Exogenous Epidermal Growth Factor Research Articles

Abstract 2029: Notch signaling inhibitor increases E-selectin/ligand interactions and alters cell migration of mesenchymal-like breast cancer cells

Abstract Activation of Notch signaling has been shown to promote cell migration properties and hematogenous metastasis by inducing a dramatic phenotypic change in breast cancer cells via the epithelial-to-mesenchymal transition (EMT). In response to these findings, Notch signaling inhibitors are being explored as therapeutics to prevent EMT in cancers. However, research examining the effects of Notch inhibitors on the activity of sialofucosylated ligands used by circulating tumor cells to adhere to vascular endothelial E-selectin during metastasis is lacking. To advance knowledge in this area, our study investigated the use of an indirect Notch signaling inhibitor (DAPT) on modifying E-selectin ligand activity of breast cancer cell lines by shear flow adhesion assays performed at physiological bone marrow shear stress. DAPT exposure to mesenchymal-like MDA-MB-231 and MDA-MB-468 (induced to the mesenchymal-like state by exogenous epidermal growth factor) breast cancer cells increased E-selectin ligand activity. Furthermore, mesenchymal-like MDA-MB-468 cells after DAPT exposure demonstrated E-selectin ligand activity similar to that of epithelial-like MDA-MB-468 cells. This supports the hypothesis by our group that epithelial-like and mesenchymal-like phenotypes determine activity of functional E-selectin ligands in breast cancer cells, as we have previously shown that epithelial cells display high E-selectin ligand activity while mesenchymal cells display little or no ligand activity. Cell migration ability was assessed using wound closure assays. Mesenchymal-like MDA-MB-468 cells exposed to DAPT exhibited decreased migration compared to mesenchymal-like MDA-MB-468 cells exposed to the DMSO diluent control, whereas mesenchymal-like MDA-MB-231 cells demonstrated no difference in migration compared to DMSO exposed cells. Altogether, our results suggest Notch inhibitors may both increase fluid-shear resistant adhesion and alter migration ability of mesenchymal-like breast cancer cells, and that factors present in the blood might impact the metastatic potential of cancer cells via regulating cellular phenotypes. Citation Format: Christian A. Showalter, Monica M. Burdick. Notch signaling inhibitor increases E-selectin/ligand interactions and alters cell migration of mesenchymal-like breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2029.

Hypoxic-induction of arginase II requires EGF-mediated EGFR activation in human pulmonary microvascular endothelial cells.

We have previously shown that hypoxia‐induced proliferation of human pulmonary microvascular endothelial cells (hPMVEC) depends on arginase II, and that epidermal growth factor receptor (EGFR) is necessary for hypoxic‐induction of arginase II. However, it remains unclear how hypoxia activates EGFR‐mediated signaling in hPMVEC. We hypothesized that hypoxia results in epidermal growth factor (EGF) production and that EGF binds to EGFR to activate the signaling cascade leading to arginase II induction and proliferation in hPMVEC. We found that hypoxia significantly increased the mRNA levels of EGF, EGFR, and arginase in hPMVEC. Hypoxia significantly increased pEGFR(Tyr845) protein levels and an EGF neutralizing antibody prevented the hypoxic induction of pEGFR. Inhibiting EGFR activation prevented hypoxia‐induced arginase II mRNA and protein induction. Treatment of hPMVEC with exogenous EGF resulted in greater levels of arginase II protein both in normoxia and hypoxia. An EGF neutralizing antibody diminished hypoxic induction of arginase II and resulted in fewer viable cells in hPMVEC. Similarly, siRNA against EGF prevented hypoxic induction of arginase II and resulted in fewer viable cells. Finally, conditioned media from hypoxic hPMVEC induced proliferation in human pulmonary artery smooth muscle cells (hPASMC), however, conditioned media from a group of hypoxic hPMVEC in which EGF were knocked down did not promote hPASMC proliferation. These findings demonstrate that hypoxia‐induced arginase II expression and cellular proliferation depend on autocrine EGF production leading to EGFR activation in hPMVEC. We speculate that EGF‐EGFR signaling may be a novel therapeutic target for pulmonary hypertensive disorders associated with hypoxia.

Photobiomodulation at 660 nm stimulates proliferation and migration of diabetic wounded cells via the expression of epidermal growth factor and the JAK/STAT pathway

Photobiomodulation (PBM) modulates cellular processes to enhance diabetic wound healing. The photon energy activates wounded cells to proliferate and migrate. However, the signalling pathways responsible for these observations remain unknown. This study aimed to determine if PBM stimulates cellular proliferation and migration via the expression of epidermal growth factor (EGF) and activation of the Janus kinase/Signal transducer and activators of transcription (JAK/STAT) signalling pathway. Normal, wounded, diabetic and diabetic wounded cell models were exposed to PBM at a wavelength of 660nm and fluence of 5J/cm2 and incubated for 48h. Non-irradiated cells (0J/cm2) and cells exposed to exogenous EGF (rh EGF) served as controls. Cellular migration was determined microscopically at 0, 24 and 48h. Flow cytometry (BrdU) was used to determine cell proliferation, while the Trypan blue exclusion assay and adenosine triphosphate (ATP) luminescence was used to determine cell viability. The enzyme linked immunosorbent assay (ELISA) was used to analyse EGF expressed in the culture media, and phosphorylated (p-) EGF receptor (p-EGFR), p-JAK2, p-STAT1 and p-STAT5 in cells. Irradiated diabetic wounded cells showed a significant increase in EGF, and activation of its receptor (p-EGFR) and JAK/STAT (p-JAK2, p-STAT1 and p-STAT5). PBM at 660nm and 5J/cm2 is able to modulate cellular autocrine signalling, particularly the EGF/EGFR loop leading to activation of the JAK/STAT pathway which in turn stimulates cell proliferation and migration.

Growth performance and gastrointestinal responses of broiler chickens fed corn-soybean meal diet without or with exogenous epidermal growth factor upon challenge with Eimeria

Epidermal growth factor (EGF), a protein known for its mitogenic and anti-apoptotic effects was fed to broiler chickens to evaluate growth performance, gastrointestinal measurements, and apparent retention (AR) of components upon challenge with Eimeria. A total of 216, d old male broiler chicks (Ross 708) were placed in cages (6 birds/cage) and allocated to treatments. The treatments were: 1) control (Lactotobacilli lactis fermentation supernatant without EGF), 2) 80 μg of EGF/kg BW/d, and 3) 160 μg of EGF/kg BW/d. A basal antibiotic-free corn-soybean diet containing TiO2 was used. Birds were offered fresh feed with respective treatments on daily basis and had free access to drinking water for 14 d. On d 5, birds (6 replicates per treatment) were challenged with 1 mL of E. acervulina and E. maxima mixture via oral gavage and the other 6 replicates were given sham. Growth performance was measured in pre- (d 0 to 5) and post- (d 6 to 14) challenge periods. Two birds per cage were necropsied on d 10 for intestinal lesion scores and tissue samples for histomorphology and expression of select intestinal genes. Excreta samples for AR of components and oocyst shedding were taken d 10 to 13 and all birds were necropsied on d 14 for gastrointestinal weight. The EGF linearly (P < 0.05) increased BWG before challenge. There was no EGF and Eimeria interaction (P > 0.05) on growth performance, AR of GE, and intestinal histomorphology; the main effects were such that Eimeria depressed (P < 0.01) BWG, FCR, AR of DM, crude fat, and GE, and villi height to crypt depth ratio. An interaction between EGF and Eimeria (P < 0.05) on indices of gut function was such that EGF improved expression of genes for nutrient transporters and tight junction proteins in Eimeria challenged birds whilst no effect in non-challenged control. In conclusion, Eimeria challenge reduced growth performance and impaired gut function; EGF showed beneficial effects on growth pre-challenge and improved indices of gut function upon Eimeria challenge.

The non-small cell lung cancer EGFR extracellular domain mutation, M277E, is oncogenic and drug-sensitive.

PurposeTo identify novel oncogenic mutations in non-small cell lung cancer patient specimens that lack mutations in known targetable genes (“pan-negative” patients).MethodsComprehensive mutational analyses were performed on 1,356 lung adenocarcinoma specimens. In this cohort of patients, common lung cancer oncogenic driver mutations were detected in the epidermal growth factor receptor (EGFR) kinase domain, the human epidermal growth factor receptor 2 kinase domain, as well as the KRAS, BRAF, ALK, ROS1 and RET genes. A sub-cohort of pan-negative patient specimens was assayed for mutations in the EGFR extracellular domain (ECD). Additionally, EGFR mutant NIH-3T3 stable cell lines were constructed and assessed for protein content, anchorage-independent growth, and tumor formation in xenograft models to identify oncogenic mutations. BaF3 lymphocytes were also used to test sensitivities of the mutations to tyrosine kinase inhibitors.ResultsIn pan-negative lung adenocarcinoma cases, a novel oncogenic EGFR ECD mutation was identified (M277E). EGFR M277E mutations encoded oncoproteins that transformed NIH-3T3 cells to grow in the absence of exogenous epidermal growth factor. Transformation was further evidenced by anchorage-independent growth and tumor formation in immunocompromised xenograft mouse models. Finally, as seen in the canonical EGFR L858R mutation, the M277E mutation conferred sensitivity to both erlotinib and cetuximab in BaF3 cell lines and to erlotinib in xenograft models.ConclusionHere, a new EGFR driver mutation, M277E, was identified in the ECD of a lung adenocarcinoma specimen. For patients with M277E-mutant lung adenocarcinoma who experienced disease recurrence, treatment with an EGFR tyrosine kinase inhibitor may predict good prognosis.

Epidermal growth factor signals attenuate phenotypic and functional development of neocortical GABA neurons.

Phenotypic development of neocortical GABA neurons is highly plastic and promoted by various neurotrophic factors such as neuregulin-1. A subpopulation of GABA neurons expresses not only neuregulin receptor (ErbB4) but also epidermal growth factor (EGF) receptor (ErbB1) during development, but the neurobiological action of EGF on this cell population is less understood than that of neuregulin-1. Here, we examined the effects of exogenous EGF on immature GABA neurons both in culture and invivo and also explored physiological consequences in adults. We prepared low density cultures from the neocortex of rat embryos and treated neocortical neurons with EGF. EGF decreased protein levels of glutamic acid decarboxylases (GAD65 and GAD67), and EGF influences on neuronal survival and glial proliferation were negligible or limited. The EGF treatment also diminished the frequency of miniature inhibitory postsynaptic currents (mIPSCs). In vivo administration of EGF to mouse pups reproduced the above GABAergic phenomena in neocortical culture. In EGF-injected postnatal mice, GAD- and parvalbumin-immunoreactivities were reduced in the frontal cortex. In addition, postnatal EGF treatment decreased mIPSC frequency in, and the density of, GABAergic terminals on pyramidal cells. Although these phenotypic influences on GABA neurons became less marked during development, it later resulted in the reduced β- and γ-powers of sound-evoked electroencephalogram in adults, which is regulated by parvalbumin-positive GABA neurons and implicated in the schizophrenia pathophysiology. These findings suggest that, in contrast to the ErbB4 ligand of neuregulin-1, the ErbB1 ligand of EGF exerts unique maturation-attenuating influences on developing cortical GABAergic neurons.

Growth factor functionalized biomaterial for drug delivery and tissue regeneration.

Elastin like polypeptides (ELPs) are a class of naturally derived and non-immunogenic biomaterials that are widely used in drug delivery and tissue engineering. ELPs undergo temperature-mediated inverse phase transitioning, which allows them to be purified in a relatively simple manner from bacterial expression hosts. Being able to genetically encode ELPs allows for the incorporation of bioactive peptides thereby functionalizing them. Here we report the synthesis of a biologically active epidermal growth factor-ELP (EGF-ELP) fusion protein that could aid in wound healing. EGF plays a crucial role in wound healing by inducing cell proliferation and migration. The use of exogenous EGF has seen success in the treatment of acute wounds, but has seen relatively minimal success in chronic wounds because the method of delivery does not prevent it from diffusing away from the application site. Our data shows that EGF-ELP retained the biological activity of EGF and the phase transitioning property of ELP. Furthermore, the ability of the EGF-ELP to self-assemble near physiological temperatures could allow for the formation of drug depots at the wound site and minimize diffusion, increasing the bioavailability of EGF and enhancing tissue regeneration.

Interdependency of EGF and GLP-2 Signaling in Attenuating Mucosal Atrophy in a Mouse Model of Parenteral Nutrition.

Background & AimsTotal parenteral nutrition (TPN), a crucial treatment for patients who cannot receive enteral nutrition, is associated with mucosal atrophy, barrier dysfunction, and infectious complications. Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) improve intestinal epithelial cell (IEC) responses and attenuate mucosal atrophy in several TPN models. However, it remains unclear whether these 2 factors use distinct or overlapping signaling pathways to improve IEC responses. We investigated the interaction of GLP-2 and EGF signaling in a mouse TPN model and in patients deprived of enteral nutrition.MethodsAdult C57BL/6J, IEC-Egfrknock out (KO) and IEC-pik3r1KO mice receiving TPN or enteral nutrition were treated with EGF or GLP-2 alone or in combination with reciprocal receptor inhibitors, GLP-2(3-33) or gefitinib. Jejunum was collected and mucosal atrophy and IEC responses were assessed by histologic, gene, and protein expression analyses. In patients undergoing planned looped ileostomies, fed and unfed ileum was analyzed.ResultsEnteral nutrient deprivation reduced endogenous EGF and GLP-2 signaling in mice and human beings. In the mouse TPN model, exogenous EGF or GLP-2 attenuated mucosal atrophy and restored IEC proliferation. The beneficial effects of EGF and GLP-2 were decreased upon Gefitinib treatment and in TPN-treated IEC-EgfrKO mice, showing epidermal growth factor–receptor dependency for these IEC responses. By contrast, in TPN-treated IEC-pi3kr1KO mice, the beneficial actions of EGF were lost, although GLP-2 still attenuated mucosal atrophy.ConclusionsUpon enteral nutrient deprivation, exogenous GLP-2 and EGF show strong interdependency for improving IEC responses. Understanding the differential requirements for phosphatidylinositol 3-kinase/phosphoAKT (Ser473) signaling may help improve future therapies to prevent mucosal atrophy.

Nuclear transportation of exogenous epidermal growth factor receptor and androgen receptor via extracellular vesicles

Epidermal growth factor receptor (EGFR) plays a central role in the progression of several human malignancies. Although EGFR is a membrane receptor, it undergoes nuclear translocation, where it has a distinct signalling pathway. Herein, we report a novel mechanism by which cancer cells can directly transport EGFR to the nucleus of other cells via extracellular vesicles (EVs). The transported receptor is active and stimulates the nuclear EGFR pathways. Interestingly, the translocation of EGFR via EVs occurs independently of the nuclear localisation sequence that is required for nuclear translocation of endogenous EGFR. Also, we found that the mutant receptor EGFRvIII could be transported to the nucleus of other cells via EVs. To assess the role of EVs in the regulation of an actual nuclear receptor, we studied the regulation of androgen receptor (AR). We found that full-length AR and mutant variant ARv7 are secreted in EVs derived from prostate cancer cell lines and could be transported to the nucleus of AR-null cells. The EV-derived AR was able to bind the androgen-responsive promoter region of prostate specific antigen, and recruit RNA Pol II, an indication of active transcription. The nuclear-translocated AR via EVs enhanced the proliferation of acceptor cells in the absence of androgen. Finally, we provide evidence that nuclear localisation of AR could occur in vivo via orthotopically-injected EVs in male SCID mice prostate glands. To our knowledge, this is the first study showing the nuclear translocation of nuclear receptors via EVs, which significantly extends the role of EVs as paracrine transcriptional regulators.

Alginate microcapsules co-embedded with MSCs and anti-EGF mAb for the induction of hair cell-like cells in guinea pigs by taking advantage of host EGF.

As the irreversibility of hair cell loss in mammals is among the main reasons giving rise to permanent hearing loss, studies have been focused on the development of biological technologies to generate new hair cells as a means of replacing lost hair cells. Bone marrow-derived mesenchymal stem cells (BMSCs) possess the capacity to differentiate into hair cell-like cells, and may find applications in the regeneration of mammalian cochlear hair cells. In order to efficiently induce BMSCs into hair cells, alginate microcapsules co-delivering rat bone marrow-derived mesenchymal stem cells (rBMSCs) and anti-EGF monoclonal antibody (mAb) were developed to examine the feasibility of differentiating rBMSC into hair cell-likes cells in vitro and in vivo by taking advantage of epidermal growth factor (EGF) ligands. In vitro analysis showed that anti-EGF mAbs bonded with exogenous EGF ligands activated the EGF receptors on rBMSCs, enhancing the expression of myosin 7a (a hair cell marker) and Notch1 (supporting cell marker) via the EGFR/Ras/Raf/ERK1/2 signal pathway. In in vivo experiments, alginate microcapsules loaded with rBMSCs (2 × 106 cells per microcapsule) together with Iso mAbs or anti-EGF mAbs were grafted into guinea pig cochlea. After 6 weeks of treatment, immunofluorescence analyses indicated that the rBMSCs embedded into anti-EGF microcapsules were more efficiently transformed into hair cells compared with the group with Iso mAb microcapsules and displayed an ordered arrangement in Reissner's membranes. The results highlighted the significance of engineering the microenvironment of stem cells for hair cell differentiation, and particularly the advantage of hair cell differentiation of rBMSCs by recruiting host EGF ligands via tethered mAbs. In conclusion, this strategy of co-embedding rBMSCs and anti-EGF mAb in alginate microcapsules is a promising modality for the regeneration of hair cell-like cells.

Epidermal growth factor enhances the developmental competence of yak (Bos grunniens) preimplantation embryos by modulating the expression of survivin and HSP70

Culture conditions can affect cleavage, zygotic genome activation, embryonic development, and blastocyst quality. Epidermal growth factor (EGF) has important autocrine and/or paracrine effects during early embryonic development. However, few studies of the effect of EGF on yak embryos have been reported. The aim of this study was to determine the effects of exogenous EGF on yak embryonic development in vitro, as well as the correlation between HSP70 and survivin expression patterns, blastocyst quality and EGF concentration. The in vitro matured cumulus–oocyte complexes (COCs) were fertilized with frozen–thawed semen, and the zygotes were incubated in culture media supplemented with different concentrations of EGF. During in vitro culture, the embryos were analyzed for development to the 4-cell, 8-cell, 16-cell, and blastocyst stages. The variation in total blastocyst cell numbers and their allocation to the inner cell mass (ICM) and trophectoderm (TE) lineages were determined by differential CDX2 staining. Relative HSP70 and survivin mRNA expression was examined by qPCR at the blastocyst stage. Compared to the control, EGF treatment (100ng/mL) significantly increased the blastocyst formation rate but had no significant effect at other stages. The total number of cells per blastocyst increased with 100ng/mL of EGF. HSP70 and survivin expression was higher in the EGF treatment groups than in the control group and highest with 100ng/mL EGF. From the present study, we conclude that EGF significantly enhances yak blastocyst formation and cell numbers, which might be associated with the increased expression of HSP70 and survivin after EGF treatment.

Epidermal growth factor mediated healing in stem cell-derived vocal fold mucosa

BackgroundThe goal of vocal fold wound healing is the reconstitution of functional tissue, including a structurally and functionally intact epithelium. Mechanisms underlying reepithelialization in vocal folds are not known, although it is suspected that healing involves the interplay between several growth factors. We used a three-dimensional human embryonic stem cell-derived model of vocal fold mucosa to examine the effects of one growth factor, exogenous epidermal growth factor (EGF), on wound healing. Materials and methodsA scratch wound was created in the in vitro model. Rate of wound healing, epidermal growth factor receptor (EGFR) activation, and cell proliferation after injury were analyzed with and without application of both exogenous EGF and an EGFR inhibitor, gefitinib. ResultsWound repair after injury was significantly hastened by application of exogenous EGF (13.3 μm/h, ±2.63) compared with absence of exogenous EGF (7.1 μm/h ±2.84), but inhibited with concurrent addition of Gefitinib (5.2 μm/h, ±2.23), indicating that EGF mediates wound healing in an EGFR-dependent manner. Immunohistochemistry revealed that EGFR activation occurred only in the presence of exogenous EGF. Although not statistically significant, increased density of Ki67 staining in the epithelium adjacent to the scratch wound was observed after treatment with EGF, suggesting a tendency for exogenous EGF to increase epithelial cell proliferation. ConclusionsExogenous EGF increases the rate of wound healing in an EGFR-dependent manner in a three-dimensional stem cell-derived model of vocal fold mucosa. This model of wound healing can be used to gain insight into the mechanisms that regulate vocal fold epithelial repair after injury.

Loss of α1,6-fucosyltransferase suppressed liver regeneration: implication of core fucose in the regulation of growth factor receptor-mediated cellular signaling

Core fucosylation is an important post-translational modification, which is catalyzed by α1,6-fucosyltransferase (Fut8). Increased expression of Fut8 has been shown in diverse carcinomas including hepatocarcinoma. In this study, we investigated the role of Fut8 expression in liver regeneration by using the 70% partial hepatectomy (PH) model, and found that Fut8 is also critical for the regeneration of liver. Interestingly, we show that the Fut8 activities were significantly increased in the beginning of PH (~4d), but returned to the basal level in the late stage of PH. Lacking Fut8 led to delayed liver recovery in mice. This retardation mainly resulted from suppressed hepatocyte proliferation, as supported not only by a decreased phosphorylation level of epidermal growth factor (EGF) receptor and hepatocyte growth factor (HGF) receptor in the liver of Fut8−/− mice in vivo, but by the reduced response to exogenous EGF and HGF of the primary hepatocytes isolated from the Fut8−/− mice. Furthermore, an administration of L-fucose, which can increase GDP-fucose synthesis through a salvage pathway, significantly rescued the delayed liver regeneration of Fut8+/− mice. Overall, our study provides the first direct evidence for the involvement of Fut8 in liver regeneration.

Tumor Growth Suppression and Enhanced Radioresponse by an Exogenous Epidermal Growth Factor in Mouse Xenograft Models with A431 Cells

PurposeThe purpose of this study was to evaluate whether an exogenous epidermal growth factor (EGF) could induce anti-tumor and radiosensitizing effects in vivo.Materials and MethodsBALB/c-nu mice that were inoculated with A431 (human squamous cell carcinoma) cells in the right hind legs were divided into five groups: I (no treatment), II (EGF for 6 days), III (EGF for 20 days), IV (radiotherapy [RT]), and V (RT plus concomitant EGF). EGF was administered intraperitoneally (5 mg/kg) once a day and the RT dose was 30 Gy in six fractions. Hematoxylin and eosin (H&E) stained sections of tumor, liver, lung, and kidney tissues were investigated. Additionally, tumors were subjected to immunohistochemistry staining with caspase-3.ResultsEGF for 6 days decreased tumor volume, but it approached the level of the control group at the end of follow-up (p=0.550). The duration of tumor shrinkage was prolonged in group V while the slope of tumor re-growth phase was steeper in group IV (p=0.034). EGF for 20 days decreased tumor volume until the end of the observation period (p < 0.001). Immunohistochemistry revealed that mice in group V showed stronger intensity than those in group IV. There were no abnormal histological findings upon H&E staining of the normal organs.ConclusionEGF-induced anti-tumor effect was ascertained in the xenograft mouse models with A431 cells. Concomitant use of EGF has the potential role as a radiosensitizer in the design of fractionated irradiation.

Persistent ERK/MAPK activation promotes lactotrope differentiation and diminishes tumorigenic phenotype.

The signaling pathways that govern the lactotrope-specific differentiated phenotype, and those that control lactotrope proliferation in both physiological and pathological lactotrope expansion, are poorly understood. Moreover, the specific role of MAPK signaling in lactotrope proliferation vs differentiation, whether activated phosphorylated MAPK is sufficient for prolactinoma tumor formation remain unknown. Given that oncogenic Ras mutations and persistently activated phosphorylated MAPK are found in human tumors, including prolactinomas and other pituitary tumors, a better understanding of the role of MAPK in lactotrope biology is required. Here we directly examined the role of persistent Ras/MAPK signaling in differentiation, proliferation, and tumorigenesis of rat pituitary somatolactotrope GH4 cells. We stimulated Ras/MAPK signaling in a persistent, long-term manner (over 6 d) in GH4 cells using two distinct approaches: 1) a doxycycline-inducible, oncogenic V12Ras expression system; and 2) continuous addition of exogenous epidermal growth factor. We find that long-term activation of the Ras/MAPK pathway over 6 days promotes differentiation of the bihormonal somatolactotrope GH4 precursor cell into a prolactin-secreting, lactotrope cell phenotype in vitro and in vivo with GH4 cell xenograft tumors. Furthermore, we show that persistent activation of the Ras/MAPK pathway not only fails to promote cell proliferation, but also diminishes tumorigenic characteristics in GH4 cells in vitro and in vivo. These data demonstrate that activated MAPK promotes differentiation and is not sufficient to drive tumorigenesis, suggesting that pituitary lactotrope tumor cells have the ability to evade the tumorigenic fate that is often associated with Ras/MAPK activation.

Epidermal growth factor attenuates tubular necrosis following mercuric chloride damage by regeneration of indigenous, not bone marrow-derived cells.

To assess effects of epidermal growth factor (EGF) and pegylated granulocyte colony-stimulating factor (P-GCSF; pegfilgrastim) administration on the cellular origin of renal tubular epithelium regenerating after acute kidney injury initiated by mercuric chloride (HgCl2). Female mice were irradiated and male whole bone marrow (BM) was transplanted into them. Six weeks later recipient mice were assigned to one of eight groups: control, P-GCSF+, EGF+, P-GCSF+EGF+, HgCl2, HgCl2+P-GCSF+, HgCl2+EGF+ and HgCl2+P-GCSF+EGF+. Following HgCl2, injection tubular injury scores increased and serum urea nitrogen levels reached uraemia after 3 days, but EGF-treated groups were resistant to this acute kidney injury. A four-in-one analytical technique for identification of cellular origin, tubular phenotype, basement membrane and S-phase status revealed that BM contributed 1% of proximal tubular epithelium in undamaged kidneys and 3% after HgCl2 damage, with no effects of exogenous EGF or P-GCSF. Only 0.5% proximal tubular cells were seen in S-phase in the undamaged group kidneys; this increased to 7–8% after HgCl2 damage and to 15% after addition of EGF. Most of the regenerating tubular epithelium originated from the indigenous pool. BM contributed up to 6.6% of the proximal tubular cells in S-phase after HgCl2 damage, but only to 3.3% after additional EGF. EGF administration attenuated tubular necrosis following HgCl2 damage, and the major cause of this protective effect was division of indigenous cells, whereas BM-derived cells were less responsive. P-GCSF did not influence damage or regeneration.

Epidermal growth factor upregulates Skp2/Cks1 and p27kip1in human extrahepatic cholangiocarcinoma cells

To evaluate the expression status of S-phase kinase-associated protein 2 (Skp2)/cyclin-dependent kinases regulatory subunit 1 (Cks1) and p27(kip1), and assess the prognostic significance of Skp2/Cks1 expression with p27(kip1) in patients with extrahepatic cholangiocarcinoma. Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December 1994 to March 2008 were enrolled. Immunohistochemical staining for Skp2, Cks1, p27(kip1), and Ki67, along with other relevant molecular biologic experiments, were performed. By Cox regression analyses, advanced age (> 65 years), advanced AJCC tumor stage, poorly differentiated histology, and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma. Exogenous epidermal growth factor (EGF, especially 0.1-10 ng/mL) significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27(kip1) in SNU-1196, SNU-1079, and SNU-245 cells. The protein levels of Skp2/Cks1 (from nuclear lysates) and p27(kip1) (from cytosolic lysate) were also significantly increased in these cells. There were significant reductions in the protein levels of Skp2/Cks1 and p27(kip1) (from nuclear lysate) after the treatment of LY294002. By chromatin immunoprecipitation assay, we found that E2F1 transcription factor directly binds to the promoter site of Skp2. Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma. EGF upregulates the mRNA and protein levels of Skp2/Cks1 and p27(kip1) via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter.

Prostaglandin E2 receptor EP1-mediated phosphorylation of focal adhesion kinase enhances cell adhesion and migration in hepatocellular carcinoma cells

The prostaglandin E₂ (PGE₂) EP1 receptor has been implicated in hepatocellular carcinoma (HCC) cell invasion. However, little is known about the mechanisms of EP1 receptor-mediated cell adhesion and migration. We previously showed that PGE₂ promotes cell adhesion and migration by activating focal adhesion kinase (FAK). The present study was designed to elucidate the association between the EP1 receptor and FAK activation in HCC cells and to investigate the related signaling pathways. The effects of PGE₂, EP1 agonist 17-phenyl trinor-PGE₂ (17-PT-PGE₂), PKC and EGFR inhibitors on FAK activation were investigated by treatment of Huh-7 cells. Phosphorylation of FAK Y397 and c-Src Y416 was investigated by western blotting. Cell adhesion and migration were analyzed by WST and transwell assays, respectively. Protein kinase C (PKC) activity was measured with a PKC assay kit. The results showed that 17-PT-PGE₂ (3 µM) increased FAK Y397 phosphorylation by more than 2-fold and promoted cell adhesion and migration in Huh-7 cells. In transfected 293 cells, expression of the EP1 receptor was confirmed to upregulate FAK phosphorylation, while the EP1 receptor antagonist sc-19220 decreased PGE₂-mediated FAK activation. PKC activity and c-Src Y416 phosphorylation were enhanced after 17-PT-PGE₂ treatment. Both PKC and c-Src inhibitor suppressed the 17-PT-PGE₂-upregulated FAK phosphorylation, as well as 17-PT-PGE₂-induced cell adhesion and migration. In addition, exogenous epidermal growth factor (EGF) treatment increased FAK phosphorylation. The EGF receptor (EGFR) inhibitor also suppressed 17-PT-PGE₂-upregulated FAK phosphorylation. Our study suggests that the PGE₂ EP1 receptor regulates FAK phosphorylation by activating the PKC/c-Src and EGFR signal pathways, which may coordinately regulate adhesion and migration in HCC.

Urothelial differentiation of human umbilical cord-derived mesenchymal stromal cells in vitro.

Human umbilical cord-derived mesenchymal stromal cells (hUCMSCs) are the most primitive of those isolated from other post-natal tissue source. The hUCMSCs possess the capability of differentiating along multi-lineage. This study aimed to investigate whether hUCMSCs can differentiate into urothelium-like cells. The hUCMSCs were isolated from fresh human umbilical cord postpartum and expanded at least to passage 3 in vitro. Subsequently, they were cultured with conditioned medium from urothelial cells (UC-CM) supplemented with 20 ng/ml exogenous epidermal growth factor (EGF). Urothelial cell specific marker uroplakin II (UPII) and cytokeratins were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence technology. During culture, hUCMSCs started to express UPII and cytokeratins weakly at 7 days and were significantly up-regulated at 2 weeks post-induction. Additionally, morphology of hUCMSCs changed from spindle-shape to a polygonal epithelial-shape similar to that of urothelial cells after 7 days. The study results indicated that hUCMSCs can differentiate into urothelium-like cells in a defined micro-environment in vitro constituted by UC-CM and exogenous EGF.

Urothelial Differentiation of Human Umbilical Cord-Derived Mesenchymal Stromal CellsIn Vitro

Human umbilical cord-derived mesenchymal stromal cells (hUCMSCs) are the most primitive of those isolated from other post-natal tissue source. The hUCMSCs possess the capability of differentiating along multi-lineage. This study aimed to investigate whether hUCMSCs can differentiate into urothelium-like cells. The hUCMSCs were isolated from fresh human umbilical cord postpartum and expanded at least to passage 3in vitro. Subsequently, they were cultured with conditioned medium from urothelial cells (UC-CM) supplemented with 20 ng/ml exogenous epidermal growth factor (EGF). Urothelial cell specific marker uroplakin II (UPII) and cytokeratins were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence technology. During culture, hUCMSCs started to express UPII and cytokeratins weakly at 7 days and were significantly up-regulated at 2 weeks post-induction. Additionally, morphology of hUCMSCs changed from spindle-shape to a polygonal epithelial-shape similar to that of urothelial cells after 7 days. The study results indicated that hUCMSCs can differentiate into urothelium-like cells in a defined micro-environmentin vitroconstituted by UC-CM and exogenous EGF.