CFP1 governs uterine epigenetic landscapes to intervene in progesterone responses for uterine physiology and suppression of endometriosis.

Progesterone (P4) is required for the preparation of the endometrium for a successful pregnancy. P4 resistance is a leading cause of the pathogenesis of endometrial disorders like endometriosis, often leading to infertility; however, the underlying epigenetic cause remains unclear. Here we demonstrate that CFP1, a regulator of H3K4me3, is required for maintaining epigenetic landscapes of P4-progesterone receptor (PGR) signaling networks in the mouse uterus. Cfp1f/f;Pgr-Cre (Cfp1d/d) mice showed impaired P4 responses, leading to complete failure of embryo implantation. mRNA and chromatin immunoprecipitation sequencing analyses showed that CFP1 regulates uterine mRNA profiles not only in H3K4me3-dependent but also in H3K4me3-independent manners. CFP1 directly regulates important P4 response genes, including Gata2, Sox17, and Ihh, which activate smoothened signaling pathway in the uterus. In a mouse model of endometriosis, Cfp1d/d ectopic lesions showed P4 resistance, which was rescued by a smoothened agonist. In human endometriosis, CFP1 was significantly downregulated, and expression levels between CFP1 and these P4 targets are positively related regardless of PGR levels. In brief, our study provides that CFP1 intervenes in the P4-epigenome-transcriptome networks for uterine receptivity for embryo implantation and the pathogenesis of endometriosis.

Peripubertal requirement of Tsg101 in maintaining the integrity of membranous structures in mouse oocytes.

OBJECTIVE: As a component of Endosomal Sorting Complex Required for Transport (ESCRT) complex I, the tumor susceptibility gene 101 (Tsg101) carries out multiple functions. In this work, we report that oocyte-specific deletion of tumor susceptibility gene 101 (Tsg101) leads to age-dependent oocyte demise in mice. MATERIALS AND METHOD: Tsg101 floxed mice (Tsg101f/f ) were bred with Zp3cre transgenic mice to examine oocyte-specific roles of Tsg101. Multiple cellular and molecular biological approaches were taken to examine what leads to oocyte demise in the absence of Tsg101. RESULTS: The death of oocytes from Zp3cre /Tsg101f/f (Tsg101d/d thereafter) mice showed a strong correlation with sexual maturation, as gonadotropin-releasing hormone antagonist injections improved the survival rate of oocytes from 5-week-old Tsg101d/d mice. Maturation of oocytes from prepubertal Tsg101d/d mice proceeded normally, but was largely abnormal in oocytes from peripubertal Tsg101d/d mice, showing shrinkage or rupture. Endolysosomal structures in oocytes from peripubertal Tsg101d/d mice showed abnormalities, with aberrant patterns of early and late endosomal markers and a high accumulation of lysosomes. Dying oocytes showed plasma membrane blebs and leakage. Blockage of endocytosis in oocytes at 4°C prevented cytoplasmic shrinkage of oocytes from Tsg101d/d mice until 9 h. The depletion of tsg-101 in Caenorhabditis elegans increased the permeability of oocytes and embryos, suggesting a conserved role of Tsg101 in maintaining membrane integrity. CONCLUSIONS: Collectively, Tsg101 plays a dual role in maintaining the integrity of membranous structures, which is influenced by age in mouse oocytes.

ADAMTS-1: a novel target gene of an estrogen-induced transcription factor, EGR1, critical for embryo implantation in the mouse uterus.

BACKGROUND: Recently, we demonstrated that estrogen (E2) induces early growth response 1 (Egr1) to mediate its actions on the uterine epithelium by controlling progesterone receptor signaling for successful embryo implantation. EGR1 is a transcription factor that regulates the spectrum of target genes in many different tissues, including the uterus. E2-induced EGR1 regulates a set of genes involved in epithelial cell remodeling during embryo implantation in the uterus. However, only few target genes of EGR1 in the uterus have been identified. RESULT: The expression of ADAM metallopeptidase with thrombospondin type 1 motif 1 (Adamts-1) was significantly downregulated in the uteri of E2-treated ovariectomized (OVX) Egr1(-/-) mice. Immunostaining of ADAMTS-1 revealed its exclusive expression in the uterine epithelium of OVX wild-type but not Egr1(-/-) mice treated with E2. The expression profiles of Adamts-1 and Egr1 were similar in the uteri of E2-treated OVX mice at various time points tested. Pre-treatment with ICI 182, 780, a nuclear estrogen receptor (ER) antagonist, effectively inhibited the E2-dependent induction of Egr1 and Adamts-1. Pharmacologic inhibition of E2-induced ERK1/2 or p38 phosphorylation interfered with the induction of EGR1 and ADAMTS-1. Furthermore, ADAMTS-1, as well as EGR1, was induced in stroma cells surrounding the implanting blastocyst during embryo implantation. Transient transfection with EGR1 expression vectors significantly induced the expression of ADAMTS-1. Luciferase activity of the Adamts-1 promoter containing EGR1 binding sites (EBSs) was increased by EGR1 in a dose-dependent manner, suggesting functional regulation of Adamts-1 transcription by EGR1. Site-directed mutagenesis of EBS on the Adamts-1 promoter demonstrated that EGR1 directly binds to the EBS at -1151/-1134 among four putative EBSs. CONCLUSIONS: Collectively, we have demonstrated that Adamts-1 is a novel target gene of E2-ER-MAPK-EGR1, which is critical for embryo implantation in the mouse uterus during early pregnancy.

Preclinical Drug Metabolism, Pharmacokinetic, and Pharmacodynamic Profiles of Ivosidenib, an Inhibitor of Mutant Isocitrate Dehydrogenase 1 for Treatment of Isocitrate Dehydrogenase 1-Mutant Malignancies.

Point mutations in isocitrate dehydrogenase 1 (IDH1) result in conversion of α-ketoglutarate to the oncometabolite, d-2-hydroxyglutarate (2-HG). Ivosidenib is a once daily (QD), orally available, potent, mutant isocitrate dehydrogenase 1 (mIDH1) inhibitor approved for the treatment of patients with relapsed or refractory acute myeloid leukemia (AML) and intensive chemotherapy-ineligible newly diagnosed AML, with a susceptible IDH1 mutation. We characterized the protein binding, metabolism, metabolites, cell permeability, and drug-drug interaction potential of ivosidenib in humans, monkeys, dogs, rats, and/or mice in in vitro experiments. In vivo pharmacokinetic (PK) profiling and assessment of drug distribution and excretion was undertaken in rats, dogs, and monkeys administered single-dose ivosidenib. The PK/pharmacodynamic (PD) relationship between ivosidenib and 2-HG was analyzed in an mIDH1 xenograft mouse model. Ivosidenib was well absorbed, showed low clearance, and moderate to long terminal half-life (5.3-18.5 hours) in rats, dogs, and monkeys. Brain to plasma exposure ratio was low (2.3%), plasma protein binding was high, and oxidative metabolism was the major elimination pathway. Ivosidenib had high cell permeability and was identified as a substrate for P-glycoprotein. There was moderate induction of cytochrome P450 (P450) enzymes CYP3A4 and CYP2B6 but minimal P450 inhibition or autoinduction. Tumor 2-HG reduction appeared to be dose- and drug-exposure-dependent. Ivosidenib showed a favorable PK profile in several animal species, along with a clear PK/PD relationship demonstrating 2-HG inhibition that translated well to patients with AML. SIGNIFICANCE STATEMENT: Ivosidenib is a mutant IDH1 (mIDH1) inhibitor approved for the treatment of certain patients with mIDH1 acute myeloid leukemia. In Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys, ivosidenib demonstrated a favorable pharmacokinetic profile, and in female BALB/c mice showed clear dose- and exposure-dependent inhibition of the oncometabolite, d-2-hydroxyglutarate, which is present at abnormal levels in mIDH1 tumors. These findings led to the further development of ivosidenib and are consistent with data from patients with mIDH1 cancers and healthy participants.

Single clonal glandular stem cells derived from human parotid glands do not attain malignant phenotype during long-term in vitro culture.

Mesenchymal stem cells (MSCs) are being intensively investigated as future therapeutics for various human diseases. One of the most important challenges to the clinical application of MSCs is the possibility of malignant transformation during long-term in vitro culturing. However, there have been no reports on the tumorigenicity of salivary gland-derived MSCs following long-term in vitro culturing. Here, we isolated a single clonal glandular stem cells from human parotid gland stem cells (hpGSCs) using a modified sub-fractionation culturing method. The possibility of malignant transformation of these cells following long-term culturing was evaluated under in vitro and in vivo culture conditions. Single clonal glandular stem cells from the human parotid gland have unique multipotent MSCs traits. hpGSCs at passage 18 stained strongly for ß-galactosidase expression and the long-term culture of hpGSCs led to a reduction in telomerase activity. hpGSCs could not survive in a soft agar environment and did not cause tumor formation in a xenograft mouse model. In addition, the expression of salivary cancer-related oncogenes was not elevated in hpGSCs following the long-term culture. In conclusion, we demonstrated that there is no possibility of acquiring a malignant transformation during long-term in vitro cell expansion of hpGSCs.

Itaconate confers tolerance to late NLRP3 inflammasome activation.

Itaconate is a unique regulatory metabolite that is induced upon Toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation after long lipopolysaccharide (LPS) priming, which establishes tolerance to late NLRP3 inflammasome activation. We show that itaconate acts synergistically with inducible nitric oxide synthase (iNOS) and that the ability of various TLR ligands to establish NLRP3 inflammasome tolerance depends on the pattern of co-expression of IRG1 and iNOS. Mechanistically, itaconate accumulation upon prolonged inflammatory stimulation prevents full caspase-1 activation and processing of gasdermin D, which we demonstrate to be post-translationally modified by endogenous itaconate. Altogether, our data demonstrate that metabolic rewiring in inflammatory macrophages establishes tolerance to NLRP3 inflammasome activation that, if uncontrolled, can result in pyroptotic cell death and tissue damage.

Different Cre systems induce differential microRNA landscapes and abnormalities in the female reproductive tracts of Dgcr8 conditional knockout mice.

OBJECTIVES: The female reproductive tract comprises several different cell types. Using three representative Cre systems, we comparatively analysed the phenotypes of Dgcr8 conditional knockout (cKO) mice to understand the function of Dgcr8, involved in canonical microRNA biogenesis, in the female reproductive tract. MATERIALS AND METHODS: Dgcr8f/f mice were crossed with Ltficre/+ , Amhr2cre/+ or PRcre/+ mice to produce mice deficient in Dgcr8 in epithelial (Dgcr8ed/ed ), mesenchymal (Dgcr8md/md ) and all the compartments (Dgcr8td/td ) in the female reproductive tract. Reproductive phenotypes were evaluated in Dgcr8 cKO mice. Uteri and/or oviducts were used for small RNA-seq, mRNA-seq, real-time RT-PCR, and/or morphologic and histological analyses. RESULT: Dgcr8ed/ed mice did not exhibit any distinct defects, whereas Dgcr8md/md mice showed sub-fertility and oviductal smooth muscle deformities. Dgcr8td/td mice were infertile due to anovulation and acute inflammation in the female reproductive tract and suffered from an atrophic uterus with myometrial defects. The microRNAs and mRNAs related to immune modulation and/or smooth muscle growth were systemically altered in the Dgcr8td/td uterus. Expression profiles of dysregulated microRNAs and mRNAs in the Dgcr8td/td uterus were different from those in other genotypes in a Cre-dependent manner. CONCLUSIONS: Dgcr8 deficiency with different Cre systems induces overlapping but distinct phenotypes as well as the profiles of microRNAs and their target mRNAs in the female reproductive tract, suggesting the importance of selecting the appropriate Cre driver to investigate the genes of interest.

Secretory phospholipase A2-X (Pla2g10) is a novel progesterone receptor target gene exclusively induced in uterine luminal epithelium for uterine receptivity in mice.

BACKGROUND: Aberration of estrogen (E2) and/or progesterone (P4) signaling pathways affects expression of their target genes, which may lead to failure of embryo implantation and following pregnancy. Although many target genes of progesterone receptors (PRs) have been identified in uterine stroma, only a few PR targets have been reported in the epithelium. Secretory phospholipase A2-(PLA2)-X, a member of the PLA2 family that releases arachidonic acids for the synthesis of prostaglandins that are important for embryo implantation, is dysregulated in the endometrium of patients suffering from repeated implantation failure. However, it is not clear whether sPLA2-X is directly regulated by ovarian steroid hormones for embryo implantation in the uterus. RESULT: P4 induced the Pla2g10 encoding of secretory PLA2-X in the apical region of uterine LE of ovariectomized mice via PR in both time- and dose-dependent manners, whereas E2 significantly inhibited it. This finding is consistent with the higher expression of Pla2g10 at the diestrus stage, when P4 is elevated during the estrous cycle, and at P4-treated delayed implantation. The level of Pla2g10 on day 4 of pregnancy (day 4) was dramatically decreased on day 5, when PRs are absent in the LE. Luciferase assays of mutagenesis in uterine epithelial cells demonstrated that four putative PR response elements in a Pla2g10 promoter region are transcriptionally active for Pla2g10. Intrauterine delivery of small interfering RNA for Pla2g10 on day 3 significantly reduced the number of implantation sites, reinforcing the critical function(s) of Pla2g10 for uterine receptivity in mice. CONCLUSIONS: Pla2g10 is a novel PR target gene whose expression is exclusively localized in the apical region of the uterine LE for uterine receptivity for embryo implantation in mice.

Comparative evaluation of itaconate and its derivatives reveals divergent inflammasome and type I interferon regulation in macrophages.

Following activation, macrophages undergo extensive metabolic rewiring1,2. Production of itaconate through the inducible enzyme IRG1 is a key hallmark of this process3. Itaconate inhibits succinate dehydrogenase4,5, has electrophilic properties6 and is associated with a change in cytokine production4. Here, we compare the metabolic, electrophilic and immunologic profiles of macrophages treated with unmodified itaconate and a panel of commonly used itaconate derivatives to examine its role. Using wild-type and Irg1-/- macrophages, we show that neither dimethyl itaconate, 4-octyl itaconate nor 4-monoethyl itaconate are converted to intracellular itaconate, while exogenous itaconic acid readily enters macrophages. We find that only dimethyl itaconate and 4-octyl itaconate induce a strong electrophilic stress response, in contrast to itaconate and 4-monoethyl itaconate. This correlates with their immunosuppressive phenotype: dimethyl itaconate and 4-octyl itaconate inhibited IκBζ and pro-interleukin (IL)-1ß induction, as well as IL-6, IL-10 and interferon-ß secretion, in an NRF2-independent manner. In contrast, itaconate treatment suppressed IL-1ß secretion but not pro-IL-1ß levels and, surprisingly, strongly enhanced lipopolysaccharide-induced interferon-ß secretion. Consistently, Irg1-/- macrophages produced lower levels of interferon and reduced transcriptional activation of this pathway. Our work establishes itaconate as an immunoregulatory, rather than strictly immunosuppressive, metabolite and highlights the importance of using unmodified itaconate in future studies.

Perivascular Stem Cell-Derived Cyclophilin A Improves Uterine Environment with Asherman's Syndrome via HIF1α-Dependent Angiogenesis.

Asherman's syndrome (AS) is characterized by intrauterine adhesions or fibrosis resulting from scarring inside the endometrium. AS is associated with infertility, recurrent miscarriage, and placental abnormalities. Although mesenchymal stem cells show therapeutic promise for the treatment of AS, the molecular mechanisms underlying its pathophysiology remain unclear. We ascertained that mice with AS, like human patients with AS, suffer from extensive fibrosis, oligo/amenorrhea, and infertility. Human perivascular stem cells (hPVSCs) from umbilical cords repaired uterine damage in mice with AS, regardless of their delivery routes. In mice with AS, embryo implantation is aberrantly deferred, which leads to intrauterine growth restriction followed by no delivery at term. hPVSC administration significantly improved implantation defects and subsequent poor pregnancy outcomes via hypoxia inducible factor 1α (HIF1α)-dependent angiogenesis in a dose-dependent manner. Pharmacologic inhibition of HIF1α activity hindered hPVSC actions on pregnancy outcomes, whereas stabilization of HIF1α activity facilitated such actions. Furthermore, therapeutic effects of hPVSCs were not observed in uterine-specific HIF1α-knockout mice with AS. Secretome analyses of hPVSCs identified cyclophilin-A as the major paracrine factor for hPVSC therapy via HIF1α-dependent angiogenesis. Collectively, we demonstrate that hPVSCs-derived cyclophilin-A facilitates HIF1α-dependent angiogenesis to ameliorate compromised uterine environments in mice with AS, representing the major pathophysiologic features of humans with AS.

A transport system based on a quantum dot-modified nanotracer is genetically and developmentally stable in pregnant mice.

The use of nanoscale materials (NMs) could cause problems such as cytotoxicity, genomic aberration, and effects on human health, but the impacts of NM exposure during pregnancy remain uncharacterized in the context of clinical applications. It was sought to determine whether nanomaterials pass through the maternal-fetal junction at any stage of pregnancy. Quantum dots (QDs) coated with heparinized Pluronic 127 nanogels and polyethyleneimine (PEI) were administered to pregnant mice. The biodistribution of QDs, as well as their biological impacts on maternal and fetal health, was evaluated. Encapsulation of QDs with a nanogel coating produces a petal-like nanotracer (PNt), which could serve as a nano-carrier of genes or drugs. PNts were injected through the tail vein and accumulated in the liver, kidneys, and lungs. QD accumulation in reproductive organs (uterus, placenta, and fetus) differed among phases of pregnancy. In phase I (7 days of pregnancy), the QDs did not accumulate in the placenta or fetus, but by phase III (19 days) they had accumulated at high levels in both tissues. Karyotype analysis revealed that the PNt-treated pups did not have genetic abnormalities when dams were treated at any phase of pregnancy. PNts have the potential to serve as carriers of therapeutic agents for the treatment of the mother or fetus and these results have a significant impact on the development and application of QD-based NPs in pregnancy.

Hyperandrogenic Milieu Dysregulates the Expression of Insulin Signaling Factors and Glucose Transporters in the Endometrium of Patients With Polycystic Ovary Syndrome.

PURPOSE: Subfertility associated with polycystic ovary syndrome (PCOS) mainly originates from oligoovulation/anovulation. Although insulin resistance and androgen excess are known to cause PCOS-associated implantation failure, the consequences of PCOS on endometrial homeostasis and pathophysiology have not been comprehensively understood. In this study, we examined whether the pathophysiologic milieu of PCOS intrinsically affects expression profiles of genes related to insulin signaling and facilitative glucose transporters (GLUTs) in the human endometrium and/or during in vitro decidualization. STUDY DESIGN: Seven healthy women with regular menstrual cycles and 13 patients with PCOS were recruited for this study. To mimic the hyperandrogenic or hyperinsulinemic milieu in the endometrium of patient with PCOS (PCOSE) in vitro, human endometrial stromal cells (hESCs) were treated with dihydrotestosterone (DHT) or insulin, respectively. RESULTS: In PCOSE, messenger RNA (mRNA) levels of insulin receptor (IR), IR substrate (IRS) 1, and IRS2 were significantly increased. Furthermore, GLUT1 and GLUT12 were aberrantly increased. Chronic exposure to insulin or DHT aberrantly increased IRS1/IRS2 phosphorylation and protein levels of GLUT1 and GLUT12 in hESCs, suggesting that not only hyperinsulinemic but also hyperandrogenic conditions affect insulin signaling and glucose metabolism. The mRNA microarrays demonstrated that DHT dysregulates various gene sets, including cell cycle and glucose metabolism, in hESCs. Furthermore, DHT suppressed the expression of GLUT1 and GLUT12 as well as decidualization markers, IGFBP1 and prolactin, during in vitro decidualization. CONCLUSIONS: The hyperandrogenic milieu affects gene expression profiles, including gene sets associated with insulin signaling, cell cycle, glucose metabolism, and/or glucose transport, in human endometrium and during in vitro decidualization.

Smoking-Related DNA Methylation is Differentially Associated with Cadmium Concentration in Blood.

Tobacco smoking, a risk factor for several human diseases, can lead to alterations in DNA methylation. Smoking is a key source of cadmium exposure; however, there are limited studies examining DNA methylation alterations following smoking-related cadmium exposure. To identify such cadmium exposure-related DNA methylation, we performed genome-wide DNA methylation profiling using DNA samples from 50 smokers and 50 non-smokers. We found that a total of 136 CpG sites (including 70 unique genes) were significantly differentially methylated in smokers as compared to that in non-smokers. The CpG site cg05575921 in the AHRR gene was hypomethylated (Δ ß > - 0.2) in smokers, which was in accordance with previous studies. The rs951295 (within RNA gene LOC105370802) and cg00587941 sites were under-methylated by > 15% in smokers, whereas cg11314779 (within CELF6) and cg02126896 were over-methylated by ≥ 15%. We analyzed the association between blood cadmium concentration and DNA methylation level for 50 smokers and 50 non-smokers. DNA methylation rates of 307 CpG sites (including 207 unique genes) were significantly correlated to blood cadmium concentration (linear regression P value < 0.001). The four significant loci (cg05575921 and cg23576855 in AHRR, cg03636183 in F2RL3, and cg21566642) were under-methylated by > 10% in smokers compared to that in non-smokers. In conclusion, our study demonstrated that DNA methylation levels of rs951295, cg00587941, cg11314779, and cg02126896 sites may be new putative indicators of smoking status. Furthermore, we showed that these four loci may be differentially methylated by cadmium exposure due to smoking.

Electroporation of AsCpf1/RNP at the Zygote Stage is an Efficient Genome Editing Method to Generate Knock-Out Mice Deficient in Leukemia Inhibitory Factor.

BACKROUND: CRISPR/Cpf1 is a class II, type V RNA-guided endonuclease that is distinct from the type II CRISPR/Cas9 nuclease, widely used for genome editing. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR/Cas9 system. The applications of CRISPR to rodent embryos for the production of knock-out (KO) mice have been achieved mainly by microinjection, which requires heavily-equipped instruments with skillful hands. Here, we evaluated the genome editing efficiency between Cpf1/mRNA and Cpf1/ribonuclear protein (RNP) in mouse embryos, and established an easy, fast, and technically less demanding method to produce KO mice using electroporation of the Cfp1/RNP system. METHODS: The efficiency of electroporation-based delivery of AsCpf1/mRNA and AsCpf1/RNP to target exon 3 of leukemia inhibitory factor (Lif) into mouse zygotes was evaluated. Embryos that developed to the two-cell stage after zygote electroporation were transferred into the oviducts of surrogate mothers to produce AsCpf1-mediated LIF KO mice. The genome editing efficiency of blastocysts and pups was tested using the T7E1 assay and/or DNA sequencing. Congenital abnormalities and reproductive phenotypes in LIF KO mice produced by electroporation with AsCpf1/RNP were examined. RESULTS: Survival and two-cell development of electroporated zygotes were comparable between the AsCpf1/mRNA and AsCpf1/RNP groups, whereas genome editing efficiency was relatively higher in the AsCpf1/RNP group (13.3% vs 18.1% at blastocyst and 33.3% vs 45.5% at offspring), respectively. Two mouse lines with a frameshift mutation in exon 3 of the Lif gene were established from the AsCpf1/RNP group. All congenital abnormalities of LIF KO mice produced by AsCpf1/RNP electroporation were observed. AsCpf1-mediated LIF KO mice showed postnatal growth retardation and implantation failure, both of which are major phenotypes of LIF KO mice generated by conventional gene targeting. CONCLUSION: Electroporation of AsCpf1/RNP at the zygote stage is an efficient genome editing method to produce KO mice.

Metabolic Profiling Using Stable Isotope Tracing Reveals Distinct Patterns of Glucose Utilization by Physiologically Activated CD8+ T Cells.

Naive CD8+ T cells differentiating into effector T cells increase glucose uptake and shift from quiescent to anabolic metabolism. Although much is known about the metabolism of cultured T cells, how T cells use nutrients during immune responses in vivo is less well defined. Here, we combined bioenergetic profiling and 13C-glucose infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. In contrast to in vitro-activated T cells, which display hallmarks of Warburg metabolism, physiologically activated CD8+ T cells displayed greater rates of oxidative metabolism, higher bioenergetic capacity, differential use of pyruvate, and prominent flow of 13C-glucose carbon to anabolic pathways, including nucleotide and serine biosynthesis. Glucose-dependent serine biosynthesis mediated by the enzyme Phgdh was essential for CD8+ T cell expansion in vivo. Our data highlight fundamental differences in glucose use by pathogen-specific T cells in vivo, illustrating the impact of environment on T cell metabolic phenotypes.

Hyperandrogenic Milieu Dysregulates the Expression of Insulin Signaling Factors and Glucose Transporters in the Endometrium of Patients With Polycystic Ovary Syndrome.

PURPOSE:: Subfertility associated with polycystic ovary syndrome (PCOS) mainly originates from oligoovulation/anovulation. Although insulin resistance and androgen excess are known to cause PCOS-associated implantation failure, the consequences of PCOS on endometrial homeostasis and pathophysiology have not been comprehensively understood. In this study, we examined whether the pathophysiologic milieu of PCOS intrinsically affects expression profiles of genes related to insulin signaling and facilitative glucose transporters (GLUTs) in the human endometrium and/or during in vitro decidualization. STUDY DESIGN:: Seven healthy women with regular menstrual cycles and 13 patients with PCOS were recruited for this study. To mimic the hyperandrogenic or hyperinsulinemic milieu in the endometrium of patient with PCOS (PCOSE) in vitro, human endometrial stromal cells (hESCs) were treated with dihydrotestosterone (DHT) or insulin, respectively. RESULTS:: In PCOSE, messenger RNA (mRNA) levels of insulin receptor (IR), IR substrate (IRS) 1, and IRS2 were significantly increased. Furthermore, GLUT1 and GLUT12 were aberrantly increased. Chronic exposure to insulin or DHT aberrantly increased IRS1/IRS2 phosphorylation and protein levels of GLUT1 and GLUT12 in hESCs, suggesting that not only hyperinsulinemic but also hyperandrogenic conditions affect insulin signaling and glucose metabolism. The mRNA microarrays demonstrated that DHT dysregulates various gene sets, including cell cycle and glucose metabolism, in hESCs. Furthermore, DHT suppressed the expression of GLUT1 and GLUT12 as well as decidualization markers, IGFBP1 and prolactin, during in vitro decidualization. CONCLUSIONS:: The hyperandrogenic milieu affects gene expression profiles, including gene sets associated with insulin signaling, cell cycle, glucose metabolism, and/or glucose transport, in human endometrium and during in vitro decidualization.

Reprogramming mechanisms influence the maturation of hematopoietic progenitors from human pluripotent stem cells.

Somatic cell nuclear transfer (SCNT) or the forced expression of transcription factors can be used to generate autologous pluripotent stem cells (PSCs). Although transcriptomic and epigenomic comparisons of isogenic human NT-embryonic stem cells (NT-ESCs) and induced PSCs (iPSCs) in the undifferentiated state have been reported, their functional similarities and differentiation potentials have not been fully elucidated. Our study showed that NT-ESCs and iPSCs derived from the same donors generally displayed similar in vitro commitment capacity toward three germ layer lineages as well as proliferative activity and clonogenic capacity. However, the maturation capacity of NT-ESC-derived hematopoietic progenitors was significantly greater than the corresponding capacity of isogenic iPSC-derived progenitors. Additionally, donor-dependent variations in hematopoietic specification and commitment capacity were observed. Transcriptome and methylome analyses in undifferentiated NT-ESCs and iPSCs revealed a set of genes that may influence variations in hematopoietic commitment and maturation between PSC lines derived using different reprogramming methods. Here, we suggest that genetically identical iPSCs and NT-ESCs could be functionally unequal due to differential transcription and methylation levels acquired during reprogramming. Our proof-of-concept study indicates that reprogramming mechanisms and genetic background could contribute to diverse functionalities between PSCs.

Oestrogen-induced expression of decay accelerating factor is spatiotemporally antagonised by progesterone-progesterone receptor signalling in mouse uterus.

Decay accelerating factor (DAF) is upregulated in the fetoplacental trophoblast, which protects the fetus from maternal complement injury. DAF was found to be downregulated in the endometrium of patients with repeated implantation failure. Thus, we examined the molecular mechanisms of DAF expression regulation by ovarian steroid hormones in the mouse uterus. Immunofluorescence staining demonstrated its exclusive localisation in the apical region of the epithelium in the uterus. Oestrogen (E2) significantly induced Daf mRNA in a time-dependent manner. Progesterone (P4) did not have any significant effect on Daf expression; however, it negatively modulated E2-induced DAF expression and RU486 effectively interfered with the inhibitory action of P4 in the uterus. During early pregnancy DAF was higher on Day 1 of pregnancy, but significantly decreased from Day 3, which is consistent with its E2-dependent regulation. Interestingly, DAF expression seemed to be influenced by the implanting blastocyst on Day 5 and it was gradually increased during preimplantation embryo development with peak levels at blastocyst stages. We demonstrated that E2-dependent DAF expression is antagonised by P4-progesterone receptor signalling in the uterine epithelium. Spatiotemporal regulation of DAF in the uterus and preimplantation embryos suggest that DAF functions as an immune modulator for embryo implantation and early pregnancy in mice.

Modulation of store-operated calcium entry and nascent adhesion by p21-activated kinase 1.

Calcium mobilization is necessary for cell movement during embryonic development, lymphocyte synapse formation, wound healing, and cancer cell metastasis. Depletion of calcium in the lumen of the endoplasmic reticulum using inositol triphosphate (IP3) or thapsigargin (TG) is known to induce oligomerization and cytoskeleton-mediated translocation of stromal interaction molecule 1 (STIM1) to the plasma membrane, where it interacts with the calcium release-activated calcium channel Orai1 to mediate calcium influx; this process is referred to as store-operated calcium entry (SOCE). Furthermore, aberrant STIM1 or SOCE regulation is associated with cancer cell motility and metastasis. The p21-activated kinases (PAKs), which are downstream effectors of GTPases, reportedly regulate cytoskeletal organization, protrusive activity, and cell migration. Although cytoskeletal remodeling apparently contributes to calcium mobilization via SOCE, and vice versa, the mechanisms by which they regulate each other remain unclear. In this study, we aimed to characterize whether PAK1 modulates calcium mobilization and STIM1 localization. Our data demonstrate that PAK1 interacts with STIM1 in vitro and that this interaction was enhanced by treatment with a nascent adhesion inducer, such as phorbol 12,13-dibutyrate (PDBu). Under basal conditions, both proteins appeared to primarily colocalize in the cytosol, whereas treatment with PDBu induced their colocalization to vinculin-positive peripheral adhesions. Downregulation of PAK1 activity via chemical inhibitors or by PAK1 shDNA expression impaired STIM1-mediated calcium mobilization via SOCE. Based on these findings, we propose that PAK1 interacts with STIM1 to regulate calcium mobilization and the formation of cellular adhesions.