Expression and activation of ephexin is altered after spinal cord injury

Failure of axon regeneration after traumatic spinal cord injury (SCI) is attributable in part to the presence of inhibitory molecular interactions. Recent evidence demonstrates that activation of Eph signaling pathways leads to modulation of growth cone dynamics and repulsion through the activation of ephexin, a novel guanine nucleotide exchange factor (GEF). However, little is known about the expression and modulation of Eph molecular targets in the injured spinal cord. In this study, we determined the expression profile of ephexin after a moderate spinal cord contusion at thoracic level (T10) in young adult rats. Western-blot studies showed increased protein expression in injured rats at 4 and 7 days postinjury (DPI) when compared with control animals. The protein levels returned to normal at 14 DPI and remained steady until 28 DPI. However, immunoprecipitation studies of the phosphorylated ephexin demonstrated that this protein is activated by day 2 until 14 DPI. Expression of ephexin was noticeable in neurons, axons, microglia/macrophages, and reactive astrocytes, and co-localized with EphA3, A4, and A7. These results demonstrate the presence of ephexin in the adult spinal cord and its activation after SCI. Therefore, we show, for the first time, the spatiotemporal pattern of ephexin expression and activation after contusive SCI. Collectively, our data support our previous findings on the putative nonpermissive roles of Eph receptors after SCI and the possible involvement of ephexin in the intracellular cascade of events.     

Eph-dependent tyrosine phosphorylation of ephexin1 modulates growth cone collapse

Ephs regulate growth cone repulsion, a process controlled by the actin cytoskeleton. The guanine nucleotide exchange factor (GEF) ephexin1 interacts with EphA4 and has been suggested to mediate the effect of EphA on the activity of Rho GTPases, key regulators of the cytoskeleton and axon guidance. Using cultured ephexin1-/- mouse neurons and RNA interference in the chick, we report that ephexin1 is required for normal axon outgrowth and ephrin-dependent axon repulsion. Ephexin1 becomes tyrosine phosphorylated in response to EphA signaling in neurons, and this phosphorylation event is required for growth cone collapse. Tyrosine phosphorylation of ephexin1 enhances ephexin1's GEF activity toward RhoA while not altering its activity toward Rac1 or Cdc42, thus changing the balance of GTPase activities. These findings reveal that ephexin1 plays a role in axon guidance and is regulated by a switch mechanism that is specifically tailored to control Eph-mediated growth cone collapse.     

Kinetic analysis of the binding of monomeric and dimeric ephrins to Eph receptors: correlation to function in a growth cone collapse assay

Eph receptors and ephrins play important roles in regulating cell migration and positioning during both normal and oncogenic tissue development. Using a surface plasma resonance (SPR) biosensor, we examined the binding kinetics of representative monomeric and dimeric ephrins to their corresponding Eph receptors and correlated the apparent binding affinity with their functional activity in a neuronal growth cone collapse assay. Our results indicate that the Eph receptor binding of dimeric ephrins, formed through fusion with disulfide-linked Fc fragments, is best described using a bivalent analyte model as a two-step process involving an initial monovalent 2:1 binding followed by a second bivalent 2:2 binding. The bivalent binding dramatically decreases the apparent dissociation rate constants with little effect on the initial association rate constants, resulting in a 30- to 6000-fold decrease in apparent equilibrium dissociation constants for the binding of dimeric ephrins to Eph receptors relative to their monomeric counterparts. Interestingly, the change was more prominent in the A-class ephrin/Eph interactions than in the B-class of ephrins to Eph receptors. The increase in apparent binding affinities correlated well with increased activation of Eph receptors and the resulting growth cone collapse. Our kinetic analysis and correlation of binding affinity with function helped us better understand the interactions between ephrins and Eph receptors and should be useful in the design of inhibitors that interfere with the interactions.     

Ephrins in reverse,park and drive

Eph receptors and their membrane-anchored ephrin ligands are thought to orchestrate cell movements by transducing bidirectional tyrosine-kinase-mediated signals into both cells expressing the receptors and cells expressing the ligands. Whether the resulting event is repulsion of an axonal growth cone, directing the orderly segmentation of hindbrain rhombomere cells or controlling angiogenic remodelling, such elaborate and diverse cell movements require intricate changes in the actin cytoskeleton, as well as precise regulation of cellular adhesion. Recent work by several groups has begun to link ephrin reverse signals to intracellular pathways that regulate actin dynamics and might help to explain how these ligands function as receptors to direct cell movement, adhesion and de-adhesion events.     

Ephrin-mediated cis-attenuation of Eph receptor signaling is essential for spinal motor axon guidance

Axon guidance receptors guide neuronal growth cones by binding in trans to axon guidance ligands in the developing nervous system. Some ligands are coexpressed in cis with their receptors, raising the question of the relative contribution of cis and trans interactions to axon guidance. Spinal motor axons use Eph receptors to select a limb trajectory in response to trans ephrins, while expressing ephrins in cis. We show that changes in motor neuron ephrin expression result in trajectory selection defects mirrored by changes in growth cone sensitivity to ephrins in vitro, arguing for ephrin cis-attenuation of Eph function. Furthermore, the relative contribution of trans-signaling and cis-attenuation is influenced by the subcellular distribution of ephrins to membrane patches containing Eph receptors. Thus, growth cone ephrins are essential for axon guidance in vivo and the balance between cis and trans modes of axon guidance ligand-receptor interaction contributes to the diversity of axon guidance signaling responses.     

Regulation of EphA 4 kinase activity is required for a subset of axon guidance decisions suggesting a key role for receptor clustering in Eph function

Signaling by receptor tyrosine kinases (RTKs) is mediated by their intrinsic kinase activity. Typically, kinase-activating mutations result in ligand-independent signaling and gain-of-function phenotypes. Like other RTKs, Ephs require kinase activity to signal, but signaling by Ephs in vitro also requires clustering by their membrane bound ephrin ligands. The relative importance of Eph kinase activity and clustering for in vivo functions is unknown. We find that knockin mice expressing a mutant form of EphA4 (EphA4(EE)), whose kinase is constitutively activated in the absence of ephrinB ligands, are deficient in the development of thalamocortical projections and some aspects of central pattern generator rhythmicity. Surprisingly, other functions of EphA4 were regulated normally by EphA4(EE), including midline axon guidance, hindlimb locomotion, in vitro growth cone collapse, and phosphorylation of ephexin1. These results suggest that signaling of Eph RTKs follows a multistep process of induced kinase activity and higher-order clustering different from RTKs responding to soluble ligands.     

The Caenorhabditis elegans Eph receptor activates NCK and N-WASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance

The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.     

Coexpressed EphA receptors and ephrin-A ligands mediate opposing actions on growth cone navigation from distinct membrane domains

Contact-dependent signaling between membrane-linked ligands and receptors such as the ephrins and Eph receptor tyrosine kinases controls a wide range of developmental and pathological processes. Paradoxically, many cell types coexpress both ligands and receptors, raising the question of how specific signaling readouts are achieved under these conditions. Here, we studied the signaling activities exerted by coexpressed EphA receptors and GPI-linked ephrin-A ligands in spinal motor neuron growth cones. We demonstrate that coexpressed Eph and ephrin proteins segregate laterally into distinct membrane domains from which they signal opposing effects on the growth cone: EphAs direct growth cone collapse/repulsion and ephrin-As signal motor axon growth/attraction. This subcellular arrangement of Eph-ephrin proteins enables axons to discriminate between cis- versus trans-configurations of ligand/receptor proteins, thereby allowing the utilization of both Ephs and ephrins as functional guidance receptors within the same neuronal growth cone.     

A subset of signal transduction pathways is required for hippocampal growth cone collapse induced by ephrin-A5

The Eph family tyrosine kinase receptors and their ligands, ephrins, play key roles in a wide variety of physiological and pathological processes including tissue patterning, angiogenesis, bone development, carcinogenesis, axon guidance, and neural plasticity. However, the signaling mechanisms underlying these diverse functions of Eph receptors have not been well understood. In this study, effects of Eph receptor activation on several important signal transduction pathways are examined. In addition, the roles of these pathways in ephrin-A5-induced growth cone collapse were assessed with a combination of biochemical analyses, pharmacological inhibition, and overexpression of dominant-negative and constitutively active mutants. These analyses showed that ephrin-A5 inhibits Erk activity but activates c-Jun N-terminal kinase. However, regulation of these two pathways is not required for ephrin-A5-induced growth cone collapse in hippocampal neurons. Artificial Erk activation by expression of constitutively active Mek1 and B-Raf failed to block ephrin-A5 effects on growth cones, and inhibitors of the Erk pathway also failed to inhibit collapse by ephrin-A5. Inhibition of JNK had no effects on ephrin-A5-induced growth cone collapse either. In addition, inhibitors to PKA and PI3-K showed no effects on ephrin-A5-induced growth cone collapse. However, pharmacological blockade of phosphotyrosine phosphatase activity, the Src family kinases, cGMP-dependent protein kinase, and myosin light chain kinase significantly inhibited ephrin-A5-induced growth cone collapse. These observations indicate that only a subset of signal transduction pathways is required for ephrin-A5-induced growth cone collapse.     

EphA4 catalytic activity causes inhibition of RhoA GTPase in Xenopus laevis embryos

The Eph family of receptor tyrosine kinases is involved in limiting cell and tissue interactions via a repulsive mechanism. The mechanism of repulsion involves reorganizing the actin cytoskeleton, but little is known of the molecular components that connect the receptor to the actin cytoskeleton. Recent studies in retinal ganglion cells have demonstrated that EphA4 activates the small GTPase Rho. We have investigated the involvement of Rho in signaling downstream from EphA4. As a model system, we have used a chimeric receptor called EPP that we express and activate in early Xenopus embryos. Previous studies demonstrated that EPP activation leads to loss of cell-cell adhesion and change in cell shape, plus loss of aspects of cell polarity in epithelial cells, such as apical microvilli and the apical/basolateral boundary. In this study, we show that injecting inhibitors of Rho GTPases into early Xenopus embryos produces a phenotype very similar to that resulting from EPP activation. More importantly, expression of a constitutively active form of Xenopus RhoA (XRhoA) concurrent with activated EPP rescued embryos from the loss of cell-cell adhesion and change in cell shape associated with EPP. These data argue that, in contrast to the case in retinal ganglion cells, EphA4 in early Xenopus embryos acts to inhibit RhoA, suggesting that this receptor may regulate Rho differently (and therefore affect the cytoskeleton differently) in neuronal and non-neuronal cells. Furthermore, overexpression of ephexin, a novel guanine nucleotide exchange factor for Rho family GTPases, also blocks EPP-induced dissociation. This suggests that EphA4, which has been demonstrated to activate ephexin in cultured neuronal cells, may also target Rho GTPase via an ephexin-independent pathway.     

New exchanges in eph-dependent growth cone dynamics

The Eph receptor tyrosine kinases and their ephrin ligands play a pivotal role during axon pathfinding and neural circuitry formation. A prominent way in which Eph receptors sculpt cellular morphology is by remodeling the actin cytoskeleton and the surrounding plasma membrane through the regulation of Rho family GTPases. Two articles in this issue of Neuron (Sahin et al. and Cowan et al.) shed light on how Eph receptors recruit guanine nucleotide exchange factors for Rho family GTPases to modulate growth cone dynamics.     

Control of cell behaviour by signalling through Eph receptors and ephrins

Eph receptor tyrosine kinases and ephrins mediate contact-dependent cell interactions that regulate the repulsion and adhesion mechanisms involved in the guidance and assembly of cells. Recent work has revealed a role of overlapping Eph receptor and ephrin expression in modulating neuronal growth cone repulsion, and has shown that bidirectional activation restricts intermingling and communication between cell populations. In addition, progress has been made in understanding how Eph receptors and ephrins control cell adhesion.     

The role of ephrins and Eph receptors in cancer

Eph receptors are the largest receptor tyrosine kinase family of transmembrane proteins with an extracellular domain capable of recognizing signals from the cells’ environment and influencing cell–cell interaction and cell migration. Ephrins are the ligands to Eph receptors and stimulate bi-directional signaling of the Eph/ephrin axis. Eph receptor and ephrin overexpression can result in tumorigenesis as related to tumor growth and survival and is associated with angiogenesis and metastasis in many types of human cancer. Recent data suggest that Eph/ephrin signaling could play an important role in the development of novel inhibition strategies and cancer treatments to potentially target this receptor tyrosine kinase and/or its ligand. A deeper understanding of the molecular basis for normal versus defective cell–cell interaction through the Eph/ephrin axis will enable the potential development of novel cancer treatments. This review emphasizes the biology of Eph/ephrin as well as the potential for novel targeted therapy through this pathway.     

EphA4-dependent axon guidance is mediated by the RacGAP alpha2-chimaerin

Neuronal network formation in the developing nervous system is dependent on the accurate navigation of nerve cell axons and dendrites, which is controlled by attractive and repulsive guidance cues. Ephrins and their cognate Eph receptors mediate many repulsive axonal guidance decisions by intercellular interactions resulting in growth cone collapse and axon retraction of the Eph-presenting neuron. We show that the Rac-specific GTPase-activating protein alpha2-chimaerin binds activated EphA4 and mediates EphA4-triggered axonal growth cone collapse. alpha-Chimaerin mutant mice display a phenotype similar to that of EphA4 mutant mice, including aberrant midline axon guidance and defective spinal cord central pattern generator activity. Our results reveal an alpha-chimaerin-dependent signaling pathway downstream of EphA4, which is essential for axon guidance decisions and neuronal circuit formation in vivo.     

Retinal axon growth cones respond to EphB extracellular domains as inhibitory axon guidance cues

Axon pathfinding relies on cellular signaling mediated by growth cone receptor proteins responding to ligands, or guidance cues, in the environment. Eph proteins are a family of receptor tyrosine kinases that govern axon pathway development, including retinal axon projections to CNS targets. Recent examination of EphB mutant mice, however, has shown that axon pathfinding within the retina to the optic disc is dependent on EphB receptors, but independent of their kinase activity. Here we show a function for EphB1, B2 and B3 receptor extracellular domains (ECDs) in inhibiting mouse retinal axons when presented either as substratum-bound proteins or as soluble proteins directly applied to growth cones via micropipettes. In substratum choice assays, retinal axons tended to avoid EphB-ECDs, while time-lapse microscopy showed that exposure to soluble EphB-ECD led to growth cone collapse or other inhibitory responses. These results demonstrate that, in addition to the conventional role of Eph proteins signaling as receptors, EphB receptor ECDs can also function in the opposite role as guidance cues to alter axon behavior. Furthermore, the data support a model in which dorsal retinal ganglion cell axons heading to the optic disc encounter a gradient of inhibitory EphB proteins which helps maintain tight axon fasciculation and prevents aberrant axon growth into ventral retina. In conclusion, development of neuronal connectivity may involve the combined activity of Eph proteins serving as guidance receptors and as axon guidance cues.     

Regulation of growth cone actin filaments by guidance cues

The motile behaviors of growth cones at the ends of elongating axons determine pathways of axonal connections in developing nervous systems. Growth cones express receptors for molecular guidance cues in the local environment, and receptor-guidance cue binding initiates cytoplasmic signaling that regulates the cytoskeleton to control growth cone advance, turning, and branching behaviors. The dynamic actin filaments of growth cones are frequently targets of this regulatory signaling. Rho GTPases are key mediators of signaling by guidance cues, although much remains to be learned about how growth cone responses are orchestrated by Rho GTPase signaling to change the dynamics of polymerization, transport, and disassembly of actin filaments. Binding of neurotrophins to Trk and p75 receptors on growth cones triggers changes in actin filament dynamics to regulate several aspects of growth cone behaviors. Activation of Trk receptors mediates local accumulation of actin filaments, while neurotrophin binding to p75 triggers local decrease in RhoA signaling that promotes lengthening of filopodia. Semaphorin IIIA and ephrin-A2 are guidance cues that trigger avoidance or repulsion of certain growth cones, and in vitro responses to these proteins include growth cone collapse. Dynamic changes in the activities of Rho GTPases appear to mediate responses to these cues, although it remains unclear what the changes are in actin filament distribution and dynamic reorganization that result in growth cone collapse. Growth cones in vivo simultaneously encounter positive and negative guidance cues, and thus, growth cone behaviors during axonal pathfinding reflect the complex integration of multiple signaling activities.     

Eph receptors and ephrins as targets for cancer therapy

Eph receptor tyrosine kinases and their ephrin ligands are involved in various signalling pathways and mediate critical steps of a wide variety of physiological and pathological processes. Increasing experimental evidence demonstrates that both Eph receptor and ephrin ligands are overexpressed in a number of human tumours, and are associated with tumour growth, invasiveness and metastasis. In this regard, the Eph/ephrin system provides the foundation for potentially exciting new targets for anticancer therapies for Eph‐expressing tumours. The purpose of this review is to outline current advances in the role of Eph receptors and ephrin ligands in cancer, and to discuss novel therapeutic approaches of anticancer therapies.     

Rho-dependent contractile responses in the neuronal growth cone are independent of classical peripheral retrograde actin flow

Rho family GTPases have been implicated in neuronal growth cone guidance; however, the underlying cytoskeletal mechanisms are unclear. We have used multimode fluorescent speckle microscopy (FSM) to directly address this problem. We report that actin arcs that form in the transition zone are incorporated into central actin bundles in the C domain. These actin structures are Rho/Rho Kinase (ROCK) effectors. Specifically, LPA mediates growth cone retraction by ROCK-dependent increases in actin arc and central actin bundle contractility and stability. In addition, these treatments had marked effects on MT organization as a consequence of strong MT-actin arc interactions. In contrast, LPA or constitutively active Rho had no effect on P domain retrograde actin flow or filopodium bundle number. This study reveals a novel mechanism for domain-specific spatial control of actin-based motility in the growth cone with implications for understanding chemorepellant growth cone responses and nerve regeneration.     

EphrinA1-induced cytoskeletal re-organization requires FAK and p130(cas)

Ephrins and Eph receptors are involved in axon guidance and cellular morphogenesis. An interaction between ephrin and Eph receptors elicits neuronal growth-cone collapse through cytoskeletal disassembly. When NIH3T3 cells were plated onto an ephrinA1-coated surface, the cells both adhered and spread. Adhesion and spreading proceeded concomitantly with changes in both the actin and microtubule cytoskeleton. EphA2, focal adhesion kinase (FAK) and p130(cas) were identified as the major ephrin-dependent phosphotyrosyl proteins during the ephrin-induced morphological changes. Mouse embryonic fibroblasts (MEFs) derived from FAK(-/-) and p130(cas-/-) mice had severe defects in ephrinA1-induced cell spreading, which were reversed after re-expression of FAK or p130(cas), respectively. Expression of a constitutively active EphA2 induced NIH3T3 cells to undergo identical, but ligand-independent, morphological changes. These data show that ephrinA1 can induce cell adhesion and actin cytoskeletal changes in fibroblasts in a FAK- and p130(cas)-dependent manner, through activation of the EphA2 receptor. The finding that ephrin Eph signalling can result in actin cytoskeletal assembly, rather than disassembly, has many implications for ephrin Eph responses in other cell types.