RESUMO
The significant role of VEGF (vascular endothelial growth factor) as an angiogenesis inducer is well recognized. Besides VEGF, EphrinB2/EphB4 also plays essential roles in vascular development and postnatal angiogenesis. Compared with classical proangiogenic factors, not only does EphrinB2/EphB4 promote sprouting of new vessels, it is also involved in the vessel maturation. Given their involvement in many physiologic and pathological conditions, EphB4 and EphrinB2 are increasingly recognized as attractive therapeutic targets for angiogenesis-related diseases through modulating their expression and function. Previous works mainly focused on the individual role of VEGF and EphrinB2/EphB4 in angiogenesis, respectively, but the correlation between EphrinB2/EphB4 and VEGF in angiogenesis has not been fully disclosed. Here, we summarize the structure and bidirectional signaling of EphrinB2/EphB4, provide an overview on the relationship between EphrinB2/EphB4 signaling and VEGF pathway in angiogenesis and highlight the associated potential usefulness in anti-angiogenetic therapy.
Assuntos
Efrina-B2/metabolismo , Neovascularização Fisiológica/fisiologia , Receptor EphB4/metabolismo , Animais , Células Cultivadas , Células Endoteliais/metabolismo , Efrina-B2/fisiologia , Humanos , Morfogênese , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/genética , Fosforilação , Receptor EphB4/fisiologia , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologia , Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Objective- Arteriovenous fistulae (AVF) are the most common access created for hemodialysis; however, many AVF fail to mature and require repeated intervention, suggesting a need to improve AVF maturation. Eph-B4 (ephrin type-B receptor 4) is the embryonic venous determinant that is functional in adult veins and can regulate AVF maturation. Cav-1 (caveolin-1) is the major scaffolding protein of caveolae-a distinct microdomain that serves as a mechanosensor at the endothelial cell membrane. We hypothesized that Cav-1 function is critical for Eph-B4-mediated AVF maturation. Approach and Results- In a mouse aortocaval fistula model, both Cav-1 mRNA and protein were increased in the AVF compared with control veins. Cav-1 KO (knockout) mice showed increased fistula wall thickening ( P=0.0005) and outward remodeling ( P<0.0001), with increased eNOS (endothelial NO synthase) activity compared with WT (wild type) mice. Ephrin-B2/Fc inhibited AVF outward remodeling in WT mice but not in Cav-1 KO mice and was maintained in Cav-1 RC (Cav-1 endothelial reconstituted) mice (WT, P=0.0001; Cav-1 KO, P=0.7552; Cav-1 RC, P=0.0002). Cavtratin-a Cav-1 scaffolding domain peptide-decreased AVF wall thickness in WT mice and in Eph-B4 het mice compared with vehicle alone (WT, P=0.0235; Eph-B4 het, P=0.0431); cavtratin also increased AVF patency (day 42) in WT mice ( P=0.0275). Conclusions- Endothelial Cav-1 mediates Eph-B4-mediated AVF maturation. The Eph-B4-Cav-1 axis regulates adaptive remodeling during venous adaptation to the fistula environment. Manipulation of Cav-1 function may be a translational strategy to enhance AVF patency.
Assuntos
Derivação Arteriovenosa Cirúrgica , Caveolina 1/fisiologia , Receptor EphB4/fisiologia , Transdução de Sinais/fisiologia , Veia Cava Inferior/fisiologia , Animais , Aorta Abdominal/cirurgia , Cavéolas/metabolismo , Caveolina 1/biossíntese , Caveolina 1/deficiência , Caveolina 1/genética , Caveolina 1/farmacologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Hemorreologia , Humanos , Pulmão/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/fisiologia , Fragmentos de Peptídeos/farmacologia , Remodelação Vascular/fisiologia , Veia Cava Inferior/cirurgiaRESUMO
PURPOSE: To evaluate the effect of stem cells from human exfoliated deciduous teeth (SHED) transfected with EphB4 gene in regulating osteogenic differentiation. METHODS: Human dental pulp tissue were harvested from extracted deciduous teeth and digested by collagenase and dispase. The SHEDs were transfected with transgenic (hEphB4-GFP) vector or empty vector (GFP-vector). Real time-polymerase chain reactionï¼real time-PCRï¼ analysis and Western blot were used to detect the expression of EphB4 in SHEDs after transfection. EphB4-SHEDs and GFP-SHEDs were subjected to osteogenic induction and assessed by alkaline phosphatase(ALP) assay and Alizarin-red S staining. SPSS 16.0 software package was used for statistical analysis. RESULTS: Real time-PCR revealed that the expression of EphB4 was significantly enhanced in EphB4-SHEDs compared to GFP-SHEDs (P<0.05). The expression of EphB4 protein was significantly higher (P<0.05) in EphB4-SHEDs compared to GFP-SHEDs. ALP assay and Alizarin-red S staining demonstrated higher ALP activity and increased mineralization with EphB4-SHEDs. CONCLUSIONS: The results indicate that transgenic expression of EphB4 in SHEDs could promote osteogenic differentiation.
Assuntos
Diferenciação Celular , Proliferação de Células , Osteogênese , Receptor EphB4 , Dente Decíduo , Células Cultivadas , Humanos , Receptor EphB4/genética , Receptor EphB4/fisiologia , Células-Tronco , TransfecçãoRESUMO
BACKGROUND AND OBJECTIVE: The goal of periodontal therapy is to regenerate/reconstruct the damaged supporting tissues of diseased teeth and to facilitate recovery of their physiological functions. Combination of stem cell transplantation and gene therapy offers a viable method for accelerating periodontal repair and regeneration. In this study, the role of the ephrinB2/EphB4 signaling pathway in regulating osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and crosstalk between PDLSCs and pre-osteoblasts within co-culture was investigated through ephrinB2 transgenic expression in PDLSCs. MATERIAL AND METHODS: PDLSCs isolated from premolar teeth of teenage patients undergoing orthodontic treatment were transfected with transgenic (hEfnB2-GFP-Bsd) vector or empty vector (GFP-Bsd). Vector-PDLSCs, EfnB2-PDLSCs, MC3T3-E1 and co-cultures of vector-PDLSCs with MC3T3-E1, and EfnB2-PDLSCs with MC3T3-E1 were subjected to osteogenic induction. The osteogenic differentiation of EfnB2-PDLSCs, vector-PDLSCs and co-cultures were assessed by reverse transcription-polymerase chain reaction, alkaline phosphatase (ALP) assay and Alizarin-red S staining. Protein expression levels of ephrinB2, EphB4, phosphorylated ephrinB2 and EphB4 were analyzed by western blot, immunoprecipitation and co-immunoprecipitation assays. RESULTS: ALP assay and Alizarin-red S staining demonstrated higher ALP activity and increased mineralization with EfnB2-PDLSCs vs. vector-PDLSCs and with co-culture of EfnB2-PDLSCs and MC3T3-E1 vs. vector-PDLSCs and MC3T3-E1. Reverse transcription-polymerase chain reaction revealed that the expression of human odonto/osteogenic markers were significantly enhanced in EfnB2-PDLSCs compared to vector-PDLSCs, and that the expression of mouse odonto/osteogenic markers were significantly higher in co-culture of EfnB2-PDLSCs with MC3T3-E1 vs. vector-PDLSCs with MC3T3-E1. The EphB4 receptor was activated through phosphorylation during osteogenic differentiation. CONCLUSION: Our data indicate that transgenic expression of ephrinB2 in PDLSCs could promote osteogenic differentiation via stimulation of the phosphorylation of ephrinB2 and EphB4, which regulates cell communication between PDLSCs and between PDLSCs and pre-osteoblasts within co-culture.
Assuntos
Efrina-B2/fisiologia , Osteoblastos/fisiologia , Osteogênese/fisiologia , Ligamento Periodontal/citologia , Receptor EphB4/fisiologia , Células-Tronco/fisiologia , Western Blotting , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Técnicas de Cocultura/métodos , Técnicas de Transferência de Genes , Humanos , Imunoprecipitação , Ligamento Periodontal/metabolismo , Ligamento Periodontal/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Members of the Eph family of receptor tyrosine kinases have been implicated in a wide array of human cancers. The EphB4 receptor is ubiquitously expressed in head and neck squamous cell carcinoma (HNSCC) and has been shown to impart tumorigenic and invasive characteristics to these cancers. In this study, we investigated whether EphB4 receptor targeting can enhance the radiosensitization of HNSCC. Our data show that EphB4 is expressed at high to moderate levels in HNSCC cell lines and patient-derived xenograft (PDX) tumors. We observed decreased survival fractions in HNSCC cells following EphB4 knockdown in clonogenic assays. An enhanced G2 cell cycle arrest with activation of DNA damage response pathway and increased apoptosis was evident in HNSCC cells following combined EphB4 downregulation and radiation compared to EphB4 knockdown and radiation alone. Data using HNSCC PDX models showed significant reduction in tumor volume and enhanced delay in tumor regrowth following sEphB4-HSA administration with radiation compared to single agent treatment. sEphB4-HSA is a protein known to block the interaction between the EphB4 receptor and its ephrin-B2 ligand. Overall, our findings emphasize the therapeutic relevance of EphB4 targeting as a radiosensitizer that can be exploited for the treatment of human head and neck carcinomas.
Assuntos
Carcinoma de Células Escamosas/enzimologia , Neoplasias de Cabeça e Pescoço/enzimologia , Proteínas de Neoplasias/fisiologia , Receptor EphB4/fisiologia , Animais , Apoptose , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/radioterapia , Linhagem Celular Tumoral/efeitos da radiação , Reparo do DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Técnicas de Silenciamento de Genes , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Queratinócitos/enzimologia , Camundongos , Terapia de Alvo Molecular , Proteínas de Neoplasias/deficiência , Interferência de RNA , RNA Interferente Pequeno/genética , Tolerância a Radiação , Receptor EphB4/deficiência , Carga Tumoral , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ischemic cardiovascular disease remains one of the leading causes of morbidity and mortality in the world. Proangiogenic therapy appears to be a promising and feasible strategy for the patients with ischemic cardiovascular disease, but the results of preclinical and clinical trials are limited due to the complicated mechanisms of angiogenesis. Facilitating the formation of functional vessels is important in rescuing the ischemic cardiomyocytes. EphrinB2/EphB4, a novel pathway in angiogenesis, plays a critical role in both microvascular growth and neovascular maturation. Hence, investigating the mechanisms of EphrinB2/EphB4 pathway in angiogenesis may contribute to the development of novel therapeutics for ischemic cardiovascular disease. Previous reviews mainly focused on the role of EphrinB2/EphB4 pathway in embryo vascular development, but their role in postnatal angiogenesis in ischemic heart disease has not been fully illustrated. Here, we summarized the current knowledge of EphrinB2/EphB4 in angiogenesis and their interaction with other angiogenic pathways in ischemic cardiovascular disease.
Assuntos
Efrina-B2/fisiologia , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/fisiopatologia , Neovascularização Fisiológica , Receptor EphB4/fisiologia , Animais , Efrina-B2/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Ligantes , Proteínas de Membrana/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Receptor EphB4/química , Receptores Notch/fisiologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
OBJECTIVES: This study aims to identify candidate genes and critical pathways involved in osteoarthritis (OA). METHODS: Gene expression data of synovial membrane from OA patients and normal controls (NCs) were downloaded from database. Totally, 15 OA and 14 NC chips were available. Differentially expressed genes (DEGs) were identified through limma package (log2 fold change >0.585, false discovery rate (FDR) < 0.05), and protein-protein interaction (PPI) network was constructed using STRING. Moreover, perturbation and pathway enrichment analyses were performed through PerturbationAnalyzer in Cytoscape (iterative criteria <1×e-10) and clusterProfiler package (FDR <0.05), respectively. RESULTS: Totally, 236 up-regulated and 290 down-regulated DEGs were identified. In PPI network, 10 hub genes were found, including VEGFA, IL6, JUN, IL1B, ICAM1, ATF3, IL8, EGR1, CDKN1A, and JUNB. After perturbation analysis, 32 DEGs were passively and significantly changed, like PISD, RARRES3, EIF4G1, and EPHA3. Furthermore, 526 DEGs were enriched in 176 pathways, and pathway cross-talk network was constructed, involving 12 pathways and 66 cross-talks. CONCLUSIONS: Pathways like rheumatoid arthritis, osteoclast differentiation, and cytokine-cytokine receptor interaction might play critical roles in OA, and previously unreported genes VEGFA, JUN, JUNB, PISD, RARRES3, EIF4G1, and EPHA3 might participate in OA, providing novel directions for drug targeting.
Assuntos
Biologia Computacional/métodos , Osteoartrite/genética , Análise por Conglomerados , Perfilação da Expressão Gênica , Humanos , Mapas de Interação de Proteínas , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Receptor EphA3 , Receptor EphB4/genética , Receptor EphB4/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Hemangiopericytoma (HPC) is a highly vascularized mesenchymal tumor. Local recurrence and distant metastasis are common features of HPC. Considering the remarkable hyper-vasculature phenotype of HPC, we assumed that dysregulated angiogenic signaling pathways were involved in HPC. The key components of angiogenic signaling pathways including VEGF-VEGF-R2, EphrinB2-EphB4 and DLL4-Notch were examined by real-time RT-PCR, Western blotting and immunostaining in 17 surgical specimens of HPC patients and in 6 controls. A significant upregulation of VEGF and VEGF-R2 associated with elevated levels of p-Akt and proliferating cell nuclear antigen (PCNA) was detected in HPC. Moreover, a dramatic increase in the mRNA and protein expression of EphB4 and its downstream factor p-Erk1/2 was found in HPC. A massive activation of core-components of DLL4-Notch signaling was detected in HPC. Double-immunofluorescent staining confirmed the expression of these upregulated key factors in the endothelial cells of tumor vessels. The present study identified the activation of multiple and crucial angiogenic signaling pathways, which could function individually and/or synergistically to stimulate angiogenesis in HPC and eventually contribute to tumor growth and progression. Our findings emphasize the importance to target multiple angiogenic signaling pathways when an anti-angiogenic therapy is considered for this highly vascularized tumor.
Assuntos
Neoplasias do Sistema Nervoso Central/irrigação sanguínea , Neoplasias do Sistema Nervoso Central/genética , Hemangiopericitoma/irrigação sanguínea , Hemangiopericitoma/genética , Neovascularização Patológica/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Ativação Transcricional/genética , Ativação Transcricional/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Proteínas de Ligação ao Cálcio , Neoplasias do Sistema Nervoso Central/patologia , Progressão da Doença , Efrina-B2/genética , Efrina-B2/fisiologia , Feminino , Hemangiopericitoma/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Receptor EphB4/genética , Receptor EphB4/fisiologia , Receptores Notch/genética , Receptores Notch/fisiologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologiaRESUMO
EFNB2 and EPHB4, which belong to a large tyrosine kinase receptor superfamily, are molecular markers of arterial and venous blood vessels, respectively. EFNB2/EPHB4 signaling plays an important role in physiological and pathological angiogenesis, and its role in tumor vessel development has been extensively studied. Pregnancy and tumors share similar features, including continuous cell proliferation and increased demand for a blood supply. Our previous studies showed that Efnb2 and Ephb4 were expressed dynamically in the spiral arteries, uterine natural killer cells, and trophoblasts during mouse gestation Days 6.5-12.5. Moreover, uterine natural killer cells and trophoblasts are required for the modification of spiral arteries. Oxygen tension within the pregnant uterus, which contributes to the vascular development, also affects EFNB2 and EPHB4 expression. Considering the role of EFNB2/EPHB4 signaling in embryonic and tumor vascular development, and its dynamic expression in the decidua and placenta, we hypothesize that EFNB2 and EPHB4 are involved in the regulation of spiral artery remodeling. Investigating this hypothesis will help clarify the mechanisms of pathological pregnancy that may underlie abnormal spiral artery development.
Assuntos
Efrina-B2/fisiologia , Gravidez/fisiologia , Receptor EphB4/fisiologia , Artéria Uterina/crescimento & desenvolvimento , Animais , Feminino , Camundongos , Camundongos Transgênicos , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Gravidez/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Artéria Uterina/metabolismo , Útero/irrigação sanguíneaRESUMO
Transmembrane tyrosine-kinase Ephrin receptors promote tumor progression and/or metastasis of several malignancies including leukemia, follicular lymphoma, glioma, malignant pleural mesothelioma, papillary thyroid carcinoma, sarcomas and ovarian, breast, bladder and non-small cell lung cancers. They also drive intestinal stem cell proliferation and positioning, control intestinal tissue boundaries and are involved in liver, pancreatic and colorectal cancers, indicating involvement in additional digestive system malignancies. We investigated the role of Ephrin-B4 receptor (EPHB4), and its ligand EFNB2, in gastric and gastroesophageal junction cancers in patient cohorts through computational, mathematical, molecular and immunohistochemical analyses. We show that EPHB4 is upregulated in preneoplastic gastroesophageal lesions and its expression further increased in gastroesophageal cancers in several independent cohorts. The closely related EPHB6 receptor, which also binds EFNB2, was downregulated in all tested cohorts, consistent with its tumor-suppressive properties in other cancers. EFNB2 expression is induced in esophageal cells by acidity, suggesting that gastroesophageal reflux disease (GERD) may constitute an early triggering event in activating EFNB2-EPHB4 signaling. Association of EPHB4 to both Barrett's esophagus and to advanced tumor stages, and its overexpression at the tumor invasion front and vascular endothelial cells intimate the notion that EPHB4 may be associated with multiple steps of gastroesophageal tumorigenesis. Analysis of oncogenomic signatures uncovered the first EPHB4-associated gene network (false discovery rate: 7 × 10(-90) ) composed of a five-transcription factor interconnected gene network that drives proliferation, angiogenesis and invasiveness. The EPHB4 oncogenomic network provides a molecular basis for its role in tumor progression and points to EPHB4 as a potential tumor aggressiveness biomarker and drug target in gastroesophageal cancers.
Assuntos
Neoplasias Esofágicas/etiologia , Junção Esofagogástrica , Redes Reguladoras de Genes , Receptor EphB4/fisiologia , Neoplasias Gástricas/etiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Efrina-B2/fisiologia , Neoplasias Esofágicas/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptores Proteína Tirosina Quinases/fisiologia , Receptor EphB4/análise , Receptor EphB4/genética , Receptores da Família Eph , Neoplasias Gástricas/genéticaRESUMO
The purpose of this paper is to investigate the possible mechanisms of resistance to chemotherapy in melanoma from the perspective of molecular biology and to discuss the strategies to overcome them. Cisplatin, a DNA-damaging compound that triggers apoptotic cell death, is commonly used in the treatment of malignant melanoma. However, most patients develop mechanisms of acquired resistance and about 25% of them do not achieve tumor regression at all, due to intrinsic resistance to therapy. In the current study, we reported the tumor xenografts of the human A375 melanoma, after 40-weeks' consecutive therapy with cisplatin that developed resistance as a result of EphB4 overexpression. Moreover, the expression of phospho-AKT and phospho-ERK were significantly increased in cisplatin-resistant tumors. In addition, combined of cisplatin with EphB4 selective inhibitor could abrogate this acquired mechanism of drug resistance due to an enhanced apoptotic effect in cisplatin-resistant xenografts. In summary, these results help to understand the mechanisms of acquired resistance to chemotherapy and provide important information for clinical treatment strategies.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma/tratamento farmacológico , Melanoma/patologia , Receptor EphB4/antagonistas & inibidores , Receptor EphB4/genética , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Xenoenxertos , Humanos , Melanoma/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular , Transplante de Neoplasias , Receptor EphB4/fisiologia , Neoplasias Cutâneas/genéticaRESUMO
EPH kinases are the largest family of receptor tyrosine kinases, and their ligands, ephrins (EFNs), are also cell surface molecules. This work presents evidence that EPHB4 on vascular smooth muscle cells (VSMCs) is involved in blood pressure regulation. We generated gene KO mice with smooth muscle cell-specific deletion of EPHB4. Male KO mice, but not female KO mice, were hypotensive. VSMCs from male KO mice showed reduced contractility when compared with their WT counterparts. Signaling both from EFNBs to EPHB4 (forward signaling) and from EPHB4 to EFNB2 (reverse signaling) modulated VSMC contractility. At the molecular level, the absence of EPHB4 in VSMCs resulted in compromised signaling from Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) to myosin light chain kinase (MLCK) to myosin light chain, the last of which controls the contraction force of motor molecule myosin. Near the cell membrane, an adaptor protein GRIP1, which can associate with EFNB2, was found to be essential in mediating EPHB4-to-EFNB reverse signaling, which regulated VSMC contractility, based on siRNA gene knockdown studies. Our research indicates that EPHB4 plays an essential role in regulating small artery contractility and blood pressure.
Assuntos
Deleção de Genes , Hipotensão/metabolismo , Músculo Liso Vascular/metabolismo , Receptor EphB4/fisiologia , Animais , Artérias/metabolismo , Pressão Sanguínea , Cálcio/metabolismo , Feminino , Genótipo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular , Fosforilação , RNA Interferente Pequeno/metabolismo , Fatores Sexuais , Transdução de SinaisRESUMO
BACKGROUND: The EphB4 receptor tyrosine kinase is overexpressed in many cancers including prostate cancer. The molecular mechanisms by which this ephrin receptor influences cancer progression are complex as there are tumor-promoting ligand-independent mechanisms in place as well as ligand-dependent tumor suppressive pathways. METHODS: We employed transient knockdown of EPHB4 in prostate cancer cells, coupled with gene microarray analysis, to identify genes that were regulated by EPHB4 and may represent linked tumor-promoting factors. We validated target genes using qRT-PCR and employed functional assays to determine their role in prostate cancer migration and invasion. RESULTS: We discovered that over 500 genes were deregulated upon EPHB4 siRNA knockdown, with integrin ß8 (ITGB8) being the top hit (29-fold down-regulated compared to negative non-silencing siRNA). Gene ontology analysis found that the process of cell adhesion was highly deregulated and two other integrin genes, ITGA3 and ITGA10, were also differentially expressed. In parallel, we also discovered that over-expression of EPHB4 led to a concomitant increase in ITGB8 expression. In silico analysis of a prostate cancer progression microarray publically available in the Oncomine database showed that both EPHB4 and ITGB8 are highly expressed in prostatic intraepithelial neoplasia, the precursor to prostate cancer. Knockdown of ITGB8 in PC-3 and 22Rv1 prostate cancer cells in vitro resulted in significant reduction of cell migration and invasion. CONCLUSIONS: These results reveal that EphB4 regulates integrin ß8 expression and that integrin ß8 plays a hitherto unrecognized role in the motility of prostate cancer cells and thus targeting integrin ß8 may be a new treatment strategy for prostate cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Cadeias beta de Integrinas/biossíntese , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptor EphB4/fisiologia , Linhagem Celular Tumoral , Humanos , Masculino , Receptores Proteína Tirosina Quinases/fisiologiaRESUMO
The Eph kinase (EPH) and ephrin (EFN) families are involved in a broad range of developmental processes. Increasing evidence is demonstrating the important roles of EPHBs and EphrinBs in the immune system. In this study on epithelial cell-specific Ephb4 knockout (KO) mice, we investigated T-cell development and function after EPHB4 deletion. KO mice presented normal thymic weight and cellularity. Their thymocyte subpopulation percentages were in the normal range. KO mice had normal T-cell numbers and percentages in the spleen, and T cells were activated and proliferated normally upon TCR ligation. Furthermore, naïve spleen CD4 cells from KO and wild type mice were capable of differentiating, in a comparable manner, into Th1, Th17 and Treg cells. In vivo, KO mice mounted effective delayed type hypersensitivity responses, indicating that thymocytes develop normally in the absence of TEC EPHB4, and T cells derived from EPHB4-deleted thymic epithelian cells (TEC) have normal function. Our data suggest that heavy redundancy and promiscuous interaction between EPHs and EFNs compensate for the missing EPHB4 in TECs, and TEC EPHB4's role in T cell development might only be revealed if multiple EPHs are ablated simultaneously. We cannot exclude the possibility that (1) some immunological parameters not examined in this study are affected by the deletion; (2) the deletion is not complete due to the leaky Cre-LoxP system, and the remaining EPHB4 in TEC is sufficient for thymocyte development; or (3) EPHB4 expression in TEC is not required for T cell development and function.
Assuntos
Receptor EphB4/fisiologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Timócitos/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células , Efrinas/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/biossíntese , Receptor EphB4/genética , Receptores da Família Eph/imunologia , Transdução de Sinais/imunologia , Baço/citologia , Baço/imunologia , Timo/crescimento & desenvolvimento , Timo/imunologiaRESUMO
Bidirectional Eph-Ephrin signaling as a focal point of research in cell-cell communications is critical for generation of nerves and vesssels as well as invation and metastasis of tumor cells. The roles for Ephrin-Eph bidirectional signaling in bone remodeling were important. EphrinB2 is expressed on osteoblasts and EphB4 is expressed on osteoclasts. Forward signaling through the EphB4 receptor into mesenchymal precursors promotes osteoblast differentiation, while reverse signaling through the EphrinB2 ligand into osteoclast suppresses differentiation. Signaling between the ligand EphrinB2 and the receptors EphB4 explains bidirectional signaling between osteoblasts and osteoclasts,bone absorption and remodeling, which may lay a theoretical foundation for identifying drug targeting and preventing and treating bone loss.
Assuntos
Remodelação Óssea/fisiologia , Efrina-B2/fisiologia , Receptor EphB4/fisiologia , Transdução de Sinais/fisiologia , Animais , Humanos , Osteoblastos/citologia , Osteoclastos/citologiaRESUMO
Neurogenesis in the adult hippocampus involves activation of quiescent neural stem cells (NSCs) to yield transiently amplifying NSCs, progenitors, and, ultimately, neurons that affect learning and memory. This process is tightly controlled by microenvironmental cues, although a few endogenous factors are known to regulate neuronal differentiation. Astrocytes have been implicated, but their role in juxtacrine (that is, cell-cell contact dependent) signaling in NSC niches has not been investigated. We found that ephrin-B2 presented from rodent hippocampal astrocytes regulated neurogenesis in vivo. Furthermore, clonal analysis in NSC fate-mapping studies revealed a previously unknown role for ephrin-B2 in instructing neuronal differentiation. In addition, ephrin-B2 signaling, transduced by EphB4 receptors on NSCs, activated ß-catenin in vitro and in vivo independently of Wnt signaling and upregulated proneural transcription factors. Ephrin-B2(+) astrocytes therefore promote neuronal differentiation of adult NSCs through juxtacrine signaling, findings that advance our understanding of adult neurogenesis and may have future regenerative medicine implications.
Assuntos
Astrócitos/fisiologia , Efrina-B2/fisiologia , Hipocampo/fisiologia , Neurogênese/fisiologia , Animais , Astrócitos/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Efrina-B2/biossíntese , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Receptor EphB4/biossíntese , Receptor EphB4/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Regulação para Cima , beta Catenina/metabolismoRESUMO
Eph-B4 determines mammalian venous differentiation in the embryo but is thought to be a quiescent marker of adult veins. We have previously shown that surgical transposition of a vein into the arterial environment is characterized by loss of venous identity, as indicated by the loss of Eph-B4, and intimal thickening. We used a mouse model of vein graft implantation to test the hypothesis that Eph-B4 is a critical determinant of venous wall thickness during postsurgical adaptation to the arterial environment. We show that stimulation of Eph-B4 signaling, either via ligand stimulation or expression of a constitutively active Eph-B4, inhibits venous wall thickening and preserves venous identity; conversely, reduction of Eph-B4 signaling is associated with increased venous wall thickness. Stimulated Eph-B4 associates with caveolin-1 (Cav-1); loss of Cav-1 or Eph-B4 kinase function abolishes inhibition of vein graft thickening. These results show that Eph-B4 is active in adult veins and regulates venous remodeling. Eph-B4-Cav-1-mediated vessel remodeling may be a venous-specific adaptive mechanism. Controlled stimulation of embryonic signaling pathways such as Eph-B4 may be a novel strategy to manipulate venous wall remodeling in adults.
Assuntos
Adaptação Fisiológica/fisiologia , Artérias/fisiologia , Receptor EphB4/fisiologia , Veias/fisiologia , Animais , Caveolina 1/fisiologia , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Ratos , Transdução de Sinais/fisiologia , Veias/transplanteRESUMO
The tyrosine kinase receptor EphB4 interacts with its ephrinB2 ligand to act as a bidirectional signaling system that mediates adhesion, migration, and guidance by controlling attractive and repulsive activities. Recent findings have shown that hematopoietic cells expressing EphB4 exert adhesive functions towards endothelial cells expressing ephrinB2. We therefore hypothesized that EphB4/ephrinB2 interactions may be involved in the preferential adhesion of EphB4-expressing tumor cells to ephrinB2-expressing endothelial cells. Screening of a panel of human tumor cell lines identified EphB4 expression in nearly all analyzed tumor cell lines. Human A375 melanoma cells engineered to express either full-length EphB4 or truncated EphB4 variants which lack the cytoplasmic catalytic domain (ΔC-EphB4) adhered preferentially to ephrinB2-expressing endothelial cells. Force spectroscopy by atomic force microscopy confirmed, on the single cell level, the rapid and direct adhesive interaction between EphB4 and ephrinB2. Tumor cell trafficking experiments in vivo using sensitive luciferase detection techniques revealed significantly more EphB4-expressing A375 cells but not ΔC-EphB4-expressing or mock-transduced control cells in the lungs, the liver, and the kidneys. Correspondingly, ephrinB2 expression was detected in the microvessels of these organs. The specificity of the EphB4-mediated tumor homing phenotype was validated by blocking the EphB4/ephrinB2 interaction with soluble EphB4-Fc. Taken together, these experiments identify adhesive EphB4/ephrinB2 interactions between tumor cells and endothelial cells as a mechanism for the site-specific metastatic dissemination of tumor cells. AACR.
Assuntos
Comunicação Celular , Movimento Celular , Endotélio Vascular/patologia , Efrina-B2/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/patologia , Melanoma/secundário , Receptor EphB4/fisiologia , Animais , Adesão Celular/genética , Comunicação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Endotélio Vascular/metabolismo , Efrina-B2/biossíntese , Efrina-B2/genética , Humanos , Melanoma/irrigação sanguínea , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptor EphB4/metabolismoRESUMO
OBJECTIVE: We investigated the mechanism responsible for imatinib (IM) resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) cell lines. METHODS: We established cell lines from a patient with Ph(+) ALL at the time of first diagnosis and relapsed phase and designated as NPhA1 and NPhA2, respectively. We also derived IM-resistant cells, NPhA2/STIR, from NPhA2 under gradually increasing IM concentrations. RESULTS: NPhA1 was sensitive to IM (IC(50) 0.05 microm) and NPhA2 showed mild IM resistance (IC(50) 0.3 microm). NPhA2/STIR could be maintained in the presence of 10 microm IM. Phosphorylation of MEK and ERK was slightly elevated in NPhA2 and significantly elevated in NPhA2/STIR compared to NPhA1 cells. After treatment with IM, phosphorylation of MEK and ERK was not suppressed but rather increased in NPhA2 and NPhA2/STIR. Active RAS was also increased markedly in NPhA2/STIR after IM treatment. The expression of BCL-2 was increased in NPhA2 compared to NPhA1, but no further increase in NPhA2/STIR. Proliferation of NPhA2/STIR was significantly inhibited by a combination of MEK inhibitor and IM. Analysis of tyrosine phosphorylation status with a protein tyrosine kinase array showed increased phosphorylation of EphB4 in NPhA2/STIR after IM treatment. Although transcription of EphB4 was suppressed in NPhA1 and NPhA2 after IM treatment, it was not suppressed and its ligand, ephrinB2, was increased in NPhA2/STIR. Suppression of EphB4 transcripts by introducing short hairpin RNA into NPhA2/STIR partially restored their sensitivity to IM. CONCLUSIONS: These results suggest a new mechanism of IM resistance mediated by the activation of RAS/MAPK pathway and EphB4.
Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Neoplasias/fisiologia , Piperazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptor EphB4/fisiologia , Proteínas ras/fisiologia , Benzamidas , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/enzimologia , Ativação Enzimática , Indução Enzimática , Efrina-B2/genética , Efrina-B2/fisiologia , Feminino , Humanos , Mesilato de Imatinib , Sistema de Sinalização das MAP Quinases/fisiologia , Pessoa de Meia-Idade , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Fosforilação/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Receptor EphB4/antagonistas & inibidores , Receptor EphB4/genética , RecidivaRESUMO
Recent findings have started to uncover the intriguing roles of the Eph family of receptor tyrosine kinases in normal epithelial cells and during oncogenic transformation. This review focuses on EphB4, an Eph receptor that has both tumor-suppressing and tumor-promoting activities in breast cancer. Understanding the multifaceted role of EphB4 in tumorigenesis may allow the development of new anticancer therapies.