Jean-Francois Mouillet

Human placental trophoblasts confer viral resistance to recipient cells

Placental trophoblasts form the interface between the fetal and maternal environments and serve to limit the maternal–fetal spread of viruses. Here we show that cultured primary human placental trophoblasts are highly resistant to infection by a number of viruses and, importantly, confer this resistance to nonplacental recipient cells by exosome-mediated delivery of specific microRNAs (miRNAs). We show that miRNA members of the chromosome 19 miRNA cluster, which are almost exclusively expressed in the human placenta, are packaged within trophoblast-derived exosomes and attenuate viral replication in recipient cells by the induction of autophagy. Together, our findings identify an unprecedented paracrine and/or systemic function of placental trophoblasts that uses exosome-mediated transfer of a unique set of placental-specific effector miRNAs to directly communicate with placental or maternal target cells and regulate their immunity to viral infections.

The levels of hypoxia-regulated microRNAs in plasma of pregnant women with fetal growth restriction

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at the post-transcriptional level. While mostly intracellular, a portion of cellular miRNAs is released to the circulation and their level in the plasma is altered in certain pathological conditions such as cancer, and also during pregnancy. We examined the circulating levels of a set of trophoblastic miRNAs, which we recently found to be regulated by hypoxia, in the plasma of pregnant women with fetal growth restriction (FGR). Pregnancy was associated with increased plasma levels of several placenta-specific miRNAs, compared to non-pregnant controls. Among pregnant women, the overall levels of miRNA species that we analyzed were increased by 1.84-fold (p < or = 0.01) in plasma of women with pregnancies complicated by FGR, but decreased in FGR placentas by 24% (p < or = 0.01) compared to values from uncomplicated pregnancies. Together, our results show that plasma concentration of miRNAs is regulated in pregnancy, and that FGR is associated with increased circulating miRNA levels, highlighting the need to explore plasma miRNAs as potential biomarkers for placental diseases.

MiR-205 silences MED1 in hypoxic primary human trophoblasts

Acting through degradation of target mRNA or inhibition of translation, microRNAs (miRNAs) regulate development, differentiation, and cellular response to diverse cues. We analyzed changes in miRNA expression in human placental trophoblasts exposed to hypoxia, which may result from hypoperfusion and placental injury. Using an miRNA microarray screen, confirmed by Northern blot analysis, we defined a set of seven miRNAs (miR‐93, miR‐205, miR‐224, miR‐335, miR‐424, miR‐451, and miR‐491) that are differentially regulated in primary trophoblasts exposed to hypoxia. We combined in silico prediction of miRNA targets with gene expression profiling data to identify a series of potential targets for the miRNAs, which were further analyzed using luciferase reporter assays. Among experimentally confirmed targets, we found that the transcriptional coactivator MED1, which plays an important role in placental development, is a target for miR‐205. Using gain‐ and loss‐of‐function assays, we confirmed that miR‐205 interacts with a specific target in the 3′‐UTR sequence of MED1 and silences MED1 expression in human trophoblasts exposed to hypoxia, suggesting that miR‐205 plays a role in trophoblast injury.—Mouillet, J.‐F., Chu, T., Nelson, D. M., Mishima, T., Sadovsky, Y. MiR‐205 silences MED1 in hypoxic primary human trophoblasts. FASEB J. 24, 2030–2039 (2010). www.fasebj.org

Review: placenta-specific microRNAs in exosomes - good things come in nano-packages.

MicroRNAs (miRNAs) are small noncoding RNA gene products that commonly regulate mRNA expression by repression of translation and/or transcript decay. Whereas common and unique types of miRNAs are expressed by the placenta during pregnancy, the functions of most placental miRNA species are unknown. In addition to their intracellular silencing function, miRNAs are also released to the extracellular space and systemic circulation, where they can potentially target cells to regulate mRNA and protein expression, providing a non-hormonal means of intercellular communication that contributes to tissue homeostasis and disease pathophysiology. This review centers on extracellular miRNAs that originate in trophoblasts and that could mediate crosstalk between the feto-placental unit and the mother during pregnancy. We specifically detail the function of miRNAs from the primate-specific chromosome 19 miRNA cluster. These miRNAs are highly expressed in human placentas and in the serum of pregnant women. They are also packaged into extracellular vesicles of diverse sizes, including exosomes, and endow non-trophoblastic cells with resistance to a variety of viruses.

A Novel Domain within the DEAD-Box Protein DP103 Is Essential for Transcriptional Repression and Helicase Activity

ABSTRACT Members of the DEAD-box family of helicases, distinguished by a core characteristic sequence of Asp-Glu-Ala-Asp, are expressed in a wide range of prokaryotes and eukaryotes and exhibit diverse cellular functions, including DNA transcription, recombination and repair, RNA processing, translation, and posttranslational regulation. Although ubiquitous, the function of most DEAD-box proteins is unknown. We and others have recently cloned DP103, which harbors conserved DEAD-box, helicase, and ATPase domains in its N terminus. DP103 (also termed Gemin3 and DDX20) interacts with SF-1, SMN, EBNA2, and EBNA3C in mammalian cells. Here we demonstrate that a discrete domain within the nonconserved C-terminal region of DP103 directly interacts with SF-1. This domain exhibits an autonomous repression function and is necessary and sufficient for repressing the transcriptional activity of SF-1. Furthermore, intact DP103 exhibits helicase activity. Importantly, the C-terminal domain is obligatory but not sufficient for this unwinding activity of DP103. Together, our results support a novel paradigm for transcriptional repression and demonstrate the bifunctional role of the C-terminal domain of DP103.

p300 Regulates the Synergy of Steroidogenic Factor-1 and Early Growth Response-1 in Activating Luteinizing Hormone-β Subunit Gene

Tight regulation of luteinizing hormone-β subunit (LHβ) expression is critical for differentiation and maturation of mammalian sexual organs and reproductive function. Two transcription factors, steroidogenic factor-1 (SF-1) and early growth response-1 (Egr-1), play a central role in activating LHβ promoter, and the synergy between these two factors is essential in mediating gonadotropin-releasing hormone stimulation of LHβ promoter. Here we demonstrate that the transcriptional co-activator p300 regulates this synergy. Overexpression of p300 results in strong stimulation of LHβ promoter but only in the presence of both SF-1 and Egr-1, and not in the presence of other Egr proteins. Mutation of the binding sites for either SF-1 or Egr-1 completely abolishes the synergy between these two factors, as well as the influence of p300. Importantly, LHβ promoter is precipitated using p300 antibodies in a chromatin immunoprecipitation assay with LβT2 gonadotropes, and this effect is enhanced by gonadotropin-releasing hormone. The influence of p300 on LHβ promoter is potentiated by steroid receptor co-activator, as well as by E1A proteins, and attenuated by Smad nuclear interacting protein 1. Taken together, these results suggest that p300 is recruited to LHβ promoter where it coordinates the functional synergy between SF-1 and Egr-1.

MicroRNAs in placental health and disease.

MicroRNAs (miRNAs) constitute a large family of small noncoding RNAs that are encoded by the genomes of most organisms. They regulate gene expression through posttranscriptional mechanisms to attenuate protein output in various genetic networks. The discovery of miRNAs has transformed our understanding of gene regulation and sparked intense efforts intended to harness their potential as diagnostic markers and therapeutic tools. Over the last decade, a flurry of studies has shed light on placental miRNAs but has also raised many questions regarding the scope of their biologic action. Moreover, the recognition that miRNAs of placental origin are released continually in the maternal circulation throughout pregnancy suggested that circulating miRNAs might serve as biomarkers for placental function during pregnancy. Although this generated much enthusiasm, recently recognized challenges have delayed the application of miRNA-based biomarkers and therapeutics in clinical practice. In this review, we summarize key findings in the field and discuss current knowledge related to miRNAs in the context of placental biology.

The Unique Expression and Function of miR-424 in Human Placental Trophoblasts

ABSTRACT Placental hypoperfusion causes cellular hypoxia and is associated with fetal growth restriction and preeclampsia. In response to hypoxia, the repertoire of genes expressed in placental trophoblasts changes, which influences key cellular processes such as differentiation and fusion. Diverse miRNAs were recently found to modulate the cellular response to hypoxia. Here we show that miR-424, which was previously shown to be upregulated by hypoxia in nontrophoblastic cell types, is uniquely downregulated in primary human trophoblasts by hypoxia or chemicals known to hinder cell differentiation. We also identify FGFR1 as a direct target of miR-424 in human trophoblasts. This effect is unique to miR-424 and is not seen with other members of this miRNA family that are expressed in trophoblasts, such as miR-15 and miR-16. Our findings establish a unique role for miR-424 during differentiation of human trophoblasts.

The role of trophoblastic microRNAs in placental viral infection

During the past decade, various types of small non-coding RNAs were found to be expressed in all kingdoms and phyla of life. Intense research efforts have begun to shed light on their biological functions, although much remains to be determined in order to fully characterize their scope of biological action. Typically, small RNAs provide sequence specificity to a protein complex that is driven to silence a long target RNA. MicroRNAs (miRNAs) are small RNAs that are coded in the genome of most eukaryotes, and contribute to the cellular identity by regulating cell-specific gene networks by translational repression or degradation of mRNA. These effects commonly fine-tune gene expression associated with developmental or environmental cues. Different cell types can be characterized by their distinctive cellular miRNA landscape. The human placenta expresses a unique set of miRNAs, a high proportion of which is derived from a large cluster located on chromosome 19, (termed chromosome 19 miRNA cluster, or C19MC). Interestingly, a fraction of these placenta-enriched miRNAs are released to the extracellular environment through exosomes that were recently found to induce an antiviral immunity. In this review, we explore relevant placental viral infections and discuss the antiviral role of exosome-packaged placental C19MC miRNAs in this context.

The Assembly of miRNA-mRNA-protein Regulatory Networks Using High-throughput Expression Data

MOTIVATION Inference of gene regulatory networks from high throughput measurement of gene and protein expression is particularly attractive because it allows the simultaneous discovery of interactive molecular signals for numerous genes and proteins at a relatively low cost. RESULTS We developed two score-based local causal learning algorithms that utilized the Markov blanket search to identify direct regulators of target mRNAs and proteins. These two algorithms were specifically designed for integrated high throughput RNA and protein data. Simulation study showed that these algorithms outperformed other state-of-the-art gene regulatory network learning algorithms. We also generated integrated miRNA, mRNA, and protein expression data based on high throughput analysis of primary trophoblasts, derived from term human placenta and cultured under standard or hypoxic conditions. We applied the new algorithms to these data and identified gene regulatory networks for a set of trophoblastic proteins found to be differentially expressed under the specified culture conditions.