RESUMO
The receptor tyrosine kinase (RTK) EphA2 is expressed in epithelial and endothelial cells and controls the assembly of cell-cell junctions. EphA2 has also been implicated in many diseases, including cancer. Unlike most RTKs, which signal predominantly as dimers, EphA2 readily forms high-order oligomers upon ligand binding. Here, we investigated if a correlation exists between EphA2 signaling properties and the size of the EphA2 oligomers induced by multiple ligands, including the widely used ephrinA1-Fc ligand, the soluble monomeric m-ephrinA1, and novel engineered peptide ligands. We used fluorescence intensity fluctuation (FIF) spectrometry to characterize the EphA2 oligomer populations induced by the different ligands. Interestingly, we found that different monomeric and dimeric ligands induce EphA2 oligomers with widely different size distributions. Our comparison of FIF brightness distribution parameters and EphA2 signaling parameters reveals that the efficacy of EphA2 phosphorylation on tyrosine 588, an autophosphorylation response contributing to EphA2 activation, correlates with EphA2 mean oligomer size. However, we found that other characteristics, such as the efficacy of AKT inhibition and ligand bias coefficients, appear to be independent of EphA2 oligomer size. Taken together, this work highlights the utility of FIF in RTK signaling research and demonstrates a quantitative correlation between the architecture of EphA2 signaling complexes and signaling features.
Assuntos
Efrina-A1 , Receptor EphA2 , Células Endoteliais/metabolismo , Efrina-A1/química , Ligantes , Fosforilação , Receptor EphA2/metabolismo , HumanosRESUMO
The EPH receptor A2 (EphA2) tyrosine kinase plays an important role in a plethora of biological and disease processes, ranging from angiogenesis and cancer to inflammation and parasitic infections. EphA2 is therefore considered an important drug target. Two short peptides previously identified by phage display, named YSA and SWL, are widely used as EphA2-targeting agents owing to their high specificity for this receptor. However, these peptides have only modest (micromolar) potency. Lack of structural information on the binding interactions of YSA and SWL with the extracellular EphA2 ligand-binding domain (LBD) has for many years precluded structure-guided improvements. We now report the high-resolution (1.53-2.20 Å) crystal structures of the YSA peptide and several of its improved derivatives in complex with the EphA2 LBD, disclosing that YSA targets the ephrin-binding pocket of EphA2 and mimics binding features of the ephrin-A ligands. The structural information obtained enabled iterative peptide modifications conferring low nanomolar potency. Furthermore, contacts observed in the crystal structures shed light on how C-terminal features can convert YSA derivatives from antagonists to agonists that likely bivalently interact with two EphA2 molecules to promote receptor oligomerization, autophosphorylation, and downstream signaling. Consistent with this model, quantitative FRET measurements in live cells revealed that the peptide agonists promote the formation of EphA2 oligomeric assemblies. Our findings now enable rational strategies to differentially modify EphA2 signaling toward desired outcomes by using appropriately engineered peptides. Such peptides could be used as research tools to interrogate EphA2 function and to develop pharmacological leads.
Assuntos
Peptídeos/metabolismo , Receptor EphA2/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Efrina-A1/química , Efrina-A1/metabolismo , Humanos , Ligantes , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/farmacologia , Fosforilação , Ligação Proteica , Engenharia de Proteínas , Multimerização Proteica , Estrutura Terciária de Proteína , Receptor EphA2/agonistas , Receptor EphA2/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Misregulation of the signaling axis formed by the receptor tyrosine kinase (RTK) EphA2 and its ligand, ephrinA1, causes aberrant cell-cell contacts that contribute to metastasis. Solid tumors are characterized by an acidic extracellular medium. We intend to take advantage of this tumor feature to design new molecules that specifically target tumors. We created a novel pH-dependent transmembrane peptide, TYPE7, by altering the sequence of the transmembrane domain of EphA2. TYPE7 is highly soluble and interacts with the surface of lipid membranes at neutral pH, while acidity triggers transmembrane insertion. TYPE7 binds to endogenous EphA2 and reduces Akt phosphorylation and cell migration as effectively as ephrinA1. Interestingly, we found large differences in juxtamembrane tyrosine phosphorylation and the extent of EphA2 clustering when comparing TYPE7 with activation by ephrinA1. This work shows that it is possible to design new pH-triggered membrane peptides to activate RTK and gain insights on its activation mechanism.
Assuntos
Efrina-A1/genética , Efrina-A2/genética , Neoplasias/genética , Peptídeos/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Efrina-A1/química , Efrina-A2/química , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Neoplasias/tratamento farmacológico , Peptídeos/administração & dosagem , Peptídeos/farmacologia , Fosforilação , Domínios Proteicos/genética , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Receptor EphA2RESUMO
This work aims at the theoretical description of EphA2-ephrin A1 inhibition by small molecules. Recently proposed ab initio-based scoring models, comprising long-range components of interaction energy, is tested on lithocholic acid class inhibitors of this proteinâ»protein interaction (PPI) against common empirical descriptors. We show that, although limited to compounds with similar solvation energy, the ab initio model is able to rank the set of selected inhibitors more effectively than empirical scoring functions, aiding the design of novel compounds.
Assuntos
Efrina-A1/metabolismo , Modelos Biológicos , Receptor EphA2/metabolismo , Sítios de Ligação , Efrina-A1/química , Receptor EphA2/química , Eletricidade Estática , TermodinâmicaRESUMO
The activation of EphA2 receptor by its natural ligand EphrinA1 causes blood brain barrier dysfunction, and inactivation of EphA2 reduces BBB damage in ischemic stroke. Thus, EphA2 targeted antagonists may serve as neuroprotective agents. We engineered four mutants of EphrinA1, EM1, EM2, EM3 and EM4, respectively. The computational analysis showed that these four mutants were capable of interacting with EphA2. Their potential neuroprotective effects were examined in mouse focal ischemia/reperfusion (I/R) model. EM2 exhibited strong neuroprotective effects, including reduced brain infarct volume, neuronal apoptosis, cerebral edema, and improved neurological scores. The EM2-mediated protection was associated with a comparative decrease in BBB leakage, inflammatory infiltration, and higher expression levels of tight junction proteins, such as zonula occludens-1 and Occludin. I/R-induced high expression of Rho-associated protein kinase 2 (ROCK2) was down-regulated after EM2 treatment. Moreover, EM2 reduced agonist doxazosin-induced EphA2 phosphorylation and cells rounding in PC3 cells, indicating EphA2-antagonizing activity of EM2. These finding provided evidences of the neuroprotection of EphA2 antagonist and a novel approach for ischemic stroke treatment. These results also suggested that a receptor agonist can be switched to an antagonist by substituting one or more relevant residues.
Assuntos
Desenho de Fármacos , Efrina-A1/química , Efrina-A1/farmacologia , Proteínas Mutantes , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Sequência de Aminoácidos , Animais , Biomarcadores , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Modelos Animais de Doenças , Efrina-A1/genética , Ataque Isquêmico Transitório/tratamento farmacológico , Ataque Isquêmico Transitório/etiologia , Ataque Isquêmico Transitório/metabolismo , Ataque Isquêmico Transitório/patologia , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptor EphA2/antagonistas & inibidores , Receptor EphA2/químicaRESUMO
Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization.
Assuntos
Efrina-A2/química , Motivo Estéril alfa , Animais , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Efrina-A1/química , Efrina-A1/metabolismo , Efrina-A2/metabolismo , Humanos , Camundongos , Fosforilação , Ligação Proteica , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Receptor EphA2RESUMO
Receptor-ligand complexes spanning a cell-cell interface inevitably establish a preferred intermembrane spacing based on the molecular dimensions and orientation of the complexes. This couples molecular binding events to membrane mechanics and large-scale spatial organization of receptors on the cell surface. Here, we describe a straightforward, epi-fluorescence-based method to precisely determine intermembrane receptor-ligand dimension at adhesions established by receptor-ligand binding between apposed membranes in vitro. Adhesions were reconstituted between planar and silica microbead supported membranes via specific interaction between cognate receptor/ligand pairs (EphA2/EphrinA1 and E-cadherin/anti-E-cadherin antibody). Epi-fluorescence imaging of the ligand enrichment zone in the supported membrane beneath the adhering microbead, combined with a simple geometrical interpretation, proves sufficient to estimate intermembrane receptor-ligand dimension with better than 1 nm precision. An advantage of this assay is that no specialized equipment or imaging methods are required.
Assuntos
Anticorpos/química , Caderinas/química , Efrina-A1/química , Efrina-A2/química , Microesferas , Imagem Óptica , Dióxido de Silício/química , Antígenos CD , Humanos , Receptor EphA2RESUMO
The EphA2 receptor and its ephrin-A1 ligand form a key cell communication system, which has been found overexpressed in many cancer types and involved in tumor growth. Recent medicinal chemistry efforts have identified bile acid derivatives as low micromolar binders of the EphA2 receptor. However, these compounds suffer from poor physicochemical properties, hampering their use in vivo. The identification of compounds able to disrupt the EphA2-ephrin-A1 complex lacking the bile acid scaffold may lead to new pharmacological tools suitable for in vivo studies. To identify the most promising virtual screening (VS) protocol aimed at finding novel EphA2 antagonists, we investigated the ability of both ligand-based and structure-based approaches to retrieve known EphA2 antagonists from libraries of decoys with similar molecular properties. While ligand-based VSs were conducted using UniPR129 and ephrin-A1 ligand as reference structures, structure-based VSs were performed with Glide, using the X-ray structure of the EphA2 receptor/ephrin-A1 complex. A comparison of enrichment factors showed that ligand-based approaches outperformed the structure-based ones, suggesting ligand-based methods using the G-H loop of ephrin-A1 ligand as template as the most promising protocols to search for novel EphA2 antagonists.
Assuntos
Descoberta de Drogas/métodos , Efrina-A1/agonistas , Inibidores de Proteínas Quinases/química , Receptor EphA2/antagonistas & inibidores , Cristalografia por Raios X , Bases de Dados de Produtos Farmacêuticos , Efrina-A1/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade , Interface Usuário-ComputadorRESUMO
Eph receptor (Eph) and ephrin signaling can play central roles in prostate cancer and other cancer types. Exposed to ephrin-A1 PC3 prostate cancer cells alter adhesion to extracellular matrix (ECM) proteins. However, whether PC3 cells increase or reduce adhesion, and by which mechanisms they change adhesion to the ECM remains to be characterized. Here, we assay how ephrin-A1 stimulates PC3 cells to adhere to ECM proteins using single-cell force spectroscopy. We find that PC3 cells binding to immobilized ephrin-A1 but not to solubilized ephrin-A1 specifically strengthen adhesion to collagen I. This Eph-ephrin-A1 signaling, which we suppose is based on mechanotransduction, stimulates ß1-subunit containing integrin adhesion via the protein kinase Akt and the guanine nucleotide-exchange factor cytohesin. Inhibiting the small GTPases, Rap1 or Rac1, generally lowered adhesion of PC3 prostate cancer cells. Our finding suggests a mechanism by which PC3 prostate cancer cells exposed to ephrins crosstalk to ß1-integrins and preferably metastasize in bone, a collagen I rich tissue.
Assuntos
Colágeno Tipo I/química , Integrina beta1/metabolismo , Receptores da Família Eph/metabolismo , Animais , Adesão Celular , Comunicação Celular , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Efrina-A1/química , Efrina-A1/farmacologia , Células HEK293 , Células HeLa , Humanos , Proteínas Imobilizadas/química , Masculino , Mecanotransdução Celular/efeitos dos fármacos , Camundongos , Microscopia de Força Atômica , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
The EPH receptor A2 (EPHA2) represents an attractive anticancer target. With the aim to identify novel EPHA2 receptor antagonists, a virtual screening campaign, combining shape-similarity and docking calculations, was conducted on a set of commercially available compounds. A combined score, taking into account both ligand- and structure-based results, was then used to identify the most promising candidates. Two compounds, selected among the best-ranked ones, were identified as EPHA2 receptor antagonists with micromolar affinity.
Assuntos
Antineoplásicos/química , Butiratos/química , Ácidos Cólicos/química , Descoberta de Drogas , Efrina-A1/antagonistas & inibidores , Naftalenos/química , Inibidores de Proteínas Quinases/química , Receptor EphA2/antagonistas & inibidores , Sítios de Ligação , Efrina-A1/química , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Receptor EphA2/química , Relação Estrutura-Atividade , Interface Usuário-ComputadorRESUMO
MicroRNAs (miRs) are small noncoding RNA sequences that negatively regulate the expression of target genes by posttranscriptional repression. miRs are dysregulated in various diseases, including cancer. let-7a miR, an antioncogenic miR, is downregulated in lung cancers. Our earlier studies demonstrated that let-7a miR inhibits tumor growth in malignant pleural mesothelioma (MPM) and could be a potential therapeutic against lung cancer. EphA2 (ephrin type-A receptor 2) tyrosine kinase is overexpressed in most cancer cells, including MPM and non-small-cell lung cancer (NSCLC) cells. Ephrin-A1, a specific ligand of the EphA2 receptor, inhibits cell proliferation and migration. In this study, to enhance the delivery of miR, the miRs were encapsulated in the DOTAP (N-[1-(2.3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium)/Cholesterol/DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethylene glycol)-2000])-PEG (polyethylene glycol)-cyanur liposomal nanoparticles (LNP) and ephrin-A1 was conjugated on the surface of LNP to target receptor EphA2 on lung cancer cells. The LNP with an average diameter of 100 nm showed high stability, low cytotoxicity, and high loading efficiency of precursor let-7a miR and ephrin-A1. The ephrin-A1 conjugated LNP (ephrin-A1-LNP) and let-7a miR encapsulated LNP (miR-LNP) showed improved transfection efficiency against MPM and NSCLC. The effectiveness of targeted delivery of let-7a miR encapsulated ephrin-A1 conjugated LNP (miR-ephrin-A1-LNP) was determined on MPM and NSCLC tumor growth in vitro. miR-ephrin-A1-LNP significantly increased the delivery of let-7a miR in lung cancer cells when compared with free let-7a miR. In addition, the expression of target gene Ras was significantly repressed following miR-ephrin-A1-LNP treatment. Furthermore, the miR-ephrin-A1-LNP complex significantly inhibited MPM and NSCLC proliferation, migration, and tumor growth. Our results demonstrate that the engineered miR-ephrin-A1-LNP complex is an effective carrier for the targeted delivery of small RNA molecules to lung cancer cells. This could be a potential therapeutic approach against tumors overexpressing the EphA2 receptor.
Assuntos
Antineoplásicos/farmacologia , Efrina-A1/química , Lipossomos/farmacologia , Neoplasias Pulmonares , MicroRNAs/farmacologia , Nanopartículas/química , Antineoplásicos/química , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Efrina-A1/metabolismo , Humanos , Lipossomos/química , MicroRNAs/química , MicroRNAs/genética , Transfecção/métodosRESUMO
The EphA2 receptor tyrosine kinase is overexpressed in a number of malignancies and is activated by ephrin ligands, most commonly by ephrin-A1. The crystal structure of the ligand-receptor complex revealed a glycosylation on the Asn-26 of ephrin-A1. Here we report for the first time the significance of the glycosylation in the biology of EphA2 and ephrin-A1. Ephrin-A1 was enzymatically deglycosylated, and its activity was evaluated in several assays using glioblastoma (GBM) cells and recombinant EphA2. We found that deglycosylated ephrin-A1 does not efficiently induce EphA2 receptor internalization and degradation, and does not activate the downstream signaling pathways involved in cell migration and proliferation. Data obtained by surface plasmon resonance confirms that deglycosylated ephrin-A1 does not bind EphA2 with high affinity. Mutations in the glycosylation site on ephrin-A1 result in protein aggregation and mislocalization. Analysis of Eph/ephrin crystal structures reveals an interaction between the ligand's carbohydrates and two residues of EphA2: Asp-78 and Lys-136. These findings suggest that the glycosylation on ephrin-A1 plays a critical role in the binding and activation of the EphA2 receptor.
Assuntos
Efrina-A1/metabolismo , Receptor EphA2/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Sítios de Ligação/genética , Ligação Competitiva , Western Blotting , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Cristalografia por Raios X , Efrina-A1/química , Efrina-A1/genética , Efrina-A2/genética , Efrina-A2/metabolismo , Glicosilação , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Receptor EphA2/química , Receptor EphA2/genética , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.
Assuntos
Efrina-A1/genética , Efrina-A1/metabolismo , Escherichia coli/genética , Engenharia Genética/métodos , Receptores da Família Eph/metabolismo , Clonagem Molecular , Efrina-A1/química , Efrina-A1/isolamento & purificação , Escherichia coli/citologia , Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Fosforilação , Estrutura Terciária de Proteína , Receptor EphA2/metabolismo , Solubilidade , Água/químicaRESUMO
The EphA2 receptor is overexpressed in glioblastoma multiforme and has been to shown to contribute to cell transformation, tumor initiation, progression, and maintenance. EphrinA1 (eA1) is a preferred ligand for the receptor. Treatment with monomeric eA1, the form of eA1 found in the extracellular environment, causes receptor phosphorylation, internalization, and down-regulation with subsequent anti-tumor effects. Here, we investigated the structure-function relationship of a monomeric eA1 focusing on its G-H loop ((108)FQRFTPFTLGKEFKE(123)G), a highly conserved region among eAs that mediates binding to their receptors. Alanine substitution mutants of the G-H loop amino acids were transfected into U-251 MG glioblastoma multiforme cells, and functional activity of each mutant in conditioned media was assessed by EphA2 down-regulation, ERK and AKT activation and cellular response assays. Alanine substitutions at positions Pro-113 Thr-115, Gly-117, Glu-122, and also Gln-109 enhanced the EphA2 receptor down-regulation and decreased p-ERK and p-AKT. Substitution mutants of eA1 at positions Phe-108, Arg-110, Phe-111, Thr-112, Phe-114, Leu-116, Lys-118, Glu-119, and Phe-120 had a deleterious effect on EphA2 down-regulation when compared with eA1-WT. Mutants at positions Phe-108, Lys-18, Lys-121, Gly-123 retained similar properties to eA1-WT. Recombinant eA1-R110A, -T115A, -G117A, and -F120A have been found to exhibit the same characteristics as the ligands contained in the conditioned media mainly due to the differences in their binding to the receptor. Thus, we have identified variants of eA1 that possess either superagonistic or antagonistic properties. These new findings will be important in the understanding of the receptor/ligand interactions and in further design of anti-cancer therapies targeting the eA/EphA system.
Assuntos
Efrina-A1/química , Regulação da Expressão Gênica , Receptor EphA2/química , Alanina/química , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Ligantes , Dados de Sequência Molecular , Mutagênese , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
Ephrin-A1 and its primary receptor, EphA2, are involved in numerous physiological processes and have been intensely studied for their roles in malignancy. Ephrin-Eph signalling is complex on its own and is also cell-type dependent, making elucidation of the exact role of ephrin-A1 in neoplasia challenging. Multiple oncogenic signalling pathways, such as MAP/ERK and PI3K are affected by ephrin-A1, and in some cases evidence suggests the promotion of a specific pathway in one cell or cancer type and inhibition of the same pathway in another type of cell or cancer. Ephrin-A1 also plays an integral role in angiogenesis and tumor neovascularization. Until recently, studies investigating ephrins focused on the ligands as GPI-anchored proteins that required membrane anchoring or artificial clustering for Eph receptor activation. However, recent studies have demonstrated a functional role for soluble, monomeric ephrin-A1. This review will focus on various forms of ephrin-A1-specific signalling in human malignancy.
Assuntos
Efrina-A1/fisiologia , Neoplasias/metabolismo , Transdução de Sinais , Animais , Fenômenos Fisiológicos Celulares , Citoesqueleto/metabolismo , Células Endoteliais/metabolismo , Efrina-A1/química , Efrina-A1/metabolismo , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/metabolismo , Estrutura Terciária de Proteína , Receptor EphA2/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The formation of a microvasculature is regulated in large part by cell-cell interactions. Ephrins and their Eph receptors mediate cell adhesion, repulsion, and migration, all critical processes in angiogenesis. (1) Here we use a covalently immobilized ephrinA1, conjugated to poly(ethylene glycol), to induce vessel formation both in vitro and in vivo in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Human umbilical vein endothelial cell (HUVEC) tubulogenesis in matrix metalloproteinase-sensitive hydrogels was visualized from 6 h to 7 days in response to three different concentrations of PEG-ephrinA1. The deposition of extracellular matrix proteins collagen IV and laminin that stabilize tubule formation were imaged, quantified, and found to be dependent on PEG-ephrinA1 concentration. To confirm the importance of the EphA2-ephrinA1 interaction in tubule formation, soluble EphA2 was used to disrupt the EphA2-ephrinA1 interaction between a coculture of HUVEC and human brain vascular pericyte cells. HUVECs seeded onto PEGDA hydrogels displayed a dose-dependent reduction in tubule formation in response to the soluble EphA2. Finally, hydrogels with releasable platelet-derived growth factor (PDGF), immobilized RGDS, and covalently immobilized PEG-ephrinA1 were implanted into the mouse cornea micropocket. These hydrogels induced a more robust vascular response with an increase in vessel density as compared with hydrogels with releasable PDGF alone. As such, PEG-ephrinA1 may represent a promising molecule to regulate cell adhesion and migration for formation of a microvasculature in tissue-engineered constructs.
Assuntos
Materiais Biomiméticos/farmacologia , Efrina-A1/farmacologia , Hidrogéis/farmacologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Efrina-A1/química , Humanos , Hidrogéis/síntese química , Hidrogéis/química , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Estrutura Molecular , Pericitos/efeitos dos fármacos , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , EstereoisomerismoRESUMO
AIMS: To slow tumor progression by reducing migratory tumor cell burden using magnetic nanoparticles (MNPs) functionalized with ligands selective for malignant cell surface receptors. MATERIALS & METHODS: Three groups of female C57BL/6 mice (control group I, control group II and experimental group) were intraperitoneally injected with a murine ovarian cancer cell line (ID8[VEGF160(+)/eGFP(+)]). Control group I received no intervention. MNPs were functionalized with ephrin-A1 mimetic peptides selective for the EphA2 receptor that is highly expressed by several cancers. Peritoneal fluids were removed by paracentesis from the experimental group and mixed with the functionalized MNPs. Magnetic filtration was used to remove particle/malignant cell conjugates and filtered peritoneal fluids were re-introduced intraperitoneally. Control group II received the same treatment as the experimental group without MNPs. RESULTS: Experimental group tumor progression was 10.77-times slower than that of control group I. CONCLUSION: Reduction of malignant cell titer significantly prolonged time to end point in a metastatic ovarian cancer model.
Assuntos
Magnetismo , Nanomedicina/métodos , Nanopartículas/química , Nanopartículas/uso terapêutico , Neoplasias Ovarianas/secundário , Neoplasias Ovarianas/terapia , Animais , Efrina-A1/química , Efrina-A1/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica/patologia , Metástase Neoplásica/terapia , Neoplasias Ovarianas/patologia , Receptor EphA2/metabolismoRESUMO
Eph receptor tyrosine kinases and their ephrin ligands regulate cell navigation during normal and oncogenic development. Signaling of Ephs is initiated in a multistep process leading to the assembly of higher-order signaling clusters that set off bidirectional signaling in interacting cells. However, the structural and mechanistic details of this assembly remained undefined. Here we present high-resolution structures of the complete EphA2 ectodomain and complexes with ephrin-A1 and A5 as the base unit of an Eph cluster. The structures reveal an elongated architecture with novel Eph/Eph interactions, both within and outside of the Eph ligand-binding domain, that suggest the molecular mechanism underlying Eph/ephrin clustering. Structure-function analysis, by using site-directed mutagenesis and cell-based signaling assays, confirms the importance of the identified oligomerization interfaces for Eph clustering.
Assuntos
Receptor EphA1/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Efrina-A1/química , Efrina-A1/genética , Efrina-A1/metabolismo , Efrina-A5/química , Efrina-A5/genética , Efrina-A5/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor EphA1/genética , Receptor EphA1/metabolismo , Receptor EphA2/química , Receptor EphA2/genética , Receptor EphA2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisAssuntos
Membrana Celular/metabolismo , Efrina-A1/metabolismo , Mecanotransdução Celular , Neoplasias/metabolismo , Receptor EphA2/química , Receptor EphA2/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Efrina-A1/química , Humanos , Ligantes , Bicamadas Lipídicas , Modelos Biológicos , Metástase Neoplásica , Neoplasias/patologia , Multimerização Proteica , Transdução de SinaisRESUMO
Activation of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands presented on apposed cell surfaces plays important roles in development and exhibits poorly understood functional alterations in cancer. We reconstituted this intermembrane signaling geometry between live EphA2-expressing human breast cancer cells and supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, clustering, and subsequent lateral transport within this junction were observed. EphA2 transport can be blocked by physical barriers nanofabricated onto the underlying substrate. This physical reorganization of EphA2 alters the cellular response to ephrin-A1, as observed by changes in cytoskeleton morphology and recruitment of a disintegrin and metalloprotease 10. Quantitative analysis of receptor-ligand spatial organization across a library of 26 mammary epithelial cell lines reveals characteristic differences that strongly correlate with invasion potential. These observations reveal a mechanism for spatio-mechanical regulation of EphA2 signaling pathways.