence of AHH in HTR cells. Computational modeling of BαP actions on IGF1, substrate-ligand binding, supports the biological premise of this work and underlines the need to determine actual biological effects rather than equating immune to bioactivity of IGF1.

Downregulation of SPARC Expression Inhibits the Invasion of Human Trophoblast Cells In Vitro

Successful pregnancy depends on the precise regulation of extravilloustrophoblast (EVT) invasion into the uterine decidua. SPARC (secreted protein acidic and rich in cysteine) is a matricellular glycoprotein that plays critical roles in the pathologies associated with obesity and diabetes, as well as tumorigenesis. The objective of this study was to investigate the role of SPARC in the process of trophoblast invasion which shares many similarities with tumor cell invasion. By Western blot, higher expression of SPARC was observed in mouse brain, ovary and uterus compared to other mouse tissues. Immunohistochemistry analysis revealed a spatio-temporal expression of SPARC in mouse uterus in the periimplantation period. At the implantation site of d8 pregnancy, SPARC mainly accumulated in the secondary decidua zone (SDZ), trophoblast cells and blastocyst. The expression of SPARC was also detected in human placental villi and trophoblast cell lines. In a Matrigel invasion assay, we found SPARC-specific RNA interference significantly reduced the invasion of human extravilloustrophoblast HTR8/SVneo cells. Microarray analysis revealed that SPARC depletion upregulated the expression of interleukin 11 (IL11), KISS1, insulin-like growth factor binding protein 4 (IGFBP4), collagen type I alpha 1 (COLIA1), matrix metallopeptidase 9 (MMP9), and downregulated the expression of the alpha polypeptide of chorionic gonadotropin (CGA), MMP1, gap junction protein alpha 1 (GJA1), et al. The gene array result was further validated by qRT-PCR and Western blot. The present data indicate that SPARC may play an important role in the regulation of normal placentation by promoting the invasion of trophoblast cells into the uterine decidua.

Oxidative stress decreases uptake of neutral amino acids in a human placental cell line (BeWo cells)

Increased oxidative stress (OS) is implicated in the pathophysiology of several pregnancy disorders. We aimed to investigate the effect of tert-butylhydroperoxide (TBHP)-induced OS upon the placental transport of the neutral amino acids l-methionine (l-Met) and l-alanine (l-Ala), by using a human trophoblast cell model (BeWo cells). TBHP reduced both total and Na+-independent 14C-l-Met intracellular steady-state accumulation over time (Amax), by reducing non-system l-mediated uptake – most probably system y+ – while having no effect on system L. Moreover, TBHP reduced total 14C-l-Ala Amax through an inhibition of system A. The effect of TBHP upon total, but not system A-mediated, 14C-l-Ala uptake was dependent upon phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) activation, and was completely prevented by the polyphenol quercetin. In conclusion, a reduction in placental uptake of neutral amino acids may contribute to the deleterious effects of pregnancy disorders associated with OS.

Hyperhomocysteinemia is detrimental to pregnancy in mice and is associated with preterm birth

Elevated levels of homocysteine produce detrimental effects in humans but its role in preterm birth is not known. Here we used a mouse model of hyperhomocysteinemia to examine the relevance of homocysteine to preterm birth. The mouse carries a heterozygous deletion of cystathionine β-synthase (Cbs+/−). Gestational period was monitored in wild type and Cbs+/− female mice. Mouse uterine and placental tissues, human primary trophoblast cells, and human myometrial and placental cell lines were used to determine the influence of homocysteine on expression of specific genes in vitro. The activity of BKCa channel in the myometrial cell line was monitored using the patch-clamp technique. We found that hyperhomocysteinemia had detrimental effects on pregnancy and induced preterm birth in mice. Homocysteine increased the expression of oxytocin receptor and Cox-2 as well as PGE2 production in uterus and placenta, and initiated premature uterine contraction. A Cox-2 inhibitor reversed these effects. Gpr109a, a receptor for niacin, induced Cox-2 in uterus. Homocysteine upregulated GPR109A and suppressed BKCa channel activity in human myometrial cells. Deletion of Gpr109a in Cbs+/− mice reversed premature birth. We conclude that hyperhomocysteinemia causes preterm birth in mice through upregulation of the Gpr109a/Cox-2/PGE2 axis and that pharmacological blockade of Gpr109a may have potential in prevention of preterm birth.

Mono-2-ethylhexyl phthalate induces oxidative stress responses in human placental cells in vitro

Di-2-ethylhexyl phthalate (DEHP) is an environmental contaminant commonly used as a plasticizer in polyvinyl chloride products. Exposure to DEHP has been linked to adverse pregnancy outcomes in humans including preterm birth, low birth-weight, and pregnancy loss. Although oxidative stress is linked to the pathology of adverse pregnancy outcomes, effects of DEHP metabolites, including the active metabolite, mono-2-ethylhexyl phthalate (MEHP), on oxidative stress responses in placental cells have not been previously evaluated. The objective of the current study is to identify MEHP-stimulated oxidative stress responses in human placental cells. We treated a human placental cell line, HTR-8/SVneo, with MEHP and then measured reactive oxygen species (ROS) generation using the dichlorofluorescein assay, oxidized thymine with mass-spectrometry, redox-sensitive gene expression with qRT-PCR, and apoptosis using a luminescence assay for caspase 3/7 activity. Treatment of HTR-8 cells with 180μM MEHP increased ROS generation, oxidative DNA damage, and caspase 3/7 activity, and resulted in differential expression of redox-sensitive genes. Notably, 90 and 180μM MEHP significantly induced mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2), an enzyme important for synthesis of prostaglandins implicated in initiation of labor. The results from the present study are the first to demonstrate that MEHP stimulates oxidative stress responses in placental cells. Furthermore, the MEHP concentrations used were within an order of magnitude of the highest concentrations measured previously in human umbilical cord or maternal serum. The findings from the current study warrant future mechanistic studies of oxidative stress, apoptosis, and prostaglandins as molecular mediators of DEHP/MEHP-associated adverse pregnancy outcomes.

Expression of the Renin-Angiotensin System in a Human Placental Cell Line

The renin-angiotensin system (RAS) is present in human placental tissue and participates in regulation of maternal-fetal blood flow during pregnancy. RAS expression in placental tissue is regulated by various hormones and is altered in various disease conditions. An in vitro system is needed to further investigate regulation of the placental RAS. To this end, we studied RAS expression in the human placenta-derived cell line, CRL-7548. CRL-7548 cells were cultured in plastic plates. Total RNA was extracted, reverse transcribed, and amplified by polymerase chain reaction (PCR) with specific primers. Angiotensin II peptide in the culture media was measured by radioimmunoassay. Renin activity was detected by radioimmunoassay measuring angiotensin I generated. Angiotensin receptor type I was detected by Western blot. Specific mRNA for angiotensin, renin, angiotensin converting enzyme, and angiotensin receptor type I was detected by real-time PCR. Renin activity was detected in the placental cell lysate, and angiotensin II peptide, the final product of the RAS system, was detected in cell culture media by radioimmunoassay. Angiotensin receptor type I was identified as a 41 kDa protein in cell lysates by Western blot. These results demonstrate that all necessary components of the classic RAS are expressed in the human placental cell line CRL-7548. This cell line may prove useful as an in vitro system for studying RAS regulation in the placenta.

Oxidative stress decreases glucose and neutral amino acid uptake in a human placental cell line (BeWo cells)

Oxidative stress decreases glucose and neutral amino acid uptake in a human placental cell line (BeWo cells)

The effects of oncostatin M on trophoblast cells: Influence on matrix metalloproteinases-2 and -9, and invasion activity

Oncostatin M (OSM), a cytokine of the interleukin-6 (IL-6) family, can either promote or inhibit cell growth in various normal and tumor cells and is expressed in rheumatoid arthritis, multiple sclerosis, multiple myeloma, and other inflammatory conditions. We investigated one of the possible mechanisms involved in trophoblast invasion using the human placental cell line derived from first trimester extravillous trophoblasts (HTR8SVneo): modulation of matrix metalloproteinase (MMP)-2 and -9 expression and enzymatic activity. And we addressed also the effects of exogenous OSM on the in vitro invasion activity of HTR8SVneo cells. We found that OSM enhanced the constitutive RNA and protein expressions of MMP-2 and MMP-9 in HTR8SVneo cell lines. Also, OSM treatment increased significantly the enzymatic activity of MMP-2 on gelatin zymography. The effects OSM on enzymatic activity of MMP-9 was not significant. We found that OSM increased invasion activities of HTR8SVneo cells in time-dependent and dose-dependent manners. This study suggests that OSM enhances invasion activities of extravillous trophoblasts during the first trimester through the increased enzyme activity of gelatinases, especially MMP-2.

Epigenetic Effects of Environmental Chemicals Bisphenol A and Phthalates

The epigenetic effects on DNA methylation, histone modification, and expression of non-coding RNAs (including microRNAs) of environmental chemicals such as bisphenol A (BPA) and phthalates have expanded our understanding of the etiology of human complex diseases such as cancers and diabetes. Multiple lines of evidence from in vitro and in vivo models have established that epigenetic modifications caused by in utero exposure to environmental toxicants can induce alterations in gene expression that may persist throughout life. Epigenetics is an important mechanism in the ability of environmental chemicals to influence health and disease, and BPA and phthalates are epigenetically toxic. The epigenetic effect of BPA was clearly demonstrated in viable yellow mice by decreasing CpG methylation upstream of the Agouti gene, and the hypomethylating effect of BPA was prevented by maternal dietary supplementation with a methyl donor like folic acid or the phytoestrogen genistein. Histone H3 was found to be trimethylated at lysine 27 by BPA effect on EZH2 in a human breast cancer cell line and mice. BPA exposure of human placental cell lines has been shown to alter microRNA expression levels, and specifically, miR-146a was strongly induced by BPA treatment. In human breast cancer MCF7 cells, treatment with the phthalate BBP led to demethylation of estrogen receptor (ESR1) promoter-associated CpG islands, indicating that altered ESR1 mRNA expression by BBP is due to aberrant DNA methylation. Maternal exposure to phthalate DEHP was also shown to increase DNA methylation and expression levels of DNA methyltransferases in mouse testis. Further, some epigenetic effects of BPA and phthalates in female rats were found to be transgenerational. Finally, the available new technologies for global analysis of epigenetic alterations will provide insight into the extent and patterns of alterations between human normal and diseased tissues. In vitro models such as human embryonic stem cells may be extremely useful in bettering the understanding of epigenetic effects on human development, health and disease, because the formation of embryoid bodies in vitro is very similar to the early stage of embryogenesis.

Development of a Model for Functional Studies of ABCG2 (Breast Cancer Resistance Protein) Efflux Employing a Standard BeWo Clone (B24)

Human choriocarcinoma-derived BeWo cells express high levels of breast cancer resistance protein (BCRP/ABCG2) with no functional P-glycoprotein (P-gp) (ABCB1) activity, making them a potential model to study bidirectional ABCG2-mediated drug transport. However, the original BeWo clone (B24) available to researchers does not form confluent monolayers with tight junctions required by the model. Our aim was to adapt culture conditions to attempt to generate confluent BeWo monolayers for drug transport studies using the standard B24 clone. BeWo cells (B24; American Type Culture collection [ATCC]) were cultured in six-well plates or polycarbonate millicell inserts in a number of media formulations, growth supplements, and basement membrane substitutes. Cells were examined for confluence by microscopy, and transepithelial electrical resistance (TEER) was measured daily; monolayer permeability was assessed when TEER had stabilized. Optimal growth rates were achieved in culture conditions consisting of Medium 199 (M199) supplemented with epidermal growth factor (EGF; 20 ng/mL), vitamin supplements, and 10% fetal calf serum (FCS) with collagen coating. A TEER of 170 Ω in 0.6 cm(2) inserts was achieved 2 weeks after seeding under optimal conditions. The cell-impermeable diffusion marker 5(6) carboxy-2,7dichlorodihydrofluorescein (C-DCDHF) had a permeability coefficient of 3.5×10(-6) cm/s, indicative of minimal paracellular permeability. ABCG2 expression, as determined by immunoblotting, remained unaffected by confluency. In conclusion, we describe culture conditions for the B24 BeWo clone that facilitate the formation of monolayers with tighter junctions and reduced paracellular transport compared to previously published models. These growth conditions provide a good model of ABCG2-mediated drug transport in a human placental cell line.

Kisspeptin-10 inhibits cell migration in vitro via a receptor-GSK3 beta-FAK feedback loop in HTR8SVneo cells

Kisspeptin inhibits cancer cell metastasis and placental trophoblast cell migration. Kisspeptin gene expression in the placenta and circulating kisspeptin levels change during normal pregnancy and they are altered in preeclampsia. We therefore assessed the effect of kisspeptin-10 on the in vitro migration of a human placental cell line derived from first trimester extravillious trophoblasts (HTR8SVneo). HTR8SVneo cells specifically bound 125I-Kisspeptin-10 but kisspeptin-10 did not induce inositol phosphate production. Cell migration was inhibited by kisspeptin-10 with a maximal inhibition at 100nM. The signaling pathways involved in inhibition of cell migration were examined. Treatment with kisspeptin-10 elicited phosphorylation of GSK3 beta at Ser9 (which inhibits activity), with a 3-fold increase at 5min. Transient phosphorylation of ERK1/2 and p38MAPK peaked at 10min. Phosphorylation of focal adhesion kinase (FAK) at Tyr925 increased 3-fold at 10min. Inhibition of GSK3 beta correlated with release of beta-catenin into the cytoplasm. These signaling events were differentially blocked by inhibitors of Gq/11, Src, EGFR, PI(3)K, PKC and MEK. The data suggest that kisspeptin/GPR54 EGF-receptor transactivation leads to phosphorylation of ERK1/2, causing activation of p90rsk which in turn inhibits GSK3 beta via Ser9 phosphorylation. Inactivation of GSK3 beta results in release of beta-catenin into the cytoplasm, affecting cell–cell adhesion and Tyr925 phosphorylation of FAK, which increases phosphorylation of ERK1/2 via RAS/Raf-1 creating a feedback loop to enhance the effects on migration. These findings indicate that kisspeptin-10 inhibits the migration of human placental trophoblast-derived HTR8SVneo cells by stimulating complex ERK1/2–p90rsk–GSK3 beta–FAK feedback interactions.

Effects of bisphenol A on the expression of cytochrome P450 aromatase (CYP19) in human fetal osteoblastic and granulosa cell-like cell lines

The effects of bisphenol A (BPA), an endocrine disruptor, on aromatase (CYP19) expression in human osteoblastic (SV-HFO) and ovarian granulosa-like (KGN) cell lines were examined. CYP19 enzyme activity was suppressed in the presence of BPA in a dose-dependent fashion in both cell lines. CYP19 gene transcript expression, as well as activities of promoter I.4 in SV-HFO and promoter II in KGN, was down-regulated by BPA, suggesting that BPA affects CYP19 at the gene-expression level. These data and the previous finding that BPA induced the down-regulation of promoter I.1 activity within the human placental cell line suggest that there may be a conserved signaling pathway that down-regulates CYP19 expression in response to BPA in both cell lines. Additionally, differences between promoter I.4 and II suggest that there may be cell- and promoter-specific down-regulating mechanisms downstream from the actions of BPA.

81 MEMBRANE AND CYTOSOLIC CALCIUM PROTEINS, TRPV6, PMCA1, NCKX3, NCX1 AND CaBP-28k, APPEAR TO BE DISTINCTLY REGULATED IN HUMAN CHORIOCARCINOMA AND PLACENTAL CELLS

Preeclampsia is a pregnancy-specific disease characterised by de novo development of concurrent hypertension, proteinuria and oxidative stress in the placenta. In the placenta, intervillous blood flow increases after 10 weeks of gestation and results in exposure of trophoblast cells to oxygen. Hypoxia occurs during the development of placenta in the first trimester and is implicated in trophoblast differentiation. Ca2+ is a universal intracellular second messenger involved in many processes such as signal transduction, hormone secretion and programmed cell death. Human placental primary cell cultures were established from first-trimester human placentas (at 7 to 12 weeks of gestation). In this study, calcium-related proteins (CRPs; TRPV6, PMCA1, NCKX3 and CaBP-28k) were investigated at normoxia (5% CO2 in 95% air) or hypoxia (2% O2/93% N2/5%CO2) for 12 h in human placental cell line (BeWo) and human placental primary cell (hPC). We confirmed mRNA expression by real-time PCR and protein expression by Western blot analysis. The data were 2 or 3 individual experiments with triplicate samples and analysed by one-way ANOVA using Tukey's multiple comparison test. In hypoxia, the level of TRPV6 mRNA and protein was not changed, however, calcium transporters' (NCKX3, CaBP-28k) mRNA and protein expressions were significantly increased in hypoxic BeWo cell compared with control (normoxia). In addition, expression of PMCA1 mRNA and protein was decreased in hypoxic BeWo cells. In hPC, CRPs (TRPV6, PMCA1, NCKX3 and CaBP-28k) mRNA and protein expressions were significantly induced by hypoxic stress compared with control. These results, taken together, indicate that alterations of calcium transporters in hypoxic stress may be involved in calcium transport in the placenta and protection of the placental trophoblasts from the oxidative stress during the pregnancy.

82 EXPRESSION OF CELL MEMBRANE Mg2+ INORGANIC PHOSPHATE CHANNELS IN THE HUMAN PLACENTA DERIVED FROM PREGNANT WOMEN AND HUMAN PLACENTAL CELL LINES (BeWo, JEG3, hPC)

Preeclampsia is a pregnancy-specific disease characterised by de novo development of concurrent hypertension and oxidative stress in the placenta. The human placenta is a highly vascularized organ whose major function is to allow maternal–fetal exchange of solutes such as Ca2+ and oxygen. The transient receptor potential cation channel subfamily is known to contain channels activated by such various stimuli as intracellular Ca2+ membrane potential, cold and pH. However, signalling mechanisms mediating hormonal regulation of Mg2+ inorganic phosphate channels during the placenta duration of pregnancy are incompletely understood. We examined the expression of cell membrane Mg2+ inorganic channels in 3 sections (fetal-, central-, maternal-) of preeclamptic placenta (PEP) and from placental cell lines, BeWo, JEG3 and hPC (isolated during the first trimester) by real-time PCR and Western blot analysis. Placental tissues (∼3 cm) from women (n = 75) between the ages of 28 to 45 years undergoing normal or Caesarean delivery were dissected into 1-cm sections. Data were analysed by one-way ANOVA, followed by Tukey's multiple comparison test. P &lt; 0.05 was considered to be statistically significant. During preterm labour, human placental expression of Mg2+ inorganic channel mRNA and protein fluctuated in the 3 sections of PEP compared with normal placenta. In hPC, the expression of Mg2+ inorganic channel (TRPM6/7, MagT1, NIPA2, SLC41A1 to 3 and SLC34A1 to 3) mRNA and protein were decreased in placental cell lines by hypoxic stress (2% O2/93% N2/5% CO2) compared with normoxia (20% O2/75% N2/5% CO2). The levels of Mg2+ inorganic channel mRNA and protein were distinctly expressed between BeWo and JEG3 cells. These results indicate that placental Mg2+ inorganic channels play potential roles in differential sections of placenta between normal and PEP, suggesting that induced Mg2+ inorganic channels of PEP may be involved in preeclamptic stress in human placenta and placental cell lines, which are a determinant factor affecting calcium transfer.

Calcium Ion Transport-Related Genes, TRPV6, PMCA1, NCKX3, CaBP-28k Are Differentially Expressed in Human Placental Cell Line (BeWo).

Preeclampsia is pregnancy-specific disease characterized de-novo development of concurrent hypertension, proteinuria and oxidative stress in placenta. Hypoxia occurs during the development of placenta in the first trimester and is implicated in trophoblast differentiation. In placenta, intervillous blood flow increases after 10 weeks of gestation and results in exposure of trophoblast cells to oxygen. During last trimester of gestation, the calcium (Ca2+) transport from mother to fetus increases dramatically in response to the accelerated demand for Ca2+ caused by bone mineralization in the fetus. Regarding the molecular basis of Ca2+ transport, several studies suggest that transcellular active Ca2+ transport is comprised of three processes: (1) the apical Ca2+ entry, (2) the binding with calbindins that serves as an intracellular Ca2+ buffer, and (3) basolateral exit. In this study, cell membrane and cytosolic calcium transporters (TRPV6, PMCA1, NCKX3 and CaBP-28k) were investigated at induced oxidative stress in human placental cell line (BeWo). The expression of calcium transporters (TRPV6, PMCA1, NCKX3 and CaBP-28k) were potential roles in BeWo cell remain to be clarified. In hypoxia, human placental cell (BeWo) expression of TRPV6 mRNA and protein level was not altered, however, calcium transporters (NCKX3, CaBP-28k) were increase at hypoxic BeWo cell compared with control group (normoxia). In addition, expression of PMCA1 mRNA and protein was decreased at hypoxic BeWo cell. Taken together, these results indicate that calcium transporters (TRPV6, PMCA1, NCKX3 and CaBP-28k) are distinctly expressed by induced oxidative stress, in BeWo cell, suggesting that alterations of calcium transporters in hypoxic stress may be involved in placenta cell is a determinant factor affecting calcium transfer in BeWo cell model. (poster)

Low amino acids affect expression of 11β-HSD2 in BeWo cells through leptin-activated JAK-STAT and MAPK pathways

Maternal protein restriction diminishes placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) activity and causes fetal growth restriction in mammals. However, it is unknown whether such effect was caused directly by nutrient deficiency, or indirectly through the mediation of maternal hormones. In the present study, a human placental cell line (BeWo) was cultured in F12K as control and F12 as low amino acids (LAA) media for 48 h to investigate the effects of amino acids deficiency on 11β-HSD2 expression and activity. Despite a significant up-regulation of 11β-HSD2 mRNA expression in LAA cells, 11β-HSD2 activity and protein content were decreased by 38 and 54%, respectively (P<0.05), indicating a mechanism of post-transcriptional regulation. Among 5 miRNAs targeting 11β-HSD2, miR-498 was expressed significantly higher in LAA cells. Leptin concentration was significantly lower (P<0.01) in LAA medium. The mRNA expression of both isoforms of leptin receptor was significantly higher in LAA cells, although no difference was detected at protein level. To further clarify whether leptin is involved in mediating the effect of LAA on 11β-HSD2 activity, leptin was supplemented to LAA medium, whereas three specific inhibitors of leptin signaling pathways, WP1066 for JAK-STAT, PD98059 for MAPK and LY294002 for PI3K, respectively were added to control medium. Leptin restored the diminished 11β-HSD2 activity in LAA cells, whereas WP1066 (5 nM) and PD98059 (50 nM) significantly decreased 11β-HSD2 activity in control cells. In conclusion, the present results indicate that LAA diminishes 11β-HSD2 expression and activity in BeWo cells through leptin-activated JAK-STAT and MAPK pathways.

Regulation of pregnancy-associated plasma protein A2 (PAPPA2) in a human placental trophoblast cell line (BeWo)

BackgroundPregnancy-associated plasma protein A2 (PAPPA2) is an insulin-like growth factor-binding protein (IGFBP) protease expressed at high levels in the placenta and upregulated in pregnancies complicated by preeclampsia and HELLP (Hemolytic anemia, Elevated Liver enzymes, and Low Platelet count) syndrome. However, it is unclear whether elevated PAPPA2 expression causes abnormal placental development, or whether upregulation compensates for placental pathology. In the present study, we investigate whether PAPPA2 expression is affected by hypoxia, oxidative stress, syncytialization factors or substances known to affect the expression of PAPPA2's paralogue, PAPPA.MethodsBeWo cells, a model of placental trophoblasts, were treated with one of the following: hypoxia (2% O2), oxidative stress (20 microM hydrogen peroxide), forskolin (10 microM and 100 microM), TGF-beta (10 and 50 ng/mL), TNF-alpha (100 ng/mL), IL-1beta (100 ng/mL) or PGE2 (1 microM). We used quantitative RT-PCR (qRT-PCR) to quantify the mRNA levels of PAPPA2, as well as those of PAPPA and ADAM12 since these proteases have similar substrates and are also highly expressed in the placenta. Where we observed significant effects on PAPPA2 mRNA levels, we tested for effects at the protein level using an in-cell Western assay.ResultsHypoxia, but not oxidative stress, caused a 47-fold increase in PAPPA2 mRNA expression, while TNF-alpha resulted in a 6-fold increase, and both of these effects were confirmed at the protein level. PGE2 resulted in a 14-fold upregulation of PAPPA2 mRNA but this was not reflected at the protein level. Forskolin, TGF-beta and IL-1beta had no significant effect on PAPPA2 mRNA expression. We observed no effects of any treatment on PAPPA or ADAM12 expression.ConclusionOur study demonstrates that factors previously known to be highly expressed in preeclamptic placentae (PGE2 and TNF-alpha), contribute to the upregulation of PAPPA2. Hypoxia, known to occur in preeclamptic placentae, also increased PAPPA2 expression. These results are consistent with the hypothesis that PAPPA2 is upregulated as a consequence of placental pathology, rather than elevated PAPPA2 levels being a cause of preeclampsia.

Wild Type and Mutant 2009 Pandemic Influenza A (H1N1) Viruses Cause More Severe Disease and Higher Mortality in Pregnant BALB/c Mice

BackgroundPregnant women infected by the pandemic influenza A (H1N1) 2009 virus had more severe disease and higher mortality but its pathogenesis is still unclear.Principal FindingsWe showed that higher mortality, more severe pneumonitis, higher pulmonary viral load, lower peripheral blood T lymphocytes and antibody responses, higher levels of proinflammatory cytokines and chemokines, and worse fetal development occurred in pregnant mice than non-pregnant controls infected by either wild type (clinical isolate) or mouse-adapted mutant virus with D222G substitution in hemagglutinin. These disease-associated changes and the lower respiratory tract involvement were worse in pregnant mice challenged by mutant virus. Though human placental origin JEG-3 cell line could be infected and proinflammatory cytokines or chemokines were elevated in amniotic fluid of some mice, no placental or fetal involvement by virus were detected by culture, real-time reverse transcription polymerase chain reaction or histopathological changes. Dual immunofluorescent staining of viral nucleoprotein and type II alveolar cell marker SP-C protein suggested that the majority of infected alveolar epithelial cells were type II pneumocytes.ConclusionThe adverse effect of this pandemic virus on maternal and fetal outcome is largely related to the severe pulmonary disease and the indirect effect of inflammatory cytokine spillover into the systemic circulation.

Identification of Zyklopen, a New Member of the Vertebrate Multicopper Ferroxidase Family, and Characterization in Rodents and Human Cells1–3

We previously detected a membrane-bound, copper-containing oxidase that may be involved in iron efflux in BeWo cells, a human placental cell line. We have now identified a gene encoding a predicted multicopper ferroxidase (MCF) with a putative C-terminal membrane-spanning sequence and high sequence identity to hephaestin (Heph) and ceruloplasmin (Cp), the other known vertebrate MCF. Molecular modeling revealed conservation of all type I, II, and III copper-binding sites as well as a putative iron-binding site. Protein expression was observed in multiple diverse mouse tissues, including placenta and mammary gland, and the expression pattern was distinct from that of Cp and Heph. The protein possessed ferroxidase activity, and protein levels decreased in cellular copper deficiency. Knockdown with small interfering RNA in BeWo cells indicates that this gene represents the previously detected oxidase. We propose calling this new member of the MCF family “zyklopen.”