RESUMEN
Neonatal mice deficient in IkappaB-alpha, an inhibitor of the ubiquitous transcription factor NF-kappaB, develop severe and widespread dermatitis shortly after birth. In humans, inflammatory skin disorders such as psoriasis are associated with accumulation in the skin of the unusual arachidonic acid metabolite 12R-hydroxyeicosatetraenoic acid (12R-HETE), a product of the enzyme 12R-lipoxygenase. To examine the etiology of the murine IkappaB-alpha-deficient skin phenotype, we investigated the expression of lipoxygenases and the metabolism of exogenous arachidonic acid in the skin. In the IkappaB-alpha-deficient animals, the major lipoxygenase metabolite was 8S-HETE, formed together with a minor amount of 12S-HETE; 12R-HETE synthesis was undetectable. Skin from the wild-type littermates formed 12S-HETE as the almost exclusive lipoxygenase metabolite. Upregulation of 8S-lipoxygenase (8-LOX) in IkappaB-alpha-deficient mice was confirmed at the transcriptional and translational level using ribonuclease protection assay and western analysis. In immunohistochemical studies, increased expression of 8-LOX was detected in the stratum granulosum of the epidermis. In the stratum granulosum, 8-LOX may be involved in the terminal differentiation of keratinocytes. Although mouse 8S-lipoxygenase and human 12R-lipoxygenase are not ortholog genes, we speculate that in mouse and humans the two different enzymes may fulfill equivalent functions in the progression of inflammatory dermatoses.
Asunto(s)
Araquidonato Lipooxigenasas/análisis , Dermatitis/enzimología , Proteínas I-kappa B/fisiología , Animales , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato Lipooxigenasas/fisiología , Ácido Araquidónico/metabolismo , Western Blotting , Humanos , Inmunohistoquímica , Ratones , Inhibidor NF-kappaB alfa , Piel/metabolismo , Regulación hacia ArribaRESUMEN
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
The Eph family of receptor tyrosine kinases and their ligands, known as ephrins, play a crucial role in vascular development during embryogenesis. The function of these molecules in adult angiogenesis has not been well characterized. Here, we report that blocking Eph A class receptor activation inhibits angiogenesis in two independent tumor types, the RIP-Tag transgenic model of angiogenesis-dependent pancreatic islet cell carcinoma and the 4T1 model of metastatic mammary adenocarcinoma. Ephrin-A1 ligand was expressed in both tumor and endothelial cells, and EphA2 receptor was localized primarily in tumor-associated vascular endothelial cells. Soluble EphA2-Fc or EphA3-Fc receptors inhibited tumor angiogenesis in cutaneous window assays, and tumor growth in vivo. EphA2-Fc or EphA3-Fc treatment resulted in decreased tumor vascular density, tumor volume, and cell proliferation, but increased cell apoptosis. However, EphA2-Fc had no direct effect on tumor cell growth or apoptosis in culture, yet inhibited migration of endothelial cells in response to tumor cells, suggesting that the soluble receptor inhibited blood vessel recruitment by the tumor. These data provide the first functional evidence for Eph A class receptor regulation of pathogenic angiogenesis induced by tumors and support the function of A class Eph receptors in tumor progression.
Asunto(s)
Neoplasias Experimentales/irrigación sanguínea , Neovascularización Patológica/prevención & control , Proteínas Tirosina Quinasas Receptoras/fisiología , Adenoma de Células de los Islotes Pancreáticos/irrigación sanguínea , Animales , Movimiento Celular , Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/citología , Femenino , Etiquetado Corte-Fin in Situ , Linfocinas/fisiología , Neoplasias Mamarias Experimentales/irrigación sanguínea , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias Experimentales/prevención & control , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Antígeno Nuclear de Célula en Proliferación/análisis , Receptor EphA1 , Receptor EphA2 , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
TGF-betas are potent inhibitors of epithelial cell proliferation. However, in established carcinomas, autocrine/paracrine TGF-beta interactions can enhance tumor cell viability and progression. Thus, we studied the effect of a soluble Fc:TGF-beta type II receptor fusion protein (Fc:TbetaRII) on transgenic and transplantable models of breast cancer metastases. Systemic administration of Fc:TbetaRII did not alter primary mammary tumor latency in MMTV-Polyomavirus middle T antigen transgenic mice. However, Fc:TbetaRII increased apoptosis in primary tumors, while reducing tumor cell motility, intravasation, and lung metastases. These effects correlated with inhibition of Akt activity and FKHRL1 phosphorylation. Fc:TbetaRII also inhibited metastases from transplanted 4T1 and EMT-6 mammary tumors in syngeneic BALB/c mice. Tumor microvessel density in a mouse dorsal skin window chamber was unaffected by Fc:TbetaRII. Therefore, blockade of TGF-beta signaling may reduce tumor cell viability and migratory potential and represents a testable therapeutic approach against metastatic carcinomas.
Asunto(s)
Apoptosis , Fragmentos Fc de Inmunoglobulinas/fisiología , Inmunoglobulina G/fisiología , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Animales/patología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Transducción de Señal , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Antígenos Transformadores de Poliomavirus/genética , Comunicación Autocrina , Mama/efectos de los fármacos , Mama/patología , Movimiento Celular , Supervivencia Celular , Femenino , Vectores Genéticos , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/genética , Virus del Tumor Mamario del Ratón , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Metástasis de la Neoplasia , Neovascularización Patológica , Proteínas Serina-Treonina Quinasas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/administración & dosificación , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Solubilidad , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1 , Células Tumorales CultivadasRESUMEN
ErbB2/Neu destabilizes the cyclin-dependent kinase (Cdk) inhibitor p27 and increases expression of cyclin D1. Therefore, we studied the roles of p27 and cyclin D1 in ErbB2-mediated mammary epithelial cell transformation. Overexpression of ErbB2 or cyclin D1 in p27(+/-) primary murine mammary epithelial cells resulted in increased proliferation, cyclin D1 nuclear localization, and colony formation in soft agar compared to those in p27(+/+) cells. In contrast, ErbB2- or cyclin D1-overexpressing p27(-/-) cells displayed reduced proliferation, anchorage-independent growth, Cdk4 activity, cyclin D1 expression, and cyclin D1 nuclear localization compared to wild-type cells. A cyclin D1 mutation in its nuclear export sequence (T286A) partially rescued nuclear localization of cyclin D1 in p27(-/-) cells but did not increase proliferation or Cdk4 kinase activity. Overexpression of E2F1, however, increased proliferation to the same degree in p27(+/+), p27(+/-), and p27(-/-) cells. Mammary glands from MMTV (mouse mammary tumor virus)-neu/p27(+/-) mice exhibited alveolar hyperplasia, enhanced proliferation, decreased apoptosis, and accelerated tumor formation compared to MMTV-neu/p27(+/+) glands. However, MMTV-neu/p27(-/-) glands showed decreased proliferation, cyclin D1 expression, and Cdk4 activity, as well as markedly prolonged tumor latency, compared to MMTV-neu/p27(+/+) glands. These results suggest that p27(+/-) mammary epithelium may be more susceptible to oncogene-induced tumorigenesis, whereas p27-null glands, due to severely impaired cyclin D1/Cdk4 function, are more resistant to transformation.
Asunto(s)
Transformación Celular Neoplásica/genética , Ciclina D1/metabolismo , Eliminación de Gen , Proteínas Proto-Oncogénicas , Receptor ErbB-2/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Secuencia de Bases , Proteínas de Ciclo Celular/genética , División Celular , Núcleo Celular/metabolismo , Transformación Celular Neoplásica/patología , Ciclina D1/genética , Quinasa 4 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Citometría de Flujo , Dosificación de Gen , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor ErbB-2/genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genéticaRESUMEN
Eph receptors are a unique family of receptor tyrosine kinases that play critical roles in embryonic patterning, neuronal targeting, vascular development and adult neovascularization. Engagement of Eph receptors by ephrin ligands mediates critical steps of angiogenesis, including juxtacrine cell-cell contacts, cell adhesion to extracellular matrix, and cell migration. Recent evidence from in vitro angiogenesis assays and analysis of mice deficient for one or more members of the Eph family establishes the role of Eph signaling in sprouting angiogenesis and blood vessel remodeling during vascular development. Furthermore, elevated expression of Eph receptors and ephrin ligands is associated with tumors and associated tumor vasculature, suggesting that Eph receptors and their ephrin ligands also play critical roles in tumor angiogenesis and tumor growth. This review will focus on the relevance of Eph receptor signaling in embryonic and adult neovascularization, and possible contributions to tumor growth and metastasis.