Yoel Sadovsky

Luteinizing Hormone Deficiency and Female Infertility in Mice Lacking the Transcription Factor NGFI-A (Egr-1)

The immediate-early transcription factor NGFI-A (also called Egr-1, zif/268, or Krox-24) is thought to couple extracellular signals to changes in gene expression. Although activins and inhibins regulate follicle-stimulating hormone (FSH) synthesis, no factor has been identified that exclusively regulates luteinizing hormone (LH) synthesis. An analysis of NGFI-A-deficient mice derived from embryonic stem cells demonstrated female infertility that was secondary to LH-β deficiency. Ovariectomy led to increased amounts of FSH-β but not LH-β messenger RNA, which suggested a pituitary defect. A conserved, canonical NGFI-A site in the LH-β promoter was required for synergistic activation by NGFI-A and steroidogenic factor-1 (SF-1). NGFI-A apparently influences female reproductive capacity through its regulation of LH-β transcription.

Nuclear Receptor DAX-1 Recruits Nuclear Receptor Corepressor N-CoR to Steroidogenic Factor 1

ABSTRACT The orphan nuclear receptor steroidogenic factor 1 (SF-1) is a critical developmental regulator in the urogenital ridge, because mice targeted for disruption of the SF-1 gene lack adrenal glands and gonads. SF-1 was recently shown to interact with DAX-1, another orphan receptor whose tissue distribution overlaps that of SF-1. Naturally occurring loss-of-function mutations of the DAX-1 gene cause the human disorder X-linked adrenal hypoplasia congenita (AHC), which resembles the phenotype of SF-1-deficient mice. Paradoxically, however, DAX-1 represses the transcriptional activity of SF-1, and AHC mutants of DAX-1 lose repression function. To further investigate these findings, we characterized the interaction between SF-1 and DAX-1 and found that their interaction indeed occurs through a repressive domain within the carboxy terminus of SF-1. Furthermore, we demonstrate that DAX-1 recruits the nuclear receptor corepressor N-CoR to SF-1, whereas naturally occurring AHC mutations of DAX-1 permit the SF-1–DAX-1 interaction, but markedly diminish corepressor recruitment. Finally, the interaction between DAX-1 and N-CoR shares similarities with that of the nuclear receptor RevErb and N-CoR, because the related corepressor SMRT was not efficiently recruited by DAX-1. Therefore, DAX-1 can serve as an adapter molecule that recruits nuclear receptor corepressors to DNA-bound nuclear receptors like SF-1, thereby extending the range of corepressor action.

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.

Type III Interferons Produced by Human Placental Trophoblasts Confer Protection against Zika Virus Infection

During mammalian pregnancy, the placenta acts as a barrier between the maternal and fetal compartments. The recently observed association between Zika virus (ZIKV) infection during human pregnancy and fetal microcephaly and other anomalies suggests that ZIKV may bypass the placenta to reach the fetus. This led us to investigate ZIKV infection of primary human trophoblasts (PHTs), which are the barrier cells of the placenta. We discovered that PHT cells from full-term placentas are refractory to ZIKV infection. In addition, medium from uninfected PHT cells protects non-placental cells from ZIKV infection. PHT cells constitutively release the type III interferon (IFN) IFNλ1, which functions in both a paracrine and autocrine manner to protect trophoblast and non-trophoblast cells from ZIKV infection. Our data suggest that for ZIKV to access the fetal compartment, it must evade restriction by trophoblast-derived IFNλ1 and other trophoblast-specific antiviral factors and/or use alternative strategies to cross the placental barrier.

Activation of Luteinizing Hormone β Gene by Gonadotropin-releasing Hormone Requires the Synergy of Early Growth Response-1 and Steroidogenic Factor-1

We have previously shown that early growth response (Egr) 1-deficient mice exhibit female infertility, reflecting a luteinizing hormone (LH) β deficiency. Egr-1 activates the LHβ gene in vitro through synergy with steroidogenic factor-1 (SF-1), a protein required for gonadotrope function. To test if this synergy is essential for gonadotropin-releasing hormone (GnRH) stimulation of LHβ, we examined the activity of the LHβ promoter in the gonadotrope cell line LβT2. GnRH markedly stimulated the LHβ promoter (15-fold). Mutation of either Egr-1 or SF-1 elements within the LHβ promoter attenuated this stimulation, whereas mutation of both promoter elements abrogated GnRH induction of the LHβ promoter. Furthermore, GnRH stimulated Egr-1 but not SF-1 expression in LβT2 cells. Importantly, overexpression of Egr-1 alone was sufficient to enhance LHβ expression. Although other Egr proteins are expressed in LβT2 cells and are capable of interacting with SF-1, GnRH stimulation of Egr-1 was the most robust. We also found that the nuclear receptor DAX-1, a repressor of SF-1 activity, reduced Egr-1–SF-1 synergy and diminished GnRH stimulation of the LHβ promoter. We conclude that the synergy between Egr-1 and SF-1 is essential for GnRH stimulation of the LHβ gene and plays a central role in the dynamic regulation of LHβ expression.

Prenatal Maternal Stress and Cord Blood Innate and Adaptive Cytokine Responses in an Inner-City Cohort

RATIONALE Stress-elicited disruption of immunity begins in utero. OBJECTIVES Associations among prenatal maternal stress and cord blood mononuclear cell (CBMC) cytokine responses were prospectively examined in the Urban Environment and Childhood Asthma Study (n = 557 families). METHODS Prenatal maternal stress included financial hardship, difficult life circumstances, community violence, and neighborhood/block and housing conditions. Factor analysis produced latent variables representing three contexts: individual stressors and ecological-level strains (housing problems and neighborhood problems), which were combined to create a composite cumulative stress indicator. CBMCs were incubated with innate (lipopolysaccharide, polyinosinic-polycytidylic acid, cytosine-phosphate-guanine dinucleotides, peptidoglycan) and adaptive (tetanus, dust mite, cockroach) stimuli, respiratory syncytial virus, phytohemagglutinin, or medium alone. Cytokines were measured using multiplex ELISAs. Using linear regression, associations among increasing cumulative stress and cytokine responses were examined, adjusting for sociodemographic factors, parity, season of birth, maternal asthma and steroid use, and potential pathway variables (prenatal smoking, birth weight for gestational age). MEASUREMENTS AND MAIN RESULTS Mothers were primarily minorities (Black [71%], Latino [19%]) with an income less than

Adrenocortical Function and Regulation of the Steroid 21-Hydroxylase Gene in NGFI-B-Deficient Mice

15,000 (69%). Mothers with the highest cumulative stress were older and more likely to have asthma and deliver lower birth weight infants. Higher prenatal stress was related to increased IL-8 production after microbial (CpG, PIC, peptidoglycan) stimuli and increased tumor necrosis factor-alpha to microbial stimuli (CpG, PIC). In the adaptive panel, higher stress was associated with increased IL-13 after dust mite stimulation and reduced phytohemagglutinin-induced IFN-gamma. CONCLUSIONS Prenatal stress was associated with altered innate and adaptive immune responses in CBMCs. Stress-induced perinatal immunomodulation may impact the expression of allergic disease in these children.

Estrogen modulates estrogen receptor alpha and beta expression, osteogenic activity, and apoptosis in mesenchymal stem cells (MSCs) of osteoporotic mice.

The immediate-early gene NGFI-B encodes an orphan nuclear receptor that binds DNA as a monomer and activates transcription through a canonical response element (NBRE). NGFI-B is expressed under basal conditions and in response to external stimuli in many mammalian tissues. In particular, NGFI-B expression is dramatically elevated in the adrenal cortex in response to stress and in Y1 adrenocortical cells in response to adrenocorticotropin. NGFI-B activates transcription through an NBRE of the gene encoding 21-hydroxylase (P450c21) in Y1 cells. Steroidogenic factor 1 (SF-1), a homolog of NGFI-B, also activates the P450c21 promoter. To examine the influence of these factors on P450c21 expression in vivo and the function of the hypothalamic-pituitary-adrenocortical axis as a whole, we generated NGFI-B (-/-) mice. These mice thrive and reproduce normally and maintain normal basal adrenocorticotropin, corticosterone, and P450c21 mRNA levels. In response to increases in adrenocorticotropin, NGFI-B (-/-) and wild-type mice demonstrated equivalent increases in serum corticosterone levels. Furthermore, and in contrast to in vitro results, no increases in P450c21 mRNA levels were observed in response to increases in adrenocorticotropin in NGFI-B (-/-) or wild-type mice. While SF-1 mRNA levels were not increased with increased steroidogenic demand, adrenal expression of Nurr1, a close homolog of NGFI-B, was induced to a greater extent by lipopolysaccharide in NGFI-B (-/-) mice than in wild-type mice. Finally, when the administration of dexamethasone for suppression was stopped, P450c21 mRNA and serum corticosterone levels recovered at the same rate in wild-type and NGFI-B (-/-) mice. Thus, while NGFI-B appears poised to affect the structure and function of the adrenal gland, the gland functions normally in its absence, suggesting that other factors, including Nurr1 and SF-1, are sufficient to drive P450c21 expression in mice and maintain normal steroidogenesis.

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

In the mouse, ovariectomy (OVX) leads to significant reductions in cancellous bone volume while estrogen (17β‐estradiol, E2) replacement not only prevents bone loss but can increase bone formation. As the E2‐dependent increase in bone formation would require the proliferation and differentiation of osteoblast precursors, we hypothesized that E2 regulates mesenchymal stem cells (MSCs) activity in mouse bone marrow. We therefore investigated proliferation, differentiation, apoptosis, and estrogen receptor (ER) α and β expression of primary culture MSCs isolated from OVX and sham‐operated mice. MSCs, treated in vitro with 10−7 M E2, displayed a significant increase in ERα mRNA and protein expression as well as alkaline phosphatase (ALP) activity and proliferation rate. In contrast, E2 treatment resulted in a decrease in ERβ mRNA and protein expression as well as apoptosis in both OVX and sham mice. E2 up‐regulated the mRNA expression of osteogenic genes for ALP, collagen I, TGF‐β1, BMP‐2, and cbfa1 in MSCs. In a comparison of the relative mRNA expression and protein levels for two ER isoforms, ERα was the predominant form expressed in MSCs obtained from both OVX and sham‐operated mice. Cumulatively, these results indicate that estrogen in vitro directly augments the proliferation and differentiation, ERα expression, osteogenic gene expression and, inhibits apoptosis and ERβ expression in MSCs obtained from OVX and sham‐operated mice. Co‐expression of ERα, but not ERβ, and osteogenic differentiation markers might indicate that ERα function as an activator and ERβ function as a repressor in the osteogenic differentiation in MSCs. These results suggest that mouse MSCs are anabolic targets of estrogen action, via ERα activation. J. Cell. Biochem. Suppl. 36: 144–155, 2001.

Hypoxia limits differentiation and up-regulates expression and activity of prostaglandin H synthase 2 in cultured trophoblast from term human placenta ☆ ☆☆ ★

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.