Compartments with predominant ephrin-B1 and EphB2/B4 expression are present alternately along the excurrent duct system in the adult mouse testis and epididymis.

BACKGROUND: Ephrin receptors (Eph) and ligands are membrane-bound cell-cell communication molecules that regulate the spatial organization of various tissues and organs by repulsive or adhesive signals arising from contact between Eph- and ephrin-bearing cells. However, the expression and functions of Eph receptors in the testis and epididymis are virtually unknown. OBJECTIVES: We aimed to investigate the expression of several EphB receptors and ephrin-B ligands in the testis and epididymis of adult mice. MATERIALS AND METHODS: mRNA and protein expression was detected via reverse transcription-polymerase chain reaction amplification and immunostaining, respectively. RESULTS: Complementary expression patterns were observed in the epithelia along the excurrent duct system in the testis and epididymis; ephrin-B1 was strongly expressed in the epithelia of the rete testis and segment I in the ductus epididymis, whereas EphB2 and/or EphB4 were strongly expressed in the epithelia of the straight tubules and efferent ductules. Moreover, ephrin-B1 was expressed in the spermatogonia, Leydig cells, and peritubular myoid cells in the testis, whereas EphB2 was expressed in elongated spermatids and EphB4 was expressed in the spermatogonia and Leydig cells. Furthermore, these receptors were found to be tyrosine-phosphorylated in the testis and/or epididymis. DISCUSSION: Receptor localization and phosphorylation patterns suggested that EphB/ephrin-B signaling might occur in the seminiferous tubules and epithelial junctions among the straight tubules, rete testis, efferent ductules, and ductus epididymis. Therefore, we propose that EphB/ephrin-B signaling may regulate epithelial boundary formation in the excurrent tubule/ductule/duct system as well as modulate spermatogenesis and spermiation. CONCLUSION: Overall, this study represents the first analysis of EphB receptor and ephrin-B ligand expression in the normal adult testis and epididymis.

A rapid and sensitive method for EphB4 identification as a diagnostic and therapeutic biomarker in invasive breast cancer.

BACKGROUND: In the roadmap to design diagnostic and therapeutic markers for breast cancer, EphB4 is of special interest due to its multiple roles in tumor initiation, progression and invasion. The aim of present study was to characterize a rapid and sensitive ELISA-based method to measure EphB4 level and its phosphorylation status following stimulation with its ligand, ephrinB2, in an invasive breast cancer cell line. MATERIALS AND METHODS: MDA-MB-231 breast cancer cells were lysed and EphB4 level was measured using ELISA. EphB4 level was measured in sub- and post-confluent states in culture dishes. Receptor phosphorylation was also detected by ELISA assay, using various concentrations of pre-clustered ephrinB2 for 20 minutes. RESULTS: Expression of EphB4 receptor was detected by ELISA in all samples. EphB4 level was significantly higher in post.confluent than sub.confluent cells. Phosphorylated receptor was also detectable with this method when cells were exogenously stimulated. CONCLUSIONS: Quantitative data from ELISA manifested a difference between levels of EphB4 in two states of different invasive properties. Moreover, ELISA method may be considered rapid and sensitive enough to detect even low levels of total and phosphorylated EphB4 Cost-effectiveness of this method for the detection of differential expression of EphB4 proteins in clinics is also noticeable.

Decreased expression of receptor tyrosine kinase of EphB1 protein in renal cell carcinomas.

Receptors tyrosine kinase of Eph superfamily plays an important role in human cancers. We previously found that EphB1 subtype is down-regulated in gastric cancer, colorectal cancer and ovary serous carcinoma. Fore the more, the decreased expression of EphB1 is related to invasion and metastasis in cancers. Although EphB1 has been revealed as an important receptor in cancers, our understanding of its roles in renal cell carcinoma (RCC) is limited. In the present study, using specific anit-EphB1 polyclonal antibody and immunohistochemistry, we evaluated EphB1 protein expression levels in RCC specimens surgically resected from 82 patients (including 62 conventional clear-cell RCC, 10 papillary, and 10 chromophobic RCC cases). We found EphB1 protein is positively expressed in the epithelium of renal tubules. Decreased expression of EphB1 was found in all RCC carcinomas compared with expression in the normal epithelium of renal tubules. EphB1 protein moderately expressed in chromophobic RCC, weakly expressed in clear-cell RCC and negatively expressed in papillary RCC. Our results indicate that EphB1 may be involved in carcinogenesis of RCC, the molecular mechanisms of down-regulation of EphB1 including genetic and epigenetic alterations and the dedicated roles of EphB1 in occurrence and progress of RCC need to be explicated in next step.

EphB1 and Ephrin-B, new potential biomarkers for squamous cell/adenosquamous carcinomas and adenocarcinomas of the gallbladder.

Squamous cell/adenosquamous carcinoma (SC/ASC) of the gallbladder are rare tumors and there are few clinical reports in the literature. Herein we report our clinical experience with 46 patients with SC/ASC and 80 with adenocarcinoma (AC). Expression of EphB1 and Ephrin-B in each tumor was determined using immunohistochemical methods for determination of correlations with prognosis. There was no difference in EphB1 and Ephrin-B expression between SC/ASC and AC tumors (P>0.05), but greater expression in those less than 3 cm in diameter, stage I or II (TNM stage), with no lymph node metastases, with no local invasion and treated with radical resection was apparent. Expression of EphB1 (P<0.05) and Ephrin-B (P<0.01) was higher in well differentiated than in poorly differentiated AC tumors. Kaplan-Meier survival analysis indicated that degree of differentiation, tumor diameter, lymph node metastases, local invasion, surgical approach and expression rate of EphB1 and Ephrin-B were closely related to the survival of SC/ASC (P<0.05) and AC patients (P<0.01). Patients with tumors that positive expressed EphB1 and Ephrin-B, whether it is SC/ASC (P SC/ASC =0.000) or AC (P AC =0.000 or P AC =0.002) had longer survival than those negative expression. Cox multivariate analysis indicated a negative correlation between expression of EphB1 or Ephrin-B and overall survival. Hence, EphB1 and Ephrin-B could be regarded as independent good prognostic factorsand important biological markers for SC/ASC and AC of gallbladder.

Blocking EphB1 receptor forward signaling in spinal cord relieves bone cancer pain and rescues analgesic effect of morphine treatment in rodents.

Treating bone cancer pain continues to be a clinical challenge and underlying mechanisms of bone cancer pain remain elusive. Here, we report that EphB1 receptor forward signaling in the spinal cord is critical to the development of bone cancer pain and morphine tolerance in treating bone cancer pain. Tibia bone cavity tumor cell implantation (TCI) produces bone cancer-related thermal hyperalgesia, mechanical allodynia, spontaneous and movement-evoked pain behaviors, and bone destruction. Production and persistence of these pain behaviors are well correlated with TCI-induced upregulation of EphB1 receptor and its ligand ephrinB2 in the dorsal horn and primary sensory neurons. Spinal administration of an EphB1 receptor blocking reagent EphB2-Fc prevents and reverses bone cancer pain behaviors and the associated induction of c-Fos and activation of astrocytes and microglial cells, NR1 and NR2B receptors, Src within the N-methyl-D-aspartate receptor complex, and the subsequent Ca(2+)-dependent signals. The exogenous ligand ephrinB2-Fc upregulates level of phosphorylation of NR1 and NR2B receptors depending on the activation of EphB1 receptor. Spinal administration of EphB2-Fc and ephrinB2-Fc induces downregulation of EphB1 and ephrinB2, respectively, accompanied with increased activity of matrix metalloproteinase (MMP)-2/9. Blocking MMP-2 or MMP-9 reverses EphB1-Fc treatment-induced downregulation of EphB1 receptor. In addition, spinal blocking or targeted mutation of EphB1 receptor reverses morphine tolerance in treating bone cancer pain in rats and defensive pain in mice. These findings show a critical mechanism underlying the pathogenesis of bone cancer pain and suggest a potential target for treating bone cancer pain and improving analgesic effect of morphine clinically.

EphB1 is underexpressed in poorly differentiated colorectal cancers.

BACKGROUND: Over- or underexpression of certain Eph receptors has been associated with tumorigenesis of some types of cancer. EphB1 is a member of receptor tyrosine kinases of the EphB subfamily involved in the development, progress and prognosis of colorectal cancers. The expression levels of EphB1 in colon cancer cell lines and human colorectal carcinoma specimens were determined and association of EphB1 expression with clinicopathological parameters was analyzed. METHODS: Quantitative real-time reverse transcription polymerase chain reaction and immunohistochemistry were used. RESULTS: The EphB1 transcript is expressed in all colon cancer cell lines tested. However, there is marked variability in the expression of the EphB1 transcripts and proteins among colorectal carcinoma specimens. Reduced expression of EphB1 in colorectal cancers more often occurred in poorly differentiated and mucinous adenocarcinomas than in well- and moderately differentiated adenocarcinomas. Further, cancer cells with a low level of EphB1 protein showed more invasive power. CONCLUSION: Our data indicate that EphB1 may have roles in the pathogenesis and development of colorectal cancer.

Zic2 regulates retinal ganglion cell axon avoidance of ephrinB2 through inducing expression of the guidance receptor EphB1.

The navigation of retinal axons to ipsilateral and contralateral targets in the brain depends on the decision to cross or avoid the midline at the optic chiasm, a critical guidance maneuver that establishes the binocular visual pathway. Previous work has identified a specific guidance receptor, EphB1, that mediates the repulsion of uncrossed axons away from its ligand, ephrinB2, at the optic chiasm midline (Williams et al., 2003), and a transcription factor Zic2, that, like EphB1, is required for formation of the ipsilateral retinal projection (Herrera et al., 2003). Although the reported similarities in localization implicated that Zic2 regulates EphB1 (Herrera et al., 2003; Williams et al., 2003; Pak et al., 2004), whether Zic2 drives expression of EphB1 protein has not been elucidated. Here we show that EphB1 protein is expressed in the growth cones of axons from ventrotemporal (VT) retina that project ipsilaterally and that repulsion by ephrinB2 is determined by the presence of this receptor on growth cones. Moreover, ectopic delivery of Zic2 into explants from non-VT retina induces expression of EphB1 mRNA and protein. The upregulated EphB1 receptor protein is localized to growth cones and is functional, because it is sufficient to change retinal ganglion cell axon behavior from extension onto, to avoidance of, ephrinB2 substrates. Our results demonstrate that Zic2 upregulates EphB1 expression and define a link between a transcription factor and expression of a guidance receptor protein essential for axon guidance at the vertebrate midline.

EphB receptors regulate stem/progenitor cell proliferation, migration, and polarity during hippocampal neurogenesis.

The adult brain maintains two regions of neurogenesis from which new neurons are born, migrate to their appropriate location, and become incorporated into the circuitry of the CNS. One of these, the subgranular zone of the hippocampal dentate gyrus, is of primary interest because of the role of this region in learning and memory. We show that mice lacking EphB1, and more profoundly EphB1 and EphB2, have significantly fewer neural progenitors in the hippocampus. Furthermore, other aspects of neurogenesis, such as polarity, cell positioning, and proliferation are disrupted in animals lacking the EphB1 receptor or its cognate ephrin-B3 ligand. Our data strongly suggest that EphB1 and ephrin-B3 cooperatively regulate the proliferation and migration of neural progenitors in the hippocampus and should be added to a short list of candidate target molecules for modulating the production and integration of new neurons as a treatment for neurodegenerative diseases or brain injury.