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Claudin-4 (CLDN4) is a tight junction protein to maintain the cancer microenvironment. We recently reported the role of the CLDN4 not forming tight junction in the induction of epithelial-mesenchymal transition (EMT). Herein, we investigated the role of CLDN4 in renal cell carcinoma (RCC), focusing on CLDN4. CLDN4 expression in 202 RCCs was examined by immunostaining. CLDN4 phosphorylation and subcellular localization were examined using high metastatic human RCC SN12L1 and low metastatic SN12C cell lines. In 202 RCC cases, the CLDN4 expression decreased in the cell membrane and had no correlation with clinicopathological factors. However, CLDN4 was localized in the nucleus in 5 cases (2%), all of which were pT3. Contrastingly, only 6 of 198 nuclear CLDN4-negative cases were pT3. CLDN4 was found in the nuclear fraction of a highly metastatic human RCC cell line, SN12L1, but not in the low metastatic SN12C cells. In SN12L1 cells, phosphorylation of tyrosine and serine residues was observed in cytoplasmic CLDN4, but not in membranous CLDN4. In contrast, phosphorylation of serine residues was observed in nuclear CLDN4. In SN12L1 cells, CLDN4 tyrosine phosphorylation by EphA2/Ephrin A1 resulted in the release of CLDN4 from tight junction and cytoplasmic translocation. Furthermore, protein kinase C (PKC)-ε phosphorylated the CLDN4 serine residue, resulting in nuclear import. Contrarily, in SN12C cells that showed decreased expression of EphA2/Ephrin A1 and PKCε, the activation of EphA2/EphrinA1 and PKCε induced cytoplasmic and nuclear translocation of CLDN4, respectively. Furthermore, the nuclear translocation of CLDN4 promoted the nuclear translocation of Yes-associated protein (YAP) bound to CLDN4, which induced the EMT phenotype. These findings suggest that the release of CLDN4 by impaired tight junction might be a mechanism underlying the malignant properties of RCC. These findings suggest that the release of CLDN4 by impaired tight junction might be one of the mechanisms of malignant properties of RCC.

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

Eph receptor tyrosine kinases and their ephrin ligands regulate cell navigation during normal and oncogenic development. Signaling of Ephs is initiated in a multistep process leading to the assembly of higher-order signaling clusters that set off bidirectional signaling in interacting cells. However, the structural and mechanistic details of this assembly remained undefined. Here we present high-resolution structures of the complete EphA2 ectodomain and complexes with ephrin-A1 and A5 as the base unit of an Eph cluster. The structures reveal an elongated architecture with novel Eph/Eph interactions, both within and outside of the Eph ligand-binding domain, that suggest the molecular mechanism underlying Eph/ephrin clustering. Structure-function analysis, by using site-directed mutagenesis and cell-based signaling assays, confirms the importance of the identified oligomerization interfaces for Eph clustering.

Eph receptors and the corresponding Eph receptor-interacting (ephrin) ligands jointly constitute a critical cell signaling network that has multiple functions. The tyrosine kinase EphA2, which belongs to the family of Eph receptors, is highly produced in tumor tissues, while found at relatively low levels in most normal adult tissues, indicating its potential application in cancer treatment. After 30 years of investigation, a large amount of data regarding EphA2 functions have been compiled. Meanwhile, several compounds targeting EphA2 have been evaluated and tested in clinical studies, albeit with limited clinical success. The present review briefly describes the contribution of EphA2-ephrin A1 signaling axis to carcinogenesis. In addition, the roles of EphA2 in resistance to molecular-targeted agents were examined. In particular, we focused on EphA2’s potential as a target for cancer treatment to provide insights into the application of EphA2 targeting in anticancer strategies. Overall, EphA2 represents a potential target for treating malignant tumors.

The biological functions of the Eph/ephrin system have been intensively investigated and well documented so far since its discovery in 1987. Although the Eph/ephrin system has been implicated in pathological settings such as Alzheimer's disease and cancer, the molecular mechanism of the Eph/ephrin system in those diseases is not well understood. Especially in cancer, recent studies have demonstrated that most of Eph and ephrin are up‐ or down‐regulated in various types of cancer, and have been implicated in tumor progression, tumor malignancy, and prognosis. However, they lack consistency and are in controversy. The localization patterns of EphA1 and EphA2 in mouse lungs are very similar, and both knockout mice showed similar phenotypes in the lungs. Ephrin‐A1 that is a membrane‐anchored ligand for EphAs was co‐localized with EphA1 and EphA2 in lung vascular endothelial cells. We recently uncovered the molecular mechanism of ephrin‐A1‐induced lung metastasis by understanding the physiological function of ephrin‐A1 in lungs. This review focuses on the function of EphA1, EphA2, and ephrin‐A1 in tumors and an establishment of pre‐metastatic microenvironment in the lungs. ADAM12‐cleaved ephrin‐A1 induces receptor endocytosis and RhoA activation leading to cell retraction. Cleaved ephrin‐A1 competes for pre‐existing Eph/ephrin interactions. Ephrin‐A1‐stimulated vascular endothelial cells are shrunk, and thereby enhances lung vascular permeability.

Accumulating evidence indicates that an elevated ephrin-A1 expression is positively correlated with a worse prognosis in some cancers such as colon and liver cancer. The detailed mechanism of an elevated ephrin-A1 expression in a worse prognosis still remains to be fully elucidated. We previously reported that ADAM12-cleaved ephrin-A1 enhanced lung vascular permeability and thereby induced lung metastasis. However, it is still unclear whether or not cleaved forms of ephrin-A1 are derived from primary tumors and have biological activities. We identified the ADAM12-mediated cleavage site of ephrin-A1 by a Matrix-assisted laser desorption ionization mass spectrometry and checked levels of ephrin-A1 in the serum and the urine derived from the primary tumors by using a mouse model. We found elevated levels of tumor-derived ephrin-A1 in the serum and the urine in the tumor-bearing mice. Moreover, inhibition of ADAM-mediated cleavage of ephrin-A1 or antagonization of the EphA receptors resulted in a significant reduction of lung metastasis. The results suggest that tumor-derived ephrin-A1 is not only a potential biomarker to predict lung metastasis from the primary tumor highly expressing ephrin-A1 but also a therapeutic target of lung metastasis.

Myocardial tissue damage that occurs during an ischemic event leads to a spiraling deterioration of cardiac muscle structural and functional integrity. Reperfusion is the only known efficacious strategy and is the most commonly used treatment to reduce injury and prevent remodeling. However, timing is critical, and the procedure is not always feasible for a variety of reasons. The complex molecular basis for cardioprotection has been studied for decades but formulation of a viable therapeutic that can significantly attenuate myocardial injury remains elusive. In this review, we address barriers to the development of a fruitful approach that will substantially improve the prognosis of those suffering from this widespread and largely unmitigated disease. Furthermore, we proffer that ephrinA1, a candidate molecule that satisfies many of the important criteria discussed, possesses robust potential to overcome these hurdles and thus offers protection that surpasses the limitations currently observed.

Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4 . EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4 . Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function. While biallelic mutations of the SLC26A4 gene cause non-syndromic hearing loss with enlarged vestibular aqueducts or Pendred syndrome, a considerable number of patients carry mono-allelic mutations. Here the authors identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4.

The activation of EphA2 receptor by its natural ligand EphrinA1 causes blood brain barrier dysfunction, and inactivation of EphA2 reduces BBB damage in ischemic stroke. Thus, EphA2 targeted antagonists may serve as neuroprotective agents. We engineered four mutants of EphrinA1, EM1, EM2, EM3 and EM4, respectively. The computational analysis showed that these four mutants were capable of interacting with EphA2. Their potential neuroprotective effects were examined in mouse focal ischemia/reperfusion (I/R) model. EM2 exhibited strong neuroprotective effects, including reduced brain infarct volume, neuronal apoptosis, cerebral edema, and improved neurological scores. The EM2-mediated protection was associated with a comparative decrease in BBB leakage, inflammatory infiltration, and higher expression levels of tight junction proteins, such as zonula occludens-1 and Occludin. I/R-induced high expression of Rho-associated protein kinase 2 (ROCK2) was down-regulated after EM2 treatment. Moreover, EM2 reduced agonist doxazosin-induced EphA2 phosphorylation and cells rounding in PC3 cells, indicating EphA2-antagonizing activity of EM2. These finding provided evidences of the neuroprotection of EphA2 antagonist and a novel approach for ischemic stroke treatment. These results also suggested that a receptor agonist can be switched to an antagonist by substituting one or more relevant residues.

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide, with a high incidence rate and mortality. The analysis of serum biomarkers for colorectal cancer diagnosis has attracted more and more attention because of its low cost, repeatability, and quantification. This study was aimed to evaluate the diagnostic performance of serum Ephrin-A1 in patients with CRC. We retrospectively analyzed CRC cases in a test cohort (121 patients and 108 controls) and validated them in a validation cohort (119 patients and 118 controls). The concentration of Ephrin-A1 in serum was detected by Enzyme-linked immunosorbent assay (ELISA) and the diagnostic performance of serum Ephrin-A1 was evaluated by receiver operating characteristic (ROC) analysis. In the test cohort, serum Ephrin-A1 levels in patients with all-stage CRC and early-stage CRC were significantly higher than those in healthy controls. The area under the ROC curve (AUC), sensitivity and specificity of all-stage CRC and early-stage CRC were 0.709 (95% CI 0.644–0.775) and 0.660 (95% CI 0.530–0.790), 48.76% and 45.00%, 81.48% and 81.48%, respectively. Similar results were observed in the validation cohort. Serum Ephrin-A1 might be served as a potential biomarker in the diagnosis of CRC.