ED-71 Improves Bone Mass in Ovariectomized Rats by Inhibiting Osteoclastogenesis Through EphrinB2-EphB4-RANKL/OPG Axis.

Purpose: Estrogen deficiency is the main reason of postmenopausal osteoporosis. Eldecalcitol (ED-71) is a new active vitamin D analogue clinically used in the treatment of postmenopausal osteoporosis. We aimed to investigate whether EphrinB2-EphB4 and RANKL/RANK/OPG signaling cooperate in mediating the process of osteoporosis by ED-71. Methods: In vivo, the ovariectomized (OVX) rats were administered orally with 30 ng/kg ED-71 once a day for 8 weeks. HE staining, Masson staining and Immunofluorescence staining were used to evaluate bone mass, bone formation, osteoclastogenesis associated factors and the expression of EphrinB2, EphB4, RANKL and OPG. In vitro, H2O2 stimulation was used to simulate the cell environment in osteoporosis. Immunofluorescence, quantitative real time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western Blot were applied to detect the expression of EphrinB2, EphB4, RANKL and OPG. In osteoblasts, EphB4 was knocked down by EphB4 small-interfering RNA (siRNA) transfection. LY294002 (PI3K inhibitor) or ARQ092 (AKT inhibitor) was used to block PI3K/AKT pathway. An indirect co-culture system of osteoblasts and osteoclasts was established. The mRNA and protein expression of osteoclastogenes is associated factors were tested by qRT-PCR and Western Blot. Results: ED-71 increased bone mass and decreased the number of osteoclasts in OVX rats. Moreover, ED-71 promoted the expression of EphrinB2, EphB4, and decreased the RANKL/OPG ratio in osteoblasts. Osteoclastogenesis was restrained when osteoclasts were indirectly co-cultured with ED-71-treated osteoblasts. After silencing of EphB4 expression in osteoblasts, ED-71 inhibited the expression of P-PI3K and P-AKT and increased the ratio of RANKL/OPG. This reversed the inhibitory effect of ED-71 on osteoclastogenes. Therefore, in ED-71-inhibited osteoclastogenes, EphB4 is a key factor affecting the secretion of RANKL and OPG by osteoblasts. EphB4 suppressed the RANKL/OPG ratio through activating PI3K/AKT signaling in osteoblasts. Conclusion: ED-71 inhibits osteoclastogenesis through EphrinB2-EphB4-RANKL/OPG axis, improving bone mass in ovariectomized rats. PI3K/AKT pathway is involved this process.

Eph-ephrin signaling couples endothelial cell sorting and arterial specification.

Cell segregation allows the compartmentalization of cells with similar fates during morphogenesis, which can be enhanced by cell fate plasticity in response to local molecular and biomechanical cues. Endothelial tip cells in the growing retina, which lead vessel sprouts, give rise to arterial endothelial cells and thereby mediate arterial growth. Here, we have combined cell type-specific and inducible mouse genetics, flow experiments in vitro, single-cell RNA sequencing and biochemistry to show that the balance between ephrin-B2 and its receptor EphB4 is critical for arterial specification, cell sorting and arteriovenous patterning. At the molecular level, elevated ephrin-B2 function after loss of EphB4 enhances signaling responses by the Notch pathway, VEGF and the transcription factor Dach1, which is influenced by endothelial shear stress. Our findings reveal how Eph-ephrin interactions integrate cell segregation and arteriovenous specification in the vasculature, which has potential relevance for human vascular malformations caused by EPHB4 mutations.

Ephrin B2 (EFNB2) potentially protects against intervertebral disc degeneration through inhibiting nucleus pulposus cell apoptosis.

Nucleus pulposus (NP) cell apoptosis is a significant indication of accelerated intervertebral disc degeneration; however, the precise mechanism is unelucidated as of yet. Ephrin B2 (EFNB2), the only gene down-regulated in the three degraded intervertebral disc tissue microarray groups (GSE70362, GSE147383 and GSE56081), was screened for examination in this study. Subsequently, EFNB2 was verified to be down-regulated in degraded NP tissue samples. Interleukin-1 (IL-1ß) treatment of NP cells to simulate the IDD environment indicated that IL-1ß treatment decreased EFNB2 expression. In degenerative NP cells stimulated by IL-1ß, EFNB2 knockdown significantly increased the rate of apoptosis as well as the apoptosis-related molecules cleaved-caspase-3 and the Bax to Bcl-2 ratio. EFNB2 was found to promote AKT, PI3K, and mTOR phosphorylation; the PI3K/AKT signaling role was investigated using the PI3K inhibitor LY294002. EFNB2 overexpression significantly increased PI3K/AKT pathway activity in IL-1ß-stimulated NP cells than the normal control. Moreover, EFNB2 partially alleviated NP cell apoptosis induced by IL-1ß, reduced the cleaved-cas3 level, and decreased the Bax/Bcl-2 ratio after the addition of the inhibitor LY294002. Additionally, EFNB2 overexpression inhibited the ERK1/2 phosphorylation; the effects of EFNB2 overexpression on ERK1/2 phosphorylation, degenerative NP cell viability, and cell apoptosis were partially reversed by ERK signaling activator Ceramide C6. EFNB2 comprehensively inhibited the apoptosis of NP cells by activating the PI3K/AKT signaling and inhibiting the ERK signaling, obviating the exacerbation of IDD. EFNB2 could be a potential target to protect against degenerative disc changes.

Inhibition of Ephrin B2 Reverse Signaling Abolishes Multiple Myeloma Pathogenesis.

Bone marrow vascular endothelial cells (BM EC) regulate multiple myeloma pathogenesis. Identification of the mechanisms underlying this interaction could lead to the development of improved strategies for treating multiple myeloma. Here, we performed a transcriptomic analysis of human ECs with high capacity to promote multiple myeloma growth, revealing overexpression of the receptor tyrosine kinases, EPHB1 and EPHB4, in multiple myeloma-supportive ECs. Expression of ephrin B2 (EFNB2), the binding partner for EPHB1 and EPHB4, was significantly increased in multiple myeloma cells. Silencing EPHB1 or EPHB4 in ECs suppressed multiple myeloma growth in coculture. Similarly, loss of EFNB2 in multiple myeloma cells blocked multiple myeloma proliferation and survival in vitro, abrogated multiple myeloma engraftment in immune-deficient mice, and increased multiple myeloma sensitivity to chemotherapy. Administration of an EFNB2-targeted single-chain variable fragment also suppressed multiple myeloma growth in vivo. In contrast, overexpression of EFNB2 in multiple myeloma cells increased STAT5 activation, increased multiple myeloma cell survival and proliferation, and decreased multiple myeloma sensitivity to chemotherapy. Conversely, expression of mutant EFNB2 lacking reverse signaling capacity in multiple myeloma cells increased multiple myeloma cell death and sensitivity to chemotherapy and abolished multiple myeloma growth in vivo. Complementary analysis of multiple myeloma patient data revealed that increased EFNB2 expression is associated with adverse-risk disease and decreased survival. This study suggests that EFNB2 reverse signaling controls multiple myeloma pathogenesis and can be therapeutically targeted to improve multiple myeloma outcomes. SIGNIFICANCE: Ephrin B2 reverse signaling mediated by endothelial cells directly regulates multiple myeloma progression and treatment resistance, which can be overcome through targeted inhibition of ephrin B2 to abolish myeloma.

Biological Significance of EphB4 Expression in Cancer.

Eph receptors and their Eph receptor-interacting (ephrin) ligands comprise a vital cell communication system with several functions. In cancer cells, there was evidence of bilateral Eph receptor signaling with both tumor-suppressing and tumor-promoting actions. As a member of the Eph receptor family, EphB4 has been linked to tumor angiogenesis, growth, and metastasis, which makes it a viable and desirable target for drug development in therapeutic applications. Many investigations have been conducted over the last decade to elucidate the structure and function of EphB4 in association with its ligand ephrinB2 for its involvement in tumorigenesis. Although several EphB4-targeting drugs have been investigated, and some selective inhibitors have been evaluated in clinical trials. This article addresses the structure and function of the EphB4 receptor, analyses its possibility as an anticancer therapeutic target, and summarises knowledge of EphB4 kinase inhibitors. To summarise, EphB4 is a difficult but potential treatment option for cancers.

M2 macrophage-derived cathepsin S promotes peripheral nerve regeneration via fibroblast-Schwann cell-signaling relay.

BACKGROUND: Although peripheral nerves have an intrinsic self-repair capacity following damage, functional recovery is limited in patients. It is a well-established fact that macrophages accumulate at the site of injury. Numerous studies indicate that the phenotypic shift from M1 macrophage to M2 macrophage plays a crucial role in the process of axon regeneration. This polarity change is observed exclusively in peripheral macrophages but not in microglia and CNS macrophages. However, the molecular basis of axonal regeneration by M2 macrophage is not yet fully understood. Herein, we aimed to identify the M2 macrophage-derived axon regeneration factor. METHODS: We established a peripheral nerve injury model by transection of the inferior alveolar nerve (IANX) in Sprague-Dawley rats. Transcriptome analysis was performed on the injured nerve. Recovery from sensory deficits in the mandibular region and histological reconnection of IAN after IANX were assessed in rats with macrophage depletion by clodronate. We investigated the effects of adoptive transfer of M2 macrophages or M2-derived cathepsin S (CTSS) on the sensory deficit. CTSS initiating signaling was explored by western blot analysis in IANX rats and immunohistochemistry in co-culture of primary fibroblasts and Schwann cells (SCs). RESULTS: Transcriptome analysis revealed that CTSS, a macrophage-selective lysosomal protease, was upregulated in the IAN after its injury. Spontaneous but partial recovery from a sensory deficit in the mandibular region after IANX was abrogated by macrophage ablation at the injured site. In addition, a robust induction of c-Jun, a marker of the repair-supportive phenotype of SCs, after IANX was abolished by macrophage ablation. As in transcriptome analysis, CTSS was upregulated at the injured IAN than in the intact IAN. Endogenous recovery from hypoesthesia was facilitated by supplementation of CTSS but delayed by pharmacological inhibition or genetic silencing of CTSS at the injured site. Adoptive transfer of M2-polarized macrophages at this site facilitated sensory recovery dependent on CTSS in macrophages. Post-IANX, CTSS caused the cleavage of Ephrin-B2 in fibroblasts, which, in turn, bound EphB2 in SCs. CTSS-induced Ephrin-B2 cleavage was also observed in human sensory nerves. Inhibition of CTSS-induced Ephrin-B2 signaling suppressed c-Jun induction in SCs and sensory recovery. CONCLUSIONS: These results suggest that M2 macrophage-derived CTSS contributes to axon regeneration by activating SCs via Ephrin-B2 shedding from fibroblasts.

Sequence basis for selectivity of ephrin-B2 ligand for Eph receptors and pathogenic henipavirus G glycoproteins.

IMPORTANCE: Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and regulates multiple cell developmental and signaling processes. It also functions as the cell entry receptor for Nipah virus and Hendra virus, zoonotic viruses that can cause respiratory and/or neurological symptoms in humans with high mortality. Here, we investigate the sequence basis of EFNB2 specificity for binding the Nipah virus attachment G glycoprotein over Eph receptors. We then use this information to engineer EFNB2 as a soluble decoy receptor that specifically binds the attachment glycoproteins of the Nipah virus and other related henipaviruses to neutralize infection. These findings further mechanistic understanding of protein selectivity and may facilitate the development of diagnostics or therapeutics against henipavirus infection.

In silico prediction of interaction between Nipah virus attachment glycoprotein and host cell receptors Ephrin-B2 and Ephrin-B3 in domestic and peridomestic mammals.

Nipah virus (NiV) is a lethal bat-borne zoonotic virus that causes mild to acute respiratory distress and neurological manifestations in humans with a high mortality rate. NiV transmission to humans occurs via consumption of bat-contaminated fruit and date palm sap (DPS), or through direct contact with infected individuals and livestock. Since NiV outbreaks were first reported in pigs from Malaysia and Singapore, non-neutralizing antibodies against NiV attachment Glycoprotein (G) have also been detected in a few domestic mammals. NiV infection is initiated after NiV G binds to the host cell receptors Ephrin-B2 and Ephrin-B3. In this study, we assessed the degree of NiV host tropism in domestic and peridomestic mammals commonly found in Bangladesh that may be crucial in the transmission of NiV by serving as intermediate hosts. We carried out a protein-protein docking analysis of NiV G complexes (n = 52) with Ephrin-B2 and B3 of 13 domestic and peridomestic species using bioinformatics tools. Protein models were generated by homology modelling and the structures were validated for model quality. The different protein-protein complexes in this study were stable, and their binding affinity (ΔG) scores ranged between -8.0 to -19.1 kcal/mol. NiV Bangladesh (NiV-B) strain displayed stronger binding to Ephrin receptors, especially with Ephrin-B3 than the NiV Malaysia (NiV-M) strain, correlating with the observed higher pathogenicity of NiV-B strains. From the docking result, we found that Ephrin receptors of domestic rat (R. norvegicus) had a higher binding affinity for NiV G, suggesting greater susceptibility to NiV infections compared to other study species. Investigations for NiV exposure to domestic/peridomestic animals will help us knowing more the possible role of rats and other animals as intermediate hosts of NiV and would improve future NiV outbreak control and prevention in humans and domestic animals.

Bioinformatic investigation of Nipah virus surface protein mutations: Molecular docking with Ephrin B2 receptor, molecular dynamics simulation, and structural impact analysis.

The SARS-CoV-2 outbreak resulted in significant challenges and loss of life. The Nipah virus, known for its high infectivity and severity, was designated an emergency concern by the World Health Organization. To understand its mutations, the Nipah virus proteins were analyzed extensively, with a focus on the essential G and F proteins responsible for viral entry into host cells. Our bioinformatics analysis unveiled multiple mutations, including simultaneous mutations within a single sequence. Notably, the G273S mutation in the F protein was identified as a potential cause of structural damage, which carries significant implications for vaccine development. Comparing the docking scores of G and F proteins with the Ephrin B2 receptor, it was found that the Y228H mutation in the G protein and the D252G mutation in the F protein likely affect virus entry into host cells. Moreover, our investigation into stability and deformability highlighted the impact of the Y228H mutation in the G protein complex. Molecular dynamics simulations revealed increased flexibility and conformational changes in the G protein complex with the Y228H mutation compared with the known complex. Furthermore, evaluating the root mean square deviation variation demonstrated greater dynamic behavior in the G protein complex and the Ephrin B2 receptor complex. This comprehensive study provides valuable insights into Nipah virus mutations, their significance for vaccine development, and the importance of understanding protein complex behavior in drug discovery. The identified mutations, especially G273S and Y228H, hold crucial implications for future research and potential interventions against the Nipah virus.

Computer-assisted identification of potential quinolone derivatives targeting Nipah virus glycoprotein attachment with human cell surface receptor ephrin-B2: Multistep virtual screening.

Nipah Virus (NiV) is a single-stranded, negative-sense, highly lethal RNA virus. Even though NiV has close to 70-80% of mortality in India and Bangladesh, still there is no available US FDA-approved drug or vaccine. NiV attachment glycoprotein (NiV-G) is critical for NiV to invade the human cell where ephrinB2 which is a crucial membrane-bound ligand that acts as a target of NiV. Most of the research has been performed targeting NiV or human ephrin-B to date. Quinolone derivatives are proven scaffolds for many approved drugs used to treat various bacterial, viral respiratory tract, and urinary tract infections, and rheumatologic disorders such as systemic lupus erythematosus, rheumatoid arthritis. Therefore, we have tried to find potential drug molecules employing quinolone scaffold-based derivatives from PubChem targeting both NiV-G and ephrin-B2 protein. A total of 1500+ quinolone derivatives were obtained from PubChem which were screened based on Drug Likeness followed by being subjected to XP docking employing Schrödinger software. The top ten best molecules were then chosen for their absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling based on the docking score ranking. Further, the top five molecules were selected for 200ns molecular dynamics (MD) simulation study with Desmond module followed by MM-GBSA study by Prime module of Schrödinger. The exhaustive analysis leads us to the top three probable lead drug molecules for NiV are PubChem CID 23646770, an analog of PubChem CID 67726448, and PubChem CID 10613168 which have predicted Ki values of 0.480 µm, 0.785 µm, and 0.380 µm, respectively. These proposed molecules can be the future drugs targeting NiV-G and human ephrin-B2 which requires further in vivo validation.

Overexpression of EphB6 and EphrinB2 controls soma spacing of cortical neurons in a mutual inhibitory way.

To establish functional circuitry, neurons settle down in a particular spatial domain by spacing their cell bodies, which requires proper positioning of the soma and establishing of a zone with unique connections. Deficits in this process are implicated in neurodevelopmental diseases. In this study, we examined the function of EphB6 in the development of cerebral cortex. Overexpression of EphB6 via in utero electroporation results in clumping of cortical neurons, while reducing its expression has no effect. In addition, overexpression of EphrinB2, a ligand of EphB6, also induces soma clumping in the cortex. Unexpectedly, the soma clumping phenotypes disappear when both of them are overexpressed in cortical neurons. The mutual inhibitory effect of EphB6/ EphrinB2 on preventing soma clumping is likely to be achieved via interaction of their specific domains. Thus, our results reveal a combinational role of EphrinB2/EphB6 overexpression in controlling soma spacing in cortical development.

Divergent Roles of Ephrin-B2/EphB4 Guidance System in Pulmonary Hypertension.

BACKGROUND: Smooth muscle cell (SMC) expansion is one key morphological hallmark of pathologically altered vasculature and a characteristic feature of pulmonary vascular remodeling in pulmonary hypertension. Normal embryonal vessel maturation requires successful coverage of endothelial tubes with SMC, which is dependent on ephrin-B2 and EphB4 ligand-receptor guidance system. In this study, we investigated the potential role of ephrin-B2 and EphB4 on neomuscularization in adult pulmonary vascular disease. METHODS AND RESULTS: Ephrin-B2 and EphB4 expression is preserved in smooth muscle and endothelial cells of remodeled pulmonary arteries. Chronic hypoxia-induced pulmonary hypertension was not ameliorated in mice with SMC-specific conditional ephrin-B2 knockout. In mice with global inducible ephrin-B2 knockout, pulmonary vascular remodeling and right ventricular hypertrophy upon chronic hypoxia exposure were significantly diminished compared to hypoxic controls, while right ventricular systolic pressure was unaffected. In contrast, EphB4 receptor kinase activity inhibition reduced right ventricular systolic pressure in hypoxia-induced pulmonary hypertension without affecting pulmonary vascular remodeling. Genetic deletion of ephrin-B2 in murine pulmonary artery SMC, and pharmacological inhibition of EphB4 in human pulmonary artery smooth muscle cells, blunted mitogen-induced cell proliferation. Loss of EphB4 signaling additionally reduced RhoA expression and weakened the interaction between human pulmonary artery smooth muscle cells and endothelial cells in a three-dimensional coculture model. CONCLUSIONS: In sum, pulmonary vascular remodeling was dependent on ephrin-B2-induced Eph receptor (erythropoietin-producing hepatocellular carcinoma receptor) forward signaling in SMC, while EphB4 receptor activity was necessary for RhoA expression in SMC, interaction with endothelial cells and vasoconstrictive components of pulmonary hypertension.

Kindlin2 enables EphB/ephrinB bi-directional signaling to support vascular development.

Direct contact between cells expressing either ephrin ligands or Eph receptor tyrosine kinase produces diverse developmental responses. Transmembrane ephrinB ligands play active roles in transducing bi-directional signals downstream of EphB/ephrinB interaction. However, it has not been well understood how ephrinB relays transcellular signals to neighboring cells and what intracellular effectors are involved. Here, we report that kindlin2 can mediate bi-directional ephrinB signaling through binding to a highly conserved NIYY motif in the ephrinB2 cytoplasmic tail. We show this interaction is important for EphB/ephrinB-mediated integrin activation in mammalian cells and for blood vessel morphogenesis during zebrafish development. A mixed two-cell population study revealed that kindlin2 (in ephrinB2-expressing cells) modulates transcellular EphB4 activation by promoting ephrinB2 clustering. This mechanism is also operative for EphB2/ephrinB1, suggesting that kindlin2-mediated regulation is conserved for EphB/ephrinB signaling pathways. Together, these findings show that kindlin2 enables EphB4/ephrinB2 bi-directional signal transmission.

Adaptive activation of EFNB2/EPHB4 axis promotes post-metastatic growth of colorectal cancer liver metastases by LDLR-mediated cholesterol uptake.

The microenvironment of distant organ plays vital roles in regulating tumor metastases. However, little is known about the crosstalk between metastasized tumor cells and target organs. Herein, we found that EFNB2 expression was upregulated in liver metastases (LM) of colorectal cancer (CRC), but not in pulmonary metastases (PM) or primary CRC tumors. EFNB2 played a tumor-promoting role in CRC LM in vitro and in vivo. Through forward signaling, EFNB2-promoted CRC LM by interacting with the EPHB4 receptor. EFNB2/EPHB4 axis enhances LDLR-mediated cholesterol uptake in CRC LM. Subsequently, EFNB2/EPHB4 axis promotes LDLR transcription by regulating STAT3 phosphorylation. Blocking LDLR reversed the role of the EFNB2/EPHB4 axis in promoting CRC LM. Using clinical data, survival analysis revealed that the survival time of patients with CRC LM was decreased in patients with high EFNB2 expression, compared with low EFNB2 expression. Inhibition of the EFNB2/EPHB4 axis markedly prolonged the survival time of BALB/c nude mice with CRC LM with a high cholesterol diet. These findings revealed a key step in the regulation of cholesterol uptake by EFNB2/EPHB4 axis and its tumor-promoting role in CRC LM.

The role of ephrinB2-EphB4 signalling in bone remodelling during orthodontic tooth movement.

OBJECTIVE: The aim of this study was to investigate the role of ephrinB2-EphB4 signalling in alveolar bone remodelling on the tension side during orthodontic tooth movement (OTM). MATERIALS AND METHODS: An OTM model was established on sixty 8-week-old male Wistar rats. They were randomly divided into the experimental group and the control group. The animals in the experimental group were administrated with subcutaneous injection of EphB4 inhibitor NVP-BHG712 every other day, whereas the control group received only the vehicle. Samples containing the maxillary first molar and the surrounding bone were collected after 0, 3, 7, 14 and 21 days of tooth movement. RESULTS: EphrinB2-EphB4 signalling was actively expressed on the tension side during tooth movement. Micro-CT analysis showed the distance of tooth movement in the experimental group was significantly greater than that of the control group (P < .05) with significantly increased trabecular separation (Tb. Sp) and decreased trabecular number (Tb. N) from day 14 to day 21. The number of osteoclasts significantly increased in the experimental group compared with the control group after 3 and 7 days of tooth movement (P < .05). The expressions of alkaline phosphatase (ALP) and osteopontin (OPN) were significantly reduced by inhibition of EphB4 (P < .05). CONCLUSION: The inhibition of EphB4 suppressed bone formation and enhanced bone resorption activities on the tension side of tooth movement. The ephrinB2-EphB4 signalling might play an important role in alveolar bone remodelling during OTM.

EphrinB2 promotes the human aortic smooth muscle cell growth and migration via mediating F-actin remodeling.

OBJECTIVES: To evaluate the potential effect of EphrinB2 in human thoracic aortic dissection (TAD) and to illustrate the mechanisms governing the role of EphrinB2 in the growth of human aortic smooth muscle cells (HASMC). METHODS: In the study, EphrinB2 expression was investigated by qRT-PCR and immunohistochemistry in 12 pairs of TAD and adjacent human tissues. HASMCs were used for in vitro experiments. Next, EphrinB2 overexpression and depletion in HASMCs were established by EphrinB2-overexpressing vectors and small interfering RNA, respectively. The transfection efficiency was evaluated by qRT-PCR and Western blot. The effects of overexpression and depletion of EphrinB2 on cell proliferation, migration, and invasion were tested in vitro. Cell Counting Kit-8, flow cytometry and transwell migration/invasion, and wound healing assay were used to explore the function of EphrinB2 on HASMC cell lines. The relationship between EphrinB2 and F-actin was assessed by Western blot, immunofluorescence, and Co-IP. RESULTS: We found that EphrinB2 was a prognostic biomarker of TAD patients. Moreover, EphrinB2 expression negatively correlated to aortic dissection tissues, and disease incidence of males, suggesting that EphrinB2 might act as a TAD suppressor by promoting proliferation or decreasing apoptosis in HASMC. Next, over-expression of EphrinB2 in HASMC lines drove cell proliferation, migration, and invasion, and inhibited apoptosis while knockdown EphrinB2 showed the opposite phenomenon, respectively. Furthermore, the level of F-actin in mRNA, protein, and distribution in HASMC cell lines highly matched with the expression of EphrinB2, which indicated that EphrinB2 could mediate the HASMC cytoskeleton via inducing F-actin. CONCLUSIONS: In conclusion, our results first provided the pivotal role of EphrinB2 in HASMC proliferation initiated by mediating F-actin and demonstrated a prognostic biomarker and the potential targets for therapy to prevent thoracic aortic dissection.

Histone deacetylase 4 inhibition ameliorates the social deficits induced by Ephrin-B2 mutation.

Deterioration of inhibitory synapse may be an essential neurological basis underlying abnormal social behaviours. Manipulations that regulate GABAergic transmission are associated with improved behavioural phenotypes in sociability. The synaptic protein, Ephrin-B2 (EB2), plays an important role in the maintenance and reconfiguration of inhibitory synapses in the medial prefrontal cortex (mPFC). However, the inhibitory cell-type specific role of EB2 in the pathophysiology and treatment of social deficits remains unknown. As expected, we revealed that tdTomato-expressing cells were only found in GABAergic neurons instead of excitatory neurons in transgenic EB2-vGATCre mice. This result indicated that depletion of EB2 would occur in those neurons, which further contribute to social deficits. In addition, specific over-expression of mPFC EB2 restored the defective social behaviour abnormalities. These results suggest that the effect of EB2 on social deficits is anatomically and cell-type specific. More importantly, the global upregulation of HDAC4 expression was found in EB2-vGATCre mice. Significant subcellular nuclear shuttling of HDAC4 in vGAT+ neurons was examined and quantified, suggesting a role of nuclear HDAC4 in mediating the mechanism underlying EB2 impairment in vGAT+ neurons. Treatment with LMK235 led to a remarkable rescue of social deficits, thus our data revealed a new domain for the potential utility of HDAC targeting agents to treat social deficits. In conclusion, these results not only revealed a novel molecular mechanism underlying the pathophysiology of social deficits, but also suggested a potential intervention avenue for the treatment of social deficits.

EPH/Ephrin-Targeting Treatment in Breast Cancer: A New Chapter in Breast Cancer Therapy.

Breast cancer (BC) is the most common malignant tumor in women. Erythropoietin-producing hepatocellular receptors (EPHs), receptor tyrosine kinases binding the membrane-bound proteins ephrins, are differentially expressed in BC, and correlate with carcinogenesis and tumor progression. With a view to examining available therapeutics targeting the EPH/ephrin system in BC, a literature review was conducted, using the MEDLINE, LIVIVO, and Google Scholar databases. EPHA2 is the most studied EPH/ephrin target in BC treatment. The targeting of EPHA2, EPHA10, EPHB4, ephrin-A2, ephrin-A4, as well as ephrin-B2 in BC cells or xenograft models is associated with apoptosis induction, tumor regression, anticancer immune response activation, and impaired cell motility. In conclusion, EPHs/ephrins seem to represent promising future treatment targets in BC.

Elevated ITGA5 facilitates hyperactivated mTORC1-mediated progression of laryngeal squamous cell carcinoma via upregulation of EFNB2.

Background: Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant tumors of the head and neck, and it has shown increasing incidence and mortality. The mechanistic target of rapamycin complex 1 (mTORC1) is frequently dysregulated in LSCC, but its underlying mechanisms remain unclear. Methods: Establishment of a novel LSCC cell line using primary LSCC tumor tissues with dysregulated mTORC1 activity and then stable knockdown of Raptor (an mTORC1 specific component) in this cell line. Transcriptomic sequencing, quantitative real-time PCR, western blot analysis, and immunofluorescence assays were used to identify the crucial downstream effector of mTORC1. A series of experiments were conducted to investigate the functions and underlying mechanisms of the mTORC1 target gene in LSCC progression. Clinical LSCC samples were used to evaluate the association of mTORC1 and its downstream targets with clinicopathological features and patient prognosis. Finally, the influence on cisplatin (CDDP) sensitivity upon depletion of the mTORC1 target gene was assessed using a cell culture system, a cell line-derived xenograft (CDX) model, and a patient-derived xenograft (PDX) model. Results: We successfully established a novel LSCC cell line with hyperactivated mTORC1 activity and then identified integrin subunit alpha 5 (ITGA5) as a novel functional downstream effector of mTORC1 in the progression of LSCC. Elevated ITGA5 promotes LSCC progression through augmentation of ephrin-B2 (EFNB2). Clinical data analysis indicated that the activation of the mTORC1-ITGA5-EFNB2 signaling pathway is associated with malignant progression and poor prognosis of LSCC patients. Inhibition of ITGA5 significantly sensitized LSCC cells to CDDP. Conclusions: Our findings highlight a novel molecular mechanism for the tumorigenesis driven by deregulated mTORC1 signaling in LSCC, suggesting that the ITGA5-EFNB2 axis may be a therapeutic target for the treatment of mTORC1-related LSCC.

A synthetic bivalent peptide ligand of EphB4 with potent agonistic activity.

Interaction between ephrin receptor EphB4 and its ligand EFNB2 mediates bidirectional signaling important for cancer: forward EFNB2-to-EphB4 signaling that is tumor suppressive, and reverse EphB4-to-EFNB2 signaling that promotes angiogenesis important for tumor growth and metastasis. Molecular agents targeting these forward and reverse signals of EphB4-EFNB2 interaction can be used to probe the molecular mechanisms of these complex signaling pathways and develop new anticancer therapeutics. In this study, we applied a bivalent ligand design strategy to synthesize a novel dimeric peptide based on an antagonist TNYL-RAW. The dimeric peptide possessed higher EphB4 receptor binding affinity than the monomeric TNYL-RAW peptide. Interestingly, the dimerization of TNYL-RAW peptide converted a monomeric antagonist of EphB4 to a dimeric agonist. This dimeric agonist promoted EphB4 phosphorylation, internalization and degradation, reduced cancer cell motility, and inhibited tube formation of HUVEC. To investigate the mechanism of action of this bivalent dimeric peptide, FRET experiments and molecular dynamic simulation were conducted and suggested that this bivalent ligand recognizes two EphB4 simultaneously which may promote receptor dimerization and oligomerization. This was further supported by the study of this bivalent ligand containing deletion of critical residues on one of its monomers which impaired its simultaneous binding to two EphB4 and ability to cause EphB4 dimerization and phosphorylation. These results demonstrate the value of this novel bivalent agonist ligand of EphB4 as a probe of the bidirectional signaling of EphB4-EFNB2 and lead for cancer drug development.