EphB4 Expression Research Articles

Cantharidin treatment inhibits hepatocellular carcinoma development by regulating the JAK2/STAT3 and PI3K/Akt pathways in an EphB4-dependent manner

Hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. The tyrosine kinase receptor EphB4 promotes oncogenesis and tumor development and progression. Its inhibition is regarded as an effective strategy for the treatment of solid tumors. In the present study, we identified cantharidin as a novel EphB4 inhibitor for HCC treatment and evaluated the underlying molecular pharmacological mechanisms of action. We observed increased expression levels of EphB4 in HCC patients and a positive correlation between EphB4 and p-JAK2 levels in HCC patient samples. Knockdown of EphB4 using small interfering RNA decreased the expression levels of p-JAK2 and p-STAT3 in HCC cells, suggesting JAK2/STAT3 being a novel downstream signaling target of EphB4. Cell viability experiments revealed that the anti-cancer effect of cantharidin was positively correlated with EphB4 expression levels in HCC cell lines. We confirmed the potent antiproliferative activity of cantharidin on HepG2 cells with high expression of EphB4 and tumor xenograft. Molecular docking assay, immunoblotting assay and quantitative reverse transcription PCR assay indicated that cantharidin bound to EphB4, and thereby resulted in EphB4 suppression at mRNA and protein levels. Hep3B and SMMC-7721 cells were with low expression of EphB4. In EphB4−/HepG2, EphB4+/HepG2, and EphB4+/Hep3B cells, EphB4 knockdown alleviated the cantharidin-induced decrease in cell viability and colony formation ability and increase in apoptosis in HepG2 cells, while its overexpression exacerbated these effects in Hep3B cells and increased the apoptosis of HepG2 cells. In nude mouse models, cantharidin suppressed tumor growth more effectively in EphB4+/SMMC-7721 xenografts than in wild-type SMMC-7721 xenografts. Underlying mechanistic study showed that by targeting EphB4, cantharidin blocked a novel target, the downstream JAK2/STAT3 pathway, and the previously known target, the PI3K/Akt signaling, resulting in intrinsic apoptosis. These results indicated that cantharidin may be a potential candidate for HCC treatment by regulating the EphB4 signaling pathway.

Synergistic antifibrotic effects of miR-451 with miR-185 partly by co-targeting EphB2 on hepatic stellate cells

Liver fibrosis is a global health problem currently without clinically approved drugs. It is characterized by the excessive accumulation of extracellular matrix (ECM) mainly produced by activated hepatic stellate cells (HSCs). Uncovering the mechanisms underlying the fibrogenic responses in HSCs may have profound translational implications. Erythropoietin-producing hepatocellular receptor B2 (EphB2) is a receptor tyrosine kinase that has been indicated to be a novel profibrotic factor involved in liver fibrogenesis. In the present study, we investigated the effects of miR-451 and miR-185 on the expression of EphB2 and their roles in liver fibrogenesis both in vitro and in vivo. We found that EphB2 upregulation is a direct downstream molecular event of decreased expression of miR-451 and miR-185 in the process of liver fibrosis. Moreover, miR-451 was unexpectedly found to upregulate miR-185 expression at the post-transcriptional level by directly targeting the nuclear export receptor exportin 1 (XPO-1) and synergistically suppress HSCs activation with miR-185. To investigate the clinical potential of these miRNAs, miR-451/miR-185 agomirs were injected individually or jointly into CCl4-treated mice. The results showed that coadministration of these agomirs synergistically alleviated liver fibrosis in vivo. These findings indicate that miR-451 and miR-451/XPO-1/miR-185 axis play important and synergistic regulatory roles in hepatic fibrosis partly through co-targeting EphB2, which provides a novel therapeutic strategy for the treatment of hepatic fibrosis.

EPH receptor B2 stimulates human monocyte adhesion and migration independently of its EphrinB ligands.

The molecular basis of atherosclerosis is not fully understood and mice studies have shown that Ephrins and EPH receptors play a role in the atherosclerotic process. We set out to assess the role for monocytic EPHB2 and its Ephrin ligands in human atherosclerosis and show a role for EPHB2 in monocyte functions independently of its EphrinB ligands. Immunohistochemical staining of human aortic sections at different stages of atherosclerosis showed that EPHB2 and its ligand EphrinB are expressed in atherosclerotic plaques and that expression proportionally increases with plaque severity. Functionally, stimulation with EPHB2 did not affect endothelial barrier function, nor did stimulation with EphrinB1 or EphrinB2 affect monocyte‐endothelial interactions. In contrast, reduced expression of EPHB2 in monocytes resulted in decreased monocyte adhesion to endothelial cells and a decrease in monocyte transmigration, mediated by an altered morphology and a decreased ability to phosphorylate FAK. Our results suggest that EPHB2 expression in monocytes results in monocyte accumulation by virtue of an increase of transendothelial migration, which can subsequently contribute to atherosclerotic plaque progression.

Kinome multigenic panel identified novel druggable EPHB4‐V871I somatic variant in high‐risk neuroblastoma

Neuroblastoma (NB) is the most common extracranial neoplasm in children. The overall outcome for high‐risk NB patients is still unacceptable, therefore, it is critical to deeply understand molecular mechanisms associated with NB, which in turn can be utilized for developing drugs towards the treatment of NB. Protein kinases (TKs) play an essential role in the regulation of cell survival and proliferation. Different kinases, such as anaplastic lymphoma kinase (ALK), Aurora kinase, RET receptor tyrosine kinase, are potential therapeutic targets in various cancers, including NB. We analysed a cohort of 45 high‐risk NB patients and 9 NB cell lines by a targeted—(t)NGS custom gene panel (genes codifying for the kinase domains of 90 TKs). We identified somatic variants in four TK genes (ALK, EPHB4, LMTK3 and EPHB6) in NB patients and we functionally characterized an interesting somatic variant, V871I, in EPHB4 gene. EPHB4 plays a crucial role in cardiovascular development and regulates vascularization in cancer‐promoting angiogenesis, tumour growth and metastasis. Several EPHB4 mutations have previously been identified in solid and haematological tumour specimens but EPHB4 mutations were not described until now in NB. Interestingly, a re‐analysis of public CGH‐array showed that the EPHB4 gain is associated with advanced diseases in NB. We further demonstrated that higher EPHB4 expression is correlated to stage 4 of NB and with poor overall survival. Additionally, we also revealed that the EPHB4‐V871I accounts for increased proliferation, migration and invasion properties in two NB cell lines by acting on VEGF, c‐RAF and CDK4 target genes and by increasing the phosphorylation of ERK1‐2 pathway. The use of two EPHB4 inhibitors, JI‐101 and NVP‐BHG712, was able to rescue the phenotype driven by the variant. Our study suggested that EPHB4 is a promising therapeutic target in high‐risk NB.

EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-\u03b1 on osteogenic differentiation

BackgroundLow concentrations of tumor necrosis factor-alpha (TNF-α) and its receptor TNFR2 are both reported to promote osteogenic differentiation of osteoblast precursor cells. Moreover, low concentrations of TNF-α up-regulate the expression of EphB4. However, the molecular mechanisms underlying TNF-α-induced osteogenic differentiation and the roles of TNFR2 and EphB4 have not been fully elucidated.ResultsThe ALP activity, as well as the mRNA and protein levels of RUNX2, BSP, EphB4 and TNFR2, was significantly elevated in MC3T3-E1 murine osteoblast precursor cells when stimulated with 0.5 ng/ml TNF-α. After TNFR2 was inhibited by gene knockdown with lentivirus-mediated shRNA interference or by a neutralizing antibody against TNFR2, the pro-osteogenic effect of TNF-α was partly reversed, while the up-regulation of EphB4 by TNF-α remained unchanged. With EphB4 forward signaling suppressed by a potent inhibitor of EphB4 auto-phosphorylation, NVP-BHG712, TNF-α-enhanced expressions of TNFR2, BSP and Runx2 were significantly decreased. Further investigation into the signaling pathways revealed that TNF-α significantly increased levels of p-JNK, p-ERK and p-p38. However, only the p-ERK level was significantly inhibited in TNFR2-knockdown cells. In addition, the ERK pathway inhibitor, U0126 (10 μM), significantly reversed the positive effect of TNF-α on the protein levels of RUNX2 and BSP.ConclusionsThe EphB4, TNFR2 and ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation.

Dysregulation of the EphrinB2\u2212EphB4 ratio in pediatric cerebral arteriovenous malformations is associated with endothelial cell dysfunction in vitro and functions as a novel noninvasive biomarker in patients

We investigated (1) EphrinB2 and EphB4 receptor expression in cerebral AVMs, (2) the impact of an altered EphrinB2:EphB4 ratio on brain endothelial cell function and (3) potential translational applications of these data. The following parameters were compared between AVM endothelial cells (AVMECs) and human brain microvascular endothelial cells (HBMVECs): quantified EphrinB2 and EphB4 expression, angiogenic potential, and responses to manipulation of the EphrinB2:EphB4 ratio via pharmacologic stimulation/inhibition. To investigate the clinical relevance of these in vitro data, Ephrin expression was assessed in AVM tissue (by immunohistochemistry) and urine (by ELISA) from pediatric patients with AVM (n = 30), other cerebrovascular disease (n = 14) and control patients (n = 29), and the data were subjected to univariate and multivariate statistical analyses. Compared to HBMVECs, AVMECs demonstrated increased invasion (p = 0.04) and migration (p = 0.08), impaired tube formation (p = 0.06) and increased EphrinB2:EphB4 ratios. Altering the EphrinB2:EphB4 ratio (by increasing EphrinB2 or blocking EphB4) in HBMVECs increased invasion (p = 0.03 and p < 0.05, respectively). EphrinB2 expression was increased in AVM tissue, which correlated with increased urinary EphrinB2 levels in AVM patients. Using the optimal urinary cutoff value (EphrinB2 > 25.7 pg/μg), AVMs were detected with high accuracy (80% vs. controls) and were distinguished from other cerebrovascular disease (75% accuracy). Post-treatment urinary EphrinB2 levels normalized in an index patient. In summary, AVMECs have an EphrinB2:EphB4 ratio that is increased compared to that of normal HBMVECs. Changing this ratio in HBMVECs induces AVMEC-like behavior. EphrinB2 is clinically relevant, and its levels are increased in AVM tissue and patient urine. This work suggests that dysregulation of the EphrinB2:EphB4 signaling cascade and increases in EphrinB2 may play a role in AVM development, with potential utility as a diagnostic and therapeutic target.

Activation of EphB receptors contributes to primary sensory neuron excitability by facilitating Ca2+ influx directly or through Src kinase-mediated N-methyl-D-aspartate receptor phosphorylation.

EphrinB-EphB receptor tyrosine kinases have been demonstrated to play important roles in pain processing after peripheral nerve injury. We have previously reported that ephrinB-EphB receptor signaling can regulate excitability and plasticity of neurons in spinal dorsal horn, and thus contribute to spinal central sensitization in neuropathic pain. How EphB receptor activation influences excitability of primary neurons in dorsal root ganglion (DRG), however, remains unknown. Here, we report that EphB receptor activation facilitates calcium influx through N-methyl-D-aspartate receptor (NMDAR) dependent and independent manners. In cultured DRG cells from adult rats, EphB1 and EphB2 receptors were expressed in neurons, but not the glial cells. Bath application of EphB receptor agonist ephrinB2-Fc induced NMDAR-independent Ca influx, which was from the extracellular space rather than endoplasmic reticulum. EphB receptor activation also greatly enhanced NMDAR-dependent Ca influx and NR2B phosphorylation, which was prevented by pretreatment of Src kinase inhibitor PP2. In nerve-injured DRG neurons, elevated expression and activation of EphB1 and EphB2 receptors contributed to the increased intracellular Ca concentration and NMDA-induced Ca influx. Repetitive intrathecal administration of EphB2-Fc inhibited the increased phosphorylation of NR2B and Ca-dependent subsequent signals Src, ERK, and CaMKII as well as behaviorally expressed pain after nerve injury. These findings demonstrate that activation of EphB receptors can modulate DRG neuron excitability by facilitating Ca influx directly or through Src kinase activation-mediated NMDA receptor phosphorylation and that EphB receptor activation is critical to DRG neuron hyperexcitability, which has been considered critical to the subsequent spinal central sensitization and neuropathic pain.

A phase II study of sEphB4-HSA in metastatic castration-resistant prostate cancer (mCRPC).

TPS274 Background: The EphB4/EphrinB2 pathway is a promising therapeutic target for patients with mCRPC. EphB4 expression is increased in prostate cancer tissue and cell lines, and retained in castration resistant states. EphB4 crosstalks with the PI3K/AKT and MAPK pathways to regulate cell survival and proliferation, and its interaction with the transmembrane ligand EphrinB2 leads to T-cell suppression and immune evasion. A soluble decoy EphB4 receptor-human serum albumin fusion protein (sEphB4-HSA) binds to EphrinB2 and blocks interaction with the cell surface EphB4 receptor to promote immune infiltration and induce tumor cell death. Here we report an ongoing phase II study exploring the preliminary efficacy and safety of sEphB4-HSA in patients with progressive disease after frontline therapy for mCRPC. Methods: Eligibility criteria include mCRPC with disease progression after second generation AR targeted therapy (i.e., abiraterone or enzalutamide), ECOG PS ≤ 2, and adequate renal, hepatic and hematological functions. Pts having received 4 or more prior treatment therapies for mCRPC are excluded. The primary objective is efficacy as reflected by PSA response using PCWG3 criteria. Secondary objectives include safety and tolerability by CTCAE v 5.0, time to PSA progression, overall response by RECIST 1.1 and PCWG3 (bone) criteria, and rPFS. Translational endpoints include expression of EphB4 and EphrinB2 in metastatic tumor samples by immunohistochemistry and correlation with alterations in MYC, PTEN/PI3K, AR, and p53 pathways. sEphB4-HSA is administered as IV infusion over 60 min every 14 days with spacing to every 21 days after 6 cycles. Therapy will continue till disease progression, unacceptable toxicity, treatment delay ≥4 weeks, or patient withdrawal. Preliminary efficacy will be assessed using PSA response rate (PR and CR) with a Simon two stage minimax trial design assuming the undesirable overall response rate (null hypothesis) to be approximately 10% or less, and the alternate hypothesis suggesting success to be approximately 30% or more. Toxicity will be evaluated by the DSMC after the first stage including 15 patients. If 2 or more respond, then an additional 10 patients will be added. Clinical trial information: NCT04033432.

Activation of EphB2 in the basolateral amygdala promotes stress vulnerability of mice by increasing NMDA-dependent synaptic function

The occurrence of major depressive disorder (MDD) has been linked to an increased vulnerability to stress. The basolateral amygdala (BLA) is one of the critical brain areas that involved in the regulation of pathological reactivity to stress. Increasing evidence indicates that the EphB2 receptor (EphB2) plays a critical role in neuropsychiatric disorders, such as Alzheimer's disease, pain and anxiety. However, whether the EphB2 in the BLA is involved in stress vulnerability is unclear. Here, we identified EphB2 in the BLA as a key regulator contributed to the modulation of stress vulnerability in adult mice. We found that the expression of EphB2 in the BLA was significantly increased in the animal model induced by chronic social stress. Knockdown of EphB2 in the BLA produced antidepressant-like behavioral effects, whereas activation of EphB2 in the BLA increased the susceptibility to subthreshold social defeat stress. Furthermore, we demonstrated that the role of EphB2 in the stress vulnerability was mediated by modulating NMDA receptors, since the knockdown of EphB2 in the BLA prevented not only the increase in the amplitudes of both the miniature and the evoked NMDAR-mediated EPSC, but also the enhancement of surface expression of NMDARs in the defeated mice. Taken together, these results suggest that EphB2 in the BLA is a critical factor contributes to the vulnerability to stress, which may be a potential target for the treatment of depression.

Prognostic and Clinicopathological Significance of EphB3 and Dysadherin Expression in Extrahepatic Cholangiocarcinoma.

AimEphB3 and dysadherin are involved in tumorigenesis and progression of many neoplasms. However, the roles of EphB3 and dysadherin in extrahepatic cholangiocarcinoma (ECC) remain to be revealed. In this study, we aimed to evaluate the expression of EphB3 and dysadherin, and investigate their clinicopathological significance in ECC.MethodsWe examined EphB3 and dysadherin expression in 100 ECC, 30 peritumoral tissues, 10 adenoma and 15 normal biliary tract tissues using EnVision immunohistochemistry. The relationship between EphB3 or dysadherin expression and clinicopathological features was evaluated using the χ2 test or Fisher’s exact test. The overall survival of ECC patients was analyzed using Kaplan-Meier univariate survival analysis and Log rank tests.ResultsWe found that EphB3 expression was significantly down-regulated and dysadherin expression was significantly up-regulated in ECC tissues compared with normal tissues (P < 0.01). EphB3 expression was negatively correlated with dysadherin expression in ECC (P < 0.01). The positive rate of EphB3 expression and negative rate of dysadherin expression was significantly higher in patients with well-differentiated type, no lymph node metastasis, no surrounding tissues and organs invasion, early TNM stages (I + II) and radical resection (P < 0.01). The survival of ECC patients with positive EphB3 or negative dysadherin expression was significantly longer than patients with negative EphB3 or positive dysadherin expression (P < 0.01). Cox multivariate analysis demonstrated that negative EphB3 or positive dysadherin expression were independent poor prognostic factors in ECC patients. The ROC curves suggested that EphB3 and dysadherin combined diagnostic efficacy (AUC=0.688, 95%CI: 0.603-0.772) was significantly higher EphB3 diagnostic efficacy (AUC=0.654, 95%CI: 0.564-0.743) or dysadherin diagnostic efficacy (AUC=0.648, 95%CI: 0.558-0.737) alone.ConclusionEphB3 and dysadherin are involved in the carcinogenesis and progression of ECC, and ECC patients with negative EphB3 or positive dysadherin expression have a poor prognosis.

Ligand-independent EphB1 signaling mediates TGF-β-activated CDH2 and promotes lung cancer cell invasion and migration.

Purpose: The initial step of cancer metastasis is that cancer cells acquire the capability to migrate and invade. Eph receptors comprise the largest family of receptor tyrosine and display dual role in tumor progression due to unique ephrin cis- or trans- signaling. The roles of EphB1 and its phosphorylation signaling in lung cancer remain to be elucidated.Patients and Methods: We analyzed the expression of EphB1 in both publicly available database and 60 cases of NSCLC patients with or without metastasis. The migration and invasion of lung cancer cells were assessed by a transwell assay. The activation of EphB1 signaling was assessed by western blot and real-time PCR. The EphB1 mutant was used to evaluate the effect of phosphorylation of EphB1.Results: Here, we showed that increased expression of EphB1 was detected in Non-Small-Cell Lung Cancer (NSCLC) biopies compared to non-cancer controls. Significant higher expression of EphB1 in lung biopsies were found in patients with metastasis compared to non-metastatic NSCLC patients. Higher EphB1 expression was correlated with poor patient survival in lung cancer. Overexpression of EphB1 promoted the migration and invasion of lung cancer cells. On the contrast, Ephrin-B2, a transmembrane ligand for EphB1 forward signaling, inhibited migration and invasion of lung cancer cells. TGF-β-activated Smad2 transcriptionally upregulated the endogenous expression of EphB1. Ligand-independent EphB1 promoted Epithelial-mesenchymal transition (EMT) through upregulating CDH2.Conclusion: Our results showed that the effect of EphB1 on the migration and invasion was context-specific and was dependent on EphB1 phosphorylation.

EphrinB/EphB Signaling Contributes to the Synaptic Plasticity of Chronic Migraine Through NR2B Phosphorylation

The specific mechanism of migraine chronification remains unclear. We previously demonstrated that synaptic plasticity was associated with migraine chronification. EphB receptors and their ligands, ephrinBs, are considered to be key molecules regulating the synaptic plasticity of the central nervous system. However, whether they can promote the chronification of migraine by regulating synaptic plasticity is unknown. Therefore, we investigated the role of ephrinB/EphB signaling in chronic migraine (CM). Male Sprague-Dawley rats were used to construct a chronic migraine model by dural infusion of an inflammatory soup for 7 days. We used qPCR, western blot, and immunofluorescence to detect the mRNA and protein levels of EphB2 and ephrinB2. The paw withdrawal latency and paw withdrawal threshold were measured after lateral ventricle treatment with EphB1-Fc (an inhibitor of EphB receptor). Changes in synaptic plasticity were explored by examining synaptic-associated proteins by western blot, dendritic spines of neurons by Golgi-Cox staining, and synaptic ultrastructure by transmission electron microscopy. We found that the expression of EphB2 and ephrinB2 increased in CM. The administration of EphB1-Fc relieved hyperalgesia and changes in synaptic plasticity induced by CM. In addition, EphB1-Fc inhibited the upregulation of NR2B phosphorylation. These results indicate that ephrinB/EphB signaling may regulate synaptic plasticity in CM via NR2B phosphorylation, which suggests the novel idea that ephrinB/EphB signaling may be a target for the treatment of migraine chronification.

EphB2 represents an independent prognostic marker in patients with gastric cancer and promotes tumour cell aggressiveness.

Dysregulated expression of ephrin type-B receptor 2 (EphB2) has been linked with the development and progression of solid tumours. In the current study, we attempted to investigate the clinical relevance in GC and the effect of EphB2 expression on gastric cancer (GC) cells. EphB2 protein levels in GC and benign gastric tissues were determined using immunohistochemistry. EphB2 transcript expression in a GC cohort with GC tissue samples (n=171) and paired adjacent normal gastric tissues (n=97) was determined using qPCR. The EphB2 expression was over-activated using a CRISPR activator for the investigation of its cellular function. The expression levels of the EphB2 protein in the tumour tissues of tissue arrays were higher than the benign non-cancerous gastric tissues (P<0.05). EphB2 mRNA expression in GC tissues was also significantly elevated when compared with adjacent non-cancerous tissues (P<0.01). EphB2 activation promoted the migration and invasion abilities of the GC cell lines (P<0.01, respectively). In contrast, EphB2 activation significantly decreased the adhesion in GC cells (P<0.0001, respectively). The enrichment analysis of the correlated genes in a GC cohort indicates that EphB2 may function through mediating the cytokine-cytokine interaction, JAK-STAT and TP53 signaling pathways. In conclusion, EphB2 represents as a novel independent prognostic marker in GC. And activation of the EphB2 gene expression elevated the levels of migration and invasion, but suppressed adhesion of GC cells, indicating that EphB2 may act as a tumour promotor in GC. Our findings thus provide fundamental evidence for the consideration of the therapeutic potential of targeting EphB2 in GC.

Tetraspanin18 regulates angiogenesis through VEGFR2 and Notch pathways.

ABSTRACTThe VEGF pathway is critically required for vasculogenesis, the formation of the primary vascular network. It is also required for angiogenesis resulting in sprouting and pruning of vessels to generate mature arborizing structures. The Notch pathway is essential for arterial–venous specification and the maturation of nascent vessels. We have determined that Tspan18, a member of the Tetraspanin family, is expressed in developing vessels but not in mature vasculature in zebrafish and mouse wound healing. Moreover, reduction at Tspan18 level resulted in aberrant vascular patterning, impaired vessel stability and defective arterial–venous specification. Tspan18 deficiency reduced VEGF, VEGFR2, Notch3 and EphrinB2, and increased EphB4, VEGFR3, Semaphorin3, Neuropilin and PlexinD1 expression. Furthermore, vascular defects of Tspan18 deficiency could be rescued by ectopic expression of VEGFR2 and Notch, but not by knockdown of Semaphorin or Plexin. Functional studies showed that knockdown of Tspan18 led to reduced endothelial cell migration, invasion and tube formation. Tspan18 has dynamic expression, regulates vascular development and maturation in the embryo with re-expression in adult life in wound healing.

Icariin Alleviates Glucocorticoid-Induced Osteoporosis through EphB4/Ephrin-B2 Axis.

Purpose Glucocorticoid (GC) is the most important risk factor for osteoporosis (OP); in the present study, we examined the potential mechanism of icariin, a natural bioactive compound isolated from the traditional Chinese herbal Epimedium, for GC-induced OP to explore its potential therapeutic effect. Methods We used a GC-induced OP mice model and treated with icariin. Pathological changes were measured by H&E staining, and the effects of icariin on osteoblasts and osteoclasts were measured by immunohistochemistry (IHC) staining and western blot (WB) analyses, while trabecular bone parameters were detected by micro-CT imaging in vivo. Results The results showed that in GC-induced OP symptoms, icariin treatment significantly increased the density of the trabecular bone when exposed to GC, revealed by H&E staining and micro-CT imaging. IHC staining showed that GC-induced OP had a lower EphB4 expression and higher Ephrin-B2 expression, but icariin could promote EphB4 while suppressing Ephrin-B2 expression. The WB results also provided evidence of the same protein expression trend, showing that the osteoblast marker OCN and the EphB4 downstream factor RhoA in the GC group were decreased, while both OCN and RhoA expression were significantly increased and the Ephrin-B2 downstream factor Grb4 in in GC group was increased after icariin treatment. Conclusion Icariin could improve the characteristics of OP through regulating the balance of the EphB4/Ephrin-B2 pathway. Further preclinical trial is needed to provide certainty of clinical benefits for OP patients.

Loss of tyrosine kinase receptor Ephb2 impairs proliferation and stem cell activity of spermatogonia in culture†.

Germline stem and progenitor cells can be extracted from the adult mouse testis and maintained long-term in vitro. Yet, the optimal culture conditions for preserving stem cell activity are unknown. Recently, multiple members of the Eph receptor family were detected in murine spermatogonia, but their roles remain obscure. One such gene, Ephb2, is crucial for maintenance of somatic stem cells and was previously found enriched at the level of mRNA in murine spermatogonia. We detected Ephb2 mRNA and protein in primary adult spermatogonial cultures and hypothesized that Ephb2 plays a role in maintenance of stem cells in vitro. We employed CRISPR-Cas9 targeting and generated stable mutant SSC lines with complete loss of Ephb2. The characteristics of Ephb2-KO cells were interrogated using phenotypic and functional assays. Ephb2-KO SSCs exhibited reduced proliferation compared to wild-type cells, while apoptosis was unaffected. Therefore, we examined whether Ephb2 loss correlates with activity of canonical pathways involved in stem cell self-renewal and proliferation. Ephb2-KO cells had reduced ERK MAPK signaling. Using a lentiviral transgene, Ephb2 expression was rescued in Ephb2-KO cells, which partially restored signaling and proliferation. Transplantation analysis revealed that Ephb2-KO SSCs cultures formed significantly fewer colonies than WT, indicating a role for Ephb2 in preserving stem cell activity of cultured cells. Transcriptome analysis of wild-type and Ephb2-KO SSCs identified Dppa4 and Bnc1 as differentially expressed, Ephb2-dependent genes that are potentially involved in stem cell function. These data uncover for the first time a crucial role for Ephb2 signaling in cultured SSCs.

The influence of Tongqiao Huashuan Decoction on the expression of VEGF and EphB2 in MCAO model rats.

To observe the effect of Miao medicine and Tongqiao Huashuan Decoction on the expression of vascular endothelial growth factor (VEGF) and ephrin-B2 (EphB2) in the frontal lobe of the involved side and the cerebellum of rat models of middle cerebral artery occlusion (MCAO) and to reveal the pharmacological mechanism of Tongqiao Huashuan Decoction in treating acute ischemic stroke. Seventy healthy male SD rats were randomly divided into sham operation, model, salvia miltiorrhiza (S. miltiorrhiza), and Miao medicine groups. Modified Longa's method was used to prepare a cerebral ischemia reperfusion model. After the operation, the rats in the sham operation group and model group were intragastrically administered with saline, those in the Miao medicine group were intragastrically administered with Tongqiao Huashuan Decoction, and those in the S. miltiorrhiza group were intraperitoneally injected with S. miltiorrhiza. After 14 days of administration, the neurological deficit scores of the rats in each group were compared before and after treatment. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of the brain tissues in the right infarcted areas of the rats. VEGF expression in the frontal lobe and cerebellum was observed through immunohistochemistry, and in situ hybridization to detect EphB2 expression in the frontal lobe and cerebellum. The neurological deficit scores were significantly improved in the Miao medicine and S. miltiorrhiza groups aftertreatment compared with those of the model group (P<0.05) and was higher in the Miao medicine group than in the S. miltiorrhiza group. The VEGF expression in the right frontal lobe and cerebellum was significantly increased in the Miao medicine and S. miltiorrhiza groups (P<0.05) with the former having higher levels than the latter (P<0.05). EphB2 expression was significantly increased in the frontal lobe and cerebellum in the Miao medicine and S. miltiorrhiza groups (P<0.05) and was higher in the frontal lobe of the Miao medicine group than that of the S. miltiorrhiza group (P<0.05) but was not significantly different in the cerebellum in the S. miltiorrhiza and Miao medicine groups (P>0.05). Tongqiao Huashuan Decoction can improve the neurological function score and promote the VEGF expression in the frontal lobe and cerebellum and the EphB2 expression in the frontal lobe of the involved side of MCAO rats. The pharmacological mechanism of Tongqiao Huashuan Decoction in treating acute ischemic stroke may be related to its regulation of VEDF and EphB2 expression in the distal part of the involved side.

Immunohistochemical assessment of Eph/ephrin expression in oral squamous cell carcinoma and precursor lesions.

To evaluate erythropoietin-producing hepatocellular carcinoma (Eph)/Eph receptor-interaction protein (ephrin) expression in oral squamous cell carcinoma (OSCC) and oral epithelial precursor lesions (OEPLs), EphA2, EphB4, and ephrinB2 were examined and compared with microvessel density (MVD) and lymphatic vessel density (LVD). Samples from 73 OSCC and 43 OEPLs patients were immunohistochemically analyzed with antibodies against EphA2, EphB4, ephrinB2, CD34, and D2-40. Results were compared with clinicopathological findings. Immunohistochemical reactivity for EphA2, EphB4, and ephrinB2 was detected in epithelial cells and some stromal vascular cells in OEPLs and OSCC, proportionately with the level of malignancy. The number of blood vessel endothelial cells stained with CD34 and lymphatic vessel endothelial cells stained with D2-40 was increased in OEPLs and OSCC. In OSCC, ephrinB2 and EphB4 exhibited significant correlation with recurrence and invasion depth, respectively. MVD was significantly lower in slight lymphocytic reaction than in prominent stromal reaction. Association was found between LVD and T classification, postoperative metastasis, survival, mode of invasion, and invasion depth. Expression of EphA2, EphB4, ephrinB2, MVD, and LVD might be associated with malignant potential of the oral epithelium. Angiogenesis and lymphangiogenesis appear to be related to progression of potentially malignant oral lesions.

EPHB4 inhibition activates ER stress to promote immunogenic cell death of prostate cancer cells

The EPHB4 receptor is implicated in the development of several epithelial tumors and is a promising therapeutic target, including in prostate tumors in which EPHB4 is overexpressed and promotes tumorigenicity. Here, we show that high expression of EPHB4 correlated with poor survival in prostate cancer patients and EPHB4 inhibition induced cell death in both hormone sensitive and castration-resistant prostate cancer cells. EPHB4 inhibition reduced expression of the glucose transporter, GLUT3, impaired glucose uptake, and reduced cellular ATP levels. This was associated with the activation of endoplasmic reticulum stress and tumor cell death with features of immunogenic cell death (ICD), including phosphorylation of eIF2α, increased cell surface calreticulin levels, and release of HMGB1 and ATP. The changes in tumor cell metabolism after EPHB4 inhibition were associated with MYC downregulation, likely mediated by the SRC/p38 MAPK/4EBP1 signaling cascade, known to impair cap-dependent translation. Together, our study indicates a role for EPHB4 inhibition in the induction of immunogenic cell death with implication for prostate cancer therapy.

EphB2 receptor cell-autonomous forward signaling mediates auditory memory recall and learning-driven spinogenesis

While ephrin-B ligands and EphB receptors are expressed to high levels in the learning centers of the brain, it remains largely unknown how their trans-synaptic interactions contribute to memory. We find that EphB2 forward signaling is needed for contextual and sound-evoked memory recall and that constitutive over-activation of the receptor’s intracellular tyrosine kinase domain results in enhanced memory. Loss of EphB2 expression does not affect the number of neurons activated following encoding, although a reduction of neurons activated after the sound-cued retrieval test was detected in the auditory cortex and hippocampal CA1. Further, spine density and maturation was reduced in the auditory cortex of mutants especially in the neurons that were dual-activated during both encoding and retrieval. Our data demonstrates that trans-synaptic ephrin-B-EphB2 interactions and forward signaling facilitate neural activation and structural plasticity in learning-associated neurons involved in the generation of memories.