Increased immortalization‐upregulated protein 2 (IMUP‐2) by hypoxia induces apoptosis of the trophoblast and pre‐eclampsia

In regulation of the developmental process, the balance between cellular proliferation and cell death is critical. Placental development tightly controls this mechanism, and increased apoptosis of placental trophoblasts can cause a variety of gynecological diseases. Members of the immortalization‐upregulated protein (IMUP) family are nuclear proteins implicated in SV40‐mediated immortalization and cellular proliferation; however, the mechanisms by which their expression is regulated in placental development are still unknown. We compared IMUP‐2 expression in normal and pre‐eclamptic placental tissues and evaluated the function of IMUP‐2 in HTR‐8/SVneo trophoblast cells under hypoxic conditions. IMUP‐2 was expressed in syncytiotrophoblasts and syncytial knots of the placental villi. IMUP‐2 expression was significantly higher in preterm pre‐eclampsia patients than in patients who went to term (P < 0.001); however, we observed no differences in IMUP‐2 expression between normal term patients with and without pre‐eclampsia. Hypoxic conditions increased apoptosis of HTR8/SVneo trophoblast cells and induced IMUP‐2 expression. Also, apoptosis of HTR‐8/SVneo trophoblast cells was increased after IMUP‐2 gene transfection. These results suggest that IMUP‐2 expression is specifically elevated in preterm pre‐eclampsia and under hypoxic conditions, and that IMUP‐2 induces apoptosis of the trophoblast. Therefore, IMUP‐2 might have functional involvement in placental development and gynecological diseases such as pre‐eclampsia. J. Cell. Biochem. 110: 522–530, 2010. © 2010 Wiley‐Liss, Inc.     

Hypoxia‐induced downregulation of XIAP in trophoblasts mediates apoptosis via interaction with IMUP‐2: Implications for placental development during pre‐eclampsia

The regulation of trophoblast apoptosis is essential for normal placentation, and increased placental trophoblast cell apoptosis is the cause of pathologies such as intrauterine growth retardation (IUGR) and pre‐eclampsia. X‐linked inhibitor of apoptosis (XIAP) is expressed in trophoblasts, but little is known about the role of XIAP in placental development. In the present study, the function of XIAP in the placenta and in HTR‐8/SVneo trophoblasts under hypoxic conditions was examined. In addition, the correlation between XIAP and immortalization‐upregulated protein‐2 (IMUP‐2) was demonstrated in HTR‐8/SVneo trophoblasts under hypoxia, based on a previous study showing that increased IMUP‐2 induces trophoblast apoptosis and pre‐eclampsia. XIAP was downregulated in pre‐eclamptic placentas (P < 0.05). In HTR‐8/SVneo trophoblasts, XIAP expression was decreased and the expression of apoptosis‐related genes was increased in response to hypoxia. Ectopic expression of hypoxia inducible factor (HIF)‐1α in HRT‐8 SV/neo cells induced the nuclear translocation of XIAP and alterations of XIAP protein stability. Furthermore, hypoxia induced nuclear translocated XIAP co‐localized with upregulated IMUP‐2 in trophoblast nuclei, and the interaction between XIAP and IMUP‐2 induced apoptosis in HRT‐8 SV/neo cells. The present results suggest that hypoxia‐induced down‐regulation of XIAP mediates apoptosis in trophoblasts through interaction with increased IMUP‐2, and that this mechanism underlies the pathogenesis of pre‐eclampsia. J. Cell. Biochem. 114: 89–98, 2012. © 2012 Wiley Periodicals, Inc.     

MicroRNA‐210 regulates human trophoblast cell line HTR‐8/SVneo function by attenuating Notch1 expression: Implications for the role of microRNA‐210 in pre‐eclampsia

Successful pregnancy depends on the precise regulation of extravillous trophoblast cell invasion ability. MicroRNA‐210‐3p (miR‐210), which is increased in the placenta of pre‐eclampsia. Furthermore, miR‐210 could inhibit trophoblasts invasion and might act as a serum biomarker for pre‐eclampsia. Previous studies have demonstrated that miR‐210 regulates HUVEC (human umbilical vein endothelial cell)‐mediated angiogenesis by regulating the NOTCH1 signaling pathway. Studies by our group have previously identified that NOTCH1 plays a positive role in regulating trophoblast functions. However, the miR‐210/NOTCH1 signaling pathway in the regulation of trophoblasts and pre‐eclampsia has not been characterized. Therefore, this study was conducted to investigate the role of miR‐210 and its relationship with NOTCH1 in trophoblasts. We first examined the expression levels of miR‐210 and NOTCH1 in pre‐eclamptic and normals placentas. Next, the expression and location of miR‐210 and NOTCH1 in the first‐trimester villi, maternal decidua, and placenta of late pregnancy were shown via in situ hybridization and immunohistochemistry. The trophoblast cell line HTR‐8/SVneo was used to investigate the effects of miR‐210 on the expression of NOTCH1 and cell bioactivity by upregulation and downregulation strategies. The results showed that miR‐210 expression was increased, whereas NOTCH1 expression was decreased in pre‐eclamptic placenta compared with controls. Upregulation of miR‐210 decreased NOTCH1 expression, impaired HTR‐8/SVneo proliferation, migration, invasion, and tube‐like formation capabilities, and promoted apoptosis. In contrast, downregulation of miR‐210 resulted in the opposite effects. These findings suggested that miR‐210 might act as a contributor to trophoblast dysfunction by attenuating NOTCH1 expression.     

Deficiency of DICER reduces the invasion ability of trophoblasts and impairs the pro‐angiogenic effect of trophoblast‐derived microvesicles

DICER is a key rate‐limiting enzyme in the canonical miRNAs biogenesis pathway, and DICER and DICER‐dependent miRNAs have been proved to play essential roles in many physiological and pathological processes. However, whether DICER is involved in placentation has not been studied. Successful spiral artery remodelling is one of the key milestones during placentation, which depends mostly on the invasion of trophoblasts and the crosstalk between trophoblasts and endothelial cells. In the present study, we show that DICER knockdown impairs the invasion ability of both primary extravillous trophoblasts (EVT) and HTR8/SVneo (HTR8) cell lines. The decreased invasion of HTR8 cells upon DICER knockdown (sh‐Dicer) was partly due to the up‐regulation of miR‐16‐2‐3p, which led to a reduced expression level of the collagen type 1 alpha 2 chain (COL1A2) protein. Moreover, microvesicles (MVs) can be secreted by HTR8 cells and promote the tube formation ability of human umbilical cord vein endothelial cells (HUVECs). However, conditioned medium and MVs derived from sh‐Dicer HTR8 cells have an anti‐angiogenic effect, due to reduced angiogenic factors and increased anti‐angiogenic miRNAs (including let‐7d, miR‐1‐6‐2 and miR‐15b), respectively. In addition, reduced protein expression of DICER is found in PE placenta by immunoblotting and immunohistochemistry. In summary, our study uncovered a novel DICER‐miR‐16‐2‐COL1A2 mediated pathway involved in the invasion ability of EVT, and DICER‐containing MVs mediate the pro‐angiogenic effect of trophoblast‐derived conditioned medium on angiogenesis, implying the involvement of DICER in the pathogenesis of PE.     

Resveratrol promotes trophoblast invasion in pre‐eclampsia by inducing epithelial‐mesenchymal transition

Impairment spiral arteries remodelling was considered to be the underlying cause of pathogenesis of pre‐eclampsia (PE). Resveratrol (RE) was reported that it could modulate cellar phenotype to ameliorate diverse human diseases. However, the biological function of RE in PE remains poorly understood. In this report, we investigated the effect of RE on trophoblast phenotype both in vivo and in vitro. We conducted MTT and transwell assays to explore cell proliferation and invasion events in HTR‐8/SVneo. In mice model, the clinical characteristics of PE were established through the injection of NG‐nitro‐l ‐arginine methyl ester (L‐NAME). Furthermore, related experiments were performed to detect cellar phenotype‐associated signalling pathway, including epithelial‐mesenchymal transition (EMT) and Wnt/β‐catenin. Cell assays indicated that RE could increase trophoblasts migration and invasion. In addition, hypertension and proteinuria were markedly ameliorated by RE compared with the controls in PE mice model. Moreover, treatment by RE in trophoblasts or in PE model, we found that RE activated EMT progress through the regulation of E‐cadherin, β‐catenin, N‐cadherin, vimentin expression, and further altered the WNT‐related gene expression, including WNT1, WNT3 and WNT5B. Our findings demonstrated that RE might stimulate the invasive capability of human trophoblasts by promoting EMT and mediating the Wnt/β‐catenin pathway in PE.     

Lysophosphatidic acid‐triggered pathways promote the acquisition of trophoblast endovascular phenotype in vitro

Successful implantation and placentation requires that extravillous cytotrophoblast acquires an endovascular phenotype and remodels uterine spiral arteries. Defects in this mechanism correlate with severe obstetric complications as implantation failure and preeclampsia. Lysophosphatidic acid (LPA) participates in embryo implantation and contributes to vascular physiology in different biological systems. However, the role of LPA on trophoblast endovascular transformation has not been studied. Due to difficulties in studying human pregnancy in vivo, we adopted a pharmacological approach in vitro to investigate LPA action in various aspects of trophoblast endovascular response, such as the formation of endothelial capillary‐like structures, migration, and proliferation. The HTR‐8/SVneo cell line established from human first trimester cytotrophoblast was used to model the acquisition of the endovascular phenotype by the invading trophoblast. LPA increased HTR‐8/SVneo tube formation, migration (wound healing assay and phalloidin staining) and proliferation (MTT assay). LPA G protein‐coupled receptors, LPA₁ and LPA₃, were expressed in HTR‐8/SVneo. By using selective antagonists, we showed that enhanced tubulogenesis was mediated by LPA₃. In addition, cyclooxygenase‐2 and inducible nitric oxide synthase pathways participated in LPA‐stimulated tubulogenesis. Inducible nitric oxide synthase was activated downstream cyclooxygenase‐2. Furthermore, prostaglandin E2 and a nitric oxide donor (SNAP) increased trophoblast tube formation in a concentration‐dependent manner. Finally, we observed that cyclooxygenase‐2 and inducible nitric oxide synthase were localized in the nucleus, and LPA did not modify their cellular distribution. Our results show that LPA‐triggered regulatory pathways promote trophoblast endovascular response in vitro, suggesting a new role for LPA during spiral artery remodeling at the maternal‐fetal interface.     

Differential Effect of DDT,DDE, and DDD on COX‐2 Expression in the Human Trophoblast Derived HTR‐8/SVneo Cells

The purpose of this study was to investigate the effect of 1,1,1‐trichloro‐2,2‐bis‐(chlorophenyl)ethane (DDT), 1,1‐bis‐(chlorophenyl)‐2,2‐dichloroethene (DDE), and 1,1‐dichloro‐2,2‐bis(chlorophenyl)ethane (DDD) isomers on COX‐2 expression in a human trophoblast‐derived cell line. Cultured HTR‐8/SVneo trophoblast cells were exposed to DDT isomers and its metabolites for 24 h, and COX‐2 mRNA and protein expression were assessed by RT‐PCR, Western blotting, and ELISA. Prostaglandin E₂ production was also measured by ELISA. Both COX‐2 mRNA and protein were detected under control (unexposed) conditions in the HTR‐8/SVneo cell line. COX‐2 protein expression and prostaglandin E₂ production but not COX‐2 mRNA levels increased only after DDE and DDD isomers exposure. It is concluded that DDE and DDD exposure induce the expression of COX‐2 protein, leading to increased prostaglandin E2 production. Interestingly, the regulation of COX‐2 by these organochlorines pesticides appears to be at the translational level. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:454‐460, 2012; View this article online at wileyonlinelibrary.com . DOI 10:1002/jbt.21444     

Curcumin stimulates angiogenesis through VEGF and expression of HLA‐G in first‐trimester human placental trophoblasts

Curcumin has a protective role in placental diseases like preeclampsia and preterm birth. Very little is known about its functional effects on growth, angiogenesis, and epigenetic activities of human first trimester placenta. HTR8/SVneo trophoblasts cells were used as model for human first trimester placenta. Effects of curcumin (≥80%) in these cells were investigated using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), radioactive thymidine uptake, quantitative real‐time polymerase chain reaction (qRT‐PCR), promoter DNA methylation, qRT‐PCR array, tube formation, wound healing, and immunoblot assays. PC3 (prostate cancer), JEG‐3 (trophoblast), and HMEC‐1 (endothelial) cells were used as control in various experiments. Unlike in PC3 cells, curcumin stimulated growth, proliferation, and viability in HTR8/SVneo cells. Curcumin increased tube formation, and messenger RNA (mRNA) expression of angiogenic factors such as vascular endothelial growth factor A (VEGFA) and protein expression of proangiogenic factor VEGF receptor‐2 and fatty acid‐binding protein‐4 (FABP4) in these cells. Curcumin‐stimulated tube formation was associated with an increased expression of VEGFR2 and FABP4. The stimulatory effects of curcumin were inhibited by VEGFR2 (SU5416) and FABP4 (BMS309403) inhibitors. Curcumin also significantly increased both mRNA and protein expression of HLA‐G in HTR8/SVneo cells. Curcumin increased mRNA expression of DNMT3A and NOTCH signaling system whereas down‐regulated mRNA expression of HSD11β2. Curcumin enhanced hypomethylation of gene promoters against oxidative stress and DNA damage pathway mediators. Curcumin promotes cell growth, migration, and thus angiogenic potential of these cells. Increased expression of HLA‐G by curcumin, hitherto unknown, is a novel finding since HLA‐G not only favors the immune environment for invasive trophoblasts but also positively modulates angiogenesis.     

ATL1 inhibits the proliferation and invasion of trophoblast cells via inhibition of the mTOR signaling pathway

Pre-eclampsia (PE) is a major cause of hypertension in maternal and fetal. Atlastin-1 (ATL1), one regulator of endoplasmic reticulum (ER) morphology, participates in tubular ER formation and protein synthesis. The objective of this study is to investigate the role and molecular mechanism of ATL1 in PE. GEO databases showed that ATL1 was upregulated in PE patients. Our data also found that ATL1 was highly expressed in PE placental tissues. The cell viability, proliferation, migration, and invasion of HTR-8/SVneo cells increased/decreased after the downregulation/upregulation of ATL1. The mTOR pathway is the downstream pathway of ATL1. The levels of p-p70S6K and p-mTOR were increased/decreased after the downregulation/upregulation of ATL1. Moreover, rapamycin, an inhibitor of mTOR pathway, reversed the promotive effect of siATL1 on proliferation, migration, and invasion in HTR-8/SVneo cells. In conclusion, ATL1 inhibits the proliferation and invasion of trophoblast cells via the inhibition of the mTOR signaling pathway in HTR-8/SVneo cells.     

Potential regulatory network in the PSG10P/miR‐19a‐3p/IL1RAP pathway is possibly involved in preeclampsia pathogenesis

Preeclampsia (PE), a pregnancy‐specific disorder, is a leading cause of perinatal maternal‐fetal mortality and morbidity. Impaired cell migration and invasion of trophoblastic cells and an imbalanced systemic maternal inflammatory response have been proposed as potential mechanisms of PE pathogenesis. Comparative analysis between PE placentas and normal placentas profiled differentially expressed miRNAs, lncRNAs, and mRNAs, including miR‐19a‐3p (miRNA), PSG10P (lncRNA), and IL1RAP (mRNA). This study was conducted to investigate their potential roles in PE pathogenesis. The expression of miR‐19a‐3p, PSG10P, and IL1RAP was examined in PE and normal placentas using RT‐qPCR. An in vitro experiment was performed in human trophoblast HET8/SVneo and TEV‐1 cells cultured in normoxic and hypoxic conditions. MiR‐19a‐3p targets were identified using Targetscan, miRanda, and PicTar analysis as well as luciferase reporter assays. The mouse model of PE was conducted using sFlt‐1 for in vivo tests. Lower levels of miR‐19a‐3p, but higher levels of PSG10P and IL1RAP were observed in PE placentas and the trophoblast cells in hypoxia. Luciferase reporter assays confirmed that PSG10P and IL1RAP were both direct targets of miR‐19a‐3p. Exposure to hypoxia inhibited cell viability, migration, and invasion of HET8/SVneo and TEV‐1 cells. Knocking out PSG10P and IL1RAP or overexpressing miR‐19a‐3p rescued the inhibition caused by hypoxia. In vivo experiments showed that IL1RAP promoted the expression of caspase‐3, a key apoptosis enzyme, but inhibited MMP9, which is responsible for degrading the extracellular matrix, suggesting a significant role of IL1RAP in cell proliferation, migration, and invasion. miR‐19a‐3p, PSG10P, and IL1RAP were all found to be involved in PE pathogenesis. With a common targeting region in their sequences, a regulatory network in the PSG10P/miR‐19a‐3p/IL1RAP pathway may contribute to PE pathogenesis during pregnancy.     

Sphingosine-1-Phosphate Promotes Extravillous Trophoblast Cell Invasion by Activating MEK/ERK/MMP-2 Signaling Pathways via S1P/S1PR1 Axis Activation

Successful placentation depends on the proper invasion of extravillous trophoblast (EVT) cells into maternal tissues. Previous reports demonstrated that S1P receptors are expressed in the EVT cells and S1P could regulate migration and function of trophoblast cells via S1P receptors. However, little is known about roles of S1P in the invasion of EVT cells. Our study was performed to investigate S1P effect on the invasion of EVT cells. We used the extravillous trophoblast cell line HTR8/SVneo cells to evaluate the effect. In vitro invasion assay was employed to determine the invasion of HTR8/SVneo cells induced by S1P. MMP-2 enzyme activity and relative level in the supernatants of HTR8/SVneo was assessed by gelatin zymography and western blot. Based on the above, siRNA and specific inhibitors were used for the intervention and study of potential signal pathways, and Real-time qPCR and western blot were used to test the mRNA and protein level of potential signal targets. We found that S1P could promote HTR8/SVneo cell invasion and upregulates activity and level of MMP-2. The promotion requires activation of MEK-ERK and is dependent on the axis of S1P/S1PR1. Our investigation of S1P may provide new insights into the molecular mechanisms of EVT invasion.     

CUL1 promotes trophoblast cell invasion at the maternal–fetal interface

Human trophoblast progenitor cells differentiate via two distinct pathways, to become the highly invasive extravillous cytotrophoblast (CTB) cells (EVT) or fuse to form syncytiotrophoblast. Inadequate trophoblast differentiation results in poor placenta perfusion, or even complications such as pre-eclampsia (PE). Cullin1 (CUL1), a scaffold protein in cullin-based ubiquitin ligases, plays an important role in early embryonic development. However, the role of CUL1 in trophoblast differentiation during placenta development has not been examined. Here we show that CUL1 was expressed in CTB cells and EVT in the first trimester human placentas by immunohistochemistry. CUL1 siRNA significantly inhibited outgrowth of extravillous explants in vitro, as well as invasion and migration of HTR8/SVneo cells of EVT origin. This inhibition was accompanied by decreased gelatinolytic activities of matrix metalloproteinase (MMP)-9 and increased expression of tissue inhibitors of MMPs (TIMP-1 and -2). Consistently, exogenous CUL1 promoted invasion and migration of HTR8/SVneo cells. Notably, CUL1 was gradually decreased during trophoblast syncytialization and CUL1 siRNA significantly enhanced forskolin-induced fusion of choriocarcinoma BeWo cells. CUL1 protein levels in human pre-eclamptic placental villi were significantly lower as compared to their matched control placentas. Taken together, our results suggest that CUL1 promotes human trophoblast cell invasion and dysregulation of CUL1 expression may be associated with PE.     

Mechanisms of pre‐apoptotic calreticulin exposure in immunogenic cell death

Dying tumour cells can elicit a potent anticancer immune response by exposing the calreticulin (CRT)/ERp57 complex on the cell surface before the cells manifest any signs of apoptosis. Here, we enumerate elements of the pathway that mediates pre‐apoptotic CRT/ERp57 exposure in response to several immunogenic anticancer agents. Early activation of the endoplasmic reticulum (ER)‐sessile kinase PERK leads to phosphorylation of the translation initiation factor eIF2α, followed by partial activation of caspase‐8 (but not caspase‐3), caspase‐8‐mediated cleavage of the ER protein BAP31 and conformational activation of Bax and Bak. Finally, a pool of CRT that has transited the Golgi apparatus is secreted by SNARE‐dependent exocytosis. Knock‐in mutation of eIF2α (to make it non‐phosphorylatable) or BAP31 (to render it uncleavable), depletion of PERK, caspase‐8, BAP31, Bax, Bak or SNAREs abolished CRT/ERp57 exposure induced by anthracyclines, oxaliplatin and ultraviolet C light. Depletion of PERK, caspase‐8 or SNAREs had no effect on cell death induced by anthracyclines, yet abolished the immunogenicity of cell death, which could be restored by absorbing recombinant CRT to the cell surface.     

Down-regulation of stearoyl-CoA desaturase-1 increases susceptibility to palmitic-acid-induced lipotoxicity in human trophoblast cells

In early pregnancy, adequate dietary factors are important for the growth of human trophoblast cells, followed by placental development. Although stearoyl-CoA desaturase 1 (SCD1) is expected to relieve palmitic acid (PA)-induced lipotoxicity by regulating diacylglycerol and ceramide, its function is unclear in human trophoblast cells. The aim was to investigate inhibitory effects of SCD1 activity on PA-induced trophoblast cell death. PA induces cell death and inhibits the invasion of human trophoblast cells (HTR8/SVneo). In addition, we demonstrate that SCD1 has a protective role against PA in human trophoblast cells by regulating AKT-mediated signaling pathway and mitochondrial membrane potential. The knockdown of SCD1 enhances the proapoptotic activity of PA in HTR8/SVneo cells. Lastly, we investigated microRNA expression predicted to target SCD1 and diacylglycerol O-acyltransferase 1 (DGAT1) by PA. Collectively, the results suggest potential roles of SCD1 and DGAT1 in alleviating the toxicity of PA and maintaining lipid homeostasis for normal placentation.     

Schistosoma japonicum Soluble Egg Antigens Attenuate Invasion in a First Trimester Human Placental Trophoblast Model

Background

Schistosomiasis affects nearly 40 million women of reproductive age, and is known to elicit a pro-inflammatory signature in the placenta. We have previously shown that antigens from schistosome eggs can elicit pro-inflammatory cytokine production from trophoblast cells specifically; however, the influence of these antigens on other characteristics of trophoblast function, particularly as it pertains to placentation in early gestation, is unknown. We therefore sought to determine the impact of schistosome antigens on key characteristics of first trimester trophoblast cells, including migration and invasion.

Methods

First trimester HTR8/SVneo trophoblast cells were co-cultured with plasma from pregnant women with and without schistosomiasis or schistosome soluble egg antigens (SEA) and measured cytokine, cellular migration, and invasion responses.

Results

Exposure of HTR8 cells to SEA resulted in a pro-inflammatory, anti-invasive signature, characterized by increased pro-inflammatory cytokines (IL-6, IL-8, MCP-1) and TIMP-1. Additionally, these cells displayed 62% decreased migration and 2.7-fold decreased invasion in vitro after treatment with SEA. These results are supported by increased IL-6 and IL-8 in the culture media of HTR8 cells exposed to plasma from Schistosoma japonica infected pregnant women.

Conclusions

Soluble egg antigens found in circulation during schistosome infection increase pro-inflammatory cytokine production and inhibit the mobility and invasive characteristics of the first trimester HTR8/SVneo trophoblast cell line. This is the first study to assess the impact of schistosome soluble egg antigens on the behavior of an extravillous trophoblast model and suggests that schistosomiasis in the pre-pregnancy period may adversely impact placentation and the subsequent health of the mother and newborn.     

Comparative proteomic analysis of trophoblast cell models reveals their differential phenotypes,potential uses,and limitations

Trophoblastic cell lines are widely used in in vitro studies of placental function as a surrogate for primary trophoblasts. To date, no reference proteomics dataset exists to directly compare the shared and unique characteristics of these cells. Here, we performed comparative proteomic profiling of the BeWo and HTR8/SVneo cell lines using label‐free quantitative MS. A total of 1557 proteins were identified, which included 338 uniquely attributed to BeWo cells, and a further 304 specifically identified in HTR8/SVneo cells. Raw data are available via ProteomeXchange, identifier PDX005045. Of the 915 proteins expressed by both cell lines, 105 were of higher abundance in BeWo cells, while 199 proteins had a significantly higher expression in HTR8/SVneo cells. Comparative GO of unique and upregulated proteins revealed principal differences in cell junction/adhesion, catenin complex, spindle and microtubule associated complex, as well as cell differentiation. Our data indicate that BeWo cells express an epithelial proteome more characteristic of villous trophoblasts, whereas HTR8/SVneo cells embrace a mesenchymal phenotype, more characteristic of extravillous trophoblasts. This novel comparative proteomic profiling of these trophoblastic cell lines provides a useful platform for future investigations of placental function.     

Endoplasmic reticulum protein 29 (ERp29): An emerging role in cancer

The endoplasmic reticulum protein 29 (ERp29) is a molecule that facilitates processing and transport of proteins in the early secretory pathway. Structural and functional analyses have suggested a biological role as a putative chaperone in the endoplasmic reticulum. The N-terminal domain of ERp29 resembles the thioredoxin domain of protein disulfide isomerase, but lacks its redox-active function due to the absence of an active motif consisting of double cysteines. In the context of carcinogenesis, the role of ERp29 in cancer progression has not been fully elucidated. However, recent studies indicate that high expression of ERp29 inversely correlates to tumor progression. In addition, over-expression of ERp29 significantly inhibits proliferation and suppresses tumorigenesis by modulating ER stress signaling and the mesenchymal-epithelial transition in breast cancer cells. In this review, we summarize the biological properties of ERp29 and its novel function as a tumor suppressor.     

Structure and assembly of the endoplasmic reticulum. Biosynthetic sorting of endoplasmic reticulum proteins

We have studied the post-translational processing and the biosynthetic sorting of three protein components of murine endoplasmic reticulum (ER), ERp60, ERp72, and ERp99. In pulse-labeled MOPC-315 (where MOPC-315 represents mineral oil-induced plasmacytoma cells) plasmacytoma cells, no precursor forms of these proteins were detected and only ERp99 was sensitive to endoglycosidase H. The ERp99 oligosaccharide remained endoglycosidase H sensitive during a 3-h chase, and analysis by high performance liquid chromatography showed the predominant structure to be Man8GlcNAc2. We have used a sucrose gradient analysis of pulse-labeled MOPC-315 plasmacytoma cells in order to directly study the biosynthetic sorting of both glycosylated and nonglycosylated ERps and have found no strong evidence to suggest these proteins ever leave the endoplasmic reticulum. In spite of their common sorting pathway, these proteins differ in their membrane orientation. Both ERp60 and ERp72 are entirely protected by the endoplasmic reticulum membrane while ERp99 appears to have a large domain exposed on the cytoplasmic face of the endoplasmic reticulum.     

The effect of zinc on human trophoblast proliferation and oxidative stress

Adequate Zinc (Zn) intake is required to prevent multiple teratogenic effects however deviations from adequate Zn intake, including high maternal Zn status, have been linked to increased incidence of pregnancy complications, including those associated with inadequate placentation. Using placental trophoblast HTR8/SVneo cells and first trimester human placental explants (n = 12), we assessed the effects of varying Zn concentrations on trophoblast proliferation, viability, apoptosis and oxidative stress. Compared to physiologically normal Zn levels (20 µM), HTR-8/SVneo cell proliferation index was significantly lower in the presence of physiologically elevated (40 µM; P = .020) and supra-physiological (80 µM; P = .007) Zn. The latter was also associated with reduced proliferation (P = .004) and viability (P < .0001) in cultured placental explants, but not apoptosis. Reactive oxygen species production in HTR8/SVneo cultures was significantly higher in the presence of 80 µM Zn compared to all physiologically relevant levels. Oxidative stress, induced by an oxidizing agent menadione, was further exacerbated by high (80 µM) Zn. Zn did not affect lipid peroxidation in either HTR8/SVneo cells or placental explants or antioxidant defense mechanisms that included glutathione reductase and superoxide dismutase. Further study should focus on elucidating mechanisms behind impaired trophoblast proliferation and increased oxidative stress as a result of elevated Zn levels.