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.

Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring 1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite 2 . Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines 2 , including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase2,3, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17-IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI-IκBζ regulatory axis could be an important new strategy for the treatment of IL-17-IκBζ-mediated autoimmune diseases.

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.

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.

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.

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.

Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

The harmonized actions of ovarian E2 and progesterone (P4) regulate the proliferation and differentiation of uterine cells in a spatiotemporal manner. Imbalances between these hormones often lead to infertility and gynecologic diseases. Whereas numerous factors that are involved in P4 signaling have been identified, few local factors that mediate E2 actions in the uterus have been revealed. Here, we demonstrate that estrogen induces the transcription factor, early growth response 1 ( Egr1), to fine-tune its actions in uterine epithelial cells (ECs) that are responsible for uterine receptivity for embryo implantation. In the presence of exogenous gonadotrophins, ovulation, fertilization, and embryonic development normally occur in Egr1-/- mice, but these animals experience the complete failure of embryo implantation with reduced artificial decidualization. Although serum levels of E2 and P4 were comparable between Egr1+/+ and Egr1-/- mice on d 4 of pregnancy, aberrantly reduced levels of progesterone receptor in Egr1-/- uterine ECs caused enhanced E2 activity and impaired P4 response. Ultrastructural analyses revealed that Egr1-/- ECs are not fully able to provide proper uterine receptivity. Uterine mRNA landscapes in Egr1-/- mice revealed that EGR1 controls the expression of a subset of E2-regulated genes. In addition, P4 signaling was unable to modulate estrogen actions, including those that are involved in cell-cycle progression, in ECs that were deficient in EGR1. Furthermore, primary coculture of Egr1-/- ECs with Egr1+/+ stromal cells, and vice versa, supported the notion that Egr1 is required to modulate E2 actions on ECs to prepare the uterine environment for embryo implantation. In contrast to its role in ECs, loss of Egr1 in stroma significantly reduced stromal cell proliferation. Collectively, our results demonstrate that E2 induces EGR1 to streamline its actions for the preparation of uterine receptivity for embryo implantation in mice.-Kim, H.-R., Kim, Y. S., Yoon, J. A., Yang, S. C., Park, M., Seol, D.-W., Lyu, S. W., Jun, J. H., Lim, H. J., Lee, D. R., Song, H. Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

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.

Estrogen-dependent expression of sine oculis homeobox 1 in the mouse uterus during the estrous cycle.

The sine oculis homeobox 1 (SIX1) is a member of the Six gene family. SIX1 is involved in tissue development by regulating proliferation, apoptosis, and differentiation. However, function of SIX1 in the uterus remains unknown. Here, we found that Six1 expression is regulated along the estrous cycle in mouse uterus. Six1 expression was significantly increased at estrus stage and decreased at the rest of stages. SIX1 is detected in the luminal and glandular epithelium of uterine endometrium at the estrus stage. Estrogen injection increased Six1 expression in the ovariectomized mouse uterus, whereas progesterone had no effect on its expression. Estrogen receptor antagonist inhibited estrogen-induced Six1 expression. Our findings imply that SIX1 may play a role as an important regulator to orchestrate the dynamic of uterine endometrium in response to estrogen level during the estrous cycle. These results will give us a better understanding of uterine biology.

Socioeconomic Inequality in mortality using 12-year follow-up data from nationally representative surveys in South Korea.

BACKGROUND: Investigations into socioeconomic inequalities in mortality have rarely used long-term mortality follow-up data from nationally representative samples in Asian countries. A limited subset of indicators for socioeconomic position was employed in prior studies on socioeconomic inequalities in mortality. We examined socioeconomic inequalities in mortality using follow-up 12-year mortality data from nationally representative samples of South Koreans. METHODS: A total of 10,137 individuals who took part in the 1998 and 2001 Korea National Health and Nutrition Examination Surveys were linked to mortality data from Statistics Korea. Of those individuals, 1,219 (12.1 %) had died as of December 2012. Cox proportional hazard models were used to estimate the relative risks of mortality according to a wide range of socioeconomic position (SEP) indicators after taking into account primary sampling units, stratification, and sample weights. RESULTS: Our analysis showed strong evidence that individuals with disadvantaged SEP indicators had greater all-cause mortality risks than their counterparts. The magnitude of the association varied according to gender, age group, and specific SEP indicators. Cause-specific analyses using equivalized income quintiles showed that the magnitude of mortality inequalities tended to be greater for cardiovascular disease and external causes than for cancer. CONCLUSION: Inequalities in mortality exist in every aspect of SEP indicators, both genders, and age groups, and four broad causes of deaths. The South Korean economic development, previously described as effective in both economic growth and relatively equitable income distribution, should be scrutinized regarding its impact on socioeconomic mortality inequalities. Policy measures to reduce inequalities in mortality should be implemented in South Korea.

Microtubule plus end-tracking proteins play critical roles in directional growth of hyphae by regulating the dynamics of cytoplasmic microtubules in Aspergillus nidulans.

Cytoplasmic microtubules (MTs) serve as a rate-limiting factor for hyphal tip growth in the filamentous fungus Aspergillus nidulans. We hypothesized that this function depended on the MT plus end-tracking proteins (+TIPs) including the EB1 family protein EBA that decorated the MT plus ends undergoing polymerization. The ebAΔ mutation reduced colony growth and the mutant hyphae appeared in an undulating pattern instead of exhibiting unidirectional growth in the control. These phenotypes were enhanced by a mutation in another +TIP gene clipA. EBA was required for plus end-tracking of CLIPA, the Kinesin-7 motor KipA, and the XMAP215 homologue AlpA. In addition, cytoplasmic dynein also depended on EBA to track on most polymerizing MT plus ends, but not for its conspicuous appearance at the MT ends near the hyphal apex. The loss of EBA reduced the number of cytoplasmic MTs and prolonged dwelling times for MTs after reaching the hyphal apex. Finally, we found that colonies were formed in the absence of EBA, CLIPA, and NUDA together, suggesting that they were dispensable for fundamental functions of MTs. This study provided a comprehensive delineation of the relationship among different +TIPs and their contributions to MT dynamics and unidirectional hyphal expansion in filamentous fungi.

Isocitrate dehydrogenase 1 (IDH1) mutation in breast adenocarcinoma is associated with elevated levels of serum and urine 2-hydroxyglutarate.

Mutations in the IDH1 and IDH2 (isocitrate dehydrogenase) genes have been discovered across a range of solid-organ and hematologic malignancies, including acute myeloid leukemia, glioma, chondrosarcoma, and cholangiocarcinoma. An intriguing aspect of IDH-mutant tumors is the aberrant production and accumulation of the oncometabolite 2-hydroxyglutarate (2-HG), which may play a pivotal oncogenic role in these malignancies. We describe the first reported case of an IDH1 p.R132L mutation in a patient with hormone receptor-positive (HR+) breast adenocarcinoma. This patient was initially treated for locally advanced disease, but then suffered a relapse and metastasis, at which point an IDH1-R132 mutation was discovered in an affected lymph node. The mutation was subsequently found in the primary tumor tissue and all metastatic sites, but not in an uninvolved lymph node. In addition, the patient's serum and urine displayed marked elevations in the concentration of 2-HG, significantly higher than that measured in six other patients with metastatic HR+ breast carcinoma whose tumors were found to harbor wild-type IDH1. In summary, IDH1 mutations may impact a rare subgroup of patients with breast adenocarcinoma. This may suggest future avenues for disease monitoring through noninvasive measurement of 2-HG, as well as for the development and study of targeted therapies against the aberrant IDH1 enzyme.

Immunolocalisation and oestrogen regulation of small proline-rich protein 2a protein in the mouse uterus.

Small proline-rich protein 2a (Sprr2a) is one of the structural components of the cornified keratinocyte cell envelope that contributes to form a protective barrier in the skin against dehydration and environmental stress. Interestingly, Sprr2a mRNA is detected in the mouse uterus and is regulated by 17ß-oestradiol (E2). In the present study, we investigated the effects of E2 and oestrogenic compounds on the regulation and localisation of Sprr2a protein in the mouse uterus. Immunohistochemical staining revealed that Sprr2a protein is detected only in the adult uterus, and not in the ovary, oviduct or testis. We also demonstrated that Sprr2a protein is tightly regulated by E2 in the mouse uterus and exclusively detected in luminal and glandular epithelial cells. Furthermore, Sprr2a is dose-dependently induced by oestrogenic compounds such as bisphenol A and 4-tert-octylphenol. Collectively, our studies suggest that Sprr2a protein may have a unique function in physiological events in the mouse uterus and can be used as an indicator to detect compounds with oestrogenic activity in the mouse uterus.

Network signatures of cellular immortalization in human lymphoblastoid cell lines.

Human lymphoblastoid cell line (LCL) has been used as an in vitro cell model in genetic and pharmacogenomic studies, as well as a good model for studying gene expression regulatory machinery using integrated genomic analyses. In this study, we aimed to identify biological networks of LCL immortalization from transcriptomic profiles of microRNAs and their target genes in LCLs. We first selected differentially expressed genes (DEGs) and microRNAs (DEmiRs) between early passage LCLs (eLCLs) and terminally differentiated late passage LCLs (tLCLs). The in silico and correlation analysis of these DEGs and DEmiRs revealed that 1098 DEG-DEmiR pairs were found to be positively (n=591 pairs) or negatively (n=507 pairs) correlated with each other. More than 41% of DEGs are possibly regulated by miRNAs in LCL immortalizations. The target DEGs of DEmiRs were enriched for cellular functions associated with apoptosis, immune response, cell death, JAK-STAT cascade and lymphocyte activation while non-miRNA target DEGs were over-represented for basic cell metabolisms. The target DEGs correlated negatively with miR-548a-3p and miR-219-5p were significantly associated with protein kinase cascade, and the lymphocyte proliferation and apoptosis, respectively. In addition, the miR-106a and miR-424 clusters located in the X chromosome were enriched in DEmiR-mRNA pairs for LCL immortalization. In this study, the integrated transcriptomic analysis of LCLs could identify functional networks of biologically active microRNAs and their target genes involved in LCL immortalization.

Trends of breastfeeding rate in Korea (1994-2012): comparison with OECD and other countries.

Breastfeeding has numerous benefits both for infants and mothers. WHO, UNICEF, and OECD report the breastfeeding rate (BR) and exclusive breastfeeding rate (EBR) at 3, 4, and 6 months of age for the international comparison. This article investigates the nationwide changes in BR and EBR in Korea from 1994 to 2012. EBR declined from 1994 to 2000, however progressively increased untill 2012. The latest data in 2012 revealed EBRs at 3, 4, and 6 months were 50.0%, 40.5%, and 11.4% respectively. The exclusive formula feeding rate (EFR) was highest in 2000 and gradually declined thereafter. In 2012, the EFRs at 3, 4 and 6 months were 21.7%, 26.5%, and 10.1%. In 2009, the EBRs at 3 and 6 months in the United States were 36.0% and 16.3% compared to 50.0% and 11.4% in Korea. In England, the EBRs were 17% and 12% in 2010. Amongst OECD countries, Hungary ranked highest EBRwith 95%, and Iceland, Norway, Slovak Republic, Australia, New Zealand followed. In conclusion, BRs were lowest in 2000, and there have been remarkable increases in BRs over the past 10 yr in Korea. Although BRs have been increasing, further efforts to increase BRs should be made continuously.

Transglutaminase 2 expression predicts progression free survival in non-small cell lung cancer patients treated with epidermal growth factor receptor tyrosine kinase inhibitor.

Transglutaminase 2 (TG2), a cross-linking enzyme, is involved in drug resistance and in the constitutive activation of nuclear factor kappa B (NF-κB). We investigated the association of non-small cell lung cancer (NSCLC) treatment efficacy with TG2 and NF-κB expression in 120 patients: 102 with adenocarcinoma and 18 with other histologic types. All patients underwent surgery; 88 received adjuvant chemotherapy, with 28 receiving platinum-based doublet chemotherapy as first-line treatment and 29 receiving epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy. Patients' TG2 and NF-κB expression values were calculated semiquantitatively. The median TG2 value was 50 (range, 0-300) and the median NF-κB value was 20 (range, 0-240). Disease-free survival did not differ between the low- and high-TG2 groups. Among patients who received palliative platinum-based doublet chemotherapy, progression free survival (PFS) was longer in the low-TG2 group than in the high-TG2 group (11.0 vs. 7.0 months; P=0.330). Among those who received EGFR-TKI therapy, PFS was also longer in the low-TG2 group than in the high-TG 2 group (11.0 vs. 2.0 months; P=0.013). Similarly, in EGFR wild-type patients treated with EGFR-TKI, PFS was longer in patients with low TG2 expression (9.0 vs. 2.0 months; P=0.013). TG2 expression levels can predict PFS in patients with NSCLC treated with EGFR-TKI.

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.