Hyunjung Jade Lim

HB-EGF: A unique mediator of embryo-uterine interactions during implantation

An implantation-competent blastocyst, several hours prior to its attachment on the uterine wall, transmits signals to surrounding uterine cells and vice-versa to initiate a two-way interaction. The language of this precocious dialogue is versatile, taking advantage of secreted molecules for long-range interactions and membrane-bound molecules for more immediate interactions. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified as an early messenger of implantation which uses both modes of communication. In this review, we discuss the footprint of HB-EGF as to how it was initially identified as a mediator of implantation and how it initiates embryo-uterine interactions during this process.

The antipsychotic agent chlorpromazine induces autophagic cell death by inhibiting the Akt/mTOR pathway in human U-87MG glioma cells.

2-Chloro-10-[3(-dimethylamino)propyl]phenothiazine mono hydrochloride (chlorpromazine; CPZ) is an antipsychotic agent that was originally developed to control psychotic disorders. The cytotoxic properties of the CPZ are well known, but its mechanism of action is poorly understood. In this study, we investigated the role of apoptosis and autophagy in CPZ-induced cytotoxicity in U-87MG glioma cells. CPZ treatment inhibited cell proliferation and long-term clonogenic survival. Additionally, CPZ triggered autophagy, as indicated by electron microscopy and accumulation of the membrane form of microtubule-associated protein 1 light chain 3 (LC3-II); however, CPZ did not induce apoptosis. Inhibition of autophagy by expression of Beclin 1 small interfering RNA (siRNA) in U-87MG cells attenuated CPZ-induced LC3-II formation. Furthermore, U-87MG cells expressing Beclin 1 siRNA attenuated CPZ-induced cell death. CPZ inhibited phosphatidylinositol 3-kinase (PI3K)/AKT/ mTOR pathway in U-87MG cells. Treatment with LY294002, a PI3K inhibitor, alone increased the accumulation of LC3-II and potentiated the effect of CPZ. In contrast, exogenous expression of AKT partially inhibited CPZ-induced LC3-II formation. When U-87MG cells were implanted into the brain of athymic nude mouse, CPZ triggered autophagy and inhibited xenograft tumor growth. These results provided the first evidence that CPZ-induced cytotoxicity is mediated through autophagic cell death in PTEN (phosphatase and tensin homolog deleted on chromosome 10)-null U-87MG glioma cells by inhibiting PI3K/AKT/mTOR pathway.

Insulin‐Secreting Cells from Human Eyelid‐Derived Stem Cells Alleviate Type I Diabetes in Immunocompetent Mice

Various attempts have been made to develop stem cell‐based therapy to alleviate type I diabetes using animal models. However, it has been a question whether human insulin produced from explanted cells is solely responsible for the normoglycemia of diabetic animals. In this study, we isolated neural crest‐like stem cells from the human eyelid fat and examined their therapeutic potentials for diabetes. The human eyelid adipose‐derived stem cells (HEACs) displayed characteristics of neural crest cells. Using a two‐step culture condition combined with nicotinamide, activin, and/or GLP‐1, we differentiated HEACs into insulin‐secreting cells and examined in vivo effects of differentiated cells by transplantation experiments. Following differentiation in vitro, HEACs released insulin and c‐peptide in a glucose‐dependent manner. Upon their transplantation under kidney capsules of streptozotocin‐treated immunocompetent mice, we observed normalization of hyperglycemia in 10 of 20 recipient mice until sacrifice after 2 months. Only the human, but not the mouse, insulin and c‐peptide were detected in the blood of recipient mice. Removal of the kidneys transplanted with HEACs resulted in a sharp increase of blood glucose level. Removed kidney tissues showed distinct expression of various human genes including insulin, and colocalization of the human insulin and the human nuclear protein in many cells. However, they showed diminished or null expression of some immune‐related genes. In conclusion, human insulin alone produced from eyelid‐derived stem cells following differentiation into insulin‐secreting cells and transplantation could normalize type I diabetes in mice. STEM CELLS 2009;27:1999–2008

Exosomal proteins in the aqueous humor as novel biomarkers in patients with neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) describes the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris and is the leading cause of blindness in people over 50. The molecular mechanisms underlying this multifactorial disease remain largely unknown. To uncover novel secretory biomarkers related to the pathogenesis of AMD, we adopted an integrated approach to compare the proteins identified in the conditioned medium (CM) of cultured RPE cells and the exosomes derived from CM and from the aqueous humor (AH) of AMD patients by LC-ESI-MS/MS. Finally, LC-MRM was performed on the AH from patients and controls, which revealed that cathepsin D, cytokeratin 8, and four other proteins increased in the AH of AMD patients. The present study has identified potential biomarkers and therapeutic targets for AMD treatment, such as proteins related to the autophagy-lysosomal pathway and epithelial-mesenchymal transition, and demonstrated a novel and effective approach to identifying AMD-associated proteins that might be secreted by RPE in vivo in the form of exosomes. The proteomics-based characterization of this multifactorial disease could help to match a particular marker to particular target-based therapy in AMD patients with various phenotypes.

Human amnion-derived mesenchymal stem cells are a potential source for uterine stem cell therapy.

Abstract.u2002 Objectives: Human amnion is an easy‐to‐obtain novel source of human mesenchymal stem cells, which poses little or no ethical dilemmas. We have previously shown that human amnion‐derived mesenchymal (HAM) cells exhibit certain mesenchymal stem cell‐like characteristics with respect to expression of stem cell markers and differentiation potentials. Materials and methods: In this study, we further characterized HAM cells’ potential for in vivo therapeutic application. Results: Flow cytometric analyses of HAM cells show that they express several stem cell‐related cell surface markers, including CD90, CD105, CD59, CD49d, CD44 and HLA‐ABC, but not CD45, CD34, CD31, CD106 or HLA‐DR. HAM cells at the 10th passage showed normal karyotype. More interestingly, the AbdB‐like HOXA genes HOXA9, HOXA10 and HOXA11 that are expressed in the mesenchyme of the developing female reproductive tract and pregnant uteri are also expressed in HAM cells, suggesting similarities between these two mesenchymal cell types. Progesterone receptor is also highly expressed in HAM cells and expression of genes or proteins in HAM cells could be manipulated with the aid of lentivirus technology or cell‐permeable peptides. To test potentials of HAM cells for in vivo application, we introduced enhanced green fluorescence protein (EGFP)‐expressing HAM cells to mice by intrauterine infusion (into uteri) or by intravenous injection (into the circulation). Presence of EGFP‐expressing cells within the uterine mesenchyme after intrauterine infusion or in lungs after intravenous injection was noted within 1–4 weeks. Conclusions: Collectively, these results suggest that HAM cells are a potential source of mesenchymal stem cells with therapeutic potential.

Egr1 is rapidly and transiently induced by estrogen and bisphenol A via activation of nuclear estrogen receptor-dependent ERK1/2 pathway in the uterus.

Coordinate actions of ovarian estrogen (E2) and progesterone (P4) via their own receptors are critical for establishing uterine receptivity for embryo implantation in the uterus. E2 regulates expression of an array of genes to mediate its major actions on heterogeneous uterine cell types. Here we have investigated regulatory mechanism(s) of E2 and bisphenol A (BPA), an endocrine disruptor with potent estrogenic activity on expression of early growth response 1 (Egr1), a zinc finger transcription factor that regulates cell growth, differentiation and apoptosis in the uterus. Egr1 was rapidly and transiently induced by E2 and BPA mainly in stromal cells via nuclear estrogen receptor (ER)-ERK1/2 pathway. ICI 182,780, an ER antagonist, effectively inhibited their actions on EGR1 expression following ERK1/2 phosphorylation. Administration of pharmacological inhibitors for ERK1/2, but not AKT significantly blocked EGR1 expression induced by E2 and BPA. P4 effectively dampened action(s) of E2 and BPA on Egr1 expression via nuclear progesterone receptor. Its antagonistic effects were partially interfered with RU486 pretreatment. Interestingly, EGR1 is specifically induced in stromal cells surrounding implanting blastocyst. Collectively, our results show that through nuclear ER-dependent ERK1/2 phosphorylation, not only E2 but also endocrine disruptors with estrogenic activity such as BPA rapidly and transiently induce Egr1 which may be important for embryo implantation and decidualization in mouse uterus.

Activation of peroxisome proliferators-activated receptor δ (PPARδ) promotes blastocyst hatching in mice

Prostaglandins participate in a variety of female reproductive processes, including ovulation, fertilization, embryo implantation and parturition. In particular, maternal prostacyclin (PGI(2)) is critical for embryo implantation and the action of PGI(2) is not mediated via its G-protein-coupled membrane receptor, IP, but its nuclear receptor, peroxisome-proliferator-activated receptor δ (PPARδ). Recently, several studies have shown that PGI(2) enhances blastocyst development and/or hatching rate in vitro, and subsequently implantation and live birth rates in mice. However, the mechanism by which PGI(2) improves preimplantation embryo development in vitro remains unclear. Using molecular, pharmacologic and genetic approaches, we show that PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice. mRNAs for PPARδ, retinoid X receptor (heterodimeric partners of PPARδ) and PGI(2) synthase (PGIS) are temporally induced after zygotic gene activation, and their expression reaches maximum levels at the blastocyst stage, suggesting that functional complex of PPARδ can be formed in the blastocyst. Carbaprostacyclin (a stable analogue of PGI(2)) and GW501516 (a PPARδ selective agonist) significantly accelerated blastocyst hatching but did not increase total cell number of cultured blastocysts. Whereas U51605 (a PGIS inhibitor) interfered with blastocyst hatching, GW501516 restored U51605-induced retarded hatching. In contrast to the improvement of blastocyst hatching by PPARδ agonists, PPAR antagonists significantly inhibited blastocyst hatching. Furthermore, deletion of PPARδ at early stages of preimplantation mouse embryos caused delay of blastocyst hatching, but did not impair blastocyst development. Taken together, PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice.

Autophagic activation in vitrified-warmed mouse oocytes

Vitrification involves the use of cryoprotectants (CPAs) and liquid nitrogen (LN2), which may cause osmotic damage and cryoinjury to oocytes. Autophagy is widely recognized as a survival or response mechanism elicited by various environmental and cellular stressors. However, the induction of autophagy in vitrified-warmed oocytes has not been examined. In this work, we investigated whether the vitrification-warming process induces autophagy in mouse oocytes. Metaphase II (MII) oocytes that were vitrified and stored in LN2 for at least 2 weeks were used in the study. In RT-PCR analyses, we observed that several Atg genes such as Atg5, Atg7, Atg12, LC3a (Map1lc3a), LC3b (Map1lc3b), and Beclin1 were expressed in MII mouse oocytes. Slight reduction in mRNA levels of Atg7 and Atg12 in vitrified-warmed oocytes was noted, and expression of these genes was not significantly influenced. Confocal live imaging analysis using oocytes from GFP-LC3 transgenic mice revealed that vitrified-warmed oocytes had a significantly higher number of GFP-LC3 puncta in comparison to fresh oocytes. The expression of BECLIN1 protein was also increased in vitrified-warmed oocytes. Treatment with 3-methyladenine, an inhibitor of autophagy, did not significantly affect the rates of oocyte survival, IVF, and embryonic development after warming and IVF. The results suggest that the observed autophagic activation in vitrified-warmed oocytes is a natural adaptive response to cold stress. Collectively, we show for the first time that vitrified-warmed mouse oocytes exhibit autophagic activation during warming and that this response is not induced by CPA-containing solutions. The induction of autophagy by cold temperature is first reported herein.

Dynamic interaction of formin proteins and cytoskeleton in mouse oocytes during meiotic maturation

Formin-2 (Fmn2) nucleates actin filaments required for spindle migration during the metaphase of meiosis I in mouse oocytes. While recent studies showed that Fmn2 is involved in the formation of a dynamic actin meshwork on meiotic spindle and the migration of chromosomes, the precise location and the mechanism of action of Fmn2 in the mouse oocyte is not known. In this work, we show that Fmn2 is colocalized with spindle during metaphase I (MI) and this pattern is lost in nocodazole-treated oocytes. Fmn2 directly interacts with polymerized microtubules (MTs) in vitro via a well-conserved domain called formin homology 2 (FH2). Microinjection of mRNA encoding formin homology 1 (FH1)FH2 domains of Fmn2 into Fmn2-/- oocytes partially rescued the defect of polar body extrusion, while mRNAs encoding FH2 domain alone could not rescue the defect. mDia1 and mDia2, Diaphanous (Dia) subfamily of formin proteins, exhibit unique patterns of expression in mouse oocytes. While mDia1 is localized on meiotic spindle, mDia2 localization is confined in spindle poles similar to γ-tubulin. Collectively, our results suggest that the ability of Fmn2 to directly interact with MTs and to polymerize actins via the conserved FH1FH2 domains is crucial for chromosomal migration in MI oocytes. We also show that mDia1 and mDia2 are dynamic components of meiotic spindle and pole complex during meiotic maturation of oocytes.

Suppression of autophagic activation in the mouse uterus by estrogen and progesterone

Autophagy is a major cellular catabolic pathway tightly associated with cell survival. The involvement of autophagy in the prolonged survival of blastocysts in the uterus is well established, and it was assumed that ovarian steroid hormones - progesterone (P4) and estrogens - have important roles in the regulation of autophagy. However, information is scarce regarding whether these hormones regulate autophagy in certain hormone-responsive cellular systems. In this study, we investigated the effects of estrogen and P4 on autophagic response in the uteri of pregnant mice and in ovariectomized (OVX) mice treated with hormones. During pregnancy, autophagic response is high on days 1 and 2 when the uterus shows an inflammatory response to mating, but it subsides around the time of implantation. Dexamethasone treatment to day 1 pregnant mice reduced autophagy in the uterus. In OVX mouse uteri, estrogen or P4 reduces autophagic response within 6u200ah. Glycogen content in OVX uteri was increased by 3-methyladenine treatment, suggesting that autophagy is involved in glycogen breakdown in the hormone-deprived uterus. The classical nuclear receptor antagonists, ICI 182u200a780 or mifepristone, lead to the recovery of the autophagic response in OVX uteri. The suppression of autophagy by 17β-estradiol is inversely correlated with the accumulation of phospho-mouse target of rapamycin, and rapamycin treatment is moderately effective in the upregulation of autophagic response in OVX mouse uteri. Collectively, this study establishes that the uterine autophagy is induced in hormone-derived environment and is suppressed by hormone treatment. Uterine autophagy may have multiple functions as a responsive mechanism to acute inflammation and as an energy provider by breaking down glycogen under hormone deprivation.