EphrinA1-Fc Attenuates Ventricular Remodeling and Dysfunction in Chronically Nonreperfused WT but not EphA2-R-M mice.

BACKGROUND: EphrinA1-Fc abolishes acute I/R injury and attenuates nonreperfused cardiac injury 4 days after permanent occlusion in mice. The goal of this study was to assess the capacity of a single intramyocardial administration of ephrinA1-Fc at the time of coronary artery ligation, to determine the degree to which early salvage effects translate to reduced adverse remodeling after 4 weeks of nonreperfused myocardial infarction (MI) in wild-type B6 and EphA2-R-M (EphA2 receptor null) mice. METHODS: At 4 weeks post-MI, echocardiography, histologic and immunohistochemical analyses of B6 mouse hearts were performed. Primary mouse cardiac fibroblasts (FBs) isolated from B6 mice cultured in the presence of low and high dose ephrinA1-Fc, both with and without pro-fibrotic TGF-ß stimulation and Western blots, were probed for relative expression of remodeling proteins MMP-2, MMP-9 and TIMP-1, in addition to DDR2 and (p)SMAD2/3/totalSMAD2/3. RESULTS: EphrinA1-Fc preserved a significant degree of contractile function, decreased adverse left ventricular remodeling, attenuated excessive compensatory hypertrophy, and decreased interstitial fibrosis in wild-type (WT) B6 mouse hearts. In contrast, most of these parameters were poorer in ephrinA1-Fc-treated EphA2-R-M mice. Of note, fibrosis was proportionately decreased, implying that other EphA receptor(s) are more important in regulating the pro-fibrotic response. Primary FBs showed disparate alteration of MMP-2, MMP-9 and TIMP-1, as well as DDR2 and p-SMAD2/3/totalSMAD2/3, which indicates that matrix remodeling and cardiac fibrosis in the injured heart are influenced by ephrinA1-Fc. CONCLUSION: This study demonstrates the capacity of a single administration of ephrinA1-Fc at the onset of injury to attenuate long-term nonreperfused post-MI ventricular remodeling that results in progressive heart failure, and the important role of EphA2 in mitigating the deleterious effects.

Intracardiac administration of ephrinA1-Fc preserves mitochondrial bioenergetics during acute ischemia/reperfusion injury.

AIMS: Intracardiac injection of recombinant EphrinA1-Fc immediately following coronary artery ligation in mice reduces infarct size in both reperfused and non-reperfused myocardium, but the cellular alterations behind this phenomenon remain unknown. MAIN METHODS: Herein, 10 wk-old B6129SF2/J male mice were exposed to acute ischemia/reperfusion (30minI/24hrsR) injury immediately followed by intracardiac injection of either EphrinA1-Fc or IgG-Fc. After 24 h of reperfusion, sections of the infarct margin in the left ventricle were imaged via transmission electron microscopy, and mitochondrial function was assessed in both permeabilized fibers and isolated mitochondria, to examine mitochondrial structure, function, and energetics in the early stages of repair. KEY FINDINGS: At a structural level, EphrinA1-Fc administration prevented the I/R-induced loss of sarcomere alignment and mitochondrial organization along the Z disks, as well as disorganization of the cristae and loss of inter-mitochondrial junctions. With respect to bioenergetics, loss of respiratory function induced by I/R was prevented by EphrinA1-Fc. Preservation of cardiac bioenergetics was not due to changes in mitochondrial JH2O2 emitting potential, membrane potential, ADP affinity, efficiency of ATP production, or activity of the main dehydrogenase enzymes, suggesting that EphrinA1-Fc indirectly maintains respiratory function via preservation of the mitochondrial network. Moreover, these protective effects were lost in isolated mitochondria, further emphasizing the importance of the intact cardiomyocyte ultrastructure in mitochondrial energetics. SIGNIFICANCE: Collectively, these data suggest that intracardiac injection of EphrinA1-Fc protects cardiac function by preserving cardiomyocyte structure and mitochondrial bioenergetics, thus emerging as a potential therapeutic strategy in I/R injury.

Functional analysis of eliciting plant response protein Epl1-Tas from Trichoderma asperellum ACCC30536.

Eliciting plant response protein (Epl) is a small Trichoderma secreted protein that acts as an elicitor to induce plant defense responses against pathogens. In the present study, the differential expression, promoter analysis, and phylogenetic tree analysis of Epl1-Tas (GenBank JN966996) from T. asperellum ACCC30536 were performed. The results showed Epl1-Tas could play an important role in the interaction between T. asperellum ACCC30536 and woody plant or woody plant pathogen. Furthermore, the effect of the Escherichia coli recombinant protein rEpl1-e and the Pichia pastoris recombinant protein rEpl1-p on Populus davidiana × P. alba var. pyramidalis (PdPap) was studied. In PdPap seedlings, rEpl1-e or rEpl1-p induction altered the expression levels of 11 genes in the salicylic acid (SA, three genes), jasmonic acid (JA, four genes) and auxin (four genes) signal transduction pathways, and five kinds of enzymes activities The induction level of rEpl1-p was significantly higher than that of rEpl1-e, indicating that rEpl1-p could be used for further induction experiment. Under 3 mg/mL rEpl1-p induction, the mean height of the PdPap seedlings increased by 57.65% and the mean lesion area on the PdPap seedlings leaves challenged with Alternaria alternata decreased by 91.22% compared with those of the control. Thus, elicitor Epl1-Tas could induce the woody plant resistance to pathogen.

Bifunctional role of ephrin A1-Eph system in stimulating cell proliferation and protecting cells from cell death through the attenuation of ER stress and inflammatory responses in bovine mammary epithelial cells.

Structural and functional development of the mammary gland is constant in the mammary gland life cycle. Eph receptors and their ligands, ephrins, control events through cell-to-cell interactions during embryonic development, and adult tissue homeostasis; however, little information on participation of ephrin A1, a representative ligand of the Eph receptor, in the development and function of normal mammary glands is known. In this study, we demonstrated functional effects of the ephrin A1-Eph system and mechanisms of its action on bovine mammary epithelial (MAC-T) cells. The in vitro cultured MAC-T cells expressed the ephrin A1 ligand and EphA1, A2, A4, A7, and A8 among the eight members of the Eph A family. Our results revealed that ephrin A1 induced MAC-T cell cycle progression and stimulated cell proliferation with abundant expression of nucleic PCNA and cyclin D1 proteins. Additionally, ephrin A1 induced activation of intracellular signaling molecules involved in PI3 K/AKT and MAPK signaling, and the proliferation-stimulating effect of ephrin A1 was mediated by activation of these pathways. Furthermore, ephrin A1 influenced expression and activation of various ER stress-related proteins and protected MAC-T cells from stress-induced cell death. Finally, ephrin A1 alleviated LPS-induced cell death through down-regulation of inflammatory cytokines. In conclusion, the results of this study suggest that the Eph A-ephrin A1 system is a positive factor in the increase and maintenance of epithelial cells in mammary glands of cows; the signaling system contributes to development, remodeling, and functionality of normal mammary glands and could overcome mastitis in cows and other mammals.

EphA receptors and ephrin-A ligands are upregulated by monocytic differentiation/maturation and promote cell adhesion and protrusion formation in HL60 monocytes.

BACKGROUND: Eph signaling is known to induce contrasting cell behaviors such as promoting and inhibiting cell adhesion/spreading by altering F-actin organization and influencing integrin activities. We have previously demonstrated that EphA2 stimulation by ephrin-A1 promotes cell adhesion through interaction with integrins and integrin ligands in two monocyte/macrophage cell lines. Although mature mononuclear leukocytes express several members of the EphA/ephrin-A subclass, their expression has not been examined in monocytes undergoing during differentiation and maturation. RESULTS: Using RT-PCR, we have shown that EphA2, ephrin-A1, and ephrin-A2 expression was upregulated in murine bone marrow mononuclear cells during monocyte maturation. Moreover, EphA2 and EphA4 expression was induced, and ephrin-A4 expression was upregulated, in a human promyelocytic leukemia cell line, HL60, along with monocyte differentiation toward the classical CD14++CD16- monocyte subset. Using RT-PCR and flow cytometry, we have also shown that expression levels of αL, αM, αX, and ß2 integrin subunits were upregulated in HL60 cells along with monocyte differentiation while those of α4, α5, α6, and ß1 subunits were unchanged. Using a cell attachment stripe assay, we have shown that stimulation by EphA as well as ephrin-A, likely promoted adhesion to an integrin ligand-coated surface in HL60 monocytes. Moreover, EphA and ephrin-A stimulation likely promoted the formation of protrusions in HL60 monocytes. CONCLUSIONS: Notably, this study is the first analysis of EphA/ephrin-A expression during monocytic differentiation/maturation and of ephrin-A stimulation affecting monocyte adhesion to an integrin ligand-coated surface. Thus, we propose that monocyte adhesion via integrin activation and the formation of protrusions is likely promoted by stimulation of EphA as well as of ephrin-A.

Enhancing Irreversible Electroporation by Manipulating Cellular Biophysics with a Molecular Adjuvant.

Pulsed electric fields applied to cells have been used as an invaluable research tool to enhance delivery of genes or other intracellular cargo, as well as for tumor treatment via electrochemotherapy or tissue ablation. These processes involve the buildup of charge across the cell membrane, with subsequent alteration of transmembrane potential that is a function of cell biophysics and geometry. For traditional electroporation parameters, larger cells experience a greater degree of membrane potential alteration. However, we have recently demonstrated that the nuclear/cytoplasm ratio (NCR), rather than cell size, is a key predictor of response for cells treated with high-frequency irreversible electroporation (IRE). In this study, we leverage a targeted molecular therapy, ephrinA1, known to markedly collapse the cytoplasm of cells expressing the EphA2 receptor, to investigate how biophysical cellular changes resulting from NCR manipulation affect the response to IRE at varying frequencies. We present evidence that the increase in the NCR mitigates the cell death response to conventional electroporation pulsed-electric fields (∼100 µs), consistent with the previously noted size dependence. However, this same molecular treatment enhanced the cell death response to high-frequency electric fields (∼1 µs). This finding demonstrates the importance of considering cellular biophysics and frequency-dependent effects in developing electroporation protocols, and our approach provides, to our knowledge, a novel and direct experimental methodology to quantify the relationship between cell morphology, pulse frequency, and electroporation response. Finally, this novel, to our knowledge, combinatorial approach may provide a paradigm to enhance in vivo tumor ablation through a molecular manipulation of cellular morphology before IRE application.

Protection by the Eph-Ephrin System Against Mesenteric Ischemia-Reperfusion Injury.

Mesenteric ischemia-reperfusion (I/R)-induced injury targets primarily endothelial and epithelial cells, leading to a cascade of inflammatory events, eventually culminating in life-threatening syndromes. Hitherto, the role of Eph, the largest family of tyrosine kinase receptors, and of their cell-bound ephrin ligands, whose interaction generates a bidirectional signaling, is still debated in I/R injury. The aim of the present work was therefore to investigate the effects produced by unidirectional activation of forward signaling (administration of chimeric protein ephrinA1-Fc), of reverse signaling (EphA2-Fc), or inhibition of both signals (monomeric EphA2 and the protein-protein interaction inhibitor UniPR1331) on the local and systemic inflammatory responses triggered by mesenteric I/R in mice.When administered at 200 µg/kg i.v., ephrin-A1-Fc prevented intestinal and lung I/R-induced injury, decreasing in the pulmonary district leukocytes recruitment, IL-1ß and TNFα levels, and EphA2 overexpression by mesenteric I/R. Blockade of Eph-ephrin signaling by equimolar EphA2 efficiently antagonized I/R-induced gut edema formation, an effect shared also by UniPR1331, mitigated lung mucosal injury, and counteracted the increase in pro-inflammatory cytokines levels. EphA2-Fc 180 µg/kg or equimolar Fc alone did not significantly modify the inflammatory responses to I/R.Our data suggest that the Eph-ephrin system is directly involved in the development of the acute inflammatory process activated in the gut by hypoxia-reoxygenation and in its amplification to distant organs, revealing that a fine pharmacological tuning of this signaling pathway may represent an attractive strategy to contain the I/R-induced inflammatory cascade.

Design and evaluation of EphrinA1 mutants with cerebral protective effect.

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.

The effect of ephrin-A1 on resistance to Photofrin-mediated photodynamic therapy in esophageal squamous cell carcinoma cells.

Esophageal squamous cell carcinoma (ESCC), the most prevalent cell type of esophageal cancer, remains a dismal disease with poor prognosis. Photodynamic therapy (PDT) is a minimally invasive treatment option for early esophageal cancer. To explore possible factors involved in resistance to PDT in esophageal cancer cells, we selected PDT-resistant subcell lines by repeated treatment of CE48T/VGH (CE48T) ESCC cells with Photofrin-PDT and then analyzed the global gene modulations in the PDT-resistant cells by whole-genome microarray. More than 700 genes reached a fold change greater than 1.5 in each of the PDT-resistant cells compared to parental cells. Among these genes, both tumor necrosis factor (TNF) and EFNA1 genes were significantly upregulated in resistant cell lines. However, they were significantly downregulated in Photofrin-PDT-treated cells compared to untreated cells. The observations made in the microarray analysis were further confirmed by quantitative PCR. We observed that recombinant tumor necrosis factor alpha (TNF-α) activated the gene expression of EFNA1 at both the messenger RNA (mRNA) level and the protein level in CE48T cells. Functional analysis showed that when incubated with oligomeric and monomeric ephrin-A1 simultaneously, ESCC cells became significantly resistant to Photofrin-PDT. Functional analysis further suggested that transmembrane and soluble ephrin-A1 may cooperate to enhance resistance to Photofrin-PDT in ESCC cells.

In PC3 prostate cancer cells ephrin receptors crosstalk to ß1-integrins to strengthen adhesion to collagen type I.

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.

Ephrin-A1-mediated dopaminergic neurogenesis and angiogenesis in a rat model of Parkinson's disease.

Cells of the neural stem cell lineage in the adult subventricular zone (SVZ) respond to brain insult by increasing their numbers and migrating through the rostral migratory stream. However, in most areas of the brain other than the SVZ and the subgranular zone of the dentate gyrus, such a regenerative response is extremely weak. Even these two neurogenic regions do not show extensive regenerative responses to repair tissue damage, suggesting the presence of an intrinsic inhibitory microenvironment (niche) for stem cells. In the present study, we assessed the effects of injection of clustered ephrin-A1-Fc into the lateral ventricle of rats with unilateral nigrostriatal dopamine depletion. Ephrin-A1-Fc clustered by anti-IgG(Fc) antibody was injected stereotaxically into the ipsilateral lateral ventricle of rats with unilateral nigrostriatal lesions induced by 6-hydroxydopamine, and histologic analysis and behavioral tests were performed. Clustered ephrin-A1-Fc transformed the subventricular niche, increasing bromodeoxyuridine-positive cells in the subventricular area, and the cells then migrated to the striatum and differentiated to dopaminergic neurons and astrocytes. In addition, clustered ephrin-A1-Fc enhanced angiogenesis in the striatum on the injected side. Along with histologic improvements, behavioral derangement improved dramatically. These findings indicate that the subventricular niche possesses a mechanism for regulating both stem cell and angiogenic responses via an EphA-mediated signal. We conclude that activation of EphA receptor-mediated signaling by clustered ephrin-A1-Fc from within the lateral ventricle could potentially be utilized in the treatment of neurodegenerative diseases such as Parkinson's disease.

Activation of erythropoietin-producing hepatocellular receptor A2 attenuates cell adhesion of human fallopian tube epithelial cells via focal adhesion kinase dephosphorylation.

Tyrosine kinase receptor erythropoietin-producing hepatocellular receptor A2 (EphA2) and its predominant ligand EphrinA1 have been studied extensively for their roles of mediating cell adhesion in epithelial cells. However, EphA2 signaling in human fallopian tube epithelial cells is poorly understood. In this study, primary cultured fallopian tube epithelial cells were used as a model treated with EphrinA1-Fc or IgG-Fc (control), to explore the role of EphA2 signal and its network involved in the regulation of cell adhesion of tubal epithelia cells. The activation of EphA2 and focal adhesion kinase (FAK) was evaluated by western blotting assay in the cultured fallopian tube epithelia cells, of which the cell adhesion activity was determined by MTT assay. A significantly negative correlation was found between phosphorylated-EphA2 (Pho-EphA2) and phosphorylated-FAK (Pho-FAK) after exposure to EphrinA1-Fc (P = 0.000; r = -0.848). EphrinA1-Fc increased Pho-EphA2 and reduced Pho-FAK in seconds, with the apex level of Pho-EphA2 and the nadir level of Pho-FAK detected at the same time (10 min). Cell adhesion of the cultured cells supplemented with EphrinA1-Fc appeared to be weaker than that of the controls at the later time points of the treatment (from 30 to 120 min) (P < 0.05). Taken together, the EphrinA1 addition directly induces an elevated Pho-EphA2 accompanied by a decreased Pho-FAK in human fallopian tube epithelia cells. Furthermore, activation of EphA2 participates in the regulation of fallopian tube cell adhesion via FAK dephosphorylation.

Eph-A2 promotes permeability and inflammatory responses to bleomycin-induced lung injury.

Stimulation by the ephrin-A1 ligand of the EphA2 receptor increases endothelial permeability. Lung injury increases the expression of EphA2, but the role of EphA2 in such injury is not well understood. To determine whether EphA2 contributes to changes in permeability and inflammation in the injured lung, we studied wild-type (WT) and EphA2 knockout (KO) mice, using isolated, perfused lung (IPL) preparations and a model of bleomycin-induced lung injury. We also studied the response of endothelial cells to ephrin-A1. In the IPL preparations, ephrin-A1 increased the filtration coefficient in WT mice, but not in EphA2 KO mice, demonstrating that EphA2 regulates vascular permeability. In early bleomycin injury in WT mice, the expression of both EphA2 and ephrin-A1 increased. EphA2 KO animals were protected from lung injury, showing less water and alveolar protein in the lungs than WT mice, consistent with reduced permeability. Bleomycin caused less accumulation of lung leukocytes in EphA2 KO animals than in WT animals, suggesting that EphA2 regulates inflammation. To determine whether EphA2 deficiency alters the production of chemokines, CXCL1 and CCL2 in the lungs were measured. After bleomycin injury, EphA2 KO animals produced less CXCL1 and CCL2 than WT animals. Because NF-κß mediates the production of chemokines, the effect of the ephrin-A1 ligand on the activation of NF-κß and the expression of chemokines was measured in endothelial cells. Ephrin-a1 significantly increased NF-κß nuclear translocation and the expression of chemokine mRNA. This study demonstrates that the expression of EphA2 increases in the injured lung, and not only contributes to changes in permeability, but also plays a previously unrecognized role in promoting inflammatory responses.

EphrinA1 inhibits malignant mesothelioma tumor growth via let-7 microRNA-mediated repression of the RAS oncogene.

EphrinA1 binding with receptor EphA2 suppresses malignant mesothelioma (MM) growth. The mechanisms whereby EphrinA1 attenuates the MM cell (MMC) growth are not clear. In this study, we report that the activation of MMCs with EphrinA1 leads to an induction of let-7 microRNA (miRNA) expression, repression of RAS proto-oncogene and the attenuation of MM tumor growth. The expression of miRNAs was determined by reverse transcription-quantitative polymerase chain reaction and in situ hybridization. RAS expression was determined by q-PCR, western blotting and immunofluorescence. MMC proliferation and tumor growth were determined by WST-1 and Matrigel assay, respectively. EphrinA1 activation induced several fold increases in let-7a1, let-7a3, let-7f1 and let-7f2 miRNA expression in MMCs. In contrast, EphrinA1 activation significantly downregulated H-RAS, K-RAS and N-RAS expression and inhibited MMC proliferation and tumor growth. In MMCs transfected with 2'-O-methyl antisense oligonucleotides to let-7 miRNA, EphrinA1 activation failed to inhibit the proliferative response and tumor growth. In mismatch antisense oligonucleotide-treated MMCs, the proliferation and tumor growth were comparable to untreated proliferating cells. Furthermore, the transfection of MMCs with let-7a miRNA precursor inhibited RAS expression and attenuated MMC tumor growth. Our data revealed that EphrinA1 signaling induces let-7 miRNA expression and attenuates MM tumor growth by targeting RAS proto-oncogene in MMCs.

Biomimetic hydrogels with immobilized ephrinA1 for therapeutic angiogenesis.

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.

Crosstalk of the EphA2 receptor with a serine/threonine phosphatase suppresses the Akt-mTORC1 pathway in cancer cells.

Receptor tyrosine kinases of the Eph family play multiple roles in the physiological regulation of tissue homeostasis and in the pathogenesis of various diseases, including cancer. The EphA2 receptor is highly expressed in most cancer cell types, where it has disparate activities that are not well understood. It has been reported that interplay of EphA2 with oncogenic signaling pathways promotes cancer cell malignancy independently of ephrin ligand binding and receptor kinase activity. In contrast, stimulation of EphA2 signaling with ephrin-A ligands can suppress malignancy by inhibiting the Ras-MAP kinase pathway, integrin-mediated adhesion, and epithelial to mesenchymal transition. Here we show that ephrin-A1 ligand-dependent activation of EphA2 decreases the growth of PC3 prostate cancer cells and profoundly inhibits the Akt-mTORC1 pathway, which is hyperactivated due to loss of the PTEN tumor suppressor. Our results do not implicate changes in the activity of Akt upstream regulators (such as Ras family GTPases, PI3 kinase, integrins, or the Ship2 lipid phosphatase) in the observed loss of Akt T308 and S473 phosphorylation downstream of EphA2. Indeed, EphA2 can inhibit Akt phosphorylation induced by oncogenic mutations of not only PTEN but also PI3 kinase. Furthermore, it can decrease the hyperphosphorylation induced by constitutive membrane-targeting of Akt. Our data suggest a novel signaling mechanism whereby EphA2 inactivates the Akt-mTORC1 oncogenic pathway through Akt dephosphorylation mediated by a serine/threonine phosphatase. Ephrin-A1-induced Akt dephosphorylation was observed not only in PC3 prostate cancer cells but also in other cancer cell types. Thus, activation of EphA2 signaling represents a possible new avenue for anti-cancer therapies that exploit the remarkable ability of this receptor to counteract multiple oncogenic signaling pathways.

Cooperative signaling between Slit2 and Ephrin-A1 regulates a balance between angiogenesis and angiostasis.

Slit proteins induce cytoskeletal remodeling through interaction with roundabout (Robo) receptors, regulating migration of neurons and nonneuronal cells, including leukocytes, tumor cells, and endothelium. The role of Slit2 in vascular remodeling, however, remains controversial, with reports of both pro- and antiangiogenic activity. We report here that cooperation between Slit2 and ephrin-A1 regulates a balance between the pro- and antiangiogenic functions of Slit2. While Slit2 promotes angiogenesis in culture and in vivo as a single agent, Slit2 potently inhibits angiogenic remodeling in the presence of ephrin-A1. Slit2 stimulates angiogenesis through mTORC2-dependent activation of Akt and Rac GTPase, the activities of which are inhibited in the presence of ephrin-A1. Activated Rac or Akt partially rescues vascular assembly and motility in costimulated endothelium. Taken together, these data suggest that Slit2 differentially regulates angiogenesis in the context of ephrin-A1, providing a plausible mechanism for the pro- versus antiangiogenic functions of Slit2. Our results suggest that the complex roles of Slit-Robo signaling in angiogenesis involve context-dependent mechanisms.

EphA2 engages Git1 to suppress Arf6 activity modulating epithelial cell-cell contacts.

ADP-ribosylation factor (Arf) 6 activity is crucially involved in the regulation of E-cadherin-based cell-cell adhesions. Erythropoietin-producing hepatocellular carcinoma (Eph)-family receptors recognize ligands, namely, ephrins, anchored to the membrane of apposing cells, and they mediate cell-cell contact-dependent events. Here, we found that Arf6 activity is down-regulated in Madin-Darby canine kidney cells, which is dependent on cell density and calcium ion concentration, and we provide evidence of a novel signaling pathway by which ligand-activated EphA2 suppresses Arf6 activity. This EphA2-mediated suppression of Arf6 activity was linked to the induction of cell compaction and polarization, but it was independent of the down-regulation of extracellular signal-regulated kinase 1/2 kinase activity. We show that G protein-coupled receptor kinase-interacting protein (Git) 1 and noncatalytic region of tyrosine kinase (Nck) 1 are involved in this pathway, in which ligand-activated EphA2, via its phosphorylated Tyr594, binds to the Src homology 2 domain of Nck1, and then via its Src homology 3 domain binds to the synaptic localizing domain of Git1 to suppress Arf6 activity. We propose a positive feedback loop in which E-cadherin-based cell-cell contacts enhance EphA-ephrinA signaling, which in turn down-regulates Arf6 activity to enhance E-cadherin-based cell-cell contacts as well as the apical-basal polarization of epithelial cells.

Endothelial EphA receptor stimulation increases lung vascular permeability.

Mediators of angiogenesis such as VEGFs and angiopoietins may regulate pulmonary vascular permeability under normal and pathological conditions. Ephrin family receptor tyrosine kinases are expressed in the vasculature and also regulate angiogenesis under some circumstances, but whether they also modulate lung vascular permeability is unknown. We hypothesized that stimulation of lung endothelial EphA receptors with ephrin-a1 ligand would alter pulmonary vascular permeability and tested this idea in vivo and in vitro. We found that ephrin-a1 ligand and EphA2 receptors are expressed in distal normal lung vasculature and that their expression is increased in injured lung, suggesting a link to mechanisms of increased permeability. Intravenous injection of ephrin-a1 caused a large increase in the leakage of labeled albumin into the lungs of rats within 30 min (293 +/- 27 vs. 150 +/- 6 ng/mg dry lung, P < 0.01), along with histological evidence of the formation of endothelial disruptions. In cultured lung vascular endothelial cells, stimulation with ephrin-a1 increased monolayer permeability by 44% (P < 0.01), a permeability change similar to that seen with VEGF stimulation of the same cells. Ephrin-a1 stimulation in vivo and in vitro was associated with histological evidence for disruptions of tight and adherens junctions. These observations describe a novel role for ephrin-a1 and EphA receptors in the regulation of vascular permeability in the lung.

Receptor EphA2 activation with ephrinA1 suppresses growth of malignant mesothelioma (MM).

The objective of this study was to understand the possible mechanisms of activation of receptor EphA2 by its ligand ephrinA1 in malignant mesothelioma cell (MMC) growth. Activation of receptor EphA2 by its ligand ephrinA1 triggered the phosphorylation of EphA2. Ligand activation of EphA2 also induced phosphorylation of ERK1/2 and significantly decreased MMC proliferation. Ligand activated and ephrinA1 vector (pcDNA/EFNA1) transfected MMC demonstrated decreased clonal growth in 3-D matrigels when compared to resting MMC. These studies suggest that EphA2 activation by its ligand ephrinA1 transmits intracellular signals from cell membrane to nucleus via ERK1/2 signaling cascade and inhibits MM growth.