Impact of chlorpyrifos on human villous trophoblasts and chorionic villi.

Placental barrier regulates maternal-fetal interchange protecting the baby from damage caused by substances found in the uterine environment or circulating in the vascular system. Organophosphate (OP) pesticides are a paramount group of environmental pollutants used in intensive agriculture for protection against diseases and pests. While many studies have reported an increased risk of pregnancy alterations in pregnant women exposed to OPs, few have analyzed the effects caused by these pesticides in the placenta. Herein, we evaluated the effects of chlorpyrifos (CPF), one of the most widely used OP insecticides, on human placenta using in vitro and ex vivo exposure models. Villous cytotrophoblast cells isolated from normal human term placentas maintained their cell viability, differentiated into syncytiotrophoblast-like structures, and increased the expression of ß-hCG, ABCG2, and P-gp in the presence of CPF at concentrations of 10 to 100µM. The same doses of CPF induced marked changes in chorionic villi samples. Indeed, CPF exposure increased stroma cell apoptosis, altered villi matrix composition, basement membrane thickness, and trophoblastic layer integrity. Histomorphological and ultrastructural alterations are compatible with those found in placentas where maternal-placenta injury is chronic and able to impair the placental barrier function and nutrient transport from mother to the fetus. Our study shows that placental ex vivo exposure to CPF produces tissue alterations and suggest that human placenta is a potential target of CPF toxicity. In addition, it highlights the importance of using different models to assess the effects of a toxic on human placenta.

Low oxygen tension induces Krüppel-Like Factor 6 expression in trophoblast cells.

The transcription factor Krüppel-Like Factor 6 (KLF6) has important roles in cell differentiation, angiogenesis, apoptosis, and proliferation. Furthermore, there is evidence that KLF6 is required for proper placental development. While oxygen is a critical mediator of trophoblast differentiation and function, the involvement of oxygen in the regulation of KLF6 expression remains unexplored. In the present study we examined the expression of KLF6 in placental tissue from uncomplicated and preeclamptic pregnancies, the latter often characterized by an inadequately perfused placenta. We also determined the effect of hypoxia and the involvement of Hypoxia-Inducible Factor 1α (HIF-1α) on the expression of KLF6 in cultured trophoblast cells and placental tissues. Results revealed that villous, interstitial and endovascular extravillous cytotrophoblasts from placentas from normal and preeclamptic pregnancies express KLF6. In addition, KLF6 immunoreactivity was higher in the placental bed of preeclamptic pregnancies than in those of uncomplicated pregnancies. We demonstrated that hypoxia induced an early and transient increase in KLF6 protein levels in HTR8/SVneo extravillous cytotrophoblast cells and in placental explants. Reoxygenation returned KLF6 protein to basal levels. Moreover, hypoxia-induced up-regulation of KLF6 expression was dependent on HIF-1α as revealed by siRNA knockdown in HTR8/SVneo cells. These results indicate that KLF6 may mediate some of the effects of hypoxia in placental development. The regulation of KLF6 protein levels by oxygen has significant implications for understanding its putative role in diseases affected by tissue hypoxia.

The organophosphate chlorpyrifos disturbs redox balance and triggers antioxidant defense mechanisms in JEG-3 cells.

INTRODUCTION: Reactive oxygen species (ROS) are produced by a number of physiological and pathological processes which influence the function of a diverse array of cellular events. An imbalance between ROS generation and elimination was reported for different environmental xenobiotics exposure. Here, we analyzed the effect of chlorpyrifos (CPF) on the JEG-3 cell antioxidant defense in conditions where cell viability and morphology were not altered. METHODS: Acetylcholinesterase (AChE) activity, reduced glutathione (GSH) content and catalase (CAT) antioxidant enzyme activity were measured by biochemical studies. ROS production was detected using the fluorogenic probe 2',7'-dichlorodihydrofluorescein diacetate. The transcript level of superoxide dismutase enzyme 1 (SOD1), glutathione reductase (GR), heme oxygenase-1 (HO-1), and nuclear factor E2-related factor 2 (Nrf2) as well as Nrf2 protein amount were analyzed by quantitative real time PCR and Western blot, respectively. RESULTS: The results showed that CPF inhibited AChE activity, induced ROS production, upregulated CAT activity, and decreased GSH concentration. In response to CPF exposure, GR and HO-1 mRNA levels were increased with no changes in SOD1 mRNA. Furthermore, CPF significantly augmented Nrf2 at both mRNA and protein levels trigging the antioxidant status by increasing nuclear Nrf2 translocation. DISCUSSION AND CONCLUSION: Taken together, these data indicate that JEG-3 cells are able to attenuate the oxidative stress induced by CPF through the adaptive activation of the Nrf2/ARE pathway.

Expression and transcriptional regulation of individual pregnancy-specific glycoprotein genes in differentiating trophoblast cells.

Human pregnancy-specific glycoproteins (PSGs), encoded by eleven highly conserved genes, are the major placental polypeptides. Low PSG levels in maternal circulation have been associated with complicated pregnancies. However, expression of each PSG gene and their regulation during cytotrophoblast cell differentiation remain poorly explored. Herein, we analyze the expression of five PSG genes and demonstrate that they are almost undetectable in undifferentiated trophoblast, but are all transcribed in differentiated cells. Among them, PSG1, PSG3 and PSG5 genes achieve high mRNA levels while PSG7 and PSG9 are poorly expressed. In addition, total PSG proteins and transcripts markedly increase during trophoblast differentiation, preceding morphological syncytialization and betahCG expression. The 5' regulatory region contributes to the transcriptional control of PSG gene induction in trophoblast cells undergoing differentiation. This responsive region in PSG3 maps within a 130 bp promoter sequence, which overlaps the transcription start site and requires a functional Retinoic Acid Responsive Element (RARE) and a GA-binding protein (GABP) consensus site for basal and differentiation-dependent promoter activity, respectively. Present findings provide novel data for understanding the control of PSG gene expression and demonstrate that their proteins and transcripts represent early markers of trophoblast differentiation.

Activation of beta-catenin signalling increases StarD7 gene expression in JEG-3 cells.

StarD7 gene encodes a protein that belongs to the StAR-related lipid transfer proteins involved in intracellular transport and metabolism of lipids. It has been previously documented that StarD7 has a wide-spread mRNA expression in trophoblastic tissues and several tumour cell lines with highest levels in both choriocarcinoma JEG-3 and JAR cells, hepatocellular carcinoma HepG2, and colorectal adenocarcinoma HT-29 cells. To understand the molecular mechanisms that regulate the expression of the human StarD7 gene, we have cloned and characterized the 5'-flanking region of the gene. Transient transfections of several 5'deleted StarD7-promoter-firefly luciferase constructs into JEG-3 cells indicated that the -312/+157 region contains the gene minimal promoter. In addition, sequence analysis of a 1.6kb gene fragment revealed the presence of a TATA-less promoter as well as multiple regulatory motifs, including one regulatory element corresponding to the T-cell factor 4 (TCF4) binding site. Inhibition of glycogen synthase kinase-3beta (GSK3beta), a component of Wnt/beta-catenin signalling, increased both StarD7 mRNA and protein expression as well as its promoter activity. Co-transfection experiments in JEG-3 cell line revealed that the StarD7 promoter is activated by TCF4 transcription factor and by its beta-catenin coactivator. Moreover, site-directed mutagenesis of the TCF4 site located -614/-608bp relative to the transcription start site markedly diminished StarD7 promoter activity. Chromatin immunoprecipitation analysis demonstrated that beta-catenin and TCF4 are bound in vivo to the StarD7 gene promoter in JEG-3 cells treated with lithium chloride. Collectively, these studies show that beta-catenin and TCF4 activate the human StarD7 gene interacting with its promoter region through Wnt/beta-catenin signalling.

Expression and localization of StarD7 in trophoblast cells.

The StAR-related lipid transfer (START) domain is defined as a motif of around 200 amino acids implicated in lipid/sterol binding. In a previous study, we identified the StarD7 transcript encoding one of the 15 family members with START domain present in the human genome. This transcript was found to be overexpressed in choriocarcinoma JEG-3 cells. In addition, we demonstrated that the recombinant StarD7 protein forms stable Gibbs and Langmuir monolayers at the air-buffer interface, showing marked surface activity and interaction with phospholipid monolayers, mainly with phosphatidylserine, cholesterol and phosphatidylglycerol. This study was undertaken to evaluate the expression and localization of StarD7 protein in trophoblastic samples. Here, we show for the first time the presence of StarD7 protein in human trophoblast cells. Western blot assays revealed a unique specific 34 kDa protein in JEG-3 cell line, choriocarcinoma tissue, complete hydatidiform mole, early and normal term placenta. Immunohistochemical data from early and normal term placentas and complete hydatidiform moles showed that this protein is abundant in the syncytiotrophoblasts, mainly at the apical side of the syncytium, with a weak and focal reaction in the cytotrophoblast cells. Furthermore, an increased StarD7 mRNA and protein expression, as well as a change in its sub-cellular localization was observed in in vitro differentiating cytotrophoblast isolated from normal term placenta. Taken together, these findings support and allow future studies to explore the possibility that StarD7 protein mediates transplacental lipid transport and/or is involved in syncytialization.

RXRalpha regulates the pregnancy-specific glycoprotein 5 gene transcription through a functional retinoic acid responsive element.

Human pregnancy-specific glycoproteins (PSG) are major placental polypeptides encoded by eleven highly conserved genes expressed by the syncytiotrophoblast. The minimal promoter region of all PSG genes contains a putative Retinoic Acid Responsive Element (RARE) though the ability of retinoids to regulate PSG gene expression has not been established. Retinoid signaling pathway plays a key role for overall placenta biology and is essential for trophoblast differentiation. In this work, we investigated the participation of the RARE motif in the regulation of PSG5 gene transcription by retinoic acid and its receptors. The minimal promoter region of PSG5 gene was activated by RXRalpha but not by RARalpha, in a ligand-dependent manner. The RARE sequence of PSG5 gene promoter was recognized by endogenous RXRalpha present in placental nuclear extracts as well as by RXRalpha either over expressed in cultured non-placental cells or in vitro translated. Mutations at specific nucleotides within the RARE motif abrogated both RXRalpha DNA binding and transcriptional activation of PSG5 promoter mediated by RXRalpha. Moreover, endogenous PSG expression was significantly induced in trophoblast-derived Jeg-3 cells upon 9-cis retinoic acid treatment. Interestingly, the induction level was higher following methotrexate-induced differentiation of Jeg-3 cells to syncytiotrophoblast-like structures. Altogether, these data provide the first evidences demonstrating that transcriptional activity of PSG5 gene is responsive to an external signal involving the retinoids-RXRalpha axis through a conserved RARE motif shared by all PSG gene family members.

Transcriptional control of the human pregnancy-specific glycoprotein 5 gene is dependent on two GT-boxes recognized by the ubiquitous specificity protein 1 (Sp1) transcription factor.

Pregnancy-specific glycoprotein 5 gene (PSG-5) belongs to the human pregnancy-specific glycoprotein family, encoded by eleven highly similar and transcriptionally active genes. High levels of PSG biosynthesis are restricted to the placenta syncytiotrophoblast and are essential for the maintenance of normal gestation in mammalian species. We have investigated here the nature of the transcription factors that recognize the FP1 (-455/-433) and the CPE (-147/-140) regulatory sequences that significantly contribute to basal PSG-5 promoter activity. Both elements bear a similar GT-box motif; and DNA-protein complex formation, as well as promoter activity, is largely dependent on the integrity of these GT-box sequences. Gel shift, super gel shift and UV-crosslinking experiments clearly demonstrate that the ubiquitous specificity protein 1 (Sp1) is the major transcription factor involved in complex formation with both cis-acting elements in normal term placenta tissue and in PSG-non-expressing COS-7 cells. Furthermore, transfection experiments indicate that Sp1 activates PSG-5 promoter constructs. In addition, we show that Sp1 is indeed co-expressed with PSG genes in the syncytiotrophoblast cells, stressing its potential role in the in vivo regulation of PSG expression.

Absence of the 9-bp deletion of mitochondrial DNA in pre-Hispanic inhabitants of Argentina.

We investigated the incidence of the Region V mitochondrial DNA 9-base-pair (bp) deletion from human remains recovered from several archaeological sites and contexts throughout Argentina. Of the 34 samples analyzed, 24 yielded DNA extractions that gave clear amplification results. All of the individuals carried two repeats of the 9 bp, one of which has been shown to be deleted in some individuals of Asian origin and defines mitochondrial lineage B. Although most of the modern Amerindian groups in the region exhibit the deletion in high frequencies, the absence of the 9-bp deletion among ancient populations of South America seems to be the rule rather than the exception, as was reported by several studies involving extinct populations. The evidence gathered until now suggests that the earliest settlers of this region of South America did not carry mitochondrial lineage B.

Mitochondrial DNA haplogroups in Amerindian populations from the Gran Chaco.

Mitochondrial DNA from 141 individuals was typed for diagnostic restriction sites and the 9-bp region V deletion to examine the distribution of the founding mtDNA lineage haplotypes in three Amerindian populations (Mataco, Toba, and Pilagá) who currently inhabit the Argentinian part of the Gran Chaco. All four lineages were identified in the three tribes and four population samples studied. Disregarding ethnic or geographic origin, haplogroups B and D exhibit high incidence among the Gran Chaco inhabitants, whereas haplogroups A and C are present in a lower frequency. Three individuals possess none of the characteristic markers and, therefore, could not be assigned to one of those lineages. A neighbor-joining representation of F(ST) distances reflects the current geographic location of the populations, and this also corresponds to their historic distribution. After separating South America into four major regions (Tropical Forest, Andes, Gran Chaco, and Patagonia-Tierra del Fuego), the Gran Chaco populations present the highest average intragroup variability (Hs = 0.64) as well as the lowest intergroup diversity (G(')(ST) = 0.06). These findings suggest high levels of gene flow among the Chaco tribes, as well as with neighbor populations from outside the region.

Transcription of genes encoding pregnancy-specific glycoproteins is regulated by negative promoter-selective elements.

The human pregnancy-specific glycoprotein (PSG) genes comprise a family of 11 highly conserved members whose expression is maximal in placental cells and marginal in other cell types. We have investigated here the molecular basis of PSG regulation by analysing a large regulatory region of the PSG-5 gene in cells that do and do not express these genes. The promoter region (-254 to -43), which does not contain a TATA-box, large GC-rich sequences or a classical initiator, was active in all cell types analysed. Additional upstream sequences up to position -3204 repressed promoter activity. Two independent repressor regions were identified and found to operate effectively in HeLa, COS-7 and HTR8/SVneo placental cells. More significantly, these negatively acting modules failed to repress a heterologous TATA-containing thymidine kinase promoter. Detailed transcriptional and DNA-protein analyses of the proximal repressor region (-605 to -254) revealed the presence of both negative and positive cis-acting elements. Disruption of the repressive functions resulted in an enhanced transcription of the reporter constructs. In conclusion, these results demonstrate that PSG-5 gene transcription is highly repressed by promoter-selective negative regulatory regions and the relief of repression allows enhanced PSG-5 gene transcription irrespective of the cell type. Furthermore, our findings suggest that PSG genes are expressed mainly through a derepression mechanism.

Differential expression of a tumor necrosis factor receptor-related transcript in gestational trophoblastic diseases in women.

Gestational trophoblastic diseases comprise a group of interrelated neoplasms, including complete hydatidiform mole (CHM), persistent gestational trophoblastic tumor (GTT), and choriocarcinoma. To better define the molecular features of these diseases, a CHM cDNA library was constructed and a novel cDNA sequence, named CHMS-1, was isolated by differential screening. The CHMS-1 sequence showed a 62% homology with the tumor necrosis factor receptor (TNF-R2) cDNA, and its amino acid deduced sequence shared a high level of homology with the "death domain" region found in various proteins, including two members of the TNF receptor superfamily, the TNF-R1 and Fas. We also determined the CHMS-1, TNF-R1, and TNF-R2 expression patterns among different CHM tissues and cell lines of trophoblastic (JEG-3) and nontrophoblastic (HeLa and COS-7) origin. Our results indicated that the CHMS-1 transcript is highly expressed in CHM in comparison with both normal early and term placenta and that it exhibits an expression profile identical to that of TNF-R1. Furthermore, the CHMS-1 transcript was undetectable in CHM-derived GTT and in the human choriocarcinoma-derived JEG-3 cells, suggesting that its expression is down-regulated in the malignant transformation of trophoblast. The presence of a potential "death domain" in CHMS-1, together with its high expression level in CHM, strongly suggests that the CHMS-1 gene encodes a protein that might be involved in tumor regression processes occurring at later stages of molar development.

A novel human zinc finger protein that interacts with the core promoter element of a TATA box-less gene.

We describe a novel human cDNA isolated by target site screening of a placental expression library, using as a probe, an essential element of a TATA box-less promoter corresponding to a pregnancy-specific glycoprotein gene. The cDNA encoded a predicted protein of 290 amino acids, designated core promoter-binding protein (CPBP), which has three zinc fingers (type Cys2-His2) at the end of its C-terminal domain, a serine/threonine-rich central region and an acidic domain lying within the N-terminal region. Additional sequence analysis and data base searches revealed that only the zinc finger domains are conserved (60-80% identity) in other transcription factors. In cotransfection assays, CPBP increased the transcription from a minimal promoter containing its natural DNA-binding site. Moreover, a chimeric protein between CPBP and Gal4 DNA binding domain also increased the activity of an heterologous reporter gene containing Gal4 DNA binding sites. The tissue distribution analysis of CPBP mRNA revealed that it is differentially expressed with an apparent enrichment in placental cells. The DNA binding and transcriptional activity of CPBP, in conjunction with its expression pattern, strongly suggests that this protein may participate in the regulation and/or maintenance of the basal expression of PSG and possibly other TATA box-less genes.

A new Comamonas testosteroni steroid-inducible gene: cloning and sequence analysis.

Comamonas testosteroni can grow on a variety of steroid compounds as the sole carbon and energy source. In a previous study, we cloned and sequenced the testosterone-inducible betahsd gene from C. testosteroni (Genti-Raimondi, S., Tolmasky, M., Patrito, L., Flury, A. and Actis, L., Molecular cloning and expression of the beta-hydroxysteroid dehydrogenase gene from Pseudomonas testosteroni. Gene, 1991, 105, 43-49.). Herein we report the cloning and characterization of another steroid-inducible gene (stdC), located 2400 bp upstream of betahsd. Nucleotide sequencing of a region encompassing the stdC gene revealed an open reading frame 546 bp long including the stop codon TGA with significant similarity to the orf4, orf1 and orf4 of unknown function described in the polyhydroxyalkanoic acid (PHA) cluster of Chromatium vinosum, Rhizobium meliloti and Thiocystis violacea, respectively. The aminoacid sequence deduced from the nucleotide sequence predicts a putative protein of 181 amino acids with a molecular weight of 20715 Da. Northern blot experiments indicate that the stdC gene was transcribed as a monocistronic mRNA with an apparent molecular size of 670 nt. The stdC transcript was abundant in C. testosteroni cells grown with different steroid carbon sources harvested in the exponential phase and was found to be under catabolite repression.

Analyses of cis-acting and trans-acting elements that are crucial to sustain pregnancy-specific glycoprotein gene expression in different cell types.

Pregnancy-specific beta 1 glycoprotein genes (PSG) are mainly expressed during human placental development, though their expression has been reported in other normal and pathological tissues, e.g. hydatidiform mole (HM), of distinct origins. However, the molecular components implicated in the regulation of PSG are not well understood. To identify some of the regulatory elements involved in the transcriptional control of PSG expression, the DNA-protein interactions and the basal activities of the TATA-box-less PSG5 promoter were determined in different tissues and cell types. In DNAse-I protection assays, DNA-binding proteins from human term placenta (HTP) protected a region of 27 bp located from nucleotides --150 to --124, overlapping the farthest 5' upstream cap site and resembling an initiator-like element. In electrophoretic mobility shift assays (EMSA), three complexes were detected using nuclear extracts from HTP and an oligonucleotide containing the 27-bp motif. In situ ultraviolet crosslinking analysis of the specific complexes revealed that two proteins of 78.0 kDa and 53.0 kDa are involved in such interactions, in accordance with the bands of 80.0 kDa and 57.5 kDa observed by Southwestern blotting. Competitive EMSA using mutant oligonucleotides with the substitution of 5'ACCCAT3' by 5'GATATC3' within the 27-bp motif revealed that this sequence is fundamental for the formation of the specific DNA-protein complexes. We show in transient transfection experiments performed in HeLa, COS-7 and JEG-3 cells, that such mutation completely abolished the transcriptional activity of the PSG5 promoter, independently of the cell type. Moreover, this mutation disrupted the formation of the specific DNA-protein complexes which were essentially the same as those displayed by HTP. We also determined the binding activities of nucleoproteins derived from placental tissues in earlier developmental and pathological stages, i.e. first trimester placenta (1-TRIM) and HM, respectively, showing that the DNA-binding patterns were different from each other and distinct from those elicited by HTP. Our results indicate that the cis-acting and trans-acting elements analyzed are indispensable to support PSG5 promoter activity in cell lines which do or do not produce PSG. In addition, these elements appear to play a role in the mechanisms involved in PSG basal expression during placental development and differentiation.

Nucleotide sequence of a pregnancy-specific beta 1 glycoprotein gene family member. Identification of a functional promoter region and several putative regulatory sequences.

The pregnancy-specific beta 1 glycoprotein (PSG) genes encode a group of heterogeneous proteins produced in large amounts by the human syncytiotrophoblast. Their expression seems to be regulated at the transcriptional level during normal pregnancy. In the present work, we isolated from a human placental library a 17 kb genomic fragment corresponding to a member of the PSG multigene family. DNA sequence analysis of 1190 nucleotides upstream of the translational start and of the first intron, revealed the presence of several putative regulatory sequences. In a transient chloramphenicol acetyltransferase expression assay, 5' flanking sequences within 123 nucleotides upstream to the first major transcription initiation site, functioned as a strong promoter in COS-7 cells. Meanwhile, sequences 5' further upstream had the ability to abolish this promoter activity. The sequence analyzed did not contain any obvious TATA-like boxes or G+C-rich regions, suggesting the existence of unique promoter elements implicated in transcription initiation and regulation of this PSG gene family member.