Lipidation and PEGylation Strategies to Prolong the in Vivo Half-Life of a Nanomolar EphA4 Receptor Antagonist.

The EphA4 receptor tyrosine kinase plays a role in neurodegenerative diseases, inhibition of nerve regeneration, cancer progression and other diseases. Therefore, EphA4 inhibition has potential therapeutic value. Selective EphA4 kinase inhibitors are not available, but we identified peptide antagonists that inhibit ephrin ligand binding to EphA4 with high specificity. One of these peptides is the cyclic APY-d3 (ßAPYCVYRßASWSC-NH2), which inhibits ephrin-A5 ligand binding to EphA4 with low nanomolar binding affinity and is highly protease resistant. Here we describe modifications of APY-d3 that yield two different key derivatives with greatly increased half-lives in the mouse circulation, the lipidated APY-d3-laur8 and the PEGylated APY-d3-PEG4. These two derivatives inhibit ligand induced EphA4 activation in cells with sub-micromolar potency. Since they retain high potency and specificity for EphA4, lipidated and PEGylated APY-d3 derivatives represent new tools for discriminating EphA4 activities in vivo and for preclinical testing of EphA4 inhibition in animal disease models.

Mapping the Universe of Eph Receptor and Ephrin Ligand Transcripts in Epithelial and Fiber Cells of the Eye Lens.

The eye lens is a transparent, ellipsoid organ in the anterior chamber of the eye that is required for fine focusing of light onto the retina to transmit a clear image. Cataracts, defined as any opacity in the lens, remains the leading cause of blindness in the world. Recent studies in humans and mice indicate that Eph-ephrin bidirectional signaling is important for maintaining lens transparency. Specifically, mutations and polymorphisms in the EphA2 receptor and the ephrin-A5 ligand have been linked to congenital and age-related cataracts. It is unclear what other variants of Ephs and ephrins are expressed in the lens or whether there is preferential expression in epithelial vs. fiber cells. We performed a detailed analysis of Eph receptor and ephrin ligand mRNA transcripts in whole mouse lenses, epithelial cell fractions, and fiber cell fractions using a new RNA isolation method. We compared control samples with EphA2 knockout (KO) and ephrin-A5 KO samples. Our results revealed the presence of transcripts for 12 out of 14 Eph receptors and 8 out of 8 ephrin ligands in various fractions of lens cells. Using specific primer sets, RT-PCR, and sequencing, we verified the variant of each gene that is expressed, and we found two epithelial-cell-specific genes. Surprisingly, we also identified one Eph receptor variant that is expressed in KO lens fibers but is absent from control lens fibers. We also identified one low expression ephrin variant that is only expressed in ephrin-A5 control samples. These results indicate that the lens expresses almost all Ephs and ephrins, and there may be many receptor-ligand pairs that play a role in lens homeostasis.

Andrographolide Derivative AL-1 Ameliorates LPS-induced Acute Lung Injury by Inhibiting NLRP3 Inflammasome and Lung Permeability.

BACKGROUND: Acute lung injury (ALI) is a serious respiratory disease with a high mortality rate, and there is an urgent need for a more effective treatment strategy. Andrographolide derivative AL-1 has been identified to possess anti-inflammatory activity. However, whether it could reduce LPS-induced lung injury in mice through inhibiting NLRP3 inflammasome activation and protecting lung permeability has not yet been elucidated. In the present research, we investigated the protective effect of AL-1 on ALI mice and demonstrated the potential mechanisms. METHODS: Male Balb/c mice were anesthetized with isoflurane, and ALI mice were induced by intratracheal instillation of LPS. The mice were euthanized after LPS administration for 12 h, then bronchoalveolar lavage fluid (BALF) and lung tissues were collected. The levels of inflammatory factors were measured by ELISA assay, and HE staining and lung injury scoring were used to evaluate the pathological changes in the pulmonary tissues. Immunohistochemistry and immunofluorescence examination were conducted to detect the expression levels of related proteins. Western blot was performed to measure the levels of NLRP3 inflammasome and tight junction proteins. RESULTS: The study indicated that AL-1 effectively alleviated lung injury by reduction of proinflammatory cytokine levels, MPO activity, lung W/D ratio, and total protein levels. Furthermore, AL-1 improved pathological changes in lung tissue and significantly reduced the infiltration of inflammatory cells. Administration with AL-1 markedly inhibited the expression of NLRP3, ASC, Caspase-1, IL-1ß, gasdermin D (GSDMD), and VCAM-1 but increased the expression of ZO-1, Occludin, JAM-A, and Claudin-1. CONCLUSION: Taken together, these results demonstrated that AL-1 ameliorated pulmonary damage by inhibiting the activation of the NLRP3 inflammasome pathway and restoring TJ protein expression.

ADAM10-cleaved ephrin-A5 contributes to prostate cancer metastasis.

A disintegrin and metalloprotease-10(ADAM10) promotes the metastasis of prostate cancer (PCa), but the specific mechanism is indistinct. Herein, DU145 cell lines with stable overexpression and knockdown of ADAM10 were constructed. We found that ectopic expression of ADAM10 not only significantly facilitated cell proliferation, migration, invasion, and inhibited apoptosis, but also could specifically hydrolyze ephrin-A5 and release the ephrin-A5 soluble ectodomain into extracellular media in vitro. These effects were reversed by ADAM10 depletion or treatment of GI254023X. Meanwhile, the co-location and physical interaction among EphA3, ephrin-A5, and ADAM10 were observed in PCa cells using immunofluorescence and immunoprecipitation techniques. Interestingly, overexpression of EphA3 exerted opposite effects in DU145 (ephrin-A5 + ) cells and PC-3 (ephrin-A5 ± ) cells. In addition, the pro-tumor function of EphA3 was reversed by the treatment with the exogenous ephrin-A5-Fc, which increased the phosphorylation level of EphA3 in PC-3 (ephrin-A5 ± ) cells. In nude mice, ADAM10 accelerated growth of the primary tumor, decreased the level of ephrin-A5 in the tumor tissue, but increased the level of ephrin-A5 in the peripheral blood, accompanied with an increase in the expression of CD31 and VEGF (vascular endothelial growth factor) in the tissue. What is more, the serum ephrin-A5 content of patients with metastatic PCa was significantly higher than that of the non-metastatic group (P < 0.05). The receiver operating characteristic curve(ROC) showed that the area under the curve(AUC) of serum ephrin-A5 as a marker of PCa metastasis was 0.843, with a sensitivity of 93.5% and a specificity of 75%. It is concluded that ADAM10-mediated ephrin-A5 shedding promotes PCa metastasis via transforming the role of EphA3 from ligand-dependent tumor suppressor to ligand-independent promoter, and ephrin-A5 in the blood can be used as a new biomarker for PCa metastasis.

Neoadjuvant therapy alters the collagen architecture of pancreatic cancer tissue via Ephrin-A5.

BACKGROUND: The treatment of pancreatic cancer (PDAC) remains clinically challenging, and neoadjuvant therapy (NAT) offers down staging and improved surgical resectability. Abundant fibrous stroma is involved in malignant characteristic of PDAC. We aimed to investigate tissue remodelling, particularly the alteration of the collagen architecture of the PDAC microenvironment by NAT. METHODS: We analysed the alteration of collagen and gene expression profiles in PDAC tissues after NAT. Additionally, we examined the biological role of Ephrin-A5 using primary cultured cancer-associated fibroblasts (CAFs). RESULTS: The expression of type I, III, IV, and V collagen was reduced in PDAC tissues after effective NAT. The bioinformatics approach provided comprehensive insights into NAT-induced matrix remodelling, which showed Ephrin-A signalling as a likely pathway and Ephrin-A5 (encoded by EFNA5) as a crucial ligand. Effective NAT reduced the number of Ephrin-A5+ cells, which were mainly CAFs; this inversely correlated with the clinical tumour shrinkage rate. Experimental exposure to radiation and chemotherapeutic agents suppressed proliferation, EFNA5 expression, and collagen synthesis in CAFs. Forced EFNA5 expression altered CAF collagen gene profiles similar to those found in PDAC tissues after NAT. CONCLUSION: These results suggest that effective NAT changes the extracellular matrix with collagen profiles through CAFs and their Ephrin-A5 expression.

Mutually Repulsive EphA7-EfnA5 Organize Region-to-Region Corticopontine Projection by Inhibiting Collateral Extension.

Coordination of skilled movements and motor planning relies on the formation of regionally restricted brain circuits that connect cortex with subcortical areas during embryonic development. Layer 5 neurons that are distributed across most cortical areas innervate the pontine nuclei (basilar pons) by protrusion and extension of collateral branches interstitially along their corticospinal extending axons. Pons-derived chemotropic cues are known to attract extending axons, but molecules that regulate collateral extension to create regionally segregated targeting patterns have not been identified. Here, we discovered that EphA7 and EfnA5 are expressed in the cortex and the basilar pons in a region-specific and mutually exclusive manner, and that their repulsive activities are essential for segregating collateral extensions from corticospinal axonal tracts in mice. Specifically, EphA7 and EfnA5 forward and reverse inhibitory signals direct collateral extension such that EphA7-positive frontal and occipital cortical areas extend their axon collaterals into the EfnA5-negative rostral part of the basilar pons, whereas EfnA5-positive parietal cortical areas extend their collaterals into the EphA7-negative caudal part of the basilar pons. Together, our results provide a molecular basis that explains how the corticopontine projection connects multimodal cortical outputs to their subcortical targets.SIGNIFICANCE STATEMENT Our findings put forward a model in which region-to-region connections between cortex and subcortical areas are shaped by mutually exclusive molecules to ensure the fidelity of regionally restricted circuitry. This model is distinct from earlier work showing that neuronal circuits within individual cortical modalities form in a topographical manner controlled by a gradient of axon guidance molecules. The principle that a shared molecular program of mutually repulsive signaling instructs regional organization-both within each brain region and between connected brain regions-may well be applicable to other contexts in which information is sorted by converging and diverging neuronal circuits.

Aggressive and recurrent ovarian cancers upregulate ephrinA5, a non-canonical effector of EphA2 signaling duality.

Erythropoietin producing hepatocellular (Eph) receptors and their membrane-bound ligands ephrins are variably expressed in epithelial cancers, with context-dependent implications to both tumor-promoting and -suppressive processes in ways that remain incompletely understood. Using ovarian cancer tissue microarrays and longitudinally collected patient cells, we show here that ephrinA5/EFNA5 is specifically overexpressed in the most aggressive high-grade serous carcinoma (HGSC) subtype, and increased in the HGSC cells upon disease progression. Among all the eight ephrin genes, high EFNA5 expression was most strongly associated with poor overall survival in HGSC patients from multiple independent datasets. In contrast, high EFNA3 predicted improved overall and progression-free survival in The Cancer Genome Atlas HGSC dataset, as expected for a canonical inducer of tumor-suppressive Eph receptor tyrosine kinase signaling. While depletion of either EFNA5 or the more extensively studied, canonically acting EFNA1 in HGSC cells increased the oncogenic EphA2-S897 phosphorylation, EFNA5 depletion left unaltered, or even increased the ligand-dependent EphA2-Y588 phosphorylation. Moreover, treatment with recombinant ephrinA5 led to limited EphA2 tyrosine phosphorylation, internalization and degradation compared to ephrinA1. Altogether, our results suggest a unique function for ephrinA5 in Eph-ephrin signaling and highlight the clinical potential of ephrinA5 as a cell surface biomarker in the most aggressive HGSCs.

The Expression of the Cancer-Associated lncRNA Snhg15 Is Modulated by EphrinA5-Induced Signaling.

The Eph receptor tyrosine kinases and their respective ephrin-ligands are an important family of membrane receptors, being involved in developmental processes such as proliferation, migration, and in the formation of brain cancer such as glioma. Intracellular signaling pathways, which are activated by Eph receptor signaling, are well characterized. In contrast, it is unknown so far whether ephrins modulate the expression of lncRNAs, which would enable the transduction of environmental stimuli into our genome through a great gene regulatory spectrum. Applying a combination of functional in vitro assays, RNA sequencing, and qPCR analysis, we found that the proliferation and migration promoting stimulation of mouse cerebellar granule cells (CB) with ephrinA5 diminishes the expression of the cancer-related lncRNA Snhg15. In a human medulloblastoma cell line (DAOY) ephrinA5 stimulation similarly reduced SNHG15 expression. Computational analysis identified triple-helix-mediated DNA-binding sites of Snhg15 in promoters of genes found up-regulated upon ephrinA5 stimulation and known to be involved in tumorigenic processes. Our findings propose a crucial role of Snhg15 downstream of ephrinA5-induced signaling in regulating gene transcription in the nucleus. These findings could be potentially relevant for the regulation of tumorigenic processes in the context of glioma.

PIWIL1 interacting RNA piR-017061 inhibits pancreatic cancer growth via regulating EFNA5.

PIWI (P element induced wimpy testis) integrating RNAs (piRNAs) are small non-coding RNAs with the length of approximately 30 nucleotides that plays crucial roles in germ cells and adult stem cells. Recently, accumulating data have shown that piRNA and PIWI proteins are involved in tumorigenesis. However, the roles of PIWI proteins and piRNAs in pancreatic cancer are still elusive. Here, we showed that piR-017061 is significantly downregulated in pancreatic cancer patients' samples and pancreatic cancer cell lines. Furthermore, we studied the function of piR-017061 in pancreatic cancer and our data revealed that piR-017061 inhibits pancreatic cancer cell growth in vitro and in vivo. Moreover, we analyzed the genomic loci around piR-017061 and identified EFNA5 as a novel target of piR-017061. Importantly, our data further revealed a direct binding between piR-017061 and EFNA5 mRNA mediated by PIWIL1. Mechanically, piR-017061 cooperates with PIWIL1 to facilitate EFNA5 mRNA degradation and loss of piR-017061 results in accumulation of EFNA5 which facilitates pancreatic cancer development. Hence, our data provided novel insights into PIWI/piRNA-mediated gene regulation and their function in pancreatic cancer. Since PIWI proteins and piRNA predominately express in germline and cancer cells, our study provided novel therapeutic strategy for pancreatic cancer treatment.

Ephrin-A5 Is Involved in Retinal Neovascularization in a Mouse Model of Oxygen-Induced Retinopathy.

Retinal neovascularization (RNV) is an important pathological feature of vitreoretinopathy that can lead to severe vision loss. The purpose of this study was to identify the role of ephrin-A5 (Efna5) in RNV and to explore its mechanism. The expression pattern and biological significance of Efna5 were investigated in a mouse model of oxygen-induced retinopathy (OIR). The expression of Efna5 and downstream signaling pathway members was determined by RT-PCR, immunofluorescence, immunohistochemistry, and western blot analyses. shRNA was used to knockdown Efna5 in the retina of the OIR mouse model. Retinal flat mounts were performed to evaluate the impact of Efna5 silencing on the RNV process. We found that the Efna5 was greatly upregulated in the retina of OIR mice. Elevated Efna5 mainly colocalized with the retinal vessels and endothelial cells. We then showed that knockdown of Efna5 in OIR mouse retinas using lentivirus-mediated shRNA markedly decreased the expression of Efna5 and reduced the retinal neovascularization and avascular retina area. We further showed hypoxia stimulation dramatically increased both total and phosphorylation levels of ERK1/2 and the phosphorylation levels of Akt in OIR mice. More importantly, knockdown of Efna5 could inhibit the p-Akt and p-ERK signaling pathways. Our results suggested that Efna5 may regulate the RNV. This study suggests that Efna5 was significantly upregulated in the retina of OIR mice and closely involved in the pathological retinal angiogenesis.

Erythropoietin-producing hepatocellular receptor A7 restrains estrogen negative feedback of luteinizing hormone via ephrin A5 in the hypothalamus of female rats.

We have previously shown that systemic injection of erythropoietin-producing hepatocellular receptor A7 (EPHA7)-Fc raises serum luteinizing hormone (LH) levels before ovulation in female rats, indicating the induction of EPHA7 in ovulation. In this study, we aimed to identify the mechanism and hypothalamus-pituitary-ovary (HPO) axis level underlying the promotion of LH secretion by EPHA7. Using an ovariectomized (OVX) rat model, in conjunction with low-dose 17ß-estradiol (E2) treatment, we investigated the association between EPHA7-ephrin (EFN)A5 signaling and E2 negative feedback. Various rat models (OVX, E2-treated OVX, and abarelix treated) were injected with the recombinant EPHA7-Fc protein through the caudal vein to investigate the molecular mechanism underlying the promotion of LH secretion by EPHA7. Efna5 was observed strongly expressed in the arcuate nucleus of the female rat by using RNAscope in situ hybridization. Our results indicated that E2, combined with estrogen receptor (ER)α, but not ERß, inhibited Efna5 and gonadotropin-releasing hormone 1 (Gnrh1) expressions in the hypothalamus. In addition, the systemic administration of EPHA7-Fc restrained the inhibition of Efna5 and Gnrh1 by E2, resulting in increased Efna5 and Gnrh1 expressions in the hypothalamus as well as increased serum LH levels. Collectively, our findings demonstrated the involvement of EPHA7-EFNA5 signaling in the regulation of LH and the E2 negative feedback pathway in the hypothalamus, highlighting the functional role of EPHA7 in female reproduction.

Spatial organization-dependent EphA2 transcriptional responses revealed by ligand nanocalipers.

Ligand binding induces extensive spatial reorganization and clustering of the EphA2 receptor at the cell membrane. It has previously been shown that the nanoscale spatial distribution of ligands modulates EphA2 receptor reorganization, activation and the invasive properties of cancer cells. However, intracellular signaling downstream of EphA2 receptor activation by nanoscale spatially distributed ligands has not been elucidated. Here, we used DNA origami nanostructures to control the positions of ephrin-A5 ligands at the nanoscale and investigated EphA2 activation and transcriptional responses following ligand binding. Using RNA-seq, we determined the transcriptional profiles of human glioblastoma cells treated with DNA nanocalipers presenting a single ephrin-A5 dimer or two dimers spaced 14, 40 or 100 nm apart. These cells displayed divergent transcriptional responses to the differing ephrin-A5 nano-organization. Specifically, ephrin-A5 dimers spaced 40 or 100 nm apart showed the highest levels of differential expressed genes compared to treatment with nanocalipers that do not present ephrin-A5. These findings show that the nanoscale organization of ephrin-A5 modulates transcriptional responses to EphA2 activation.

Polycomb-mediated repression of EphrinA5 promotes growth and invasion of glioblastoma.

Glioblastoma (GBM) is the most common and most aggressive intrinsic brain tumour in adults. Integrated transcriptomic and epigenomic analyses of glioblastoma initiating cells (GIC) in a mouse model uncovered a novel epigenetic regulation of EfnA5. In this model, Bmi1 enhances H3K27me3 at the EfnA5 locus and reinforces repression of selected target genes in a cellular context-dependent fashion. EfnA5 mediates Bmi1-dependent proliferation and invasion in vitro and tumour formation in an allograft model. Importantly, we show that this novel Polycomb feed-forward loop is also active in human GIC and we provide pre-clinical evidence of druggability of the EFNA5 signalling pathway in GBM xenografts overexpressing Bmi1.

Binding of EphrinA5 to RET receptor tyrosine kinase: An in vitro study.

Eph/Ephrin signaling pathways are crucial in regulating a large variety of physiological processes during development, such as cell morphology, proliferation, migration and axonal guidance. EphrinA (efn-A) ligands, in particular, can be activated by EphA receptors at cell-cell interfaces and have been proposed to cause reverse signaling via RET receptor tyrosine kinase. Such association has been reported to mediate spinal motor axon navigation, but conservation of the interactive signaling pathway and the molecular mechanism of the interaction are unclear. Here, we found Danio rerio efn-A5b bound to Mus musculus EphA4 with high affinity, revealing structurally and functionally conserved EphA/efn-A signaling. Interestingly, we observed no interaction between efn-A5b and RET from zebrafish, unlike earlier cell-based assays. Their lack of association indicates how complex efn-A signaling is and suggests that there may be other molecules involved in efn-A5-induced RET signaling.

Epha2 and Efna5 participate in lens cell pattern-formation.

Ephrin type-A receptor 2 (EPHA2) and one of its ligands, ephrin-A5 (EFNA5), have been associated with loss of eye lens transparency, or cataract, - an important cause of visual impairment. Here we show that mice functionally lacking EPHA2 (Epha2-null), EFNA5 (Efna5-null), or both receptor and ligand (Epha2/Efna5-null) consistently develop mostly transparent lenses with an internal refractive disturbance and a grossly disturbed cellular architecture. In situ hybridization localized Epha2 and Efna5 transcripts to lens epithelial cells and nascent fiber cells at the lens equator. In vivo labeling of Epha2-null lenses with a thymidine analog detected a significant decrease in lens epithelial cell proliferation within the germinative zone resulting in impaired early lens growth. Ex vivo imaging of Epha2-null, Efna5-null, and Epha2/Efna5-null lenses labelled in vivo with a membrane-targeted red fluorescent protein revealed misalignment of elongating fiber cells at the lens equator and loss of Y-suture pattern formation near the anterior and posterior poles of the lens. Immuno-fluorescent labeling of lens major intrinsic protein or aquaporin-0 (MIP/AQP0) showed that the precise, radial column patterning of hexagonal fiber cells throughout the cortex region was disrupted in Epha2-null, Efna5-null and Epha2/Efna5-null lenses. Collectively, these data suggest that Epha2 and Efna5 participate in the complex, global patterning of lens fiber cells that is necessary for maximal optical quality.

Abnormal expression of ephrin-A5 affects brain development of congenital hypothyroidism rats.

EphA5 and its ligand ephrin-A5 interaction can trigger synaptogenesis during early hippocampus development. We have previously reported that abnormal EphA5 expression can result in synaptogenesis disorder in congenital hypothyroidism (CH) rats. To better understand its precise molecular mechanism, we further analyzed the characteristics of ephrin-A5 expression in the hippocampus of CH rats. Our study revealed that ephrin-A5 expression was downregulated by thyroid hormone deficiency in the developing hippocampus and hippocampal neurons in rats. Thyroxine treatment for hypothyroid hippocampus and triiodothyronine treatment for hypothyroid hippocampal neurons significantly improved ephrin-A5 expression but could not restore its expression to control levels. Hypothyroid hippocampal neurons in-vitro showed synaptogenesis disorder characterized by a reduction in the number and length of neurites. Furthermore, the synaptogenesis-associated molecular expressions of NMDAR-1 (NR1), PSD95 and CaMKII were all downregulated correspondingly. These results suggest that ephrin-A5 expression may be decreased in CH, and abnormal activation of ephrin-A5/EphA5 signaling affects synaptogenesis during brain development. Such findings provide an important basis for exploring the pathogenesis of CH genetically.

Regulatory roles of ephrinA5 and its novel signaling pathway in mouse primary granulosa cell apoptosis and proliferation.

Recent findings suggest that ephrinA5 (Efna5) has a novel role in female mouse fertility, in addition to its well-defined role as a neurogenesis factor. Nevertheless, its physiological roles in ovarian granulosa cells (GC) have not been determined. In this study, mouse GC were cultured and transfected with ephrin A5 siRNA and negative control to determine the effects of Efna5 on GC apoptosis, proliferation, cell cycle progression, and related signaling pathways. To understand the mode signaling, the mRNA expression levels of Efna5 receptors (Eph receptor A5, Eph receptor A3, Eph receptor A8, and Eph receptor B2) were examined. Both mRNA and protein expressions of apoptosis-related factors (Bax, Bcl-2, Caspase 8, Caspase 3, and Tnfα) and a proliferation marker, Pcna, were investigated. Additionally, the role of Efna5 on paracrine oocyte-secreted factors and steroidogenesis hormones were also explored. Efna5 silencing suppressed GC apoptosis by downregulating Bax and upregulating Bcl-2 in a Caspase 8-dependent manner. Efna5 knockdown promoted GC proliferation via p-Akt and p-ERK pathway activation. The inhibition of Efna5 enhanced BMH15 and estradiol expression, but suppressed GDF9, while progesterone level remained unaltered. These results demonstrated that Efna5 is a pro-apoptotic agent in GC and plays important role in folliculogenesis by mediating apoptosis, proliferation, and steroidogenesis in female mouse. Therefore Efna5 might be potential therapeutic target for female fertility disorders.

Ligand-dependent EphA7 signaling inhibits prostate tumor growth and progression.

The downregulation of receptor tyrosine kinase EphA7 is frequent in epithelial cancers and linked to tumor progression. However, the detailed mechanism of EphA7-mediated prostate tumor progression remains elusive. To test the role of EphA7 receptor in prostate cancer (PCa) progression directly, we generated EphA7 receptor variants that were either lacking the cytoplasmic domain or carrying a point mutation that inhibits its phosphorylation by site-directed mutagenesis. Overexpression of wild-type (WT) EphA7 in PCa cells resulted in decreased tumor volume and increased tumor apoptosis in primary tumors. In addition, ectopic expression of WT EphA7 both can delay PCa cell proliferation and could inhibit PCa cell migration and invasion. This protein can also induce PCa cell apoptosis that correlated with increasing the protein expression levels of Bax, elevating the caspase-3 activities, reducing the protein expression levels of Bcl-2 and facilitating the dephosphorylation of Akt, which is further increased by the stimulation of ephrinA5-Fc. However, expression of these EphA7 mutants in PCa cells has no effect in vivo and in vitro. The expression of EphA7 and ephrinA5 was significantly decreased in PCa specimens compared with BPH tissues or paired normal tissues. Moreover, the phosphorylation of EphA7 was positively related with ephrinA5 expression in human prostate tissues. In sum, receptor phosphorylation of EphA7, at least in part, suppress PCa tumor malignancy through targeting PI3K/Akt signaling pathways.

EphA2 and ephrin-A5 are not a receptor-ligand pair in the ocular lens.

Eph-ephrin bidirectional signaling is essential for eye lens transparency in humans and mice. Our previous studies in mouse lenses demonstrate that ephrin-A5 is mainly expressed in the anterior epithelium, where it is required for maintaining the anterior epithelial monolayer. In contrast, EphA2 is localized in equatorial epithelial and fiber cells where it is essential for equatorial epithelial and fiber cell organization and hexagonal cell shape. Immunostaining of lens epithelial and fiber cells reveals that EphA2 and ephrin-A5 are also co-expressed in anterior fiber cell tips, equatorial epithelial cells and newly formed lens fibers, although they are not precisely colocalized. Due to this complex expression pattern and the promiscuous interactions between Eph receptors and ephrin ligands, as well as their complex bidirectional signaling pathways, cataracts in ephrin-A5(-/-) or EphA2(-/-) lenses may arise from loss of function or abnormal signaling mechanisms. To test whether abnormal signaling mechanisms may play a role in cataractogenesis in ephrin-A5(-/-) or EphA2(-/-) lenses, we generated EphA2 and ephrin-A5 double knockout (DKO) mice. We compared the phenotypes of EphA2(-/-) and ephrin-A5(-/-) lenses to that of DKO lenses. DKO lenses displayed an additive lens phenotype that was not significantly different from the two single KO lens phenotypes. Similar to ephrin-A5(-/-) lenses, DKO lenses had abnormal anterior epithelial cells leading to a large mass of epithelial cells that invade into the underlying fiber cell layer, directly resulting in anterior cataracts in ephrin-A5(-/-) and DKO lenses. Yet, similar to EphA2(-/-) lenses, DKO lenses also had abnormal packing of equatorial epithelial cells with disorganized meridional rows, lack of a lens fulcrum and disrupted fiber cells. The DKO lens phenotype rules out abnormal signaling by EphA2 in ephrin-A5(-/-) lenses or by ephrin-A5 in EphA2(-/-) lenses as possible cataract mechanisms. Thus, these results indicate that EphA2 and ephrin-A5 do not form a lens receptor-ligand pair, and that EphA2 and ephrin-A5 have other binding partners in the lens to help align differentiating equatorial epithelial cells or maintain the anterior epithelium, respectively.