The lack of EphB3 receptor prevents bone loss in mouse models of osteoporosis.

Bone homeostasis is a complex process in which some Eph kinase receptors and their ephrin ligands appear to be involved. In the present study, we address this issue by examining, both in vitro and in vivo, the role of EphB2 and EphB3 in mesenchymal stromal/stem cell (MSC) differentiation into bone tissue. This was first evaluated by quantitative reverse transcription PCR (RT-qPCR) and histological staining in MSCs cultured in specific mediums revealing that although EphB2-/- MSCs mainly expressed pro-adipogenic transcription factors, EphB3-/- MSCs showed abundant osteogenic transcripts, such as Runx2, Msx2, and Sp7. To clarify the underlying molecular mechanisms, we found that the lack of EphB3 signaling alters the genetic profile of differentiating MSCs, reducing the expression of many inhibitory molecules and antagonists of the BMP signaling pathway, and increasing Bmp7 expression, a robust bone inductor. Then, to confirm the osteogenic role of EphB3 in vivo, we studied the condition of 2 mouse models of induced bone loss (ovariectomy or long-term glucocorticoid treatment). Interestingly, in both models, both WT and EphB2-/- mice equally developed the disease but EphB3-/- mice did not exhibit the typical bone loss, nor an increase in urine Ca2+ or blood serum CTX-1. This phenotype in EphB3-KO mice could be due to their significantly higher proportions of osteoprogenitor cells and preosteoblasts, and their lower number of osteoclasts, as compared with WT and EphB2-KO mice. Thus, we conclude that EphB3 acts as a negative regulator of the osteogenic differentiation, and its absence prevents bone loss in mice subjected to ovariectomy or dexamethasone treatment.

Osteoporosis affects more than 200 million people, mostly women. Our work shows that the EphB3 receptor restricts bone formation, and its absence prevents bone loss in osteoporotic mice. The bone protection observed in EphB3-deficient mice is due to the presence of more bone-forming cells and fewer bone-degrading cells. Molecularly, we found that when there's no EphB3 in mesenchymal stem cells, some bone-promoting genes are increased while many inhibitors are reduced. Therefore, this receptor could become a key target for new therapies that would help to improve the quality of life for those suffering from bone diseases. We're really excited to share our findings with a broad audience, including patients, healthcare professionals, researchers, and the life sciences industry.

Peripheral T-cell responses of EphB2- and EphB3-deficient mice in a model of collagen-induced arthritis.

Both EphB2- and EphB3-deficient mice exhibit profound histological alterations in the thymic epithelial network but few changes in T-cell differentiation, suggesting that this organization would be sufficient to produce functional T lymphocytes. Also, other antigen-presenting cells involved in immunological education could substitute the thymic epithelium. Accordingly, we found an increased frequency of plasmacytoid dendritic cells but not of conventional dendritic cells, medullary fibroblasts or intrathymic B lymphocytes. In addition, there are no lymphoid infiltrates in the organs of mutant mice nor do they contain circulating autoantibodies. Furthermore, attempts to induce arthritic lesions after chicken type II collagen administration fail totally in EphB2-deficient mice whereas all WT and half of the immunized EphB3-/- mice develop a typical collagen-induced arthritis. Our results point out that Th17 cells, IL4-producing Th2 cells and regulatory T cells are key for the induction of disease, but mutant mice appear to have deficits in T cell activation or cell migration properties. EphB2-/- T cells show reduced in vitro proliferative responses to anti-CD3/anti-CD28 antibodies, produce low levels of anti-type II collagen antibodies, and exhibit low proportions of T follicular helper cells. On the contrary, EphB3-/- lymph node cells respond accurately to the different immune stimuli although in lower levels than WT cells but show a significantly reduced migration in in vitro transwell assays, suggesting that no sufficient type II collagen-dependent activated lymphoid cells reached the joints, resulting in reduced arthritic lesions.

EphB3 receptor suppressor invasion, migration and proliferation in glioma by inhibiting EGFR-PI3K/AKT signaling pathway.

Eph receptors are the largest subfamily of receptor tyrosine kinases, and they have been shown to play a crucial role in glioma. The EphB3 receptor is a member of this family, and its effect on the invasion, migration and proliferation of glioma cells was examined in this study. It was found that the expression of EphB3 was decreased in glioma specimens with increasing tumor grade. Additionally, the U87MG and U251 cell lines showed low levels of EphB3 expression. This finding was consistent with the negative correlation between EphB3 expression in glioma tissues and tumor grade. Depletion of EphB3 gene in U87MG and U251 cell lines resulted in a substantial enhancement of their invasion, migration, and proliferation capacities in vitro. Furthermore, the knockdown of EphB3 led to an upregulation of EGFR, p-PI3K, and p-AKT protein levels. On the other hand, EphB3 overexpression reduced the invasiveness, proliferative capacity and migration rate of U87MG and U251 cells, and downregulated EGFR, p-PI3K and p-AKT. These findings indicate that EphB3 functions as a tumor suppressor in glioma, and its downregulation enhances the malignant potential of glioma cells by activating the EGFR-PI3K/AKT pathway. Thus, EphB3 is a promising diagnostic marker for glioma, and the EphB3-EGFR-PI3K / AKT axis deserves further investigation as a potential therapeutic target.

Barcoded viral tracing of single-cell interactions in central nervous system inflammation.

Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets.

Pygo1 regulates pathological cardiac hypertrophy via a ß-catenin-dependent mechanism.

Wnt/ß-catenin signaling plays a key role in pathological cardiac remodeling in adults. The identification of a tissue-specific Wnt/ß-catenin interaction factor may provide a tissue-specific clinical targeting strategy. Drosophila Pygo encodes the core interaction factor of Wnt/ß-catenin. Two Pygo homologs (Pygo1 and Pygo2) have been identified in mammals. Different from the ubiquitous expression profile of Pygo2, Pygo1 is enriched in cardiac tissue. However, the role of Pygo1 in mammalian cardiac disease is yet to be elucidated. In this study, we found that Pygo1 was upregulated in human cardiac tissues with pathological hypertrophy. Cardiac-specific overexpression of Pygo1 in mice spontaneously led to cardiac hypertrophy accompanied by declined cardiac function, increased heart weight/body weight and heart weight/tibial length ratios, and increased cell size. The canonical ß-catenin/T-cell transcription factor 4 (TCF4) complex was abundant in Pygo1-overexpressing transgenic (Pygo1-TG) cardiac tissue, and the downstream genes of Wnt signaling, that is, Axin2, Ephb3, and c-Myc, were upregulated. A tail vein injection of ß-catenin inhibitor effectively rescued the phenotype of cardiac failure and pathological myocardial remodeling in Pygo1-TG mice. Furthermore, in vivo downregulated pygo1 during cardiac hypertrophic condition antagonized agonist-induced cardiac hypertrophy. Therefore, our study is the first to present in vivo evidence demonstrating that Pygo1 regulates pathological cardiac hypertrophy in a canonical Wnt/ß-catenin-dependent manner, which may provide new clues for tissue-specific clinical treatment via targeting this pathway.NEW & NOTEWORTHY In this study, we found that Pygo1 is associated with human pathological hypertrophy. Cardiac-specific overexpression of Pygo1 in mice spontaneously led to cardiac hypertrophy. Meanwhile, cardiac function was improved when expression of Pygo1 was interfered in hypertrophy-model mice. Our study is the first to present in vivo evidence demonstrating that Pygo1 regulates pathological cardiac hypertrophy in a canonical Wnt/ß-catenin-dependent manner, which may provide new clues for a tissue-specific clinical treatment targeting this pathway.

Expression Profile and Prognostic Significance of EPHB3 in Colorectal Cancer.

The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) is expressed in cells at the base of intestinal crypts, acting as a cellular guide in the maintenance of intestinal crypt architecture. We aimed to investigate the expression profile of EPHB3 in colorectal precancerous lesions and colorectal cancers (CRCs), and assess its prognostic value. EPHB3 expression was higher in CRCs than in normal mucosa and was associated with the intestinal stem cell markers EPHB2, OLFM4, LRIG1, and a proposed cancer stem cell marker, CD44. Enhanced EPHB3 expression significantly declined during the transformation from adenoma to carcinoma and as the tumor invaded into deeper tissue layers. Namely, a substantial reduction of EPHB3 expression was observed in the budding cancer cells at the invasive tumor fronts, which was more extensive than E-cadherin downregulation. In an azoxymethane/dextran sulfate sodium-induced, colitis-associated, CRC model, EPHB3 expression increased along with tumor development. In a large cohort of CRC patients, EPHB3 positivity was observed in 24% of 610 CRCs and was negatively correlated with tumor differentiation, lympho-vascular invasion, and tumor, node, and metastasis stages. EPHB3 was positively associated with microsatellite instability but was associated with neither CpG island methylation, nor with KRAS and BRAF mutations. Notably, EPHB3 positivity was associated with better clinical outcomes, although it was not an independent prognostic marker. Overexpression of EPHB3 in the colon cancer cell line, DLD1, led to decreased cell growth and migration and reduced mitogen-activated protein kinase signaling. Taken together, our data demonstrate the suppressive role of EPHB3 in CRC progression.

Polycistronic Delivery of IL-10 and NT-3 Promotes Oligodendrocyte Myelination and Functional Recovery in a Mouse Spinal Cord Injury Model.

One million estimated cases of spinal cord injury (SCI) have been reported in the United States and repairing an injury has constituted a difficult clinical challenge. The complex, dynamic, inhibitory microenvironment postinjury, which is characterized by proinflammatory signaling from invading leukocytes and lack of sufficient factors that promote axonal survival and elongation, limits regeneration. Herein, we investigated the delivery of polycistronic vectors, which have the potential to coexpress factors that target distinct barriers to regeneration, from a multiple channel poly(lactide-co-glycolide) (PLG) bridge to enhance spinal cord regeneration. In this study, we investigated polycistronic delivery of IL-10 that targets proinflammatory signaling, and NT-3 that targets axonal survival and elongation. A significant increase was observed in the density of regenerative macrophages for IL-10+NT-3 condition relative to conditions without IL-10. Furthermore, combined delivery of IL-10+NT-3 produced a significant increase of axonal density and notably myelinated axons compared with all other conditions. A significant increase in functional recovery was observed for IL-10+NT-3 delivery at 12 weeks postinjury that was positively correlated to oligodendrocyte myelinated axon density, suggesting oligodendrocyte-mediated myelination as an important target to improve functional recovery. These results further support the use of multiple channel PLG bridges as a growth supportive substrate and platform to deliver bioactive agents to modulate the SCI microenvironment and promote regeneration and functional recovery. Impact statement Spinal cord injury (SCI) results in a complex microenvironment that contains multiple barriers to regeneration and functional recovery. Multiple factors are necessary to address these barriers to regeneration, and polycistronic lentiviral gene therapy represents a strategy to locally express multiple factors simultaneously. A bicistronic vector encoding IL-10 and NT-3 was delivered from a poly(lactide-co-glycolide) bridge, which provides structural support that guides regeneration, resulting in increased axonal growth, myelination, and subsequent functional recovery. These results demonstrate the opportunity of targeting multiple barriers to SCI regeneration for additive effects.

Neural crest cells bulldoze through the microenvironment using Aquaporin 1 to stabilize filopodia.

Neural crest migration requires cells to move through an environment filled with dense extracellular matrix and mesoderm to reach targets throughout the vertebrate embryo. Here, we use high-resolution microscopy, computational modeling, and in vitro and in vivo cell invasion assays to investigate the function of Aquaporin 1 (AQP-1) signaling. We find that migrating lead cranial neural crest cells express AQP-1 mRNA and protein, implicating a biological role for water channel protein function during invasion. Differential AQP-1 levels affect neural crest cell speed and direction, as well as the length and stability of cell filopodia. Furthermore, AQP-1 enhances matrix metalloprotease activity and colocalizes with phosphorylated focal adhesion kinases. Colocalization of AQP-1 with EphB guidance receptors in the same migrating neural crest cells has novel implications for the concept of guided bulldozing by lead cells during migration.

Receptor Tyrosine Kinase EphB3: a Prognostic Indicator in Colorectal Carcinoma.

Although EphB3 expression is down-regulated in colorectal cancer (CRC) cells compared with normal intestinal epithelial cells, the relationship between EphB3 expression and clinicopathological parameters in CRC is unclear. We examined EphB3 expression in 128 CRC tissue specimens and in 19 adenoma specimens using immunohistochemistry. The relationships between EphB3 expression and clinicopathological parameters, KRAS mutations, BRAF V600E mutation, MSI and survival were evaluated using Spearman's rank correlation and Kaplan-Meier survival analyses, respectively. CpG methylation in the EphB3 promoter was examined in four human CRC cell lines and tissues. EphB3 was strongly expressed in all normal intestinal epithelial cells (128/128) and adenoma cells (19/19). In CRC tumor cells, EphB3 expression was negative or weak in 41.4% (53/128), moderate in 26.6% (34/128), and strong in 32.0% (41/128) of samples. EphB3 expression was negatively associated with invasive depth (P = 0.016, rs = -0.213), lymph node metastasis (P = 0.000, rs = -0.490), and TNM stage (P = 0.000, rs = -0.390), and was positively associated with poor differentiation (P = 0.001, rs = 0.290), BRAF V600E mutation (P = 0.008, rs = 0.235), and longer overall survival (P < 0.001). In multivariate analysis, EphB3 expression (P = 0.007) and lymph node metastasis (P < 0.001) were independent prognostic factors for poor survival. Hypermethylation of the EphB3 promoter was detected in cell lines and CRC tissues. EphB3 is down-regulated in CRC compared to normal mucosa. Hypermethylation of CpG island is contributed to downregulation of EphB3 in CRC. EphB3 expression in tumor cells may be a useful prognostic indicator for patients with CRC.

Design, synthesis, biological evaluation and molecular docking study of arylcarboxamido piperidine and piperazine-based hydroxamates as potential HDAC8 inhibitors with promising anticancer activity.

HDAC8 has been established as one of the vital targets as far as the cancer is concerned. Different compounds having potential HDAC inhibitory activity have been approved by USFDA. However, none of these compounds are selective towards specific HDAC isoform. In this current study, some new hydroxamate derivatives with alkylpiperidine and alkylpiperazine linker moieties have been designed, synthesized and biologically evaluated. All these compounds are effective HDAC8 inhibitors comprising more or less similar cytotoxic potential against different cancer cell lines. It is observed that the piperazine scaffold containing compound is more active than the compound with piperidine scaffold for exerting HDAC8 inhibitory activity. Moreover, the 4-quinolyl cap group is better than the biphenyl group which is better than the benzyl group for producing higher HDAC8 inhibition as well as cytotoxicity. These compounds displayed selective HDAC8 inhibition over HDAC3. Moreover, these compounds showed an increased caspase3/7 activity suggesting their anticancer potential through modulation of apoptotic pathways. Molecular docking study with three potent compounds was performed with both HDAC3 and HDAC8 enzymes to understand the selectivity profile of these compounds. Compound containing 4-quinolyl cap group with alkyl piperazinyl urea linker moiety has been emerged out as the lead molecule that may be further modified to design more effective and selective HDAC8 inhibitors in future.

Luteolin-7-O-ß-d-Glucoside Inhibits Cellular Energy Production Interacting with HEK2 in Keratinocytes.

Flavonoids have been demonstrated to affect the activity of many mammalian enzyme systems. Their functional phenolic groups are able to mediate antioxidant effects by scavenging free radicals. Molecules of this class have been found able to modulate the activity of kinases, phospholipase A2, cyclooxygenases, lipoxygenase, glutathione S-transferase, and many others. Recently, it has been demonstrated that luteolin, in the form of Luteolin-7-O-ß-d-glucoside (LUT-7G) is able to induce the keratinocyte differentiation process in vitro. This flavonoid is able to counteract the proliferative effects of IL-22/IL6 pathway by the inhibition of STAT3 activity also in vivo in a psoriatic mouse model. Observations on energy metabolism changes of differentiating cells led us to perform a complete metabolomics analysis using human primary keratinocytes treated with LUT-7G. Our results show that LUT-7G, is not only able to impair the nuclear translocation of STAT3, but it also blocks the energy metabolism pathway, depressing the glycolytic and Krebs pathway by the inhibition of hexokinase 2 activity. These data confirm that LUT-7G can be proposed as a potential candidate for the treatment of inflammatory and proliferative diseases, but its role as a hexokinase 2 (HEK2) inhibitor opens new perspectives in nutritional science, and especially in cancer therapy, in which the inhibition of the Warburg effect could be relevant.

Sonic hedgehog pathway activation is associated with cetuximab resistance and EPHB3 receptor induction in colorectal cancer.

A major problem of colorectal cancer (CRC) targeted therapies is relapse caused by drug resistance. In most cases of CRC, patients develop resistance to anticancer drugs. Cetuximab does not show many of the side effects of other anticancer drugs and improves the survival of patients with metastatic CRC. However, the molecular mechanism of cetuximab resistance is not fully understood. Methods: EPHB3-mediated cetuximab resistance was confirmed by in vitro western blotting, colony-forming assays, WST-1 colorimetric assay, and in vivo xenograft models (n = 7 per group). RNA-seq analysis and receptor tyrosine kinase assays were performed to identify the cetuximab resistance mechanism of EPHB3. All statistical tests were two-sided. Results: The expression of EFNB3, which upregulates the EPHB3 receptor, was shown to be increased via microarray analysis. When resistance to cetuximab was acquired, EPHB3 protein levels increased. Hedgehog signaling, cancer stemness, and epithelial-mesenchymal transition signaling proteins were also increased in the cetuximab-resistant human colon cancer cell line SW48R. Despite cells acquiring resistance to cetuximab, STAT3 was still responsive to EGF and cetuximab treatment. Moreover, inhibition of EPHB3 was associated with decreased STAT3 activity. Co-immunoprecipitation confirmed that EGFR and EPHB3 bind to each other and this binding increases upon resistance acquisition, suggesting that STAT3 is activated by the binding between EGFR and EPHB3. Protein levels of GLI-1, SOX2, and Vimentin, which are affected by STAT3, also increased. Similar results were obtained in samples from patients with CRC. Conclusion: EPHB3 expression is associated with anticancer drug resistance.

Role of EphB3 Receptor in Mediating Head and Neck Tumor Growth, Cell Migration, and Response to PI3K Inhibitor.

Eph proteins have emerged as critical drivers affecting tumor growth and progression in human malignancies. Our The Cancer Genome Atlas (TCGA) data analysis showed that EphB3, a receptor tyrosine kinase, is frequently coamplified with PIK3CA in head and neck squamous cell carcinoma (HNSCC). We therefore hypothesized that EphB3 amplification plays a protumorigenic role in HNSCC and that EphB3 and PIK3CA are cooperating oncogenes that contribute toward its pathogenesis. This hypothesis was not experimentally supported, because EphB3 knockdown failed to alter HNSCC tumor cell growth in vitro or in vivo with an orthotopic model. However, responsiveness of EphB3 knockdown tumors to the PI3K inhibitor, BKM120, was significantly decreased in terms of both tumor growth delay and survival. This is correlated with an increase in prosurvival proteins, S6 and BcL-XL, in the EphB3 shRNA tumors treated with BKM120 compared with controls. We further observed that EphB3 knockdown resulted in increased migration in vitro and increased EMT gene signature in vivo To explain these results, we examined EphB3 phosphorylation levels in HNSCC at baseline. Although total EphB3 levels were high, we found low phospho-EphB3 levels in HNSCCs. Forced EphB3 phosphorylation with an ephrin-B2-Fc fusion protein resulted in decreased HNSCC migration and cell growth, and enhanced response to BKM120 in vitro These data collectively indicate that progression of HNSCC selects for low/inhibited EphB3 activity to enhance their survival and migratory abilities and decrease response to PI3K signaling. Therefore, strategies focused on activating EphB3 might be helpful to inhibit tumor growth and enhance sensitivity to PI3K inhibitors in HNSCC. Mol Cancer Ther; 17(9); 2049-59. ©2018 AACR.

Upregulation of EphB3 in gastric cancer with acquired resistance to a FGFR inhibitor.

Amplification of fibroblast growth factor receptor2 (FGFR2) has been regarded as a druggable target in gastric cancer (GC). Despite known potential of AZD4547, a selective inhibitor of FGFR 1-3, to suppress tumorigenic effects of activated FGFR2, resistance to the targeted agent has been an unresolved issue. This study was performed to elucidate the mechanism of AZD4547 resistance in GC cells. SNU-16 cells were used to establish an AZD4547-resistant GC cell line, SNU-16R. Elevated phosphorylation of EphB3 was confirmed using the Human Phospho-Receptor Tyrosine Kinase Array kit. A tyrosine kinase inhibitor (TKI) of EphB3 was used to investigate the effects of suppressed EphB3 activity in the SNU-16R cell line. SNU-16R cells exhibited upregulated phosphorylation of EphB3. Treatment of SNU-16R cells with the EphB3 TKI resulted in induction of apoptosis, decreased cellular viability, and cell cycle arrest at sub-G1 phase. SNU-16R cells expressed upregulated levels of N-cadherin, vimentin, Snail, matrix metalloproteinase 2 (MMP-2), and MMP-9, and reduced levels of E-cadherin, characteristic of epithelial to mesenchymal transition (EMT). Matrigel invasion assay also demonstrated the increased invasiveness of SNU-16R cells. EphB3 TKI treatment inhibited EMT of SNU-16R cells. Activation of mammalian target of rapamycin (mTOR) through the Ras-ERK1/2 pathway was suggested as the signal transduction mechanism downstream EphB3 by showing enhanced phosphorylation of Raf-1, MEK1/2, ERK1/2, mTOR and its downstream substrates in SNU-16R cells. As expected, EphB3 TKI decreased phosphorylation of these proteins. Our data suggest phosphorylation of mTOR through signaling by EphB3 is a potential mechanism of AZD4547 resistance in GC cells.

EphB3 signaling induces cortical endothelial cell death and disrupts the blood-brain barrier after traumatic brain injury.

Damage to the cerebrovascular network is a major contributor to dysfunction in patients suffering from traumatic brain injury (TBI). Vessels are composed of lumen-forming endothelial cells that associate closely with both glial and neuronal units to establish a functional blood-brain barrier (BBB). Under normal physiological conditions, these vascular units play important roles in central nervous system (CNS) homeostasis by delivering oxygen and nutrients while filtering out molecules and cells that could be harmful; however, after TBI this system is disrupted. Here, we describe a novel role for a class of receptors, called dependence receptors, in regulating vessel stability and BBB integrity after CCI injury in mice. Specifically, we identified that EphB3 receptors function as a pro-apoptotic dependence receptor in endothelial cells (ECs) that contributes to increased BBB damage after CCI injury. In the absence of EphB3, we observed increased endothelial cell survival, reduced BBB permeability and enhanced interactions of astrocyte-EC membranes. Interestingly, the brain's response to CCI injury is to reduce EphB3 levels and its ligand ephrinB3; however, the degree and timing of those reductions limit the protective response of the CNS. We conclude that EphB3 is a negative regulator of cell survival and BBB integrity that undermine tissue repair, and represents a protective therapeutic target for TBI patients.

Cell type-specific localization of Ephs pairing with ephrin-B2 in the rat postnatal pituitary gland.

Sox2-expressing stem/progenitor cells in the anterior lobe of the pituitary gland form two types of micro-environments (niches): the marginal cell layer and dense cell clusters in the parenchyma. In relation to the mechanism of regulation of niches, juxtacrine signaling via ephrin and its receptor Eph is known to play important roles in various niches. The ephrin and Eph families are divided into two subclasses to create ephrin/Eph signaling in co-operation with confined partners. Recently, we reported that ephrin-B2 localizes specifically to both pituitary niches. However, the Ephs interacting with ephrin-B2 in these pituitary niches have not yet been identified. Therefore, the present study aims to identify the Ephs interacting with ephrin-B2 and the cells that produce them in the rat pituitary gland. In situ hybridization and immunohistochemistry demonstrated cell type-specific localization of candidate interacting partners for ephrin-B2, including EphA4 in cells located in the posterior lobe, EphB1 in gonadotropes, EphB2 in corticotropes, EphB3 in stem/progenitor cells and EphB4 in endothelial cells in the adult pituitary gland. In particular, double-immunohistochemistry showed cis-interactions between EphB3 and ephrin-B2 in the apical cell membranes of stem/progenitor cell niches throughout life and trans-interactions between EphB2 produced by corticotropes and ephrin-B2 located in the basolateral cell membranes of stem/progenitor cells in the early postnatal pituitary gland. These data indicate that ephrin-B2 plays a role in pituitary stem/progenitor cell niches by selective interaction with EphB3 in cis and EphB2 in trans.

Increased epithelial-free areas in thymuses with altered EphB-mediated thymocyte-thymic epithelial cell interactions.

Epithelial-free areas, present in both thymic cortex and medulla, have been studied in WT and EphB-deficient mice that have important alterations in the development of thymic epithelium due to the lack of proper thymocyte-thymic epithelial cell interactions. In both WT and mutant thymuses, the number and size of epithelial-free areas are significantly larger in the medulla than in the cortex. The two parameters show a reverse correlation: low numbers of these areas course with large epithelial-free areas and vice versa. However, their structure and cell content are similar in mutant and WT thymuses. Cortical epithelial-free areas just contain DP thymocytes, while the medullary ones consist of SP cells, blood vessels, mesenchyme-derived ER-TR7+ cells and components of the extracellular matrix (i.e., collagen IV, fibronectin, laminin). Other components, such as desmin, αSMA, PDGFRß and Ng2, frequently associated with blood vessel walls, also appear. Vimentin, although present in medullary epithelial-free areas, does not co-express with epithelial cells. Other markers related to epithelial-mesenchymal transitions, such as Snail, Slug or FSP1, are not expressed. These results suggest that alterations in the cell interactions between distinct thymic cell components that induce both increased proportions of apoptotic thymic epithelial cells and altered behavior of the mesenchyme associated with the medullary vasculature could explain the appearance of these areas and their differences in the cortex and medulla.

EphB3-targeted regulation of miR-149 in the migration and invasion of human colonic carcinoma HCT116 and SW620 cells.

microRNAs play key roles during various crucial cell processes such as proliferation, migration, invasion and apoptosis. Also, microRNAs have been shown to possess oncogenic and tumor-suppressive functions in human cancers. Here, we describe the regulation and function of miR-149 in colorectal cancer cell lines. miR-149 expression patterns were detected in human colorectal cell lines and tissue samples, and then focused on its role in regulation of cell growth, migration, invasion, and its target gene identification. Furthermore, the function of the target gene of miR-149 was analyzed in vitro and in vivo. miR-149 expression was downregulated in human colorectal cancer HCT116 and SW620 cell lines compared to the normal colon epithelial NCM460 cell line using quantitative real-time polymerase chain reaction methods. Further studies indicated that introduction of miR-149 was able to suppress cell migration and invasion. Then, EphB3 was identified as a direct target gene of miR-149 in colorectal cancer cells. Moreover, experiments in vitro showed that knockdown expression of EphB3 could suppress cell proliferation and invasion, and ectopic expression of EphB3 restored the phenotypes of CRC cell lines transfected with miR149. In addition, silencing of EphB3 significantly affected cycle progression distribution and increased apoptosis in CRC cell lines. Finally, in vivo results demonstrated that knockdown of EphB3 by siRNA inhibited tumor growth. In conclusion,the important role of miR-149 in colorectal cancer progression suggesting that miR-149 may serve as a therapeutic target for colorectal cancer treatment.

EphB3 protein is associated with histological grade and FIGO stage in ovarian serous carcinomas.

Eph (Erythropoietin-producing human hepatocellular carcinoma cell) is the largest subfamily of receptor tyrosine kinases. Eph receptors and their ephrin ligands are involved in embryonic development and physiological processes. Aberrant expression of Eph/ephrin may contribute to a variety of diseases including cancer. EphB3 is a member of Eph receptors and has been found to play roles in carcinogenesis of some types of human cancer. But, its expression and clinical significance in ovarian serous carcinoma have not been well investigated and are unknown. In this study, a set of ovarian tissues including normal fallopian tube, serous borderline tumor, and serous carcinoma were subjected to immunohistochemistry using a specific polyclonal antibody for EphB3. The relationship between EphB3 expression and clinicopathological parameters was statistically analyzed. EphB3 was strongly expressed in all fallopian tube specimens (19/19, 100%). EphB3 was negatively or weekly expressed in 1 of 17 (5.8%) in borderline tumors and 26 of 50 (52.0%) in serous carcinomas, moderately expressed in 7 of 17 (41.2%) in borderline tumors and 14 of 50 (28%) in serous carcinomas, and strongly expressed in 9 17 (52.9%) in borderline tumors and 10 of 50 (20%) in serous carcinomas. EphB3 expression is significantly reduced in serous carcinomas compared with normal fallopian tubes and borderline tumors (p < 0.001). EphB3 expression is negatively associated with histological grade (p < 0.001, rs = -0.613) and FIGO stage (p = 0.001, rs = -0.464) of serous carcinomas. Our results show EphB3 protein lost in ovarian serous carcinoma and is associated with tumor grade and FIGO stage, which indicate EphB3 protein may play a role in carcinogenesis of ovarian serous carcinoma and may be used as a molecular marker for prognosis.

EphB3 Stimulates Cell Migration and Metastasis in a Kinase-dependent Manner through Vav2-Rho GTPase Axis in Papillary Thyroid Cancer.

Eph receptors, the largest subfamily of transmembrane tyrosine kinase receptors, have been increasingly implicated in various physiologic and pathologic processes, and the roles of the Eph family members during tumorigenesis have recently attracted growing attentions. In the present study, we explored the function of EphB3, one member of Eph family, in papillary thyroid cancer (PTC). We found that the expression of EphB3 was significantly elevated in PTC. Either overexpression of EphB3 or activation of EphB3 by EfnB1-Fc/EfnB2-Fc stimulated in vitro migration of PTC cells. In contrast, siRNA-mediated knockdown of EphB3 or EphB3-Fc treatment, which only blocked EphB3-mediated forward signaling, inhibited migration and metastasis of PTC cells. A mechanism study revealed that EphB3 knockdown led to suppressed activity of Rac1 and enhanced activity of RhoA. Moreover, we found that Vav2, an important regulator of Rho family GTPases, was activated by EphB3 in a kinase-dependent manner. Altogether, our work suggested that EphB3 acted as a tumor promoter in PTC by increasing the in vitro migration as well as the in vivo metastasis of PTC cells through regulating the activities of Vav2 and Rho GTPases in a kinase-dependent manner.