Extraembryonic heparin-binding epidermal growth factor-like growth factor deficiency compromises placentation in mice.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) is expressed in the embryo and uterus at the implantation site, stimulating trophoblast invasive activity essential for placentation. The effect of extraembryonic HBEGF deficiency on placental development was investigated by breeding mice heterozygous for the Hbegf null mutation. On gestation day 13.5, the average placental weights of the wild-type (Hbegf+/+) and heterozygous (Hbegf+/-) mice were approximately 76 and 77 mg, respectively, as opposed to reduced average placental weights of approximately 61 mg in homozygous null (Hbgef-/-) females. In contrast, fetal weights were not significantly affected by genotype. HBEGF immunostaining in placental sections was Hbegf gene dosage-dependent, while expression of other EGF family members was comparable in Hbegf+/+ and Hbegf-/- placentas. Histological analysis revealed no apparent differences in trophoblast giant cells, but the spongiotrophoblast region was reduced compared to labyrinth (P < 0.05) in Hbegf null placentas. While no differences in cell apoptosis were noted, proliferation as assessed by nuclear Ki67 staining was elevated in the labyrinth and decreased in the spongiotrophoblast region of Hbegf-/- placentas. Labyrinth morphology appeared disrupted in Hbegf -/- placentas stained with laminin, a marker for capillary basement membrane, and the capillary density was reduced. Immunohistochemical staining revealed reduced vascular endothelial growth factor (VEGF) levels in both spongiotrophoblast and labyrinth (P < 0.01) regions of Hbegf-/- placentas. In vitro, HBEGF supplementation increases the expression of VEGF in a human trophoblast cell line. These findings suggest that trophoblast HBEGF promotes placental capillary formation by inducing VEGF in the developing placenta of mice.

Polycomb repressive complex 1 controls uterine decidualization.

Uterine stromal cell decidualization is an essential part of the reproductive process. Decidual tissue development requires a highly regulated control of the extracellular tissue remodeling; however the mechanism of this regulation remains unknown. Through systematic expression studies, we detected that Cbx4/2, Rybp, and Ring1B [components of polycomb repressive complex 1 (PRC1)] are predominantly utilized in antimesometrial decidualization with polyploidy. Immunofluorescence analyses revealed that PRC1 members are co-localized with its functional histone modifier H2AK119ub1 (mono ubiquitination of histone-H2A at lysine-119) in polyploid cell. A potent small-molecule inhibitor of Ring1A/B E3-ubiquitin ligase or siRNA-mediated suppression of Cbx4 caused inhibition of H2AK119ub1, in conjunction with perturbation of decidualization and polyploidy development, suggesting a role for Cbx4/Ring1B-containing PRC1 in these processes. Analyses of genetic signatures by RNA-seq studies showed that the inhibition of PRC1 function affects 238 genes (154 up and 84 down) during decidualization. Functional enrichment analyses identified that about 38% genes primarily involved in extracellular processes are specifically targeted by PRC1. Furthermore, ~15% of upregulated genes exhibited a significant overlap with the upregulated Bmp2 null-induced genes in mice. Overall, Cbx4/Ring1B-containing PRC1 controls decidualization via regulation of extracellular gene remodeling functions and sheds new insights into underlying molecular mechanism(s) through transcriptional repression regulation.

Control of regional decidualization in implantation: Role of FoxM1 downstream of Hoxa10 and cyclin D3.

Appropriate regulation of regional uterine stromal cell decidualization in implantation, at the mesometrial triangle and secondary decidual zone (SDZ) locations, is critical for successful pregnancy, although the regulatory mechanisms remain poorly understood. In this regard, the available animal models that would specifically allow mechanistic analysis of site-specific decidualization are strikingly limited. Our study found that heightened expression of FoxM1, a Forkhead box transcription factor, is regulated during decidualization, and its conditional deletion in mice reveals failure of implantation with regional decidualization defects such as a much smaller mesometrial decidua with enlarged SDZ. Analysis of cell cycle progression during decidualization both in vivo and in vitro demonstrates that the loss of FoxM1 elicits diploid cell deficiency with enhanced arrests prior to mitosis and concomitant upregulation of polyploidy. We further showed that Hoxa10 and cyclin D3, two decidual markers, control transcriptional regulation and intra-nuclear protein translocation of FoxM1 in polyploid cells, respectively. Overall, we suggest that proper regional decidualization and polyploidy development requires FoxM1 signaling downstream of Hoxa10 and cyclin D3.

Synthesis and evaluation of NS5A inhibitors containing diverse heteroaromatic cores.

Inhibitors of the HCV NS5A nonstructural protein are showing promising clinical potential in the treatment of hepatitis C when used in combination with other direct-acting antiviral agents. Current NS5A clinical candidates such as daclatasvir, ledipasvir, and ombitasvir share a common pharmacophore that features a pair of (S)-methoxycarbonylvaline capped pyrrolidines linked to various cores by amides, imidazoles and/or benzimidazoles. In this Letter, we describe the evaluation of NS5A inhibitors which contain alternative heteroaromatic replacements for these amide mimetics. The SAR knowledge gleaned in the optimization of scaffolds containing benzoxazoles was parlayed toward the identification of potent NS5A inhibitors containing other heteroaromatic replacements such as indoles and imidazopyridines.

Benzimidazole-containing HCV NS5A inhibitors: effect of 4-substituted pyrrolidines in balancing genotype 1a and 1b potency.

The treatment of HCV with highly efficacious, well-tolerated, interferon-free regimens is a compelling clinical goal. Trials employing combinations of direct-acting antivirals that include NS5A inhibitors have shown significant promise in meeting this challenge. Herein, we describe our efforts to identify inhibitors of NS5A and report on the discovery of benzimidazole-containing analogs with subnanomolar potency against genotype 1a and 1b replicons. Our SAR exploration of 4-substituted pyrrolidines revealed that the subtle inclusion of a 4-methyl group could profoundly increase genotype 1a potency in multiple scaffold classes.

Discovery of thienoimidazole-based HCV NS5A inhibitors. Part 1: C2-symmetric inhibitors with diyne and biphenyl linkers.

The discovery of C2-symmetric bis-thienoimidazoles HCV NS5A inhibitors is herein reported. Two straightforward approaches to access the requisite diyne and biphenyl linker moieties are described. This study revealed the paramount importance of the aromatic character of the linker to achieve high genotype 1a potency.

Discovery of thienoimidazole-based HCV NS5A inhibitors. Part 2: non-symmetric inhibitors with potent activity against genotype 1a and 1b.

The discovery of non-symmetric thienoimidazole-containing HCV NS5A inhibitors is described. The inhibitors herein reported display high potencies against both genotype 1a and 1b. In this follow-up manuscript, we discuss the importance of the linker aromaticity to achieve high potency, particularly against genotype 1a.

Estrogen mediated epithelial proliferation in the uterus is directed by stromal Fgf10 and Bmp8a.

To define endometrial stromal-derived paracrine mediators that participate in estradiol-17ß (E2)-induced epithelial proliferation, microarray analysis of gene expression was carried out in mouse uterine epithelial-stromal co-culture systems under the condition of E2 or vehicle (control). Our results demonstrated gene alteration by E2: in epithelial cells, we found up-regulation of 119 genes and down-regulation of 28 genes, while in stroma cells we found up-regulation of 144 genes and down-regulation of 184 genes. A functional enrichment analysis of the upregulated epithelial genes implicated them for proliferation, while upregulated stromal genes were associated with extracellular functions. Quantitative RT-PCR and in situ hybridization results confirmed differential gene expression in both cell cultures and ovariectomized uteri after the above treatments. Based on our identification of stromal secretory factors, we found evidence that suppression by siRNA specifically for Bmp8a and/or Fgf10 in the stromal layer caused significant inhibition of proliferation by E2 in the co-culture system, suggesting Bmp8a and Fgf10 act as paracrine mediators during E2-dependent control of uterine proliferation. The localization of receptors and receptor activation signaling in epithelial cells in both the co-culture system and uteri was consistent with their involvement in ligand-receptor signaling. Interestingly, loss of Bmp8a or Fgf10 also caused abrogation of E2-regulated epithelial receptor signaling in co-culture systems, suggesting that stroma-derived Fgf10 and Bmp8a are responsible for epithelial communication. Overall, stromal Fgf10 and Bmp8a serve as potential paracrine factors for E2-dependent regulation of epithelial proliferation in the uterus.

Epigenetic regulations through DNA methylation and hydroxymethylation: clues for early pregnancy in decidualization.

DNA methylation at cytosines is an important epigenetic modification that participates in gene expression regulation without changing the original DNA sequence. With the rapid progress of high-throughput sequencing techniques, whole-genome distribution of methylated cytosines and their regulatory mechanism have been revealed gradually. This has allowed the uncovering of the critical roles played by DNA methylation in the maintenance of cell pluripotency, determination of cell fate during development, and in diverse diseases. Recently, rediscovery of 5-hydroxymethylcytosine, and other types of modification on DNA, have uncovered more dynamic aspects of cell methylome regulation. The interaction of DNA methylation and other epigenetic changes remodel the chromatin structure and determine the state of gene transcription, not only permanently, but also transiently under certain stimuli. The uterus is a reproductive organ that experiences dramatic hormone stimulated changes during the estrous cycle and pregnancy, and thus provides us with a unique model for studying the dynamic regulation of epigenetic modifications. In this article, we review the current findings on the roles of genomic DNA methylation and hydroxymethylation in the regulation of gene expression, and discuss the progress of studies for these epigenetic changes in the uterus during implantation and decidualization.

Discovery of Thienoimidazole-Based HCV NS5A Genotype 1a and 1b Inhibitors.

The discovery of potent thienoimidazole-based HCV NS5A inhibitors is herein reported. A novel method to access the thienoimidazole [5,5]-bicyclic system is disclosed. This method gave access to a common key intermediate (6) that was engaged in Suzuki or Sonogashira reactions with coupling partners bearing different linkers. A detailed study of the structure-activity relationship (SAR) of the linkers revealed that aromatic linkers with linear topologies are required to achieve high potency for both 1a and 1b HCV genotypes. Compound 20, with a para-phenyl linker, was identified as a potential lead displaying potencies of 17 and 8 pM against genotype 1a and 1b replicons, respectively.

Epigenetic changes through DNA methylation contribute to uterine stromal cell decidualization.

Embryo-uterine interaction during early pregnancy critically depends on the coordinated expression of numerous genes at the site of implantation. The epigenetic mechanism through DNA methylation (DNM) plays a major role in the control of gene expression, although this regulatory event remains unknown in uterine implantation sites. Our analysis revealed the presence of DNA methyltransferase 1 (Dnmt1) in mouse endometrial cells on the receptive d 4 of pregnancy and early postattachment (d 5) phase, whereas Dnmt3a had lower abundant expression. Both Dnmt1 and Dnmt3a were coordinately expressed in decidual cells on d 6-8. 5-Methycytosine showed a similar expression pattern to that of Dnmt1. The preimplantation inhibition of DNM by 5-aza-2'-deoxycytodine was not antagonistic for embryonic attachment, although endometrial stromal cell proliferation at the site of implantation was down-regulated, indicating a disturbance with the postattachment decidualization event. Indeed, the peri- or postimplantation inhibition of DNM caused significant abrogation of decidualization, with concomitant loss of embryos. We next identified decidual genes undergoing alteration of DNM using methylation-sensitive restriction fingerprinting. One such gene, Chromobox homolog 4, an epigenetic regulator in the polycomb group protein family, exhibited hypomethylation in promoter DNA and increased expression with the onset of decidualization. Furthermore, inhibition of DNM resulted in enhanced expression of hypermethylated genes (Bcl3 and Slc16a3) in the decidual bed as compared with control, indicating aberration of gene expression may be associated with DNM-inhibition-induced decidual perturbation. Overall, these results suggest that uterine DNM plays a major role for successful decidualization and embryo development during early pregnancy.

Overexpression of cyclin D3 improves decidualization defects in Hoxa-10(-/-) mice.

Uterine decidualization, a crucial process for implantation, is a tightly regulated process encompassing proliferation, differentiation, and polyploidization of uterine stromal cells. Hoxa (Homeobox A)-10, a homeobox transcription factor, is highly expressed in decidualizing stromal cells. Targeted gene deletion experiments have demonstrated marked infertility resulting from severely compromised decidualization in Hoxa-10(-/-) mice. However, the underlying mechanism by which Hoxa-10 regulates stromal cell differentiation remains poorly understood. Cyclin D3, a G(1) phase cell-cycle regulatory protein involved in stromal cell proliferation and decidualization, is significantly reduced in Hoxa-10(-/-) mice. The expression of cyclin D3 in the pregnant mouse uterus parallels stromal cell decidualization. Here, we show that adenovirus-driven cyclin D3 replacement in Hoxa-10(-/-) mice improves stromal cell decidualization. To address our question of whether cyclin D3 replacement in Hoxa-10(-/-) mice can improve decidualization, both in vitro and in vivo studies were completed after the addition of cyclin D3 or empty (control) viral vectors. Immunostaining demonstrated increased proliferation and decidualization in both in vitro and in vivo studies, and in situ hybridization confirmed increased expression of decidualization markers in vivo. Placentation was demonstrated as well in vivo in the cyclin D3-replaced animals. However, fertility was not restored in Hoxa-10(-/-) mice after d 10 of pregnancy. Finally, we identified several downstream targets of cyclin D3 during decidualization in vitro via proteomics experiments, and these were confirmed using in situ hybridization in vivo. Collectively, these results demonstrate that cyclin D3 expression influences a host of genes involved in decidualization and can improve decidualization in Hoxa-10(-/-) mice.

Developmental regulation of decidual cell polyploidy at the site of implantation.

Polyploidy has been reported in several animal cells, as well as within humans; however the mechanism of developmental regulation of this process remains poorly understood. Polyploidy occurs in normal biologic processes as well as in pathologic states. Decidual polyploid cells are terminally differentiated cells with a critical role in continued uterine development during embryo implantation and growth. Here we review the mechanisms involved in polyploidy cell formation in normal developmental processes, with focus on known regulatory aspects in decidual cells.

Nucleolar Sik-similar protein (Sik-SP) is required for the maintenance of uterine estrogen signaling mechanism via ERα.

Sik-similar protein (Sik-SP), a small nucleolar ribonucleoprotein, has been shown to be primarily involved in ribosome biogenesis. However, its role in the hormone-directed nuclear receptor signaling is largely unknown. Here, we provide novel evidence that Sik-SP is required for appropriate regulation of estrogen receptor (ER)α-mediated estradiol-17ß (E2)-dependent uterine physiologic responses in mice. Studies by Western blotting using the newly developed antibodies for Sik-SP showed that this protein is up-regulated in both the ovariectomized wild-type and ERα null uteri by E2. Immunohistochemical analyses in uterine sections showed that this protein is induced in the epithelial and stromal cells. Coimmunoprecipitation studies revealed that E2 directs molecular interaction between Sik-SP and ERα. Furthermore, gel-mobility shift and chromatin immunoprecipitation analyses provided evidence that Sik-SP is recruited with ERα to estrogen-responsive uterine gene promoters. Overexpression of Sik-SP in vitro demonstrated a role for Sik-SP in cellular growth and viability. In a primary uterine epithelial-stromal coculture system, E2 exhibited early induction of Sik-SP in both the epithelial and stromal cells. Interestingly, suppression of Sik-SP in this coculture model, for the stromal but not epithelial cells, caused perturbation of E2-dependent proliferation in the epithelial cell layer. Similarly, in vivo uterine suppression of Sik-SP also caused inhibition of epithelial cell proliferation and aberrant prolongation of water imbibition in the late phase by E2. Finally, studies showed that Sik-SP is physiologically important during the onset of implantation by E2. In conclusion, Sik-SP, an early E2-responsive nucleolar protein, is necessary to induce E2-dependent ERα-mediated appropriate physiologic responses in the uterus.

Decidual cell polyploidization necessitates mitochondrial activity.

Cellular polyploidy has been widely reported in nature, yet its developmental mechanism and function remain poorly understood. In the present study, to better define the aspects of decidual cell polyploidy, we isolated pure polyploid and non-polyploid decidual cell populations from the in vivo decidual bed. Three independent RNA pools prepared for each population were then subjected to the Affymetrix gene chip analysis for the whole mouse genome transcripts. Our data revealed up-regulation of 1015 genes and down-regulation of 1207 genes in the polyploid populations, as compared to the non-polyploid group. Comparative RT-PCR and in situ hybridization results indeed confirmed differential expressional regulation of several genes between the two populations. Based on functional enrichment analyses, up-regulated polyploidy genes appeared to implicate several functions, which primarily include cell/nuclear division, ATP binding, metabolic process, and mitochondrial activity, whereas that of down-regulated genes primarily included apoptosis and immune processes. Further analyses of genes that are related to mitochondria and bi-nucleation showed differential and regional expression within the decidual bed, consistent with the pattern of polyploidy. Consistently, studies revealed a marked induction of mitochondrial mass and ATP production in polyploid cells. The inhibition of mitochondrial activity by various pharmacological inhibitors, as well as by gene-specific targeting using siRNA-mediated technology showed a dramatic attenuation of polyploidy and bi-nucleation development during in vitro stromal cell decidualization, suggesting mitochondria play a major role in positive regulation of decidual cell polyploidization. Collectively, analyses of unique polyploidy markers and molecular signaling networks may be useful to further characterize functional aspects of decidual cell polyploidy at the site of implantation.

Mouse primary uterine cell coculture system revisited: ovarian hormones mimic the aspects of in vivo uterine cell proliferation.

Previously, the uterine epithelial-stromal coculture system had limited success mimicking in vivo ovarian hormone-dependent cell-specific proliferation. Here, we established a mouse primary uterine coculture system, in which cells collected in pseudopregnancy specifically on d 4 are conducive to supporting hormone-induced cell-specific proliferation. When two cell types are placed in coculture without direct contact via cell culture inserts (nonadjacent), as opposed to with contact (adjacent), epithelial cells exhibit significant proliferation by estradiol-17ß (E2), whereas progesterone in combination with E2 caused inhibition of epithelial cell proliferation and a major shift in proliferation from epithelial to stromal cells. Epithelial cell integrity, with respect to E-cadherin expression, persisted in nonadjacent, but not adjacent, conditions. In subsequent studies of nonadjacent cocultures, localization of estrogen receptor (ER)α and progesterone receptor (PR), but not ERß, appeared to be abundant, presumably indicating that specific ER or PR coregulator expression might be responsible for this difference. Consistently, an agonist of ERα, but not ERß, was supportive of proliferation, and antagonists of ER or PR totally eliminated cell-specific proliferation by hormones. RT-PCR analyses also revealed that hormone-responsive genes primarily exhibit appropriate regulation. Finally, suppression of immunoglobulin heavy chain binding protein, a critical regulator of ERα signaling, in epithelial and/or stromal cells caused dramatic inhibition of E2-dependent epithelial cell proliferation, suggesting that a molecular perturbation approach is applicable to mimic in vivo uterine control. In conclusion, our established coculture system may serve as a useful alternative model to explore in vivo aspects of cell proliferation via communication between the epithelial and stromal compartments under the direction of ovarian hormones.

GPR30 activation opposes estrogen-dependent uterine growth via inhibition of stromal ERK1/2 and estrogen receptor alpha (ERα) phosphorylation signals.

Although estradiol-17ß (E2)-regulated early and late phase uterine responses have been well defined, the molecular mechanisms linking the phases remain poorly understood. We have previously shown that E2-regulated early signals mediate cross talk with estrogen receptor (ER)-α to elicit uterine late growth responses. G protein-coupled receptor (GPR30) has been implicated in early nongenomic signaling mediated by E2, although its role in E2-dependent uterine biology is unclear. Using selective activation of GPR30 by G-1, we show here a new function of GPR30 in regulating early signaling events, including the inhibition of ERK1/2 and ERα (Ser118) phosphorylation signals and perturbation of growth regulation under the direction of E2 in the mouse uterus. We observed that GPR30 primarily localizes in the uterine epithelial cells, and its activation alters gene expression and mediates inhibition of ERK1/2 and ERα (Ser118) phosphorylation signals in the stromal compartment, suggesting a paracrine signaling is involved. Importantly, viral-driven manipulation of GPR30 or pharmacological inhibition of ERK1/2 activation effectively alters E2-dependent uterine growth responses. Overall, GPR30 is a negative regulator of ERα-dependent uterine growth in response to E2. Our work has uncovered a novel GPR30-regulated inhibitory event, which may be physiologically relevant in both normal and pathological situations to negatively balance ERα-dependent uterine growth regulatory functions induced by E2.

Regional development of uterine decidualization: molecular signaling by Hoxa-10.

Uterine decidualization, a key event in implantation, is critically controlled by stromal cell proliferation and differentiation. Although the molecular mechanism that controls this event is not well understood, the general consensus is that the factors derived locally at the site of implantation influence aspects of decidualization. Hoxa-10, a developmentally regulated homeobox transcription factor, is highly expressed in decidualizing stromal cells, and targeted deletion of Hoxa-10 in mice shows severe decidualization defects, primarily due to the reduced stromal cell responsiveness to progesterone (P(4)). While the increased stromal cell proliferation is considered to be an initiator of decidualization, the establishment of a full-grown functional decidua appears to depend on the aspects of regional proliferation and differentiation. In this regard, this article provides an overview of potential signaling mechanisms mediated by Hoxa-10 that can influence a host of genes and cell functions necessary for propagating regional decidual development.