RESUMO
There are 13 known endogenous ligands for the epidermal growth factor receptor (EGFR) and its closely related ErbB receptor family members. We previously reported that betacellulin (BTC) is more efficacious than epidermal growth factor (EGF) in mediating corneal wound healing, although the molecular basis for this difference was unknown. For the most part, differences between ligands can be attributed to variability in binding properties, such as the unique rate of association and dissociation, pH sensitivity, and selective binding to individual ErbB family members of each ligand. However, this was not the case for BTC. Despite being better at promoting wound healing via enhanced cell migration, BTC has reduced receptor affinity and weaker induction of EGFR phosphorylation. These data indicate that the response of BTC is not due to enhanced affinity or kinase activity. Receptor phosphorylation and proximity ligation assays indicate that BTC treatment significantly increases ErbB3 phosphorylation and EGFR-ErbB3 heterodimers when compared with EGF treatment. We observed that EGFR-ErbB3 heterodimers contribute to cell migration, because the addition of an ErbB3 antagonist (MM-121) or RNA interference-mediated knockdown of ErbB3 attenuates BTC-stimulated cell migration compared with EGF. Thus, we demonstrate that, despite both ligands binding to the EGFR, BTC biases the EGFR to dimerize with ErbB3 to regulate the biologic response.
Assuntos
Betacelulina/metabolismo , Receptor ErbB-3/metabolismo , Linhagem Celular , Movimento Celular/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Humanos , Ligantes , Fosforilação/fisiologia , Transdução de Sinais/fisiologiaRESUMO
PURPOSE: The antiangiogenic receptor tyrosine kinase inhibitor (RTKi), 3-[(4-bromo-2,6-difluorophenyl)methoxy]-5-[[[[4-(1-pyrrolidinyl) butyl] amino] carbonyl]amino]-4-isothiazolecarboxamide hydrochloride, targets VEGFR2 (half maximal inhibitory concentration [IC50] = 11 nM); however, off-target inhibition of epidermal growth factor receptor (EGFR) occurs at higher concentrations. (IC50 = 5.8 µM). This study was designed to determine the effect of topical RTKi treatment on EGF-mediated corneal epithelial wound healing and to develop new strategies to minimize off-target EGFR inhibition. METHODS: In vitro corneal epithelial wound healing was measured in response to EGF using a transformed human cell line (hTCEpi cells). In vivo corneal wound healing was assessed using a murine model. In these complementary assays, wound healing was measured in the presence of varying RTKi concentrations. Immunoblot analysis was used to examine EGFR and VEGFR2 phosphorylation and the kinetics of EGFR degradation. An Alamar Blue assay measured VEGFR2-mediated cell biology. RESULTS: Receptor tyrosine kinase inhibitor exposure caused dose-dependent inhibition of EGFR-mediated corneal epithelial wound healing in vitro and in vivo. Nanomolar concentrations of menadione, a vitamin K3 analog, when coadministered with the RTKi, slowed EGFR degradation and ameliorated the inhibitory effects on epithelial wound healing both in vitro and in vivo. Menadione did not alter the RTKi's IC50 against VEGFR2 phosphorylation or its inhibition of VEGF-induced retinal endothelial cell proliferation. CONCLUSIONS: An antiangiogenic RTKi exhibited off-target effects on the corneal epithelium that can be minimized by menadione without deleteriously affecting its on-target VEGFR2 blockade. These data indicate that menadione has potential as a topical supplement for individuals suffering from perturbations in corneal epithelial homeostasis, especially as an untoward side effect of kinase inhibitors.
Assuntos
Epitélio Corneano/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Vitamina K 3/farmacologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Fator de Crescimento Epidérmico/metabolismo , Epitélio Corneano/metabolismo , Receptores ErbB/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologiaRESUMO
Detection of orthotopic xenograft tumors is difficult due to poor spatial resolution and reduced image fidelity with traditional optical imaging modalities. In particular, light scattering and attenuation in tissue at depths beyond subcutaneous implantation hinder adequate visualization. We evaluate the use of multispectral optoacoustic tomography (MSOT) to detect upregulated epidermal growth factor (EGF) receptor in orthotopic pancreatic xenografts using a near-infrared EGF-conjugated CF-750 fluorescent probe. MSOT is based on the photoacoustic effect and thus not limited by photon scattering, resulting in high-resolution tomographic images. Pancreatic tumor-bearing mice with luciferase-transduced S2VP10L tumors were intravenously injected with EGF-750 probe before MSOT imaging. We characterized probe specificity and bioactivity via immunoblotting, immunocytochemistry, and flow cytometric analysis. In vitro data along with optical bioluminescence/fluorescence imaging were used to validate acquired MSOT in vivo images of probe biodistribution. Indocyanine green dye was used as a nonspecific control to define specificity of EGF-probe accumulation. Maximum accumulation occurred at 6 hours postinjection, demonstrating specific intratumoral probe uptake and minimal liver and kidney off-target accumulation. Optical bioluminescence and fluorescence imaging confirmed tumor-specific probe accumulation consistent with MSOT images. These studies demonstrate the utility of MSOT to obtain volumetric images of ligand probe biodistribution in vivo to detect orthotopic pancreatic tumor lesions through active targeting of the EGF receptor.
Assuntos
Receptores ErbB/biossíntese , Neoplasias Pancreáticas/diagnóstico por imagem , Técnicas Fotoacústicas/métodos , Tomografia/métodos , Animais , Receptores ErbB/isolamento & purificação , Corantes Fluorescentes , Humanos , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Radiografia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: In many cell types, the E3 ubiquitin ligase, c-Cbl, induces ligand-dependent ubiquitylation of the epidermal growth factor receptor (EGFR) and targets the receptor for lysosomal degradation. The goal of this study was to determine whether c-Cbl is a negative regulator of EGFR in the corneal epithelium and if it can be inhibited to promote corneal epithelial homeostasis. METHODS: Expression and activity of c-Cbl were blocked in immortalized human corneal epithelial cells (hTCEpi) using RNAi and pharmacological agents ([4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine] or PP1). Following c-Cbl inhibition, cells were assessed for ligand-dependent receptor ubiquitylation, receptor phosphorylation, and in vitro wound healing. Subsequent experiments used PP1 in hTCEpi cells and monitored in vivo murine corneal epithelial wound healing. RESULTS: Knockdown and inhibition of c-Cbl decreased ligand-dependent ubiquitylation of the EGFR and prolonged receptor activity as measured by tyrosine phosphorylation. Further, these treatments also increased the extent of ligand-dependent corneal epithelial wound healing in vitro and in vivo. CONCLUSION: Manipulating the duration of EGFR activity can enhance the rate of restoration of the corneal epithelial layer. Based on our findings, c-Cbl is a new therapeutic target to enhance EGFR-mediated corneal epithelial homeostasis that bypasses the limitations of previous approaches.
Assuntos
Epitélio Corneano/metabolismo , Receptores ErbB/metabolismo , Traumatismos Oculares/metabolismo , Homeostase/fisiologia , Proteínas Proto-Oncogênicas c-cbl/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Inibidores Enzimáticos , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/lesões , Epitélio Corneano/patologia , Traumatismos Oculares/patologia , Feminino , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Transdução de Sinais , Ubiquitinação , Cicatrização/fisiologia , Quinases da Família src/antagonistas & inibidoresRESUMO
Both constitutive and ligand-mediated membrane trafficking regulate Epidermal Growth Factor Receptor (EGFR) signaling. The constitutive endocytosis and recycling of the unliganded EGFR is a critical determinant of cell surface EGFR expression and the cell's sensitivity to ligands. We report that two proteins with established roles in trafficking the EGF:EGFR complex to the lysosome also regulate the recycling of the unliganded EGFR. Knock down of either Tumor suppressor gene 101 (TSG101) or RAB7 causes the endosomal accumulation of the inactive, unliganded receptor in morphologically and biochemically distinct organelles. Knock down of TSG101 causes the EGFR to accumulate in low density endosomes whereas RAB7 knock down results in EGFR accumulation in high density endosomes. Knock down of either protein caused the receptor to co-localize primarily with LAMP-1, but not EEA1. These two proteins regulate EGFR slow, perinuclear recycling, via distinct mechanism and are new molecular targets that regulate cell surface EGFR expression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Receptores ErbB/metabolismo , Fatores de Transcrição/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Endossomos , Células HeLa , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Transporte Proteico , Receptores da Transferrina/metabolismo , proteínas de unión al GTP Rab7RESUMO
Endocytosis positively and negatively regulates cell surface receptor signaling by temporally and spatially controlling interactions with downstream effectors. This process controls receptor-effector communication. However, the relationship between receptor endocytic trafficking and cell physiology is unclear. In MDA-MB-468 cells, cell surface EGF receptors (EGFRs) promote cell growth, whereas intracellular EGFRs induce apoptosis, making these cells an excellent model for studying the endocytic regulation of EGFR signaling. In addition, MDA-MB-468 cells have limited EGFR degradation following stimulation. Here, we report that in MDA-MB-468 cells the phosphorylated EGFR accumulates on the limiting membrane of the endosome with its carboxyl terminus oriented to the cytoplasm. To determine whether perturbation of EGFR trafficking is sufficient to cause apoptosis, we used pharmacological and biochemical strategies to disrupt EGFR endocytic trafficking in HeLa cells, which do not undergo EGF-dependent apoptosis. Manipulation of HeLa cells so that active EGF·EGFRs accumulate on the limiting membrane of endosomes reveals that receptor phosphorylation is sustained and leads to apoptosis. When EGF·EGFR complexes accumulated in the intraluminal vesicles of the late endosome, phosphorylation of the receptor was not sustained, nor did the cells undergo apoptosis. These data demonstrate that EGFR-mediated apoptosis is initiated by the activated EGFR from the limiting membrane of the endosome.