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.

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.

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.