RESUMEN
Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4(CRBN) E3 ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumour activity. The anti-tumour activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degradation of the translation termination factor GSPT1. Patient-derived acute myeloid leukaemia tumour cells exhibit high sensitivity to CC-885, indicating the clinical potential of this mechanism. Crystallographic studies of the CRBN-DDB1-CC-885-GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn containing a glycine residue at a key position, interacting with both CC-885 and a 'hotspot' on the cereblon surface. Although GSPT1 possesses no obvious structural, sequence or functional homology to previously known cereblon substrates, mutational analysis and modelling indicate that the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for substrate recruitment. These findings define a structural degron underlying cereblon 'neosubstrate' selectivity, and identify an anti-tumour target rendered druggable by cereblon modulation.
Asunto(s)
Antineoplásicos/farmacología , Péptido Hidrolasas/metabolismo , Factores de Terminación de Péptidos/metabolismo , Compuestos de Fenilurea/farmacología , Talidomida/análogos & derivados , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Antineoplásicos/química , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Factor de Transcripción Ikaros/química , Factor de Transcripción Ikaros/metabolismo , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Péptido Hidrolasas/química , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/deficiencia , Compuestos de Fenilurea/química , Unión Proteica , Proteolisis/efectos de los fármacos , Especificidad por Sustrato , Talidomida/química , Talidomida/farmacología , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept (ACE-011, and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with ß-thalassemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by sequestering lefty1 from erythroid cells. These findings identify lefty1 as a signaling component in the development of erythroid cells and rationalize the use of sotatercept in DBA patients.
Asunto(s)
Anemia de Diamond-Blackfan/tratamiento farmacológico , Eritropoyesis/efectos de los fármacos , Factores de Determinación Derecha-Izquierda/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/uso terapéutico , Proteínas de Pez Cebra/antagonistas & inhibidores , Receptores de Activinas Tipo II/antagonistas & inhibidores , Receptores de Activinas Tipo II/sangre , Anemia de Diamond-Blackfan/sangre , Anemia de Diamond-Blackfan/genética , Animales , Modelos Animales de Enfermedad , Eritropoyesis/genética , Técnicas de Silenciamiento del Gen , Genes p53 , Humanos , Factores de Determinación Derecha-Izquierda/sangre , Factores de Determinación Derecha-Izquierda/genética , Ligandos , Proteínas Ribosómicas/sangre , Proteínas Ribosómicas/deficiencia , Proteínas Ribosómicas/genética , Transducción de Señal/efectos de los fármacos , Pez Cebra , Proteínas de Pez Cebra/sangre , Proteínas de Pez Cebra/genética , Talasemia beta/sangre , Talasemia beta/tratamiento farmacológicoRESUMEN
Cereblon (CRBN), a substrate receptor of the Cullin 4 RING E3 ubiquitin ligase complex, is the target of the immunomodulatory drugs lenalidomide and pomalidomide. Recently, it was demonstrated that binding of these drugs to CRBN promotes the ubiquitination and subsequent degradation of 2 common substrates, transcription factors Aiolos and Ikaros. Here we report that CC-122, a new chemical entity termed pleiotropic pathway modifier, binds CRBN and promotes degradation of Aiolos and Ikaros in diffuse large B-cell lymphoma (DLBCL) and T cells in vitro, in vivo, and in patients, resulting in both cell autonomous as well as immunostimulatory effects. In DLBCL cell lines, CC-122-induced degradation or short hairpin RNA-mediated knockdown of Aiolos and Ikaros correlates with increased transcription of interferon (IFN)-stimulated genes independent of IFN-α, -ß, and -γ production and/or secretion and results in apoptosis in both activated B-cell (ABC) and germinal center B-cell DLBCL cell lines. Our results provide mechanistic insight into the cell-of-origin independent antilymphoma activity of CC-122, in contrast to the ABC subtype selective activity of lenalidomide.
Asunto(s)
Antineoplásicos/farmacología , Linfocitos B/efectos de los fármacos , Factor de Transcripción Ikaros/genética , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Péptido Hidrolasas/genética , Piperidonas/farmacología , Quinazolinonas/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Animales , Antineoplásicos/química , Linfocitos B/metabolismo , Linfocitos B/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Factor de Transcripción Ikaros/metabolismo , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Interferones/genética , Interferones/metabolismo , Lenalidomida , Lentivirus/genética , Lentivirus/metabolismo , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones SCID , Imitación Molecular , Péptido Hidrolasas/metabolismo , Piperidonas/química , Proteolisis/efectos de los fármacos , Quinazolinonas/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Linfocitos T/patología , Talidomida/análogos & derivados , Talidomida/farmacología , Ubiquitina-Proteína Ligasas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pomalidomide is an IMiD(®) immunomodulatory agent, which has shown clinically significant benefits in relapsed and/or refractory multiple myeloma (rrMM) patients when combined with dexamethasone, regardless of refractory status to lenalidomide or bortezomib. (Schey et al, ; San Miguel et al, 2013; Richardson et al, 2014; Scott, ) In this work, we present preclinical data showing that the combination of pomalidomide with dexamethasone (PomDex) demonstrates potent anti-proliferative and pro-apoptotic activity in both lenalidomide-sensitive and lenalidomide-resistant MM cell lines. PomDex also synergistically inhibited tumour growth compared with single-agent treatment in xenografts of lenalidomide-resistant H929 R10-1 cells. Typical hallmarks of IMiD compound activity, including IKZF3 (Aiolos) degradation, and the downregulation of interferon regulatory factor (IRF) 4 and MYC, seen in lenalidomide-sensitive H929 MM cell lines, were also observed in PomDex-treated lenalidomide-resistant H929 MM cells. Remarkably, this resulted in strong, synergistic effects on the induction of apoptosis in both lenalidomide-sensitive and resistant MM cells. Furthermore, gene expression profiling revealed a unique differential gene expression pattern in PomDex-treated samples, highlighted by the modulation of pro-apoptotic pathways in lenalidomide-resistant cells. These results provide key insights into molecular mechanisms of PomDex in the lenalidomide-resistant setting.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Dexametasona/administración & dosificación , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunomodulación/efectos de los fármacos , Lenalidomida , Ratones SCID , Mieloma Múltiple/genética , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Talidomida/administración & dosificación , Talidomida/análogos & derivados , Talidomida/uso terapéutico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Cereblon (CRBN), the molecular target of lenalidomide and pomalidomide, is a substrate receptor of the cullin ring E3 ubiquitin ligase complex, CRL4(CRBN) . T cell co-stimulation by lenalidomide or pomalidomide is cereblon dependent: however, the CRL4(CRBN) substrates responsible for T cell co-stimulation have yet to be identified. Here we demonstrate that interaction of the transcription factors Ikaros (IKZF1, encoded by the IKZF1 gene) and Aiolos (IKZF3, encoded by the IKZF3 gene) with CRL4(CRBN) is induced by lenalidomide or pomalidomide. Each agent promotes Aiolos and Ikaros binding to CRL4(CRBN) with enhanced ubiquitination leading to cereblon-dependent proteosomal degradation in T lymphocytes. We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin-2 expression. The findings link lenalidomide- or pomalidomide-induced degradation of these transcriptional suppressors to well documented T cell activation. Importantly, Aiolos could serve as a proximal pharmacodynamic marker for lenalidomide and pomalidomide, as healthy human subjects administered lenalidomide demonstrated Aiolos degradation in their peripheral T cells. In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4(CRBN) , leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.
Asunto(s)
Factor de Transcripción Ikaros/metabolismo , Péptido Hidrolasas/metabolismo , Linfocitos T/efectos de los fármacos , Talidomida/análogos & derivados , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Inhibidores de la Angiogénesis/farmacología , Humanos , Factor de Transcripción Ikaros/genética , Factores Inmunológicos/farmacología , Lenalidomida , Péptido Hidrolasas/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Linfocitos T/metabolismo , Talidomida/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Eph receptors and their ligands (ephrins) play an important role in axonal guidance, topographic mapping, and angiogenesis. The signaling pathways mediating these activities are starting to emerge and are highly cell- and receptor-type specific. Here we demonstrate that activated EphB1 recruits the adaptor proteins Grb2 and p52Shc and promotes p52Shc and c-Src tyrosine phosphorylation as well as MAPK/extracellular signal-regulated kinase (ERK) activation. EphB1-mediated increase of cell migration was abrogated by the MEK inhibitor PD98059 and Src inhibitor PP2. In contrast, cell adhesion, which we previously showed to be c-jun NH2-terminal kinase (JNK) dependent, was unaffected by ERK1/2 and Src inhibition. Expression of dominant-negative c-Src significantly reduced EphB1-dependent ERK1/2 activation and chemotaxis. Site-directed mutagenesis experiments demonstrate that tyrosines 600 and 778 of EphB1 are required for its interaction with c-Src and p52Shc. Furthermore, phosphorylation of p52Shc by c-Src is essential for its recruitment to EphB1 signaling complexes through its phosphotyrosine binding domain. Together these findings highlight a new aspect of EphB1 signaling, whereby the concerted action of c-Src and p52Shc activates MAPK/ERK and regulates events involved in cell motility.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Quimiotaxis/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Receptor EphB1/metabolismo , Animales , Proteína Tirosina Quinasa CSK , Movimiento Celular , Proteína Adaptadora GRB2 , Humanos , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Tirosina/metabolismo , Familia-src QuinasasRESUMEN
Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in beta-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. beta-Catenin-null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin-beta-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell-cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin-null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.
Asunto(s)
Cadherinas/metabolismo , Inhibición de Contacto , Proteínas del Citoesqueleto/metabolismo , Factores de Crecimiento Endotelial/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Linfocinas/farmacología , Proteínas Tirosina Fosfatasas/metabolismo , Transactivadores/metabolismo , Animales , Antígenos CD , Cadherinas/genética , División Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Unión Proteica , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Factor A de Crecimiento Endotelial Vascular , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial Vascular , beta CateninaRESUMEN
The drugs lenalidomide and pomalidomide bind to the protein cereblon, directing the CRL4-CRBN E3 ligase toward the transcription factors Ikaros and Aiolos to cause their ubiquitination and degradation. Here we describe CC-220 (compound 6), a cereblon modulator in clinical development for systemic lupus erythematosis and relapsed/refractory multiple myeloma. Compound 6 binds cereblon with a higher affinity than lenalidomide or pomalidomide. Consistent with this, the cellular degradation of Ikaros and Aiolos is more potent and the extent of substrate depletion is greater. The crystal structure of cereblon in complex with DDB1 and compound 6 reveals that the increase in potency correlates with increased contacts between compound 6 and cereblon away from the modeled binding site for Ikaros/Aiolos. These results describe a new cereblon modulator which achieves greater substrate degradation via tighter binding to the cereblon E3 ligase and provides an example of the effect of E3 ligase binding affinity with relevance to other drug discovery efforts in targeted protein degradation.
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Factor de Transcripción Ikaros/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Proteolisis/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Línea Celular Tumoral , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Humanos , Lenalidomida/química , Lenalidomida/metabolismo , Morfolinas , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Ftalimidas , Piperidonas , Unión Proteica , Ubiquitina-Proteína LigasasRESUMEN
Vascularization defects in genetic recombinant mice have defined critical roles for a number of specific receptor tyrosine kinases. Here we evaluated whether an endothelium-expressed receptor tyrosine phosphatase, CD148 (DEP-1/PTPeta), participates in developmental vascularization. A mutant allele, CD148(DeltaCyGFP), was constructed to eliminate CD148 phosphatase activity by in-frame replacement of cytoplasmic sequences with enhanced green fluorescent protein sequences. Homozygous mutant mice died at midgestation, before embryonic day 11.5 (E11.5), with vascularization failure marked by growth retardation and disorganized vascular structures. Structural abnormalities were observed as early as E8.25 in the yolk sac, prior to the appearance of intraembryonic defects. Homozygous mutant mice displayed enlarged vessels comprised of endothelial cells expressing markers of early differentiation, including VEGFR2 (Flk1), Tal1/SCL, CD31, ephrin-B2, and Tie2, with notable lack of endoglin expression. Increased endothelial cell numbers and mitotic activity indices were demonstrated. At E9.5, homozygous mutant embryos showed homogeneously enlarged primitive vessels defective in vascular remodeling and branching, with impaired pericyte investment adjacent to endothelial structures, in similarity to endoglin-deficient embryos. Developing cardiac tissues showed expanded endocardial projections accompanied by defective endocardial cushion formation. These findings implicate a member of the receptor tyrosine phosphatase family, CD148, in developmental vascular organization and provide evidence that it regulates endothelial proliferation and endothelium-pericyte interactions.
Asunto(s)
Endotelio Vascular/enzimología , Mutación , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/fisiología , Alelos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Northern Blotting , Western Blotting , Catálisis , Dominio Catalítico , División Celular , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Endotelio Vascular/citología , Efrina-B2/metabolismo , Proteínas Fluorescentes Verdes , Homocigoto , Riñón/metabolismo , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Electrónica , Modelos Genéticos , Miocardio/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Pruebas de Precipitina , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor TIE-2 , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 1 de la Leucemia Linfocítica T Aguda , Factores de Tiempo , Factores de Transcripción/metabolismo , Tirosina/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Angiogenesis is a multistep process involving a diverse array of molecular signals. Ligands for receptor tyrosine kinases (RTKs) have emerged as critical mediators of angiogenesis. Three families of ligands, vascular endothelial cell growth factors (VEGFs), angiopoietins, and ephrins, act via RTKs expressed in endothelial cells. Recent evidence indicates that VEGF cooperates with angiopoietins to regulate vascular remodeling and angiogenesis in both embryogenesis and tumor neovascularization. However, the relationship between VEGF and ephrins remains unclear. Here we show that interaction between EphA RTKs and ephrinA ligands is necessary for induction of maximal neovascularization by VEGF. EphA2 RTK is activated by VEGF through induction of ephrinA1 ligand. A soluble EphA2-Fc receptor inhibits VEGF-, but not basic fibroblast growth factor-induced endothelial cell survival, migration, sprouting, and corneal angiogenesis. As an independent, but complementary approach, EphA2 antisense oligonucleotides inhibited endothelial expression of EphA2 receptor and suppressed ephrinA1- and VEGF-induced cell migration. Taken together, these data indicate an essential role for EphA receptor activation in VEGF-dependent angiogenesis and suggest a potential new target for therapeutic intervention in pathogenic angiogenesis.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Endotelio Vascular/efectos de los fármacos , Activación Enzimática/fisiología , Linfocinas/antagonistas & inhibidores , Neovascularización Fisiológica/efectos de los fármacos , Receptor EphA2/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Neovascularización de la Córnea , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Efrina-A1/metabolismo , Efrina-A1/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/farmacología , Fosforilación , Receptor EphA2/metabolismo , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
CXC chemokines, which induce angiogenesis, have glutamine-leucine-arginine amino acid residues (ELR motif) in the amino terminus and bind CXCR2 and the Duffy antigen chemokine-binding protein. Duffy, a seven transmembrane protein that binds CXC and CC chemokines, has not been shown to couple to trimeric G proteins or to transduce intracellular signals, although it is highly expressed on red blood cells, endothelial cells undergoing neovascularization, and neuronal cells. The binding of chemokines by Duffy could modulate chemokine responses positively or negatively. Positive regulation could come through the presentation of chemokine to functional receptors, and negative regulation could come through Duffy competition with functional chemokine receptors for chemokine binding, thus serving as a decoy receptor. To determine whether Duffy has a role in angiogenesis and/or maintenance of homeostasis, we developed transgenic mice expressing mDuffy under the control of the preproendothelin promoter/enhancer (PPEP), which directs expression of the transgene to the endothelium. Two PPEP-mDuffy-transgenic founders were identified, and expression of the transgene in the endothelium was verified by Northern blot, RT-PCR, and immunostaining of tissues. The phenotype of the mice carrying the transgene appeared normal by all visual parameters. However, careful comparison of transgenic and nontransgenic mice revealed two phenotypic differences: mDuffy-transgenic mice exhibited a diminished angiogenic response to MIP-2 in the corneal micropocket assay, and mDuffy-transgenic mice exhibited enhanced hepatocellular toxicity and necrosis as compared with nontransgenic littermates in response to overdose of acetaminophen (APAP; 400 mg/kg body weight). Morover, APAP treatment was lethal in 50% of the mDuffy-transgenic mice 24 h post challenge, and 100% of the nontransgenic littermates survived this treatment at the 24 h time point. Our data suggest that enhanced expression of mDuffy on endothelial cells can lead to impaired angiogenic response to chemokines and impaired maintenance of homeostasis in response to toxic stresses.
Asunto(s)
Antígenos de Protozoos , Proteínas Portadoras/genética , Quimiocinas CXC/fisiología , Homeostasis/genética , Neovascularización Patológica/genética , Proteínas Protozoarias , Receptores de Superficie Celular/genética , Estrés Fisiológico/fisiopatología , Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Animales , Proteínas Portadoras/fisiología , Endotelio Corneal/fisiología , Hígado/patología , Hígado/fisiopatología , Ratones , Ratones Transgénicos , Necrosis , Receptores de Superficie Celular/fisiología , Receptores de Interleucina-8B/fisiología , Estrés Fisiológico/inducido químicamenteRESUMEN
Contact inhibition of cell proliferation evokes a unique cellular program of growth arrest compared with stress, age, or other physical constraints. The last decade of research on genes activated by cell-cell contact has uncovered features of transmembrane signaling, cytoskeletal reorganization, and transcriptional control that initiate and maintain a quiescent phenotype. This review will focus on mechanisms controlling contact inhibition of cell proliferation, highlighting specific gene expression responses that are activated by cell-cell contact. Although a temporal framework for imposition of these mechanisms has not yet been well described, contact inhibition of cell proliferation clearly requires their coordinated function. Novel targets for intervention in proliferative disorders are emerging from these studies.
Asunto(s)
Comunicación Celular/fisiología , Proliferación Celular , Comunicación Celular/genética , Expresión Génica , Biología Molecular , Receptores de Superficie Celular/fisiologíaRESUMEN
The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in the regulation of growth in many animal cells, including cancer cells. Phosphorylation of specific tyrosine residues within the cytoplasmic domain of EGFR is part of the initial activation process that occurs upon ligand binding, and these phosphotyrosine residues subsequently serve as docking sites for intracellular signaling molecules. To study the phosphorylation on each individual site, EGFR generated from a human epidermoid carcinoma cell line (A431) was analyzed by mass spectrometry. Liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) was used to identify the tryptic phosphopeptides and their sites of phosphorylation (Y992, Y1045, Y1068, Y1086, S1142, Y1148, and Y1173). Ion intensities for the phosphorylated and unphosphorylated tryptic peptides containing the sites of phosphorylation were measured, and the intensity ratios were used to assess the degree of phosphorylation at each site. Ligand concentrations were varied for epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) as stimuli, and all of the EGFR tyrosine sites were consequently found to exhibit increased levels of phosphorylation, although at different rates and to different extents. Phosphorylation of Y992 appeared to plateau at lower concentrations of ligand, whereas the other sites continued to have increased phosphorylation throughout a wide range of concentrations. Only small differences could be detected between the EGF and the TGF alpha-induced EGFR phosphorylation. Pretreatment of A431 cells with a small molecule EGFR inhibitor nearly eliminated the ligand-induced phosphorylation on all of the sites except for Y992 and Y1068.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Secuencia de Aminoácidos , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/química , Receptores ErbB/genética , Humanos , Ligandos , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Factor de Crecimiento Transformador alfa/farmacología , Células Tumorales CultivadasRESUMEN
Recent evidence has demonstrated that inflammation plays an important role in the progression of coronary atherosclerosis. This review focuses on acute coronary syndromes and examines some novel therapeutic strategies aimed at manipulating the inflammatory environment in these patients in order to reduce the subsequent major adverse coronary event rate following myocardial infarction.
Asunto(s)
Antiinflamatorios/uso terapéutico , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Enfermedad de la Arteria Coronaria/complicaciones , Humanos , Inflamación/complicaciones , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
The Cul4-Rbx1-DDB1-Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon (CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degradation. Here we present the crystal structure of human CRBN bound to DDB1 and the drug lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are critical for antiproliferative effects.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Proteínas de Unión al ADN/metabolismo , Péptido Hidrolasas/metabolismo , Talidomida/análogos & derivados , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/química , Animales , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Humanos , Lenalidomida , Ratones , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Péptido Hidrolasas/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Talidomida/química , Talidomida/farmacología , Ubiquitina-Proteína LigasasRESUMEN
The pathophysiology of ineffective erythropoiesis in ß-thalassemia is poorly understood. We report that RAP-011, an activin receptor IIA (ActRIIA) ligand trap, improved ineffective erythropoiesis, corrected anemia and limited iron overload in a mouse model of ß-thalassemia intermedia. Expression of growth differentiation factor 11 (GDF11), an ActRIIA ligand, was increased in splenic erythroblasts from thalassemic mice and in erythroblasts and sera from subjects with ß-thalassemia. Inactivation of GDF11 decreased oxidative stress and the amount of α-globin membrane precipitates, resulting in increased terminal erythroid differentiation. Abnormal GDF11 expression was dependent on reactive oxygen species, suggesting the existence of an autocrine amplification loop in ß-thalassemia. GDF11 inactivation also corrected the abnormal ratio of immature/mature erythroblasts by inducing apoptosis of immature erythroblasts through the Fas-Fas ligand pathway. Taken together, these observations suggest that ActRIIA ligand traps may have therapeutic relevance in ß-thalassemia by suppressing the deleterious effects of GDF11, a cytokine which blocks terminal erythroid maturation through an autocrine amplification loop involving oxidative stress and α-globin precipitation.
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Receptores de Activinas Tipo II/metabolismo , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Eritroblastos/metabolismo , Eritropoyesis/efectos de los fármacos , Factores de Diferenciación de Crecimiento/antagonistas & inhibidores , Hematínicos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Talasemia beta/metabolismo , Animales , Apoptosis/fisiología , Comunicación Autocrina/fisiología , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Modelos Animales de Enfermedad , Proteína Ligando Fas , Amplificación de Genes/fisiología , Factores de Diferenciación de Crecimiento/metabolismo , Ligandos , Ratones , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno , Transducción de Señal , Receptor fasRESUMEN
Faded, or actively removed text on thermally printed paper samples may be enhanced and retrieved through the use of a simple iodine fuming procedure. The recovery of printed documentation evidence in this fashion is neither affected by prior fingerprint enhancement techniques (such as ninhydrin or DFO), nor by sample age. This method allows, for the first time, evidence to be obtained from completely faded thermal paper samples (receipts, for example) as well as allowing deliberately removed printed text (a consequence of solvent washing pre-treatment in latent fingerprint enhancement procedures) to be recovered.
RESUMEN
Angiogenesis contributes to a wide range of neoplastic, ischemic, and inflammatory disorders. Definition of the intrinsic molecular controls in angiogenic vessel growth promises novel therapeutic approaches for angiogenesis-related diseases. CD148 (also named DEP-1/PTP eta) is a receptor-like protein tyrosine phosphatase that is abundantly expressed in vascular endothelial cells. To explore a role of CD148 in endothelial vessel formation, we generated a monoclonal antibody, Ab1, against the ectodomain sequence of CD148 and examined its effects on endothelial-cell growth and vessel formation. Here we report that a bivalent, but not a monovalent, form of the Ab1 antibody inhibits endothelial-cell growth and blocks angiogenesis in mouse cornea in vivo. We further demonstrate that (1) bivalent Ab1 arrests cell-cycle progression of CD148-transfected CHO cells at G(0)/G(1) phase, (2) coexpression of catalytically inactive CD148 mutants attenuates the Ab1-cell growth inhibition, and (3) bivalent Ab1 suppresses phosphorylation of ERK1/2 kinases and Met tyrosine kinase as activated CD148 does, with an increase in CD148-associated tyrosine phosphatase activity. Taken together, these findings demonstrate that Ab1-induced ectodomain oligomerization arrests endothelial-cell growth through catalytic activity of the CD148 cytoplasmic domain. The present study defines CD148 as a valuable molecular target for antiangiogenesis therapy.
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Anticuerpos Monoclonales/farmacología , Células Endoteliales/metabolismo , Inhibidores Enzimáticos/farmacología , Neovascularización Patológica/tratamiento farmacológico , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Células CHO , Córnea/irrigación sanguínea , Córnea/inmunología , Córnea/metabolismo , Córnea/patología , Cricetinae , Cricetulus , Células Endoteliales/patología , Inhibidores Enzimáticos/inmunología , Fase G1/efectos de los fármacos , Fase G1/genética , Fase G1/inmunología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Neovascularización Patológica/genética , Neovascularización Patológica/inmunología , Neovascularización Patológica/metabolismo , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/inmunología , Estructura Terciaria de Proteína/genética , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/inmunología , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/inmunología , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Fase de Descanso del Ciclo Celular/genética , Fase de Descanso del Ciclo Celular/inmunologíaRESUMEN
Glomerular development proceeds through the spatially ordered and sequential recruitment, proliferation, assembly, and differentiation of endothelial, mesangial, and epithelial progenitors. The molecular determinants of cell-cell recognition and targeting in this process have yet to be defined. The Eph/ephrin family of membrane receptors and counter-receptors are critical participants of developmental vascular assembly in extrarenal sites. Renal expression patterns of ephrin-B2 and EphB4 were investigated using mice expressing beta-galactosidase under control of ephrin-B2 or EphB4 promoters. The earliest glomerular expression of ephrin-B2 was identified in a subset of differentiating comma-stage glomerular epithelial cells (podocyte progenitors) adjacent to the vascular cleft where endothelial progenitors are subsequently recruited. Epithelial ephrin-B2 expression was accompanied by expression in endothelial and mesangial cells as capillary assembly progressed. At or near completion of glomerular maturation, epithelial ephrin-B2 expression was extinguished, with persistence in glomerular endothelial cells. Throughout development, one of several ephrin-B2 receptors, EphB4, was persistently and exclusively expressed in endothelial cells of venous structures. The findings show sequential ephrin-B2 expression across glomerular lineages, first in a distinct subset of podocyte progenitors and subsequently in endothelial cells of the developing glomerulus. Given targeting functions for Eph/ephrin family proteins, the findings suggest that ephrin-B2 expression marks podocyte progenitors at the site of vascular cleft formation, where expression may establish an "address" to which endothelial and mesangial progenitors are recruited. Thus, the present results suggest that ephrin-B2 and EphB interactions play an important role in glomerular microvascular assembly.
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Glomérulos Renales/embriología , Proteínas de la Membrana/metabolismo , Animales , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario y Fetal , Endotelio Vascular/embriología , Efrina-B2 , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos/genética , Microcirculación , Circulación Renal , Factores de Tiempo , Distribución TisularRESUMEN
Ephrin-B/EphB family proteins are implicated in bidirectional signaling and were initially defined through the function of their ectodomain sequences in activating EphB receptor tyrosine kinases. Ephrin-B1-3 are transmembrane proteins sharing highly conserved C-terminal cytoplasmic sequences. Here we use a soluble EphB1 ectodomain fusion protein (EphB1/Fc) to demonstrate that ephrin-B1 transduces signals that regulate cell attachment and migration. EphB1/Fc induced endothelial ephrin-B1 tyrosine phosphorylation, migration and integrin-mediated (alpha(v)beta(3) and alpha(5)beta(1)) attachment and promoted neovascularization, in vivo, in a mouse corneal micropocket assay. Activation of ephrin-B1 by EphB1/Fc induced phosphorylation of p46 JNK but not ERK-1/2 or p38 MAPkinases. By contrast, mutant ephrin-B1s bearing either a cytoplasmic deletion (ephrin-B1DeltaCy) or a deletion of four C-terminal amino acids (ephrin-B1DeltaPDZbd) fail to activate p46 JNK. Transient expression of intact ephin-B1 conferred EphB1/Fc migration responses on CHO cells, whereas the ephrin-B1DeltaCy and ephrin-B1DeltaPDZbd mutants were inactive. Thus ephrin-B1 transduces 'outside-in' signals through C-terminal protein interactions that affect integrin-mediated attachment and migration.