Repulsive axon guidance cues ephrin-A2 and slit3 stop protrusion of the growth cone leading margin concurrently with inhibition of ADF/cofilin and ERM proteins.

Axonal growth cones turn away from repulsive guidance cues. This may start with reduced protrusive motility in the region the growth cone leading margin that is closer to the source of repulsive cue. Using explants of E7 chick temporal retina, we examine the effects of two repulsive guidance cues, ephrin-A2 and slit3, on retinal ganglion cell growth cone protrusive activity, total F-actin, free F-actin barbed ends, and the activities (phosphorylation states) of actin regulatory proteins, ADF/cofilin and ezrin, radixin, moesin (ERM) proteins. Ephrin-A2 rapidly stops protrusive activity simultaneously with reducing F-actin, free barbed ends and the activities of ADF/cofilin and ERM proteins. Slit3 also stops protrusion and reduces the activities of ADF/cofilin and ERM proteins. We interpret these results as indicating that repulsive guidance cues inhibit actin polymerization and actin-membrane linkage to stop protrusive activity. Retrograde F-actin flow withdraws actin to the C-domain, where F-actin bundles interact with myosin II to generate contractile forces that can collapse and retract the growth cone. Our results suggest that common mechanisms are used by repulsive guidance cue to disable growth cone motility and remodel growing axon terminals.

Green tea catechin inhibits ephrin-A1-mediated cell migration and angiogenesis of human umbilical vein endothelial cells.

Angiogenesis, the formation of new blood vessels from preexisting capillaries, is essential for tumor progression and metastasis. During tumor neovascularization, vascular endothelial growth factor and ephrin (Eph) families emerge as critical mediators of angiogenesis. The green tea catechin epigallocatechin gallate (EGCG), a tyrosine kinase inhibitor, has been demonstrated in previous studies to be an effective antiangiogenesis agent. However, the inhibitory effect of green tea catechins on ephrin-A1-mediated tumor angiogenesis has not been demonstrated yet. Thus, in this study, we investigated the molecular mechanism of ephrin-A1-mediated cell migration and angiogenesis, as well as the inhibitory effects of EGCG. Here we show that ephrin-A1 mediates endothelial cell migration and regulates vascular remodeling in tumor neovascularization in vitro. We also demonstrated that ephrin-A1-mediated cell migration required the activation of extracellular-regulated kinase (ERK-1/2) but not of phosphatidylinositol-3-kinase. The green tea catechin EGCG inhibited ephrin-A1-mediated endothelial cell migration, as well as tumor angiogenesis, in a dose-dependent manner. Furthermore, EGCG inhibited the ephrin-A1-mediated phosphorylation of EphA2 and ERK-1/2. Taken together, these data indicated that activation of ERK-1/2 plays an essential role in ephrin-A1-mediated cell migration. EGCG inhibited ephrin-A1-mediated endothelial migration and angiogenesis. It suggests a novel antiangiogenesis application of EGCG in cancer chemoprevention.

Retinal axon response to ephrin-as shows a graded, concentration-dependent transition from growth promotion to inhibition.

Ephrin-As act as retinal topographic mapping labels, but the molecular basis for two key aspects of mapping remains unclear. First, although mapping is believed to require balanced opposing forces, ephrin-As have been reported to be retinal axon repellents, and the counterbalanced force has not been molecularly identified. Second, although graded responsiveness across the retina is required for smooth mapping, a sharp discontinuity has instead been reported. Here, an axon growth assay was developed to systematically vary both retinal position and ephrin concentration and test responses quantitatively. Responses varied continuously with retinal position, fulfilling the requirement for smooth mapping. Ephrin-A2 inhibited growth at high concentrations but promoted growth at lower concentrations. Moreover, the concentration producing a transition from promotion to inhibition varied topographically with retinal position. These results lead directly to a mapping model where position within a concentration gradient may be specified at the neutral point between growth promotion and inhibition.

Actin turnover is required to prevent axon retraction driven by endogenous actomyosin contractility.

Growth cone motility and guidance depend on the dynamic reorganization of filamentous actin (F-actin). In the growth cone, F-actin undergoes turnover, which is the exchange of actin subunits from existing filaments. However, the function of F-actin turnover is not clear. We used jasplakinolide (jasp), a cell-permeable macrocyclic peptide that inhibits F-actin turnover, to study the role of F-actin turnover in axon extension. Treatment with jasp caused axon retraction, demonstrating that axon extension requires F-actin turnover. The retraction of axons in response to the inhibition of F-actin turnover was dependent on myosin activity and regulated by RhoA and myosin light chain kinase. Significantly, the endogenous myosin-based contractility was sufficient to cause axon retraction, because jasp did not alter myosin activity. Based on these observations, we asked whether guidance cues that cause axon retraction (ephrin-A2) inhibit F-actin turnover. Axon retraction in response to ephrin-A2 correlated with decreased F-actin turnover and required RhoA activity. These observations demonstrate that axon extension depends on an interaction between endogenous myosin-driven contractility and F-actin turnover, and that guidance cues that cause axon retraction inhibit F-actin turnover.