Genetic and epigenetic mechanisms collaborate to control SERPINA3 expression and its association with placental diseases.

SERPINA3 (Serpin peptidase inhibitor clade A member 3), also known as a1-antichymotrypsin, is a serine protease inhibitor involved in a wide range of biological processes. Recently, it has been shown to be up-regulated in human placental diseases in association with a hypomethylation of the 5' region of the gene. In the present study, we show that the promoter of SERPINA3 is transcriptionally activated by three transcription factors (TFs) (SP1, MZF1 and ZBTB7B), the level of induction being dependent on the rs1884082 single nucleotide polymorphism (SNP) located inside the promoter, the T allele being consistently induced to a higher level than the G, with or without added TFs. When the promoter was methylated, the response to ZBTB7B was allele specific (the G allele was strongly induced, while the T allele was strongly down-regulated). We propose an adaptive model to explain the interest of such a regulation for placental function and homeostasis. Overexpression of SERPINA3 in JEG-3 cells, a trophoblast cell model, decreased cell adhesion to the extracellular matrix and to neighboring cells, but protects them from apoptosis, suggesting a way by which this factor could be deleterious at high doses. In addition, we show in different human populations that the T allele appears to predispose to Intra Uterine Growth Restriction (IUGR), while a G allele at a second SNP located in the second exon (rs4634) increases the risk of preeclampsia. Our results provide mechanistic views inside the involvement of SERPINA3 in placental diseases, through its regulation by a combination of epigenetic, genetic and TF-mediated regulations.

Combination of promoter hypomethylation and PDX1 overexpression leads to TBX15 decrease in vascular IUGR placentas.

Preeclampsia (PE) and vascular intra-uterine growth restriction (vIUGR) are two pathological obstetrical conditions originating from placental dysfunction. Recently, methylation changes at the placental level have been shown to be indicative of these diseases. The alteration of such epigenetic marks is therefore a novel pathway that might be critical for these pathologies. Here, we identified a region located in the distal promoter of the T-box-containing transcription factor TBX15 that is differentially methylated in pathological placentas. The level of methylation correlated significantly with the weight and stature of the newborn. The promoter was found to be hypomethylated in vIUGR coinciding with the down-regulation of its expression. PDX1, a transcription factor important for the regulation of insulin metabolism regulation was able to repress the TBX15 promoter in a methylation-dependent manner, which might, at least partially, explain the specific mRNA decrease of TBX15 observed in vIUGR placentas. Overall, the data presented herein suggest that TBX15 might be involved in the pathophysiology of placental diseases.

Re-evaluation of the role of STOX1 transcription factor in placental development and preeclampsia.

Preeclampsia is a common disease of pregnancy, characterized by high blood pressure and proteinuria appearing from the second trimester of gestation. Preeclampsia has been shown to have a strong genetic component. In 2005 a positional cloning project led to the discovery of the STOX1 transcription factor, and mutations of this gene were proposed as causal for preeclampsia in Dutch families. Despite the publication of three contradictory studies, we have shown by analyzing the functional effects of STOX1 that its overexpression in choriocarcinoma cells recapitulates several transcriptomic aspects of preeclampsia. In this review, the current literature is analyzed to evaluate the possible involvement of STOX1 in the pathogenesis of this disease. While preeclampsia obviously cannot be considered as a disease caused by mutation in a single gene, we argue that STOX1 may be at the center of common pathways leading to preeclampsia.

STOX1 overexpression in choriocarcinoma cells mimics transcriptional alterations observed in preeclamptic placentas.

BACKGROUND: Mutations in STOX1 were proposed to be causal for predisposing to preeclampsia, a hypertensive disorder originating from placental defects, affecting up to 10% of human pregnancies. However, after the first study published in 2005 three other groups have dismissed the polymorphism described in the first paper as a causal mutation. METHODOLOGY AND PRINCIPAL FINDINGS: In the present study, we have produced a choriocarcinoma cell line overexpressing STOX1. This overexpression results in transcriptional modification of 12.5% of the genes, some of them being direct targets as shown by chromatin immunoprecipitation. STOX1 overexpression correlates strongly and specifically with transcriptomic alterations in preeclamptic placentas (r = 0.30, p = 9.10(-7)). Numerous known key modulators of preeclampsia (such as Endoglin, Syncytin, human chorionic gonadotrophin -hCG-, and Glial Cell Missing Homolog -GCM1-) were modified in these transformed choriocarcinoma cells. CONCLUSIONS: Our results contribute to reconcile contradictory data concerning the involvement of STOX1 in preeclampsia. In addition, they strongly suggest that anomalies in STOX1 expression are associated with the onset of preeclampsia, thus indicating that this gene should be the target of future studies. Our cellular model could constitute an invaluable resource for studying specific aspects of this human disease.

Expressional and epigenetic alterations of placental serine protease inhibitors: SERPINA3 is a potential marker of preeclampsia.

Preeclampsia is the major pregnancy-induced hypertensive disorder. It modifies the expression profile of placental genes, including several serine protease inhibitors (SERPINs). The objective of this study was to perform a systematic expression analysis of these genes in normal and pathological placentas and to pinpoint epigenetic alterations inside their promoter regions. Expression of 18 placental SERPINs was analyzed by quantitative RT-PCR on placentas from pregnancies complicated by preeclampsia, intrauterine growth restriction, or both and was compared with normal controls. SERPINA3, A5, A8, B2, B5, and B7 presented significant differences in expression in >or=1 pathological situation. In parallel, the methylation status of the CpG islands located in their promoter regions was studied on a sample of control and preeclamptic placentas. Ten SERPIN promoters were either totally methylated or totally unmethylated, whereas SERPINA3, A5, and A8 presented complex methylation profiles. For SERPINA3, the analysis was extended to 81 samples and performed by pyrosequencing. For the SERPINA3 CpG island, the average methylation level was significantly diminished in preeclampsia and growth restriction. The hypomethylated CpGs were situated at putative binding sites for developmental and stress response (hypoxia and inflammation) factors. Our results provide one of the first observations of a specific epigenetic alteration in human placental diseases and provide new potential markers for an early diagnosis.

Hypoxia-activated genes from early placenta are elevated in preeclampsia, but not in Intra-Uterine Growth Retardation.

BACKGROUND: As a first step to explore the possible relationships existing between the effects of low oxygen pressure in the first trimester placenta and placental pathologies developing from mid-gestation, two subtracted libraries totaling 2304 cDNA clones were constructed. For achieving this, two reciprocal suppressive/subtractive hybridization procedures (SSH) were applied to early (11 weeks) human placental villi after incubation either in normoxic or in hypoxic conditions. The clones from both libraries (1440 hypoxia-specific and 864 normoxia-specific) were spotted on nylon macroarrays. Complex cDNAs probes prepared from placental villi (either from early pregnancy, after hypoxic or normoxic culture conditions, or near term for controls or pathological placentas) were hybridized to the membranes. RESULTS: Three hundred and fifty nine clones presenting a hybridization signal above the background were sequenced and shown to correspond to 276 different genes. Nine of these genes are mitochondrial, while 267 are nuclear. Specific expression profiles characteristic of preeclampsia (PE) could be identified, as well as profiles specific of Intra-Uterine Growth Retardation (IUGR). Focusing on the chromosomal distribution of the fraction of genes that responded in at least one hybridization experiment, we could observe a highly significant chromosomal clustering of 54 genes into 8 chromosomal regions, four of which containing imprinted genes. Comparative mapping data indicate that these imprinted clusters are maintained in synteny in mice, and apparently in cattle and pigs, suggesting that the maintenance of such syntenies is requested for achieving a normal placental physiology in eutherian mammals. CONCLUSION: We could demonstrate that genes induced in PE were also genes highly expressed under hypoxic conditions (P = 5 x 10(-5)), which was not the case for isolated IUGR. Highly expressed placental genes may be in syntenies conserved interspecifically, suggesting that the maintenance of such clusters is requested for achieving a normal placental physiology in eutherian mammals.

Regulation of the endothelin/endothelin receptor system by interleukin-1{beta} in human myometrial cells.

Proinflammatory cytokines produced at the fetomaternal interface, such as IL-1beta, have been implicated in preterm and term labor. The present study was performed to evaluate the influence of IL-1beta on the endothelin (ET)/ET receptor system in human myometrial cells. We report that myometrial cells under basal conditions not only respond to but also secrete ET-1, one of the main regulators of uterine contractions. Prolonged exposure of the cells to IL-1beta led to a decrease in prepro-ET-1 and ET-3 mRNA correlated with a decrease in immunoreactive ET-1 and ET-3 levels in the culture medium. Whereas ETA receptor expression at both protein and mRNA levels was not affected by IL-1beta treatment, we demonstrated an unexpected predominance of the ETB receptor subtype under this inflammatory condition. Whereas the physiological function of ETB remains unclear, we confirmed that only ETA receptors mediate ET-1-induced myometrial cell contractions under basal conditions. By contrast, prolonged exposure of the cells to IL-1beta abolished the contractile effect induced by ET-1. Such a regulation of IL-1beta on the ET release and the balance of ETA to ETB receptors leading to a loss of ET-1-induced myometrial cell contractions suggest that complex regulatory mechanisms take place to constraint the onset of infection-induced premature contractions.

Profiling of oxygen-modulated gene expression in early human placenta by systematic sequencing of suppressive subtractive hybridization products.

Villi from first-trimester human placenta were exposed to oxygen concentrations of either 2 or 20% during 3 h to construct two reciprocally subtracted libraries using the suppressive subtractive hybridization (SSH) methodology. After cloning, sequencing, and gene identification, the genes (1,071 clones corresponding to 822 different sequences) were classified according to 1) the subtracted library from which they originated and 2) within 58 groups of gene functions. We then developed a logarithm of the odds (LOD) test to identify a possible excess of genes in each group. We show that genes involved in angiogenesis are significantly overrepresented in the "hypoxic" condition (2% O2), whereas apoptotic genes are overrepresented in the "normoxic" condition (20% O2). Furthermore, we observed an excess of kinases relative to phosphatases and an excess of genes involved in proliferation over genes involved in cell growth in the hypoxic condition. To validate our results, we used quantitative RT-PCR to analyze the set of eight genes involved in angiogenesis on six independent placentas. Finally, we studied the distribution of gene clusters on human chromosomes to check whether their chromosomal distribution was random or not. We observed on human chromosome 11 a clear clustering of genes regulated similarly by O2 tension, and we also discovered indications that such clustering exists on chromosomes 6 and 12.