RESUMEN
Platelet-derived growth factor (PDGF)-BB and PDGF-DD mediate mesangial cell proliferation in vitro and in vivo. While PDGF-BB is a ligand for the PDGF alpha- and beta-receptor chains, PDGF-DD binds more selectively to the beta-chain, suggesting potential differences in the biological activities. Signal transduction and regulation of gene expression induced by PDGF-BB and -DD were compared in primary human mesangial cells (HMCs), which expressed PDGF alpha- and beta-receptor subunits. The growth factor concentrations used were chosen based on their equipotency in inducing HMCs proliferation and binding to the betabeta-receptor. Both growth factors, albeit at different concentrations induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. In addition, PDGFs led to the phosphorylation and activation of signal transducers and activators of transcription 1 (STAT1) and STAT3. HMCs proliferation induced by either PDGF-BB or -DD could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase-, Janus kinase (JAK)/STAT-, or phosphatidyl-inositol 3-kinase pathways. Using a gene chip array and subsequent verification by real-time reverse transcriptase (RT)-polymerase chain reaction, we found that in HMC genes for matrix metalloproteinase 13 (MMP-13) and MMP-14 and, to a low extent, cytochrome B5 and cathepsin L were exclusively regulated by PDGF-BB, whereas no exclusive gene regulation was detected by PDGF-DD. However, at the protein level, both MMP-13 and -14 were equally induced by PDGF-BB and -DD. PDGF-BB and -DD effect similar biological responses in HMCs albeit at different potencies. Rare apparently differential gene regulation did not result in different protein expression, suggesting that in HMCs both PDGFs exert their biological activity almost exclusively via the PDGF beta-receptor.
Asunto(s)
Inductores de la Angiogénesis/farmacología , Células Mesangiales/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Anticuerpos Monoclonales/metabolismo , Becaplermina , Western Blotting , Línea Celular , Proliferación Celular/efectos de los fármacos , Colagenasas/metabolismo , Densitometría , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Metaloproteinasa 13 de la Matriz , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Factor de Crecimiento Derivado de Plaquetas/genética , Análisis por Matrices de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacología , Proteínas Proto-Oncogénicas c-sis , ARN Mensajero/metabolismo , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-alpha, whereas PDGF-B activates both PDGFR-alpha and PDGFR-beta. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-beta but not PDGFR-alpha. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-alpha activation may result from PDGFR-alpha/beta heterodimerization.
Asunto(s)
Linfocinas , Factor de Crecimiento Derivado de Plaquetas/química , Factor de Crecimiento Derivado de Plaquetas/fisiología , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Bromodesoxiuridina/metabolismo , ADN Complementario/metabolismo , Dimerización , Relación Dosis-Respuesta a Droga , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Reacción en Cadena de la Polimerasa , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Distribución Tisular , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
Hepatocyte growth factor/scatter factor (HGF/SF) can stimulate growth of gastrointestinal epithelial cells in vitro; however, the physiological role of HGF/SF in the digestive tract is poorly understood. To elucidate this in vivo function, mice were analyzed in which an HGF/SF transgene was overexpressed throughout the digestive tract. Nearly a third of all HGF/SF transgenic mice in this study (28 of 87) died by 6 months of age as a result of sporadic intestinal obstruction of unknown etiology. Enteric ganglia were not overtly affected, indicating that the pathogenesis of this intestinal lesion was different from that operating in Hirschsprung's disease. Transgenic mice also exhibited a rectal inflammatory bowel disease (IBD) with a high incidence of anorectal prolapse. Expression of interleukin-2 was decreased in the transgenic colon, indicating that HGF/SF may influence regulation of the local intestinal immune system within the colon. These results suggest that HGF/SF plays an important role in the development of gastrointestinal paresis and chronic intestinal inflammation. HGF/SF transgenic mice may represent a useful model for the study of molecular mechanisms associated with a subset of IBD and intestinal pseudo-obstruction. Moreover, our data identify previously unappreciated side effects that may be encountered when using HGF/SF as a therapeutic agent.
Asunto(s)
Colitis Ulcerosa/patología , Factor de Crecimiento de Hepatocito/genética , Seudoobstrucción Intestinal/patología , Prolapso Rectal/patología , Animales , Colitis Ulcerosa/mortalidad , Femenino , Expresión Génica , Humanos , Interleucina-2/genética , Seudoobstrucción Intestinal/mortalidad , Intestino Grueso/patología , Intestino Grueso/fisiología , Masculino , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-kit/genética , Prolapso Rectal/mortalidad , Tasa de SupervivenciaRESUMEN
The fibroblast growth factor (FGF) family of signaling molecules has been implicated in normal developmental and physiological processes, as well as in human malignancy. Using a homology-based genomic DNA mining process, we identified a human gene encoding a novel member of the FGF family, that we designate FGF-20. The FGF-20 cDNA was isolated, and its sequence confirmed the gene prediction. FGF-20 is expressed in normal brain, particularly the cerebellum, and in some cancer cell lines. Recombinant FGF-20 protein induces DNA synthesis in a variety of cell types and is recognized by multiple FGF receptors. Ectopic expression of FGF-20 in NIH 3T3 cells renders the cells transformed in vitro and tumorigenic in nude mice. These results underscore the utility of mining genomic DNA databases and reveal FGF-20 to be a novel oncogene that may play a role in human cancer.
Asunto(s)
Transformación Celular Neoplásica , Factores de Crecimiento de Fibroblastos/fisiología , Células 3T3/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , División Celular/efectos de los fármacos , División Celular/genética , Transformación Celular Neoplásica/genética , ADN/biosíntesis , ADN/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/farmacología , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transfección , XenopusRESUMEN
Expression of platelet-derived growth factor (PDGF)-A and PDGF-B is increased in patients with proliferative retinopathies in which traction retinal detachments occur. Previous studies have demonstrated that increased expression of PDGF-A in the retina of transgenic mice results in retinal gliosis due to proliferation of astrocytes with different retinal phenotypes based on the time of onset and location of the PDGF-A production. In this study, we investigated the effects of PDGF-B in the retina using gain-of-function transgenic mice that express PDGF-B in photoreceptors. These mice show proliferation of astrocytes, pericytes, and, to a lesser extent, endothelial cells, resulting in ectopic cells on the surface and extending into the retina. The sheets of cells exert traction on the retina resulting in traction retinal detachments similar to those seen in humans with proliferative retinopathies. These studies suggest that PDGF-B has more dramatic effects in the retina than PDGF-A, because it acts on additional cell types, in particular on pericytes, which have a highly developed contractile apparatus. These studies in the retina suggest a means that could be used in other tissues throughout the body to achieve graded PDGF effects. They also provide a new model of traction retinal detachment that can be used to investigate new treatments for patients with proliferative retinopathies.
Asunto(s)
Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas Proto-Oncogénicas c-sis/biosíntesis , Desprendimiento de Retina/etiología , Animales , Astrocitos/metabolismo , Astrocitos/patología , Northern Blotting , Bovinos , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Técnica del Anticuerpo Fluorescente Directa , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Pericitos/metabolismo , Pericitos/patología , Células Fotorreceptoras de Vertebrados/patología , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Proto-Oncogénicas c-sis/genética , ARN Mensajero/metabolismo , Desprendimiento de Retina/metabolismo , Desprendimiento de Retina/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Using a differential display RT-PCR strategy to identify novel growth-factor-induced transcripts, we cloned and characterized the human homolog of yeast NOP5/NOP58, whose gene product has been implicated in the execution of early pre-rRNA processing steps. Human NOP5 cDNA was isolated from an M426 fibroblast cDNA library. Determination of the cDNA nucleotide sequence revealed an open reading frame of 1587 nucleotides encoding a predicted gene product of 529 amino acids and mass of 59554Da. The yeast and human NOP5 gene products were found to share 63% homology and 46% identity. NOP5 mRNA was induced within 2h of platelet-derived growth factor (PDGF) treatment of human M426 fibroblasts. Pretreatment with cycloheximide enhanced, while actinomycin blocked induction of the NOP5 transcript. In vitro translational analysis of the cDNA revealed a 60kDa species, consistent with the predicted molecular weight of the gene product. Ubiquitous, but differential NOP5 mRNA expression was revealed after Northern blot analysis of total RNA from several human tissues. Moreover, NOP5 mRNA expression was also demonstrated in cell lines of fibroblast, epithelial, and myeloid origin. A highly charged carboxy terminal domain and consensus phosphorylation sites were identified. The presence of potential regulatory elements, together with growth factor induction and widespread expression is consistent with the hypothesis that the NOP5 gene product may play a role in fundamental cellular growth processes.
Asunto(s)
ADN Complementario/aislamiento & purificación , Proteínas Nucleares , Factor de Crecimiento Derivado de Plaquetas/farmacología , Ribonucleoproteínas Nucleolares Pequeñas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Línea Celular , ADN Complementario/química , ADN Complementario/genética , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células K562 , Datos de Secuencia Molecular , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae , Análisis de Secuencia de ADN , Distribución Tisular , Células Tumorales CultivadasRESUMEN
A sensitive method for staining proteins after transfer from polyacrylamide gels to nitrocellulose paper is described. Transferred proteins are first derivatized by reaction of the nitrocellulose replica with sulfosuccinimidobiotin and are then reacted sequentially with streptavidin, rabbit anti-streptavidin, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Incubation with the enzyme substrate alpha-chloronaphthol, produces dark protein bands against a white background. The binding of streptavidin to the proteins is dependent on biotin derivatization as demonstrated by competition with biotinylated bovine serum albumin or 10 nM biotin. The procedure detects less than 5 ng of transferred protein in a single band and is thus 5-10 times more sensitive than horseradish peroxidase-conjugated avidin alone. For bovine serum albumin, the method is comparable in sensitivity to silver staining of protein in polyacrylamide gels.
Asunto(s)
Antígenos de Superficie/análisis , Avidina , Biotina , Técnicas para Inmunoenzimas/normas , Proteínas de la Membrana/análisis , Coloración y Etiquetado/métodos , Estreptavidina , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/inmunología , Colodión , Electroforesis en Gel de Poliacrilamida , Proteínas de la Membrana/inmunología , ConejosRESUMEN
Interleukin (IL)-4 plays an important role in the differentiation of naive T helper (Th) cells into Th2. Mast cells can produce a significant amount of IL-4 and have been proposed to play a major role in the induction of Th2 responses. Recently, it has been reported that mast cells have a distinct IL-15 receptor system different from that of T or natural killer cells. In the present study, we demonstrated that IL-15 induced IL-4 production from a mouse mast cell line, MC/9, and bone marrow-derived mast cells. IL-4 mRNA expression was increased by IL-15, suggesting that IL-15 promotes IL-4 expression at the transcriptional level. In these mast cells, signal transducer and activator of transcription (STAT) 6 were rapidly tyrosine-phosphorylated in response to IL-15. In MC/9 cells, the expression of a C-terminally truncated dominant negative form of STAT6 significantly suppressed the IL-4 mRNA up-regulation by IL-15, suggesting that STAT6 activation is essential for the IL-15-mediated IL-4 production. Additionally, tyrosine phosphorylation of Tyk2 was rapidly increased by IL-15 treatment in this cell line. Altogether, our results suggest that IL-15 plays an important role in stimulating early IL-4 production in mast cells that may be responsible for the initiation of Th2 response.
Asunto(s)
Interleucina-15/farmacología , Interleucina-4/biosíntesis , Mastocitos/fisiología , Transactivadores/fisiología , Animales , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Línea Celular , Ratones , Fosforilación , Factor de Transcripción STAT6 , Transducción de Señal/efectos de los fármacos , Células Th2/fisiología , TirosinaRESUMEN
Identification of the transcriptionally activated targets of receptor tyrosine kinases is critical to understanding biologic programs directing both normal and neoplastic growth. To elucidate these molecular processes, we identified genes induced by a potent mesenchymal mitogen, platelet-derived growth factor (PDGF). Using differential display reverse transcription-polymerase chain reaction technology, we isolated a novel growth factor-induced cDNA, San5. San5 transcript induction occurred within 60 min in NIH 3T3 fibroblasts and proceeded in the presence of cycloheximide. Maximal induction of the San5 transcript occurred between 8 and 16 h, concurrent with passage of fibroblasts through G(1). San5 message was potently induced by PDGF AA and BB and acidic and basic fibroblast growth factors, all strong activators of fibroblast proliferation, but not by epidermal growth factor and interleukin-4. In a murine hematopoietic progenitor cell line, San5 transcript induction strictly correlated with [(3)H]thymidine uptake. Isolation and sequencing of the murine San5 cDNA revealed amino acid sequence homology to yeast Nop5p, a nucleolar protein required for pre-rRNA processing and ribosome assembly. Strikingly, SAN5 was able to rescue a nop5 null mutant, implicating SAN5 in the process of ribosome biogenesis. Consistent with this result, SAN5 was localized to the nucleolus in both yeast and mouse. Thus, San5 may provide a link between growth factor receptor activation and the cellular translational machinery.
Asunto(s)
ADN Complementario/genética , Regulación Fúngica de la Expresión Génica , Factor de Crecimiento Derivado de Plaquetas/genética , Ribosomas/fisiología , Saccharomyces cerevisiae/fisiología , Células 3T3 , Secuencia de Aminoácidos , Animales , ADN Complementario/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Alineación de SecuenciaRESUMEN
Hepatocyte growth factor/scatter factor (HGF/SF) stimulates numerous cellular activities capable of contributing to the metastatic phenotype, including growth, motility, invasiveness, and morphogenetic transformation. When inappropriately expressed in vivo, an HGF/SF transgene induces numerous hyperplastic and neoplastic lesions. NK1 and NK2 are natural splice variants of HGF/SF; all interact with a common receptor, Met. Although both agonistic and antagonistic properties have been ascribed to each isoform in vitro, NK1 retains the full spectrum of HGF/SF-like activities when expressed as a transgene in vivo. Here we report that transgenic mice broadly expressing NK2 exhibit none of the phenotypes characteristic of HGF/SF or NK1 transgenic mice. Instead, when coexpressed in NK2-HGF/SF bitransgenic mice, NK2 antagonizes the pathological consequences of HGF/SF and discourages the subcutaneous growth of transplanted Met-containing melanoma cells. Remarkably, the metastatic efficiency of these same melanoma cells is dramatically enhanced in NK2 transgenic host mice relative to wild-type recipients, rivaling levels achieved in HGF/SF and NK1 transgenic hosts. Considered in conjunction with reports that in vitro NK2 induces scatter, but not other activities, these data strongly suggest that cellular motility is a critical determinant of metastasis. Moreover, our results demonstrate how alternatively structured ligands can be exploited in vivo to functionally dissociate Met-mediated activities and their downstream pathways.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Hígado/patología , Hígado/fisiología , Animales , División Celular , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia , Isoformas de Proteínas/genética , Empalme del ARNRESUMEN
Retinal astrocytes are located in the nerve fiber layer and along retinal blood vessels and have been hypothesized to participate in the induction and maintenance of the blood-retinal barrier. Platelet-derived growth factor-A (PDGF-A) is normally produced by retinal ganglion cells and is involved in astrocyte recruitment and proliferation. We used gain-of-function transgenic mice that express PDGF-A in photoreceptors to explore the roles of PDGF-A and astrocytes in the retina. Transgene-positive mice developed glial infiltration of the inner retina and had significantly less oxygen-induced retinal vascular closure and no neovascularization compared with littermate controls, which had prominent vascular closure and neovascularization. The increased survival of endothelial cells in transgenic mice in the face of oxygen-induced down-regulation of vascular endothelial growth factor was accompanied by an increase in astrocyte-derived fibroblast growth factor-2. Therefore, PDGF-A increases retinal astrocytes, which promote the survival of endothelial cells as well as their expression of barrier characteristics.
Asunto(s)
Gliosis/inducido químicamente , Isquemia/prevención & control , Factor de Crecimiento Derivado de Plaquetas/farmacología , Retina/efectos de los fármacos , Vasos Retinianos/efectos de los fármacos , Animales , Capilares/fisiopatología , Supervivencia Celular/efectos de los fármacos , Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/patología , Endotelio Vascular/fisiopatología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Hiperoxia/inducido químicamente , Hiperoxia/fisiopatología , Isquemia/inducido químicamente , Linfocinas/fisiología , Ratones , Ratones Transgénicos/genética , Neovascularización Patológica/prevención & control , Oxígeno , Fenotipo , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , ARN Mensajero/metabolismo , Retina/metabolismo , Vasos Retinianos/fisiopatología , Rodopsina/genética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Aberrant communication among growth factors and cytokines that regulate tissue homeostasis often results in malignancy. Among the many cell types that participate in this process, stromal fibroblasts communicate in a paracrine and juxtracrine manner with cells of epithelial, endothelial, and hematopoietic origin. For fibroblasts, platelet-derived growth factor (PDGF) is a major proliferative and differentiation agent. Interleukin-4 (IL-4), however, possesses only modulating functions in this cell type. Here, we investigated the consequences of deleting Stat6 on PDGF and IL-4 signaling, proliferation, and transcriptional activation by establishing and characterizing early passage fibroblasts from wild-type and Stat6 null mice. Both wild-type and Stat6-/- fibroblasts showed nearly identical PDGFR and IL-4R activation, gross substrate tyrosine phosphorylation, PI 3-kinase activation, as well as Stat1, 3 and 5 DNA binding activities. Unexpectedly, IL-4's enhancement of PDGF-induced [3H]thymidine incorporation was greatly diminished in Stat6-/-, but not wild-type fibroblasts. PDGF-induced [3H]thymidine uptake was largely unaffected. Strikingly, IL-4, but not PDGF induction of the proinflammatory gene products, IL-6 and MCP-1 was markedly reduced in Stat6-/- fibroblasts. Thus, Stat6 is an important and specific mediator of IL-4-enhanced PDGF-induced proliferation as well as IL-4's transcriptional activation of IL-6 and MCP-1.
Asunto(s)
Fibroblastos/patología , Fibroblastos/fisiología , Interleucina-4/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Transducción de Señal/genética , Transactivadores/genética , Activación Transcripcional/genética , Animales , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular Transformada , Eliminación de Gen , Ratones , Factor de Transcripción STAT6 , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacosRESUMEN
Keratinocyte growth factor (KGF) is an unusual fibroblast growth factor (FGF) family member in that its activity is largely restricted to epithelial cells, and added heparin/heparan sulfate inhibits its activity in most cell types. The effects of heparan sulfate proteoglycan (HSPG) on binding and signaling by acidic FGF (aFGF) and KGF via the KGFR were studied using surface-bound and soluble receptor isoforms expressed in wild type and mutant Chinese hamster ovary (CHO) cells lacking HSPG. Low concentrations of added heparin (1 microgram/mL) enhanced the affinity of ligand binding to surface-bound KGFR in CHO mutants, as well as ligand-stimulated MAP kinase activation and c-fos induction, but had little effect on binding or signaling in wild type CHO cells. Higher heparin concentrations inhibited KGF, but not aFGF, binding and signaling. In addition to the known interaction between HSPG and KGF, we found that the KGFR also bound heparin. The biphasic effect of heparin on KGF, but not aFGF, binding and signaling suggests that occupancy of the HSPG binding site on the KGFR may specifically inhibit KGF signaling. In contrast to events on the cell surface, added heparin was not required for high-affinity soluble KGF-KGFR interaction. These results suggest that high-affinity ligand binding is an intrinsic property of the receptor, and that the difference between the HSPG-dependent ligand binding to receptor on cell surfaces and the HSPG-independent binding to soluble receptor may be due to other molecule(s) present on cell surfaces.
Asunto(s)
Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/fisiología , Proteoglicanos de Heparán Sulfato/fisiología , Queratinocitos/fisiología , Receptores de Factores de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento/fisiología , Transducción de Señal , Animales , Sitios de Unión/genética , Células CHO , Cricetinae , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Heparina/metabolismo , Heparina/farmacología , Heparitina Sulfato/metabolismo , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Queratinocitos/metabolismo , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratas , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , TransfecciónRESUMEN
The molecular and genetic events that contribute to the genesis and progression of cutaneous malignant melanoma, a complex and aggressive disease with a high propensity for metastasis, are poorly understood due in large part to the dearth of relevant experimental animal models. Here we used transgenic mice ectopically expressing hepatocyte growth factor/scatter factor (HGF/SF) to show that the Met signaling pathway is an important in vivo regulator of melanocyte function, whose subversion induces malignant melanoma. Tumorigenesis occurred in stages, beginning with the abnormal accumulation of melanocytes in the epidermis and dermis and culminating in the development of metastatic melanoma. Oncogenesis in this model was driven by creation of HGF/SF-Met autocrine loops through forced expression of the transgenic ligand and apparent selection of melanocytes overexpressing endogenous receptor, rather than paracrine stimulation or mutational activation of c-met. Preference for liver as a metastatic target correlated with high HGF/SF-Met autocrine activity, consistent with the notion that such activity may influence colonization. Although basic fibroblast growth factor and its receptor were both weakly expressed in the majority of melanomas examined, high levels were found only in those rare neoplasms with low or undetectable HGF/SF and Met expression, suggesting that these two tyrosine kinase receptor autocrine loops serve a critical overlapping function in melanocytic tumorigenesis. Our data support a causal role for HGF/SF-Met signaling in the development of melanoma and acquisition of the metastatic phenotype. Moreover, this transgenic mouse should serve as a highly useful model, facilitating our understanding of mechanisms by which human melanoma progresses to malignancy and expediting the development of efficacious therapeutic modalities designed to constrain metastasis.
Asunto(s)
Factor de Crecimiento de Hepatocito/metabolismo , Melanocitos/metabolismo , Melanoma Amelanótico/etiología , Melanoma Amelanótico/secundario , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/patología , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-met/metabolismoRESUMEN
Stat6 signaling pathways have been correlated with functional responses induced by IL-4 and PDGF that may play a role in human malignancy. Utilizing fluorescence in situ hybridization, we mapped the human Stat6 gene to chromosome 12q bands 13.3-14.1, a breakpoint region implicated in a wide variety of solid tumors. To understand the genesis of three human Stat6 variant cDNAs, including a naturally occurring dominant negative species, we further characterized the genomic structure and flanking regions of the human Stat6 gene. The human Stat6 gene encompassed over 19 kb and contained 23 exons. For promoter studies, we introduced flanking sequence 5' of Stat6 exon 1 into a promoterless luciferase reporter vector and characterized basal promoter activity by deletion analysis. DNA sequence analysis revealed potential transcriptional regulation of the putative promoter through numerous consensus binding elements. Finally, we conclude that selective exon deletion and utilization of alternative donor/acceptor sites appear to explain best human Stat6 variant mRNAs.
Asunto(s)
Cromosomas Humanos Par 12/genética , Genes/genética , Neoplasias/genética , Transactivadores/genética , Células 3T3/citología , Células 3T3/metabolismo , Animales , Secuencia de Bases , Mapeo Cromosómico , Exones/genética , Humanos , Hibridación Fluorescente in Situ , Intrones/genética , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Factor de Transcripción STAT6 , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
Despite a wealth of experimental data implicating fibroblast growth factor (FGF) signaling in various developmental processes, genetic inactivation of individual genes encoding specific FGFs or their receptors (FGFRs) has generally failed to demonstrate their role in vertebrate organogenesis due to early embryonic lethality or functional redundancy. Here we show that broad mid-gestational expression of a novel secreted kinase-deficient receptor, specific for a defined subset of the FGF superfamily, caused agenesis or severe dysgenesis of kidney, lung, specific cutaneous structures, exocrine and endocrine glands, and craniofacial and limb abnormalities reminiscent of human skeletal disorders associated with FGFR mutations. Analysis of diagnostic molecular markers revealed that this soluble dominant-negative mutant disrupted early inductive signaling in affected tissues, indicating that FGF signaling is required for growth and patterning in a broad array of organs and in limbs. In contrast, transgenic mice expressing a membrane-tethered kinase-deficient FGFR were viable. Our results demonstrate that secreted FGFR mutants are uniquely effective as dominant-negative agents in vivo, and suggest that related soluble receptor isoforms expressed in wild-type mouse embryos may help regulate FGF activity during normal development.
Asunto(s)
Tipificación del Cuerpo/fisiología , Inducción Embrionaria/fisiología , Factores de Crecimiento de Fibroblastos/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Células 3T3 , Animales , Línea Celular , Desarrollo Embrionario y Fetal , Epidermis/embriología , Genes Dominantes , Ratones , Ratones Transgénicos , Mutación , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/genética , Proteínas Recombinantes de Fusión , Transducción de Señal , Solubilidad , TransgenesRESUMEN
Hepatocyte growth factor/scatter factor (HGF/SF) is a potent mitogen, motogen, and morphogen for epithelial cells expressing its tyrosine kinase receptor, the c-met proto-oncogene product, and is required for normal development in the mouse. Inappropriate stimulation of Met signal transduction induces aberrant morphogenesis and oncogenesis in mice and has been implicated in human cancer. NK1 is a naturally occurring HGF/SF splice variant composed of only the amino terminus and first kringle domain. While the biological activities of NK1 have been controversial, in vitro data suggest that it may have therapeutic value as an HGF/SF antagonist. Here, we directly test this hypothesis in vivo by expressing mouse NK1 in transgenic mice and comparing the consequent effects with those observed for mice carrying an HGF/SF transgene. Despite robust expression, NK1 did not behave as an HGF/SF antagonist in vivo. Instead, NK1-transgenic mice displayed most of the phenotypic characteristics associated with HGF/SF-transgenic mice, including enlarged livers, ectopic skeletal-muscle formation, progressive renal disease, aberrant pigment cell localization, precocious mammary lobuloalveolar development, and the appearance of mammary, hepatocellular, and melanocytic tumors. And like HGF/SF-transgenic livers, NK1 livers had higher levels of tyrosine-phosphorylated complexes associated with Met, suggesting that the mechanistic basis for the effects of NK1 overexpression in vivo was autocrine activation of Met. We conclude that NK1 acts in vivo as a partial agonist. As such, the efficacy of NK1 as a therapeutic HGF/SF antagonist must be seriously questioned.
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Empalme Alternativo , Factor de Crecimiento de Hepatocito/agonistas , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Expresión Génica , Factor de Crecimiento de Hepatocito/biosíntesis , Factor de Crecimiento de Hepatocito/genética , Humanos , Riñón/crecimiento & desarrollo , Hígado/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Neoplasias/etiología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-met/metabolismoRESUMEN
Interleukin (IL)-4-mediated nuclear signaling by Stat6 has been implicated in lymphoid cell proliferation and the transcriptional activation of genes encoding major histocompatability complex (MHC) class II molecules and Fc receptors. To investigate IL-4-mediated transcriptional events, we cloned two naturally occurring human Stat6 isoforms, Stat6b and Stat6c, that encoded an NH2-terminal truncation or an SH2 domain deletion, respectively. Stat6 variant mRNAs were differentially expressed in many human tissues. To elucidate the biologic role of each isoform, we examined the consequences of overexpression in IL-4-responsive FDC-P2 cells. Stat6 and Stat6b (to a lesser extent) enhanced DNA synthesis, up-regulated endogenous MHC class II and Fcgamma receptors, and became tyrosine phosphorylated in response to IL-4 stimulation. In contrast, Stat6c, which lacks functionally critical SH2 domain residues, unexpectedly inhibited IL-4-mediated mitogenesis and cell surface antigen expression and was not tyrosine phosphorylated. Although Stat6c only modestly diminished endogenous Stat6 tyrosine phosphorylation, it abolished endogenous Stat6 FcgammaRI and Iepsilon DNA binding activity and FcgammaRI-luciferase reporter transcriptional activation. Our results indicate that the molecular mechanism of inhibition by Stat6c was due to suppression of endogenous Stat6 dimer formation. Thus, Stat6b and Stat6c are naturally occurring attenuated and dominant negative Stat6 variants, respectively, that affect IL-4-mediated biologic responses through differential transcriptional regulation.
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Interleucina-4/fisiología , Transducción de Señal , Transactivadores/antagonistas & inhibidores , Secuencia de Aminoácidos , Línea Celular , ADN/metabolismo , ADN Complementario/aislamiento & purificación , Dimerización , Humanos , Datos de Secuencia Molecular , Fosforilación , ARN Mensajero/metabolismo , Factor de Transcripción STAT6 , Espectrometría de Fluorescencia , Timidina/metabolismo , Transactivadores/genética , Activación Transcripcional , Tirosina/metabolismoRESUMEN
Hepatocyte growth factor/scatter factor (HGF/SF) has been implicated as a renotrophic agent, capable of stimulating renal regeneration after unilateral nephrectomy or acute kidney failure. However, evaluation of the therapeutic utility of HGF/SF requires thorough analysis of its effects in an appropriate in vivo model system. To this end, the renal structure and function in HGF/SF transgenic mice were examined. Mice overexpressing HGF/SF in the kidney and serum demonstrated prominent tubular cystic disease and progressive glomerulosclerosis, and were susceptible to premature death from renal failure. The tubular phenotype appeared to result from HGF/SF-Met autocrine stimulation of the tubular epithelium and consequent hyperplasia. Electron microscopic examination of glomeruli, which also showed enhanced cellular proliferation, revealed ultrastructural features consistent with focal segmental glomerulosclerosis: an increase in mesangial matrix, effacement of foot processes, and thickening of basement membrane. These changes were not present at birth but developed progressively with age, which is consistent with the notion that HGF/SF may not be essential for the early stages of nephrogenesis, but may play an important role in renal epithelial renewal and regeneration. Thus, HGF/SF transgenic mice could be a useful model for dissecting the molecular mechanisms leading to polycystic disease and focal segmental glomerulosclerosis. Moreover, our results suggest that caution should be used when considering HGF/SF as a future therapeutic agent.
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Factor de Crecimiento de Hepatocito/fisiología , Glomérulos Renales/patología , Túbulos Renales/patología , Enfermedades Renales Poliquísticas/etiología , Animales , Femenino , Hiperplasia , Masculino , Ratones , Ratones TransgénicosRESUMEN
The mouse homolog of a novel human protein tyrosine kinase encoding gene, A6, was cloned and characterized. The human A6 cDNA is unique in that its gene product exhibited in vitro kinase activity but its predicted amino acid (aa) sequence revealed no consensus motifs commonly found within the kinase domain of protein kinase family members. Here, we isolated a mouse A6 cDNA clone from a murine myeloid progenitor 32D cell library using a 1.1 kb cDNA probe containing the entire human A6 open reading frame (ORF). Determination of the mouse A6 cDNA nucleotide (nt) sequence revealed an ORF of 1050 nt encoding a protein of 350 aa and a molecular mass of 40,201 Da. The mouse and human A6 gene products shared 93% identity. In vitro translation, as well as immunoprecipitation of 32D cell lysates confirmed expression of mouse A6 as a 40 kDa protein. Northern blot analysis of total RNA from mouse cell lines derived from diverse tissues including NIH 3T3 fibroblasts, L cell fibroblasts, C2C12 myoblasts, M1 myeloblasts, BALB/MK cells, 70Z/3 preB lymphocytes, and p388D1 monocytes demonstrated widespread A6 mRNA expression. A6 mRNA was also ubiquitously expressed at varying levels in all tissues examined. The identification of a potential actin/phosphoinositide binding domain and consensus phosphorylation sites, coupled with A6's expression in a variety of cell types suggest that the A6 gene product may play a role in basic cellular processes.