Alice Davy

Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis

In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.

Eph/ephrin signaling: networks

Bidirectional signaling has emerged as an important signature by which Ephs and ephrins control biological functions. Eph/ephrin signaling participates in a wide spectrum of developmental processes, and cross-regulation with other communication pathways lies at the heart of the complexity underlying their function in vivo. Here, we review in vitro and in vivo data describing molecular, functional, and genetic interactions between Eph/ephrin and other cell surface signaling pathways. The complexity of Eph/ephrin function is discussed in terms of the pathways that regulate Eph/ephrin signaling and also the pathways that are regulated by Eph/ephrin signaling.

Ephrin‐A5 modulates cell adhesion and morphology in an integrin‐dependent manner

The ephrins are membrane‐tethered ligands for the Eph receptor tyrosine kinases, which play important roles in patterning of the nervous and vascular systems. It is now clear that ephrins are more than just ligands and can also act as signalling‐competent receptors, participating in bidirectional signalling. We have recently shown that ephrin‐A5 signals within caveola‐like domains of the plasma membrane upon engagement with its cognate Eph receptor, leading to increased adhesion of the cells to fibronectin. Here we show that ephrin‐A5 controls sequential biological events that are consistent with its role in neuronal guidance. Activation of ephrin‐A5 induces an initial change in cell adhesion followed by changes in cell morphology. Both effects are dependent on the activation of β1 integrin involving members of the Src family of protein tyrosine kinases. The prolonged activation of ERK‐1 and ERK‐2 is required for the change in cell morphology. Our work suggests a new role for class A ephrins in specifying the affinity of the cells towards various extracellular substrates by regulating integrin function.

EphB–ephrin-B interactions suppress colorectal cancer progression by compartmentalizing tumor cells

The genes encoding tyrosine kinase receptors EphB2 and EphB3 are β-catenin and Tcf4 target genes in colorectal cancer (CRC) and in normal intestinal cells. In the intestinal epithelium, EphB signaling controls the positioning of cell types along the crypt-villus axis. In CRC, EphB activity suppresses tumor progression beyond the earliest stages. Here we show that EphB receptors compartmentalize the expansion of CRC cells through a mechanism dependent on E-cadherin–mediated adhesion. We demonstrate that EphB-mediated compartmentalization restricts the spreading of EphB-expressing tumor cells into ephrin-B1–positive territories in vitro and in vivo. Our results indicate that CRC cells must silence EphB expression to avoid repulsive interactions imposed by normal ephrin-B1–expressing intestinal cells at the onset of tumorigenesis.

Ephrin signaling in vivo: look both ways.

Eph receptors and ephrins have captured the interest of the developmental biology community in recent years for their pleiotropic functions during embryogenesis. Loss‐of‐function studies using various animal models have demonstrated the involvement of Ephs and ephrins in many aspects of embryogenesis including segmentation, neural crest cells migration, angiogenesis, and axon guidance. An essential property of this signaling pathway is the ability of both Ephs and ephrins to behave as receptors or ligands and their consequent cell autonomous and nonautonomous mode of action. While many reports did not discriminate between Eph autonomous signaling (forward) and ephrin autonomous signaling (reverse), recent genetic and in vivo studies have shown that both forward and reverse signaling play important roles during embryogenesis. Developmental Dynamics 232:1–10, 2005.

Inhibition of Gap Junction Communication at Ectopic Eph/ephrin Boundaries Underlies Craniofrontonasal Syndrome

Mutations in X-linked ephrin-B1 in humans cause craniofrontonasal syndrome (CFNS), a disease that affects female patients more severely than males. Sorting of ephrin-B1–positive and –negative cells following X-inactivation has been observed in ephrin-B1+/− mice; however, the mechanisms by which mosaic ephrin-B1 expression leads to cell sorting and phenotypic defects remain unknown. Here we show that ephrin-B1+/− mice exhibit calvarial defects, a phenotype autonomous to neural crest cells that correlates with cell sorting. We have traced the causes of calvarial defects to impaired differentiation of osteogenic precursors. We show that gap junction communication (GJC) is inhibited at ectopic ephrin boundaries and that ephrin-B1 interacts with connexin43 and regulates its distribution. Moreover, we provide genetic evidence that GJC is implicated in the calvarial defects observed in ephrin-B1+/− embryos. Our results uncover a novel role for Eph/ephrins in regulating GJC in vivo and suggest that the pleiotropic defects seen in CFNS patients are due to improper regulation of GJC in affected tissues.

Differential activation of ERKs to focal adhesions by PKC ε is required for PMA-induced adhesion and migration of human glioma cells

Protein kinase C (PKC) is a family of serine/threonine kinases involved in the transduction of a variety of signals. There is increasing evidence to indicate that specific PKC isoforms are involved in the regulation of distinct cellular processes. In glioma cells, PKC α was found to be a critical regulator of proliferation and cell cycle progression, while PKC ε was found to regulate adhesion and migration. Herein, we report that specific PKC isoforms are able to differentially activate extracellular-signal regulated kinase (ERK) in distinct cellular locations: while PKC α induces the activation of nuclear ERK, PKC ε induces the activation of ERK at focal adhesions. Inhibition of the ERK pathway completely abolished the PKC-induced integrin-mediated adhesion and migration. Thus, we present the first evidence that PKC ε is able to activate ERK at focal adhesions to mediate glioma cell adhesion and motility, providing a molecular mechanism to explain the different biological functions of PKC α and ε in glioma cells.

Ephrin-B1 reverse signaling controls a posttranscriptional feedback mechanism via miR-124.

ABSTRACT Eph receptors and ephrins exhibit complex and highly dynamic expression patterns during embryonic development. In addition, changes in their expression levels are often associated with pathological situations in adults. Yet, little is known about the mechanisms regulating their expression. Here we report that the expression of ephrin-B1 is controlled by a feedback loop involving posttranscriptional regulatory mechanisms. We observed that the EfnB1 3′ untranslated region (3′-UTR) confers instability to mRNA transcripts, and we identified miR-124 as a posttranscriptional repressor of EfnB1 expression. Furthermore, we showed that miR-124 is itself regulated by ephrin-B1 reverse signaling, thus revealing the existence of a mutually repressive interaction between ephrin-B1 and this microRNA (miRNA). Lastly, we demonstrated the relevance of this mutual inhibition for neuronal differentiation. Our results suggest that miRNAs could be important effectors of Eph/ephrin signaling to refine domains of expression and to regulate function.

Regulation of neural progenitor cell state by ephrin-B

Maintaining a balance between self-renewal and differentiation in neural progenitor cells during development is important to ensure that correct numbers of neural cells are generated. We report that the ephrin-B–PDZ-RGS3 signaling pathway functions to regulate this balance in the developing mammalian cerebral cortex. During cortical neurogenesis, expression of ephrin-B1 and PDZ-RGS3 is specifically seen in progenitor cells and is turned off at the onset of neuronal differentiation. Persistent expression of ephrin-B1 and PDZ-RGS3 prevents differentiation of neural progenitor cells. Blocking RGS-mediated ephrin-B1 signaling in progenitor cells through RNA interference or expression of dominant-negative mutants results in differentiation. Genetic knockout of ephrin-B1 causes early cell cycle exit and leads to a concomitant loss of neural progenitor cells. Our results indicate that ephrin-B function is critical for the maintenance of the neural progenitor cell state and that this role of ephrin-B is mediated by PDZ-RGS3, likely via interacting with the noncanonical G protein signaling pathway, which is essential in neural progenitor asymmetrical cell division.

Loss of functional caveolae during senescence of human fibroblasts.

Primary human fibroblasts have a finite replicative lifespan in culture that culminates in a unique state of growth arrest, termed senescence that is accompanied by distinct morphological and biochemical alterations. Senescent cell responses to extracellular stimuli are believed to be altered at a point after receptors are bound by ligand, leading to improper integration of the signals which initiate DNA replication. In this study we demonstrate that one of the key organizing membrane microdomains for receptor signaling, caveolae, are absent in senescent cells. A comparison of young and senescent cells indicated that senescent cells contained a higher total amount of caveolins 1 and 2 but had significantly less of both proteins in the caveolar fraction. Additionally, caveolar fractions from senescent cells completely lacked the tyrosine‐kinase activity associated with functional caveolae. Furthermore, old cells had little caveolar protein exposed to the outer plasma membrane as estimated by using an in vivo biotinylation assay and no detectable caveolin 1 on the cell surface when processed for immunofluoresence and confocal microscopy. Together, these data suggest that a fundamental loss of signal integration at the plasma membrane of senescent cells is due to the loss of signaling competent caveolae.