The expression of messenger RNAs coding for growth factors, their receptors, and eph-class receptor tyrosine kinases in normal and ototoxically damaged chick cochleae

Messenger RNAs coding for growth factors and receptor tyrosine kinases were measured by quantitative competitive and by semi-quantitative reverse-transcription polymerase chain reaction in whole and dissected chick inner ears. The fibroblast growth factor (FGF) receptor 1 chick embryonic kinase (CEK) 1 was expressed in all structures examined (otocyst, hatchling whole cochlea, cochlear nerve ganglion, and cochlear and vestibular sensory epithelia), although slightly more heavily in the otocyst. The related fibroblast growth factor receptors CEK 2 and 3 were preferentially expressed in the nerve ganglion and in the vestibular sensory epithelium, respectively. FGF 1 mRNA was low in early development, increasing to mature levels at around embryonic age 11 days, while FGF2, mRNA was expressed at constant levels at all ages. In response to ototoxic damage, FGF1 mRNA levels were increased in the early damaged cochlear sensory epithelium. Immunohistochemistry for CEK1 showed that normal hair cells expressed the receptor heavily on the hair cell stereocilia, while with early damage, CEK1 came to be expressed heavily on the apical surfaces of the supporting cells. In normal chicks, the CEK4 and CEK8 eph-class receptor tyrosine kinases were expressed relatively heavily by the cochlear nerve ganglion, and CEK10 was expressed relatively heavily by the cochlear hair cell sensory epithelium. The results suggest that the FGF system may be involved in the response of the cochlear epithelium to ototoxic damage. The eph-class receptor tyrosine kinase CEK10 may be involved in cell interactions in the cochlear sensory epithelium, while CEK4 and CEK8 may play a role in the cochlear innervation.

Signals from Eph and ephrin proteins: a developmental tool kit

Interactions between Eph receptors and their ligands the ephrin proteins are critically important in many key developmental processes. Emerging evidence also supports a role for these molecules in postembryonic tissues, particularly in pathological processes, including tissue injury and tumor metastasis. We review the signaling mechanisms that allow the 14 Eph and nine ephrin proteins to deliver intracellular signals that regulate cell shape and movement. What emerges is that the initiation of these signals is critically dependent on which Eph and ephrin proteins are expressed, the level of their expression, and, in some cases, which splice variants are expressed. Diversity at the level of initial interaction and in the downstream signaling processes regulated by Eph-ephrin signaling provides a subtle, versatile system of regulation of intercellular adhesion, cell shape, and cell motility.

The role of the Eph-ephrin signalling system in the regulation of developmental patterning

The Eph and ephrin system, consisting of fourteen Eph receptor tyrosine kinase proteins and nine ephrin membrane proteins in vertebrates, has been implicated in the regulation of many critical events during development. Binding of cell surface Eph and ephrin proteins results in bi-directional signals, which regulate the cytoskeletal, adhesive and motile properties of the interacting cells. Through these signals Eph and ephrin proteins are involved in early embryonic cell movements, which establish the germ layers, cell movements involved in formation of tissue boundaries and the pathfinding of axons. This review focuses on two vertebrate models, the zebrafish and mouse, in which experimental perturbation of Eph and/or ephrin expression in vivo have provided important insights into the role and functioning of the Eph/ephrin system.

Attenuation of Eph Receptor Kinase Activation in Cancer Cells by Coexpressed Ephrin Ligands

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting “in trans” with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral “cis” associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis-inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans (Falivelli et al. 2013).

Immunmodulatorische Konsequenzen einer Eph-Rezeptorspaltung durch MMP-7

Tumore haben die Fähigkeit ihr Mikromilieu zu modulieren, um so ihre Entwicklung und ihre Ausbreitung zu fördern oder sich vor Angriffen des Immunsystems zu schützen. Die Expression der Matrix Metalloproteinasen 7 (MMP-7) wurde in vielen verschiedenen Tumoren analysiert. Neben prometastatischen und wachstumsfördernden Funktionen wurden auch antiapoptotische Wirkungen von MMP-7 auf die Tumorzellen belegt (Strand et al., 2004). Doch noch sind nicht alle immunmodulatorischen Eigenschaften von MMP-7 aufgeklärt worden. Ziel der vorliegenden Arbeit war es, die immunologischen Konsequenzen einer MMP-7 Expression durch Tumorzellen zu untersuchen.rnIm Rahmen dieser Arbeit konnte gezeigt werden, dass MMP-7 über die Spaltung der Rezeptortyrosinkinase EphB2 die Aktinpolymerisation und dadurch auch die Endozytose in Zellen verändern kann. EphB2 wurde als Target einer MMP-7 vermittelten Spaltung identifiziert. Die Untersuchungen mit MMP-7 überexprimierenden Hek 293 EcR Zellen und MMP-7 behandelten DCs zeigten unter dem Einfluss von MMP-7 eine wesentlich geringere EphB2 Expression auf deren Zelloberflächen. Zudem konnte durch in vitro Spaltversuche und anschließende Sequenzierung die Schnittstelle der MMP-7 induzierten Spaltung von EphB2 bestimmt werden. Anschließende Analysen belegten, dass durch die MMP-7 vermittelte Spaltung von EphB2 die Aktivierung der kleinen GTPasen Rac1 und Cdc42 stark reduziert wurden. Die Funktion von Cdc42 und Rac1 während der Aktinpolymerisation, als auch innerhalb der EphB2- Signalkaskade wurde bereits beschrieben (Irie and Yamaguchi, 2002). In der vorliegenden Arbeit wurde gezeigt, dass MMP-7 die Aktinpolymerisation in Zellen reduzierte, was warscheinlich eine direkte Auswirkung der EphB2 Spaltung war. rnWeitere Versuche ließen einen Zusammenhang zwischen der reduzierten Aktinpolymerisation und der verminderten Endozytose in MMP-7 behandelten Zellen erkennen. Unter dem Einfluss von MMP-7 konnte sowohl in Hek 293 EcR MMP7 Zellen als auch in unreifen DCs eine Reduktion der endozytotischen Aktivität ermittelt werden.rnUntersuchungen mit humanen T-Zellen zeigten auch hier einen verminderten Nachweis von EphB2 auf den Zellen, wenn diese vorher mit MMP-7 inkubiert wurden. Zudem führte die Anwesenheit von MMP-7 in T-Zellen ebenfalls zu einer verminderten Aktinpolymerisation. Die mit der Aktinpolymerisation verbundene Restrukturierung des Zytoskeletts gilt als essentieller Prozess für die T-Zellaktivität (Tskvitaria-Fuller et al., 2003; Krummel et al., 2000). Anschließende Versuche konnte daher nicht nur eine Beeinträchtigung von MMP-7 auf die zytotoxische Aktivität von T-Zellen belegen, sondern deuteten auch auf eine verminderten Proliferation nach Antigenstimulation unter dem Einfluss von MMP-7 hin.rnDie Rolle von MMP-7 aber auch die von EphB2 in der Tumorimmunologie wurde bereits untersucht. So konnte eine induzierte Überexpression von EphB2 das Krebszellwachstum, die Adhäsion und die Migration inhibieren, während der Verlust der EphB2 Expression zu einer verstärkten Invasion und Metastisierung von Tumorzellen führte (Guo et al., 2006). Zudem konnte gezeigt werde, dass Tumorzellen die Funktion von DCs beeinflussen können. DCs aus tumortragenden Mäusen zeigten im Vergleich zu Kontrollzellen eine reduzierte Aktivierung von Cdc42 und Rac1 und zudem eine verminderte Endozytoseaktivität (Tourkova et al., 2007). Die in der vorliegenden Arbeit gezeigten MMP-7 bedingten Veränderungen der Aktinpolymerisation stellen womöglich eine Verbindung zwischen den genannten Untersuchungen her und offenbaren weitere immunologische Konsequenzen einer MMP-7 Expression im Tumor. rnrn

The multifaceted roles of Eph/ephrin signaling in breast cancer

http://www.ncbi.nlm.nih.gov/pubmed/22568950

Hypoxia up-regulates expression of Eph receptors and ephrins in mouse skin.

Eph receptor tyrosine kinases and their ligands (ephrins) are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Both receptors and ligands have been shown to be up-regulated in a variety of tumors. To address the hypothesis that hypoxia is an important regulator of Ephs/ephrins expression, we developed a mouse skin flap model of hypoxia. We demonstrate that our model truly represents segmental skin hypoxia by applying four independent methods: continuous measurement of partial cutaneous oxygen tension, monitoring of tissue lactate/pyruvate ratio, time course of hypoxia-inducible factor-1alpha (HIF-1alpha) induction, and localization of stabilized HIF-1alpha by immunofluorescence in the hypoxic skin flap. Our experiments indicate that hypoxia up-regulates not only HIF-1alpha and vascular endothelial growth factor (VEGF) expression, but also Ephs and ephrins of both A and B subclasses in the skin. In addition, we show that in Hep3B and PC-3 cells, the hypoxia-induced up-regulation of Ephs and ephrins is abrogated by small interfering RNA-mediated down-regulation of HIF-1alpha. These novel findings shed light on the role of this versatile receptor/ligand family in adult angiogenesis. Furthermore, our model offers considerable potential for analyzing distinct mechanisms of neovascularization in gene-targeted mice.

Nck-interacting Ste20 kinase couples Eph receptors to c-Jun N-terminal kinase and integrin activation.

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62(dok), RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62(dok) most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.

Nck recruitment to Eph receptor, EphB1/ELK, couples ligand activation to c-Jun kinase.

Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. We mapped the EphB1 subdomain that binds Nck and its Drosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr594 was required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruited Nck to native receptor complexes and activated c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocked Nck recruitment to EphB1, attenuated downstream JNK activation, and blocked cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK.

Discutiendo la terciarización de la clase trabajadora en el Gran Buenos Aires (1974-2010) : Una aproximación a partir de las bases usuarias de la EPH

Este trabajo forma parte de una investigación más amplia, a ser desarrollada por el autor en futuros estudios de posgrado, sobre las transformaciones experimentadas por lo que podríamos llamar la 'composición', 'morfología' o 'estructura' de la clase obrera en Argentina, a partir del profundo proceso de reestructuración capitalista que viene afectando a las relaciones de clase desde los inicios de la última dictadura militar, en marzo de 1976. En primer lugar, procuraremos analizar los enfoques y argumentos más influyentes que, tanto en el debate internacional como en el nacional, se han apoyado en la terciarización como suerte de 'base objetiva' para explicar [o postular] la pérdida de centralidad de la clase obrera. Luego nos enfocaremos en las variaciones que ha presentado la composición sectorial del empleo asalariado en el Gran Buenos Aires [GBA], a lo largo del período en estudio [1974-2010]. Lo haremos utilizando las bases usuarias de la Encuesta Permanente de Hogares [EPH]. Finalmente, analizaremos los posibles solapamientos estadísticos entre la modificación en la composición sectorial del empleo del GBA y otros dos fenómenos: la tercerización de actividades [outsourcing], y las políticas de promoción industrial regionales implementadas en el país

Discutiendo la terciarización de la clase trabajadora en el Gran Buenos Aires (1974-2010) : Una aproximación a partir de las bases usuarias de la EPH

Este trabajo forma parte de una investigación más amplia, a ser desarrollada por el autor en futuros estudios de posgrado, sobre las transformaciones experimentadas por lo que podríamos llamar la 'composición', 'morfología' o 'estructura' de la clase obrera en Argentina, a partir del profundo proceso de reestructuración capitalista que viene afectando a las relaciones de clase desde los inicios de la última dictadura militar, en marzo de 1976. En primer lugar, procuraremos analizar los enfoques y argumentos más influyentes que, tanto en el debate internacional como en el nacional, se han apoyado en la terciarización como suerte de 'base objetiva' para explicar [o postular] la pérdida de centralidad de la clase obrera. Luego nos enfocaremos en las variaciones que ha presentado la composición sectorial del empleo asalariado en el Gran Buenos Aires [GBA], a lo largo del período en estudio [1974-2010]. Lo haremos utilizando las bases usuarias de la Encuesta Permanente de Hogares [EPH]. Finalmente, analizaremos los posibles solapamientos estadísticos entre la modificación en la composición sectorial del empleo del GBA y otros dos fenómenos: la tercerización de actividades [outsourcing], y las políticas de promoción industrial regionales implementadas en el país

Discutiendo la terciarización de la clase trabajadora en el Gran Buenos Aires (1974-2010) : Una aproximación a partir de las bases usuarias de la EPH

Este trabajo forma parte de una investigación más amplia, a ser desarrollada por el autor en futuros estudios de posgrado, sobre las transformaciones experimentadas por lo que podríamos llamar la 'composición', 'morfología' o 'estructura' de la clase obrera en Argentina, a partir del profundo proceso de reestructuración capitalista que viene afectando a las relaciones de clase desde los inicios de la última dictadura militar, en marzo de 1976. En primer lugar, procuraremos analizar los enfoques y argumentos más influyentes que, tanto en el debate internacional como en el nacional, se han apoyado en la terciarización como suerte de 'base objetiva' para explicar [o postular] la pérdida de centralidad de la clase obrera. Luego nos enfocaremos en las variaciones que ha presentado la composición sectorial del empleo asalariado en el Gran Buenos Aires [GBA], a lo largo del período en estudio [1974-2010]. Lo haremos utilizando las bases usuarias de la Encuesta Permanente de Hogares [EPH]. Finalmente, analizaremos los posibles solapamientos estadísticos entre la modificación en la composición sectorial del empleo del GBA y otros dos fenómenos: la tercerización de actividades [outsourcing], y las políticas de promoción industrial regionales implementadas en el país

Discutiendo la terciarización de la clase trabajadora en el Gran Buenos Aires (1974-2010) : Una aproximación a partir de las bases usuarias de la EPH

Este trabajo forma parte de una investigación más amplia, a ser desarrollada por el autor en futuros estudios de posgrado, sobre las transformaciones experimentadas por lo que podríamos llamar la 'composición', 'morfología' o 'estructura' de la clase obrera en Argentina, a partir del profundo proceso de reestructuración capitalista que viene afectando a las relaciones de clase desde los inicios de la última dictadura militar, en marzo de 1976. En primer lugar, procuraremos analizar los enfoques y argumentos más influyentes que, tanto en el debate internacional como en el nacional, se han apoyado en la terciarización como suerte de 'base objetiva' para explicar [o postular] la pérdida de centralidad de la clase obrera. Luego nos enfocaremos en las variaciones que ha presentado la composición sectorial del empleo asalariado en el Gran Buenos Aires [GBA], a lo largo del período en estudio [1974-2010]. Lo haremos utilizando las bases usuarias de la Encuesta Permanente de Hogares [EPH]. Finalmente, analizaremos los posibles solapamientos estadísticos entre la modificación en la composición sectorial del empleo del GBA y otros dos fenómenos: la tercerización de actividades [outsourcing], y las políticas de promoción industrial regionales implementadas en el país

An Eph receptor regulates integrin activity through R-Ras

The ability of integrins to mediate cell attachment to extracellular matrices and to blood proteins is regulated from inside the cell. Increased ligand-binding activity of integrins is critical for platelet aggregation upon blood clotting and for leukocyte extravasation to inflamed tissues. Decreased adhesion is thought to promote tumor cell invasion. R-Ras, a small intracellular GTPase, regulates the binding of integrins to their ligands outside the cell. Here we show that the Eph receptor tyrosine kinase, EphB2, can control integrin activity through R-Ras. Cells in which EphB2 is activated become poorly adherent to substrates coated with integrin ligands, and a tyrosine residue in the R-Ras effector domain is phosphorylated. The R-Ras phosphorylation and loss of cell adhesion are causally related, because forced expression of an R-Ras variant resistant to phosphorylation at the critical site made cells unresponsive to the anti-adhesive effect of EphB2. This is an unusual regulatory pathway among the small GTPases. Reduced adhesiveness induced through the Eph/R-Ras pathway may explain the repulsive effect of the Eph receptors in axonal pathfinding and may facilitate tumor cell invasion and angiogenesis.