RESUMEN
Cell migration is a stepwise process that coordinates multiple molecular machineries. Using in vitro angiogenesis screens with short interfering RNA and chemical inhibitors, we define here a MAP4K4-moesin-talin-ß1-integrin molecular pathway that promotes efficient plasma membrane retraction during endothelial cell migration. Loss of MAP4K4 decreased membrane dynamics, slowed endothelial cell migration, and impaired angiogenesis in vitro and in vivo. In migrating endothelial cells, MAP4K4 phosphorylates moesin in retracting membranes at sites of focal adhesion disassembly. Epistasis analyses indicated that moesin functions downstream of MAP4K4 to inactivate integrin by competing with talin for binding to ß1-integrin intracellular domain. Consequently, loss of moesin (encoded by the MSN gene) or MAP4K4 reduced adhesion disassembly rate in endothelial cells. Additionally, α5ß1-integrin blockade reversed the membrane retraction defects associated with loss of Map4k4 in vitro and in vivo. Our study uncovers a novel aspect of endothelial cell migration. Finally, loss of MAP4K4 function suppressed pathological angiogenesis in disease models, identifying MAP4K4 as a potential therapeutic target.
Asunto(s)
Movimiento Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Integrinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencias de Aminoácidos , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Forma de la Célula/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Epistasis Genética , Adhesiones Focales/metabolismo , Humanos , Integrina alfa1/efectos de los fármacos , Integrina alfa1/metabolismo , Integrina beta1/química , Integrina beta1/efectos de los fármacos , Integrina beta1/metabolismo , Integrinas/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Neovascularización Patológica , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Talina/química , Talina/metabolismoRESUMEN
Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling.
Asunto(s)
Proteínas Portadoras/fisiología , Endotelio Vascular/embriología , Endotelio Vascular/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Uniones Intercelulares/fisiología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/metabolismo , Neovascularización Fisiológica , Embarazo , Interferencia de ARN , Transducción de Señal , Pez Cebra , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/fisiologíaRESUMEN
EGFL7 was identified by a number of groups as a putative secreted factor produced by the vascular endothelial cells (ECs). In a recent publication, we showed that EGFL7 regulates midline angioblast migration in zebrafish embryos-a key step in vascular tubulogenesis. In this study, we further characterized the zebrafish vasculature in the Egfl7 knockdown embryos at the ultrastructural level, and found that malformation of axial vessels is indeed due to the accumulation of angioblasts and aberrant connection among themselves, but not abnormal interaction between ECs and other cell types. Using in vitro biochemical assays, we demonstrated that EGFL7 is tightly associated with the extracellular matrix (ECM), and it supports EC migration either as a single factor or in combination with other ECM molecules. In order to evaluate if the biological function of EGFL7 is evolutionarily conserved, we generated Egfl7 knockout mice and analysed vascular development in a number of tissues. We found that vascular coverage of a given tissue is reduced or delayed, and vascular morphogenesis is defective in the Egfl7 mutant mice. Taken together, we conclude that EGFL7 provides a proper microenvironment for endothelial cell migration, thereby enabling accurate patterning. Our study indicates that the molecular composition of the ECM influences vascular morphogenesis.
Asunto(s)
Vasos Sanguíneos/embriología , Embrión de Mamíferos/irrigación sanguínea , Embrión de Mamíferos/metabolismo , Morfogénesis , Neovascularización Fisiológica , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Vasos Sanguíneos/ultraestructura , Tipificación del Cuerpo , Movimiento Celular , Embrión no Mamífero/irrigación sanguínea , Embrión no Mamífero/ultraestructura , Células Endoteliales/citología , Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Ratones , Neoplasias/irrigación sanguínea , Neovascularización Patológica , Proteínas de Pez Cebra/genéticaRESUMEN
In recent years, the elucidation of the structures of many signalling molecules has allowed new insights into the molecular mechanisms that govern signal transduction events. In the field of cytokine signalling, the solved structures of cytokine/receptor complexes and of key components involved in signal transduction such as STAT factors or the tyrosine phosphatase SHP2 have broadened our understanding of the molecular basis of the signalling events and provided key information for the rational design of therapeutic approaches to modulate or block cytokine signal transduction. Unfortunately, no structural data on the intracellular parts of cytokine receptors are available. The exact molecular mechanism underlying one of the first steps in signal transduction, namely the recruitment of signalling components to the cytoplasmic parts of cytokine receptors, remains elusive. Here we investigated possible mechanisms underlying the different potency of the STAT3-activating motifs of gp130 after IL-6 stimulation. Our data indicate that the extent of STAT3 activation by the different receptor motifs is not influenced by structural features such as contacts between the two gp130 chains. In addition, the proximity of the negatively regulating motif around tyrosine Y759 to the different STAT3-recruiting motifs does not seem to be responsible for their differential capacity to activate STAT3. However, the potency of a specific motif to activate STAT3 directly reflects the affinity for the binding of STAT3 to this motif.
Asunto(s)
Receptor gp130 de Citocinas/metabolismo , Interleucina-6/farmacología , Factor de Transcripción STAT3/metabolismo , Secuencias de Aminoácidos/efectos de los fármacos , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Receptor gp130 de Citocinas/efectos de los fármacos , Receptor gp130 de Citocinas/genética , Regulación de la Expresión Génica , Ratones , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Ratas , Factor de Transcripción STAT3/efectos de los fármacos , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tirosina/efectos de los fármacos , Tirosina/metabolismoRESUMEN
Diverse biological roles for mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) have necessitated the identification of potent inhibitors in order to study its function in various disease contexts. In particular, compounds that can be used to carry out such studies in vivo would be critical for elucidating the potential for therapeutic intervention. A structure-based design effort coupled with property-guided optimization directed at minimizing the ability of the inhibitors to cross into the CNS led to an advanced compound 13 (GNE-495) that showed excellent potency and good PK and was used to demonstrate in vivo efficacy in a retinal angiogenesis model recapitulating effects that were observed in the inducible Map4k4 knockout mice.
RESUMEN
Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is a serine/threonine kinase implicated in the regulation of many biological processes. A fragment-based lead discovery approach was used to generate potent and selective MAP4K4 inhibitors. The fragment hit pursued in this article had excellent ligand efficiency (LE), an important attribute for subsequent successful optimization into drug-like lead compounds. The optimization efforts eventually led us to focus on the pyridopyrimidine series, from which 6-(2-fluoropyridin-4-yl)pyrido[3,2-d]pyrimidin-4-amine (29) was identified. This compound had low nanomolar potency, excellent kinase selectivity, and good in vivo exposure, and demonstrated in vivo pharmacodynamic effects in a human tumor xenograft model.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/síntesis química , Animales , Descubrimiento de Drogas , Femenino , Péptidos y Proteínas de Señalización Intracelular/química , Ratones , Modelos Moleculares , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/química , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
Many oncology drugs are administered at their maximally tolerated dose without the knowledge of their optimal efficacious dose range. In this study, we describe a multifaceted approach that integrated preclinical and clinical data to identify the optimal dose for an antiangiogenesis agent, anti-EGFL7. EGFL7 is an extracellular matrix-associated protein expressed in activated endothelium. Recombinant EGFL7 protein supported EC adhesion and protected ECs from stress-induced apoptosis. Anti-EGFL7 antibodies inhibited both of these key processes and augmented anti-VEGF-mediated vascular damage in various murine tumor models. In a genetically engineered mouse model of advanced non-small cell lung cancer, we found that anti-EGFL7 enhanced both the progression-free and overall survival benefits derived from anti-VEGF therapy in a dose-dependent manner. In addition, we identified a circulating progenitor cell type that was regulated by EGFL7 and evaluated the response of these cells to anti-EGFL7 treatment in both tumor-bearing mice and cancer patients from a phase I clinical trial. Importantly, these preclinical efficacy and clinical biomarker results enabled rational selection of the anti-EGFL7 dose currently being tested in phase II clinical trials.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Anticuerpos/farmacología , Apoptosis , Factores de Crecimiento Endotelial/inmunología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Bevacizumab , Proteínas de Unión al Calcio , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Ensayos Clínicos Fase I como Asunto , Familia de Proteínas EGF , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Insulinoma/irrigación sanguínea , Insulinoma/tratamiento farmacológico , Insulinoma/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Ratones Transgénicos , Células Neoplásicas Circulantes/efectos de los fármacos , Células Neoplásicas Circulantes/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Carga Tumoral/efectos de los fármacos , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
During sprouting angiogenesis, groups of endothelial cells (ECs) migrate together in units called sprouts. In this study, we demonstrate that the vascular-specific secreted factor EGFL7 regulates the proper spatial organization of ECs within each sprout and influences their collective movement. In the homozygous Egfl7-knockout mice, vascular development is delayed in many organs despite normal EC proliferation, and 50% of the knockout embryos die in utero. ECs in the mutant vasculatures form abnormal aggregates and the vascular basement membrane marker collagen IV is mislocalized, suggesting that ECs fail to recognize the proper spatial position of their neighbors. Although the migratory ability of individual ECs in isolation is not affected by the loss of EGFL7, the aberrant spatial organization of ECs in the mutant tissues decreases their collective movement. Using in vitro and in vivo analyses, we showed that EGFL7 is a component of the interstitial extracellular matrix deposited on the basal sides of sprouts, a location suitable for conveying positional information to neighboring ECs. Taken together, we propose that EGFL7 defines the optimal path of EC movement by assuring the correct positioning of each EC in a nascent sprout.
Asunto(s)
Vasos Sanguíneos/embriología , Tipificación del Cuerpo , Movimiento Celular/genética , Células Endoteliales/citología , Proteínas/fisiología , Animales , Vasos Sanguíneos/anomalías , Proteínas de Unión al Calcio , Células Cultivadas , Embrión de Pollo , Familia de Proteínas EGF , Endotelio Vascular/anomalías , Viabilidad Fetal/genética , Humanos , Ratones , Ratones Noqueados , Modelos Biológicos , Proteínas/genéticaRESUMEN
The presence of a Src homology 2 (SH2) domain sequence similarity in the sequence of Janus kinases (Jaks) has been discussed since the first descriptions of these enzymes. We performed an in depth study to determine the function of the Jak1 SH2 domain. We investigated the functionality of the Jak1 SH2 domain by stably reconstituting Jak1-defective human fibrosarcoma cells U4C with endogenous amounts of Jak1 in which the crucial arginine residue Arg466 within the SH2 domain has been replaced by lysine. This mutant still binds to the receptor subunits gp130 and OSMR. Moreover, the SH2 R466K mutation does not affect the subcellular distribution of Jak1 as assessed by cell fractionation and confocal microscopy of cells expressing endogenous levels of non-tagged or a yellow fluorescent protein (YFP)-tagged Jak1-R466K, respectively. Likewise, the signaling capacity of Jak1 was not affected by this point mutation. However, we found that the SH2 domain is structurally important for cytokine receptor binding and surface expression of the OSMR.