Epidermal Growth Factor-like Factor Research Articles

Decreased Expression of EZH2 in Granulosa Cells Contributes to Endometriosis-Associated Infertility by Targeting IL-1R2.

The mechanism by which endometriosis, a common gynecological disease characterized by chronic pelvic pain and infertility, causes infertility remains elusive. Luteinized unruptured follicle syndrome, the most common type of ovulatory dysfunction, is a cause of endometriosis-associated infertility involving reduced numbers of retrieved and mature oocytes. Ovulation is controlled by luteinizing hormone and paracrine signals produced within the follicle microenvironment. Generally, interleukin (IL)-1β is elevated in endometriosis follicular fluid, whereby it amplifies ovulation signals by activating extracellular-regulated kinase 1/2 and CCAAT/enhancer binding protein β pathways. However, this amplification of ovulation by IL-1β does not occur in patients with endometriosis. To illuminate the mechanism of ovulatory dysfunction in endometriosis, we analyzed the effect of oxidative stress and IL-1β expression on endometriosis follicles. We found that oxidative stress decreased EZH2 expression and reduced H3K27Me3 levels in endometriosis ovarian granulosa cells (GCs). Selective Ezh2 depletion in mice ovarian GCs reduced fertility by disturbing cumulus-oocyte complex expansion and reducing epidermal growth factor-like factor expression. Gene expression and H3K27Me3 ChIP-sequencing (ChIP-Seq) of GCs revealed IL-1 receptor 2 (IL-1R2), a high-affinity IL-1β-receptor that suppresses IL-1β-mediated inflammatory cascades during ovulation, as a crucial target gene of the EZH2-H3K27Me3 axis. Moreover, IL-1β addition did not restore ovulation upon Ezh2 knockdown, indicating a vital function of IL-1R2 in endometriosis. Thus, our findings show that reducing EZH2 and H3K27Me3 in GCs suppressed ovulatory signals by increasing IL-1R2 expression, which may ultimately contribute to endometriosis-associated infertility.

Gonadotropin Dependent Neuregulin1 Signaling Regulates Luteal Cell Survival

The formation of a functional corpus luteum (CL) is an absolute requirement for reproductive success and is induced by the mid-cycle surge of luteinizing hormone (LH). The CL is a transient ovarian endocrine structure that maintains pregnancy in primate during the first trimester and in rodents during the entire pregnancy by producing steroid hormone progesterone (P4). CL growth and differentiation are tightly regulated by both survival and cell death signals, including endocrine (LH), intra-ovarian regulators, and cell-cell interactions. Neuregulin-1 (NRG1) is a member of the epidermal growth factor-like factor family that mediates it’s effect through the erythroblastoma (ErbB) family. However, the detailed mechanisms associated with the interplay of NRG1 and its receptors in CL function is not known. Therefore, we examined the role and action of NRG1 and its receptors in the gonadotropin signaling pathway that impacts CL functions. Immunocolocalization of NRG1 and ErbB2/3 in pregnant rat CL on day 14 and 21 suggest that both NRG1 and ErbB2/3 are differentially expressed in CL. Moreover, both NRG1 and ErbB2/3 are highly expressed in rat CL on day 14 compared to day 21. Furthermore, in vitro studies revealed that rat luteal cells (LCs) treated with exogenous tumor necrosis factor-α (TNFα, an inflammatory cytokine) promoted apoptosis in LCs in a dose and time-dependent manner. However, the effects of TNFα was attenuated in presence of exogenous NRG1. Under these experimental conditions, immunoblot analysis indicated that exogenous TNFα treatment in the presence of NRG1 inhibits apoptosis through increased levels of the anti-apoptotic proteins Bcl2 and Bclxl, and activation of ErbB2-ErbB3-PI3K-Akt signaling pathway. Collectively, these studies provide new insights on the NRG1-mediated anti-apoptotic mechanism in LCs through ErbB3-ErbB2-PI3K-Akt→Bcl/Bcl-xL pathway and may have important clinical implications.Acknowledgements: This study was supported in part by National Institutes of Health Grants 1 SC1 GM130544-01A1, 1SC3GM113751 and G12RR03034. This research was conducted in a facility constructed with support from the Research Facilities Improvement Grant C06RR018386 from the National Institutes of Health National Center for Research Resources.

166 Paracrine Regulation of Epidermal Growth Factor-Like Factors in Oviduct Cells and its Effect on Oocyte Maturation and Subsequent Embryo Development

Progesterone (P4) and progesterone receptor signalling appears essential for maintenance of a proper cumulus cell expansion during the oocyte maturation by regulating the epidermal growth factor-like factors (EGF-F) related pathway during the ovulatory process. It is known that expression of EGF-F including amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC) is critical for cumulus–oocyte complex (COC) expansion and resumption of meiosis. Therefore, we hypothesised that oviduct cells might be involved in nonexclusive mechanisms of actions of P4 that in turn modulate oocyte meiosis resumption by regulating the levels of EGF-F. First, we added different concentrations of P4 (0, 0.5, 1, and 2 μg mL−1) to oviduct cell culture medium and assessed the effect of P4 on expression of AREG, EREG, and BTC in oviduct cells by immunocytochemical analysis. Then, the oviduct cells were used for co-culturing under the proper concentration of P4 with porcine oocytes. The COC were randomly cultured in 3 groups: (1) culturing without oviduct cells, (2) co-culturing with oviduct cells, and (3) co-culturing with oviduct cells treated with P4. After IVM, extrusion of the 1st polar body was observed under the microscope. To evaluate embryo development competence, the matured oocytes were activated with electrical stimulus and parthenotes were cultured in porcine zygote medium-5 for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The cleavage and blastocyst formation rates were observed under the microscope to evaluate developmental competence. To count the total cell number of blastocysts, they were stained with 5 μg mL−1 of Hoechst 33342 for 10 min. The data were analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA). Values are means ± standard error of mean (P < 0.05). Significantly higher levels of EGF-F were observed in oviduct cells treated with 1 μg mL−1 progesterone. The oocyte maturation rate of co-culture group treated with P4 (80.7 ± 1.6%) was significantly higher than that of the control (69.7 ± 2.1%). There was a significant difference between co-culture treated with P4 and the control in cleavage rate (67.2 ± 2.4% and 82.0 ± 1.6%). However, no significant difference was observed between the co-culture groups. The co-culture treated with P4 group showed significantly higher rate of blastocyst formation (37.7 ± 0.8%) and total cell number of blastocyst (72.8 ± 1.0) than control and co-culture groups. In conclusion, co-culturing with oviduct cell treated with P4 improved oocyte maturation and subsequent embryo development. Thus, we suggested that oviduct cells induce the expression of EGF-F under the treatment of P4, which has a beneficial effect on porcine oocyte development. This research was supported by NRF-20142A1021187, Korea IPET (#316002-05-2-SB010), RDA (#PJ010928032017) and Research Institute for Veterinary Science, the BK21 plus program.

Gonadotropin-Dependent Neuregulin-1 Signaling Regulates Female Rat Ovarian Granulosa Cell Survival.

Mammalian ovarian follicular development and maturation of an oocyte competent to be fertilized and develop into an embryo depends on tightly regulated, spatiotemporally orchestrated crosstalk among cell death, survival, and differentiation signals through extra- and intraovarian signals, as well as on a permissive ovarian follicular microenvironment. Neuregulin-1 (NRG1) is a member of the epidermal growth factor-like factor family that mediates its effects by binding to a member of the erythroblastoma (ErbB) family. Our experimental results suggest gonadotropins promote differential expression of NRG1 and erbB receptors in granulosa cells (GCs), and NRG1 in theca cells during follicular development, and promote NRG1 secretions in the follicular fluid (FF) of rat ovaries. During the estrous cycle of rat, NRG1 and erbB receptors are differentially expressed in GCs and correlate positively with serum gonadotropins and steroid hormones. Moreover, in vitro experimental studies suggest that the protein kinase C inhibitor staurosporine (STS) causes the physical destruction of GCs by the activation of caspase-3. Exogenous NRG1 treatment of GCs delayed onset of STS-induced apoptosis and inhibited cleaved caspase-3 expressions. Moreover, exogenous NRG1 treatment of GCs alters STS-induced death by maintaining the expression of ErbB2, ErbB3, pAkt, Bcl2, and BclxL proteins. Taken together, these studies demonstrate that NRG1 is gonadotropin dependent, differentially regulated in GCs and theca cells, and secreted in ovarian FF as an intracellular survival factor that may govern follicular maturation.

Mechanisms underlying interleukin-22-induced expression of heparin-binding epidermal growth factor-like growth factor in HaCaT cells

Objective To investigate the mechanisms underlying interleukin-22（IL-22）-induced expression of heparin-binding epidermal growth factor-like growth factor（HB-EGF）in HaCaT cells. Methods Some HaCaT cells were divided into several inverention groups treated with IL-22 at concentrations of 12.5, 25, 50, 100 μg/L, respectively and a control group treated with phosphate buffer saline（PBS）. After 24-hour culture, total proteins were extracted from the HaCaT cells, and Western blot was performed to measure the expression of phosphorylated extracellular signal-regulated kinase 1/2（P-ERK1/2）in the mitogen-activated protein kinase（MAPK）-ERK1/2 pathway, as well as phosphorylated-JAK2（P-JAK2）and phosphorylated-signal transducer and activator of transcription 3（P-STAT3）in the JAK2/STAT3 pathway. In a blocking experiment, some HaCaT cells were divided into 4 groups to be treated with PBS, IL-22, PD98059（an inhibitor of MAPK-ERK1/2）combined with IL-22（PD98059 group）, AG490（an inhibitor of JAK2/STAT3）combined with IL-22（AG490 group）, respectively. After 24-hour treatment, total proteins and mRNAs were extracted from the HaCaT cells followed by Western blot and real-time quantitative reverse transcription-PCR for the measurement of protein and mRNA expressions of HB-EGF respectively. Statistical analysis was carried out with the software SPSS 16.0 by one-way analysis of variance（ANOVA）for intergroup comparisons and by Bonferroni's test for multiple comparisons. Results After treatment with IL-22 at the above 4 concentrations, the expressions of P-ERK1/2, P-JAK2 and P-STAT3 in HaCaT cells were all increased compared with the control group（all P< 0.05）. The protein and mRNA expression levels（expressed as the HB-EGF/β-actin ratio and 2-△△Ct respectively）of HB-EGF were both significantly decreased in the PD98059 group and AG490 group than in the IL-22 group（protein: 0.183 ± 0.020 and 0.199 ± 0.011 vs. 0.924 ± 0.032, F= 37.700, 36.400, respectively, both P< 0.05; mRNA: 1.034 ± 0.072 and 0.989 ± 0.038 vs. 1.844 ± 0.135, F= 11.271, 13.429, respectively, both P< 0.05）. Conclusions IL-22 can activate the MAPK-ERK1/2 and JAK2/STAT3 signaling pathways in HaCaT cells, which may contribute to IL-22-induced expression of HB-EGF in HaCaT cells. Key words: Interleukin-22; Fibroblast growth factor 1; Keratinocytes; Mitogen-activated protein kinases; Janus kinase 2; STAT3 transcription factor; Psoriasis; HaCaT cells

Regulation of ovulatory genes in bovine granulosa cells: lessons from siRNA silencing of PTGS2

Prostaglandin endoperoxide synthase-2 (PTGS2), tumour necrosis factor-alpha-induced protein-6 (TNFAIP6), pentraxin-3 (PTX3), epidermal growth factor-like factors: amphiregulin (AREG) and epiregulin (EREG) are essential for successful ovulation. In this study, we compared the induction of these ovulatory genes in bovine granulosa cells (GCs) in vivo (after LH surge) and in vitro (forskolin (FRS) treatment). These genes were markedly stimulated in GCs isolated from cows 21 h after LH-surge. In isolated GCs, FRS induced a distinct temporal profile for each gene. Generally, there was a good agreement between the in vivo and in vitro inductions of these genes except for PTX3. Lack of PTX3 induction in isolated GCs culture suggests that other follicular compartments may mediate its induction by LH. Next, to study the role of PTGS2 and prostaglandins (PGs) in the cascade of ovulatory genes, PTGS2 was silenced with siRNA. PTGS2 siRNA caused a marked and specific knockdown of PTGS2 mRNA and PGE2 production (70% compared with scrambled siRNA) in bovine GCs. Importantly, PTGS2 silencing also reduced AREG, EREG and TNFAIP6 mRNA levels but not PTX3. Exogenous PGE2 increased AREG, EREG and TNFAIP6 mRNA levels, further confirming that these genes are prostanoid dependent. A successful and specific knockdown of PTGS2 was also achieved in endometrial cells (EndoCs) expressing PTGS2. Then, cholesterol-conjugated PTGS2 (chol-PTGS2) siRNA that facilitates cells' entry was investigated. In EndoCs, but not in GCs, chol-PTGS2 siRNA succeeded to reduce PTGS2 and PGE2 levels even without transfection reagent. PTGS2 knockdown is a promising tool to critically examine the functions of PTGS2 in the reproductive tract.

Lactadherin/MFG‐E8 is essential for microglia‐mediated neuronal loss and phagoptosis induced by amyloid β

Nanomolar β-amyloid peptide (Aβ) can induce neuronal loss in culture by activating microglia to phagocytose neurons. We report here that this neuronal loss is mediated by the bridging protein lactadherin/milk-fat globule epidermal growth factor-like factor 8 (MFG-E8), which is released by Aβ-activated microglia, binds to co-cultured neurons and opsonizes neurons for phagocytosis by microglia. Aβ stimulated microglial phagocytosis, but did not opsonize neurons for phagocytosis. Aβ (250nM) induced delayed neuronal loss in mixed glial-neuronal mouse cultures that required microglia and occurred without increasing neuronal apoptosis or necrosis. This neuronal death/loss was prevented by antibodies to MFG-E8 and was absent in cultures from Mfge8 knockout mice (leaving viable neurons), but was reconstituted by addition of recombinant MFG-E8. Thus, nanomolar Aβ caused neuronal death by inducing microglia to phagocytose otherwise viable neurons via MFG-E8. The direct neurotoxicity of micromolar Aβ was not affected by MFG-E8. The essential role of MFG-E8 in Aβ-induced phagoptosis, suggests this bridging protein as a potential therapeutic target to prevent neuronal loss in Alzheimer's disease.

ADAM17 silencing by adenovirus encoding miRNA-embedded siRNA revealed essential signal transduction by angiotensin II in vascular smooth muscle cells

Small interfering RNA (siRNA) mediated gene silencing has been utilized as a powerful molecular tool to study the functional significance of a specific protein. However, due to transient gene silencing and insufficient transfection efficiency, this approach can be problematic in primary cell culture such as vascular smooth muscle cells. To overcome this weakness, we utilized an adenoviral-encoded microRNA (miRNA)-embedded siRNA “mi/siRNA”-based RNA interference. Here, we report the results of silencing a disintegrin and metalloprotease 17 (ADAM17) in cultured rat vascular smooth muscle cells and its functional mechanism in angiotensin II signal transduction. 3 distinct mi/siRNA sequences targeting rat ADAM17 were inserted into pAd/CMV/V5-DEST and adenoviral solutions were obtained. Nearly 90% silencing of ADAM17 was achieved when vascular smooth muscle cells were infected with 100 multiplicity of infection of each ADAM17 mi/siRNA encoding adenovirus for 3days. mi/siRNA-ADAM17 but not mi/siRNA-control inhibited angiotensin II-induced epidermal growth factor receptor trans-activation and subsequent extracellular signal-regulated kinase activation and hypertrophic response in the cells. mi/siRNA-ADAM17 also inhibited angiotensin II-induced heparin-binding epidermal growth factor-like factor shedding. This inhibition was rescued with co-infection of adenovirus encoding mouse ADAM17 but not by its cytosolic domain deletion mutant or cytosolic Y702F mutant. As expected, angiotensin II induced tyrosine phosphorylation of ADAM17 in the cells. In conclusion, ADAM17 activation via its tyrosine phosphorylation contributes to heparin-binding epidermal growth factor-like factor shedding and subsequent growth promoting signals induced by angiotensin II in vascular smooth muscle cells. An artificial mi/siRNA-based adenoviral approach appears to be a reliable gene-silencing strategy for signal transduction research in primary cultured vascular cells.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus–oocyte complexes

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2.

Effects of heparin-binding epidermal growth factor-like growth factor on mitochondrial pathway of apoptosis in neonatal rats with necrotizing enterocolitis

Objective To investigate the effect of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on mitochondrial pathway of apoptosis in rats with neonatal necrotizing enterocolitis (NEC).Methods Sprague-Dawley neonatal rats were randomly divided into three groups with ten in each.NEC group rats were formula fed,and hypoxia exposed by 100％ N2 for 90 s and cold stress at 4 ℃ for 10 min twice a day for three days.Additionally,rats in HB-EGF group received HB-EGF 800μg/kg by gavage four times a day for three days.Rats in control group were given breast milk feeding for three days without any interventions.Seventy-two hours after born,all neonatal rats were sacrificed after fasting for 12 h,from which the terminal ileum was removed.HE-staining was done for histologic evaluation.Mitochondrial ultrastructure was observed under electron microscopy.Cytochrome C was detected by immunohistochemical analysis and apoptosis inducing factor (AIF) and apoptotic protease activating factor-1 (APAF-1) were measured by Western blot.Analysis of variance and q-test were used to compare the difference among groups.Results (1) The incidence of NEC in HB-EGF group was lower than that in NEC group (2/10 vs 9/10,x2 =7.27,P＜0.01).(2) In NEC group,mitochondria in epithelial cells and muscle cells of intestine were significantly swelling,appearing many electron-lucent zones in matrix.Ultrastructure of mitochondria were severely damaged.In HB-EGF group,mitochondria were less swelling and showed milder damage than those in NEC group.(3) The expression of cytochrome C in ileal tissue in NEC group was higher than that in control group (0.030±0.018 vs 0.002±0.001,q=6.15,P＜0.01).The expression of cytoehrome C in ileal tissue in HB-EGF group was lower than that in NEC group (0.014±0.018 vs 0.030±0.018,q=3.53,P＜0.05).The expression of APAF-1 and AIF in NEC group was higher than those in control group (1.364±0.299 vs 0.215±0.033,q=15.31,P＜0.05;0.181±0.050 vs0.127±0.045,q=3.71,P＜0.05).Compared to NEC group,the expression of APAF-1 was lower (0.455±0.123 vs 1.364±0.299,q=4.04,P＜0.05) and the expression of AIF was higher (0.289±0.045 vs 0.181±0.050,q=7.32,P＜0.05) in HB-EGF group.Conclusions HB-EGF could reduce the incidence of NEC in neonatal rats by inhibiting the mitochondrial pathway related apoptosis through down regulation of APAF-1. Key words: Enterocolitis, necrotizing; Intercellular signaling peptides and proteins; Apoptotic protease-activating factor 1 ; Apoptosis ; Mitochondria ; Rats

Selective Smad4 Knockout in Ovarian Preovulatory Follicles Results in Multiple Defects in Ovulation

The TGF-β signaling pathway is involved with multiple processes in the mammalian ovary, including primordial follicle formation, granulosa cell (GC) proliferation, follicle atresia, ovulation, and feedback regulation between the pituitary and ovary. The transcriptional factor SMAD4 (Sma- and Mad-related protein 4) is the central component of the canonical TGF-β signaling pathway. Smad4 knockout (KO) using Amhr2-Cre, which is expressed in GCs of immature developing follicles, causes premature luteinization. In this study, we specifically depleted Smad4 in GCs of preovulatory follicles using Cyp19-Cre mice. As different from results with Smad4(fl/fl);Amhr2-Cre mice, Smad4 depletion in preovulatory follicles did not cause premature luteinization or suppress GC proliferation; rather, it increased follicle atresia. In addition, Nppc and Npr2 expressions were reduced by Smad4 depletion; thus, their effect of maintaining oocyte meiotic arrest was weakened in Smad4 conditional KO mice. Smad4(fl/fl);Cyp19-Cre female mice were subfertile and had irregular estrous cycles and ovulation defects. Smad4 KO also blocked LH-induced cumulus expansion and follicle rupture, but not oocyte meiotic resumption. Our results also indicated that SMAD4 was required for LH-stimulated activation of ERK1/2 and the expressions of ovulation-related genes. The defects arising from SMAD4 depletion could not be rescued by intraovarian mediators of LH actions, such as epidermal growth factor-like factors and prostaglandin E2. Furthermore, corpus lutea did not form in Smad4(fl/fl);Cyp19-Cre female mice, indicating that SMAD4 was crucial for GCs terminal differentiation. Thus, by characterizing the ovarian phenotypes of preovulatory follicle-specific Smad4 KO mice, we identified the developmental stage-specific functions of the canonical TGF-β signaling pathway in ovulation and luteinization.

Androgen/androgen receptor pathway regulates expression of the genes for cyclooxygenase-2 and amphiregulin in periovulatory granulosa cells

It is well known that the androgen/androgen receptor (AR) pathway is involved in both male and female fertility in mammals. AR knockout female mice are reported to exhibit various abnormalities in follicle development, and a subfertile phenotype. In exogenous gonadotropin-induced superovulation, serum androgen levels were robustly elevated in female mice at the periovulatory stage after human chorionic gonadotropin (hCG) treatment. At this stage, ovarian AR proteins were strongly expressed in cumulus cells. Because these results suggested that the androgen/AR pathway is involved in ovulation, we investigated the expression of ovulation-related genes in the mouse ovary treated with the nonaromatizable androgen, 5α-dihydrotestosterone (DHT). DHT treatment induced the expression of the genes for cyclooxyganase-2 (Cox-2 or prostaglandin endoperoxidase synthase 2) and the epidermal growth factor-like factor, amphiregulin (Areg), in the ovary, whereas their hCG-induced expression was suppressed by the AR antagonist flutamide. These genes were also induced by DHT in AR-expressing primary granulosa and granulosa tumor-derived cells. Reporter assays, electrophoretic shift mobility assays and chromatin immunoprecipitation assays demonstrated that androgen response sequence(s) existing upstream of each gene were responsible for androgen responsiveness and were occupied by the AR in periovulatory granulosa cells. Our results suggest that the androgen/AR pathway is involved in the ovulatory process via expression of the Cox-2 and Areg genes in periovulatory granulosa cells.

How to Tailor Giant Proteoliposomes

In this project we address the challenge of incorporation of transmembrane proteins with defined orientation in biomimetic giant unilamellar vesicles (GUVs). The reconstitution of biologically relevant transmembrane proteins, like receptors or channel proteins, into GUVs makes them easily accessible to biophysical investigation using patch-clamp or optical microscopy while preserving their natural configuration. Our strategy combines two approaches: cell-free eukaryotic protein expression and biomimetic model systems. The cell-free protein expression system is based on insect lysates [1]. It provides endoplasmic reticulum (ER)-based vesicles which enable signal-induced translocation and post-translational modification, e.g. glycosylation and phosphorylation. Further the expressed protein is inserted into the ER membrane corresponding to the natural process in a living cell. Starting from small ER vesicles of approximately one micrometer diameter we applied electro-swelling to achieve giant proteoliposomes [2]. Our recent work showed that the efficiency of this method can be improved substantially by the presence of synthetic lipids in the electro-swelling process. As an example, we introduced the one-transmembrane protein heparin-binding epidermal growth factor-like factor Hb-EGF-eYFP in GUV membranes aided by the lipid DOPC. We applied single-molecule fluorescence microscopy to detect and localize the protein, and characterized their mobility in the mixed natural-synthetic membrane. In addition, we introduced biotinylated lipids that enabled us to immobilize the protein-decorated GUVs to streptavidin coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces. [1] Kubick et al. Current Topics in Membranes 09. [2] Shaklee et al. ChemBioChem 10.

β-Catenin (CTNNB1) Promotes Preovulatory Follicular Development but Represses LH-Mediated Ovulation and Luteinization

Wingless-type mouse mammary tumor virus integration site family (WNT)/beta-catenin (CTNNB1) pathway components are expressed in ovarian granulosa cells, direct female gonad development, and are regulated by the pituitary gonadotropins. However, the in vivo functions of CTNNB1 during preovulatory follicular development, ovulation, and luteinization remain unclear. Using a mouse model Ctnnb1((Ex3)fl/fl);Cyp19-Cre (Ctnnb1((Ex3)gc-/-)), expressing dominant stable CTNNB1 in granulosa cells of small antral and preovulatory follicles, we show that CTNNB1 facilitates FSH-induced follicular growth and decreases the follicle atresia (granulosa cell apoptosis). At the molecular level, WNT signaling and FSH synergistically promote the expression of genes required for cell proliferation and estrogen biosynthesis, but decrease FOXO1, which negatively regulates proliferation and steroidogenesis. Conversely, dominant stable CTNNB1 represses LH-induced oocyte maturation, ovulation, luteinization, and progesterone biosynthesis. Specifically, granulosa cells in the Ctnnb1((Ex3)gc)(-/-) mice showed compromised responses to the LH surge and decreased levels of the epidermal growth factor-like factors (Areg and Ereg) that in vivo and in vitro mediate LH action. One underlying mechanism by which CTNNB1 prevents LH responses is by reducing phosphorylation of cAMP-responsive element-binding protein, which is essential for the expression of Areg and Ereg. By contrast, depletion of Ctnnb1 using the Ctnnb1(fl/fl);Cyp19-Cre mice did not alter FSH regulation of preovulatory follicular development or female fertility but dramatically enhanced LH induction of genes in granulosa cells in culture. Thus, CTNNB1 can enhance FSH and LH actions in antral follicles but overactivation of CTNNB1 negatively effects LH-induced ovulation and luteinization, highlighting the cell context-dependent and developmental stage-specific interactions of WNT/CTNNB1 pathway and G protein-coupled gonadotropin receptors in female fertility.

Prophase I arrest and progression to metaphase I in mouse oocytes: comparison of resumption of meiosis and recovery from G2-arrest in somatic cells

Mammalian oocytes are arrested at prophase I until puberty when luteinizing hormone (LH) induces resumption of meiosis of follicle-enclosed oocytes. Resumption of meiosis is tightly coupled with regulating cyclin-dependent kinase 1 (CDK1) activity. Prophase I arrest depends on inhibitory phosphorylation of CDK1 and anaphase-promoting complex-(APC-CDH1)-mediated regulation of cyclin B levels. Prophase I arrest is maintained by endogenously produced cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) that in turn phosphorylates (and activates) the nuclear kinase WEE2. In addition, PKA-mediated phosphorylation of the phosphatase CDC25B results in its cytoplasmic retention. The combined effect maintains low levels of CDK1 activity that are not sufficient to initiate resumption of meiosis. LH triggers synthesis of epidermal growth factor-like factors in mural granulosa cells and leads to reduced cGMP transfer from cumulus cells to oocytes via gap junctions that couple the two cell types. cGMP inhibits oocyte phosphodiesterase 3A (PDE3A) and a decline in oocyte cGMP results in increased PDE3A activity. The ensuing decrease in oocyte cAMP triggers maturation by alleviating the aforementioned phosphorylations of WEE2 and CDC25B. As a direct consequence CDC25B translocates into the nucleus. The resulting activation of CDK1 also promotes extrusion of WEE2 from the nucleus thereby providing a positive amplification mechanism for CDK1 activation. Other kinases, e.g. protein kinase B, Aurora kinase A and polo-like kinase 1, also participate in resumption of meiosis. Mechanisms governing meiotic prophase I arrest and resumption of meiosis share common features with DNA damage-induced mitotic G2-checkpoint arrest and checkpoint recovery, respectively. These common features include CDC14B-dependent activation of APC-CDH1 in prophase I arrested oocytes or G2-arrested somatic cells, and CDC25B-dependent cell cycle resumption in both oocytes and somatic cells.

Prospective evaluation of candidate urine and cell markers in patients with interstitial cystitis enrolled in a randomized clinical trial of Bacillus Calmette Guerin (BCG)

We measured candidate urine biomarkers and bladder cell DNA cytometry in interstitial cystitis (IC) patients randomized to receive intravesical Bacillus Calmette Guerin (BCG) or placebo in a multicenter trial. Participants received 6 weekly instillations and were followed for 34 weeks. Urine was collected at baseline, prior to fourth treatment, and at study end. Antiproliferative factor (APF) activity was determined by 3H-thymidine incorporation assay; heparin-binding epidermal growth factor-like growth factor (HB-EGF) and epidermal growth factor-like growth factor (EGF) levels were determined by ELISA. Cellular DNA content was measured by image analysis to determine the mean hyperdiploid fraction (HDF) of the urine cell pellet. Associations between marker levels, and treatment or symptoms, were examined. Baseline APF positivity rate and mean levels of the other biomarkers were similar to previous smaller studies. During the week 34 follow-up, mean HDF decreased (P = 0.0003) and HB-EGF increased (P < 0.0001); both correlated weakly with decreased urgency. There was no difference in any biomarker between symptom responders and non-responders, but the percentage of responders was low and not significantly different for BCG versus placebo. APF positivity, decreased HB-EGF, increased EGF, and increased HDF were confirmed at baseline in IC patients. Changes in HDF and HB-EGF levels correlated weakly with changes in urgency, but the low BCG response rate prevented identification of additional associations between biomarker changes and treatment or symptoms.

Paracrine and Autocrine Regulation of Epidermal Growth Factor-Like Factors in Cumulus Oocyte Complexes and Granulosa Cells: Key Roles for Prostaglandin Synthase 2 and Progesterone Receptor

The molecular bridges that link the LH surge with functional changes in cumulus cells that possess few LH receptors are being unraveled. Herein we document that epidermal growth factor (EGF)-like factors amphiregulin (Areg), epiregulin (Ereg), and betacellulin (Btc) are induced in cumulus oocyte complexes (COCs) by autocrine and paracrine mechanisms that involve the actions of prostaglandins (PGs) and progesterone receptor (PGR). Areg and Ereg mRNA and protein levels were reduced significantly in COCs and ovaries collected from prostaglandin synthase 2 (Ptgs2) null mice and Pgr null (PRKO) mice at 4 h and 8 h after human chorionic gonadotropin, respectively. In cultured COCs, FSH/forskolin induced Areg mRNA within 0.5 h that peaked at 4 h, a process blocked by inhibitors of p38MAPK (SB203580), MAPK kinase (MEK) 1 (PD98059), and PTGS2 (NS398) but not protein kinase A (PKA) (KT5720). Conversely, AREG but not FSH induced Ptsg2 mRNA at 0.5 h with peak expression of Ptgs2 and Areg mRNAs at 4 h, processes blocked by the EGF receptor tyrosine kinase inhibitor AG1478 (AG), PD98059, and NS398. PGE2 reversed the inhibitory effects of AG on AREG-induced expression of Areg but not Ptgs2, placing Ptgs2 downstream of EGF-R signaling. Phorbol 12-myristate 13-acetate (PMA) and adenovirally expressed PGRA synergistically induced Areg mRNA in granulosa cells. In COCs, AREG not only induced genes that impact matrix formation but also genes involved in steroidogenesis (StAR, Cyp11a1) and immune cell-like functions (Pdcd1, Runx1, Cd52). Collectively, FSH-mediated induction of Areg mRNA via p38MAPK precedes AREG induction of Ptgs2 mRNA via ERK1/2. PGs acting via PTGER2 in cumulus cells provide a secondary, autocrine pathway to regulate expression of Areg in COCs showing critical functional links between G protein-coupled receptor and growth factor receptor pathways in ovulating follicles.

Heparin-binding epidermal growth factor-like growth factor and intestinal ischemia-reperfusion injury

Intestinal ischemia/reperfusion (I/R) injury affects patients of different ages, especially premature babies and the elderly. The outcome after intestinal I/R is often dismal, which may be attributed to loss of the barrier and immune functions of the intestines, as well as development of secondary injury in remote organs. The available treatment for advanced gut ischemia mandates extensive resection, which may cause growth retardation in infants and nutritional problems in the elderly. Throughout the past decade we have been investigating the potential therapeutic role of heparin-binding epidermal growth factor-like factor (HB-EGF) in intestinal I/R. The mitogenic and chemoattractant functions of HB-EGF formed the initial rationale for our investigations. In addition, HB-EGF is a potent antiapoptotic protein that enables cells and tissues exposed to different apoptotic stimuli to survive hypoxic, oxidative, and nutritional stresses. HB-EGF is known to have a vital role in wound healing and postischemic regeneration in different organs. In the current review, we summarize the results of our findings of the beneficial effects of HB-EGF in intestinal I/R, supported by additional evidence from the literature and an explanation of different possible mechanisms of its actions. Collectively, the data strongly suggest a potential therapeutic role for the use of HB-EGF to treat intestinal ischemic diseases such as I/R and necrotizing enterocolitis.

Involvement of epidermal growth factor receptor in chemically induced mouse bladder tumour progression.

This study was designed to investigate the role of the epidermal growth factor receptor (EGFR) and its ligands in chemically induced mouse bladder cancer. Bladder tumours were induced in C57Bl/6 and B6D2F1 mice by treatment with the carcinogen, N:-butyl-N:-(4-hydroxybutyl) nitrosamine (BBN). The levels of mRNA for EGFR and its ligands were analysed by reverse transcription-polymerase chain reaction (RT-PCR) in bladder tumours and in normal bladder urothelia. EGFR mRNA was detected in all tumours, transforming growth factor alpha (TGFalpha) mRNA levels were similar to those in normal bladder urothelia or were decreased and mRNA levels for amphiregulin, heparin-binding epidermal growth factor-like factor (HB-EGF) and betacellulin were significantly higher than those in normal urothelia. Seven cell lines were derived from chemically induced tumours. These cell lines were able to grow in serum-free conditions. All the cell lines tested expressed the genes encoding EGFR and at least one of its ligands. Proliferation of these cell lines was inhibited by AG1478, a specific EGFR tyrosine kinase inhibitor, strongly suggesting that EGFR was involved in cell growth. As expected, EGFR was found to be phosphorylated in serum-free medium, this phosphorylation being inhibited by AG1478. Conditioned medium of a bladder cancer cell line had EGFR-stimulating activity and an antibody directed against EGFR inhibited proliferation by 45%. This suggests that tumour cell growth is stimulated by an autocrine loop involving EGFR and secreted growth factors. AG1478 decreased the expression of genes for amphiregulin, HB-EGF and betacellulin, showing that EGFR activation induces up-regulation of the EGFR ligands. These results suggest that EGFR plays a critical role in bladder tumour progression.

Characterization of a factor(s) from partially purified human gonadotrophin preparations which inhibit(s) the binding of radiolabelled human LH and human chorionic gonadotrophin to Candida albicans

We have shown previously that partially purified human chorionic gonadotrophin (hCG) preparations inhibited the specific binding of 125I-labelled hLH or hCG to Candida albicans membranes at much lower concentrations than did highly purified hLH or hCG preparations. We now describe the characterization and partial purification of a heat-labile glycoprotein from commercially available gonadotrophin preparations. The factor strongly inhibited LH binding to Candida membranes, but not to sheep or pig luteal LH receptors. This material had a molecular weight of 16,000-21,000 daltons, bound strongly to CM-Sepharose at physiological pH, and could be resolved completely from hCG and from epidermal growth factor-like factors present in commercial gonadotrophin preparations. Its activity was not attenuated by a range of inhibitors specific for the four major classes of proteolytic enzymes, nor did it inhibit hormone binding by causing degradation of 125I-labelled hLH or hCG tracers. Factors which inhibited hLH binding to Candida membranes were also present in partially purified human urinary and equine serum gonadotrophin preparations and in placental extracts, but were not detected in highly purified CG of hLH preparations. The properties of this factor were similar to those described for beta-core protein, a cleavage product of the beta subunit of hCG which is a contaminant of commercial gonadotrophin preparations. Highly purified beta-core protein inhibited 125I-labelled hLH binding to Candida membranes, but not to sheep luteal binding sites. Preparations of hCG depleted of inhibitor activity could stimulate adenylate cyclase activity in Candida membranes almost five fold. In contrast, partially purified inhibitor preparations strongly inhibited basal adenylate cyclase activity (to 18% of control levels). These observations suggest that endogenous LH-like factors, perhaps similar to beta-core proteins of hCG, may play a role in the regulation of morphogenesis in Candida species.