RESUMEN

Tumors are thought to be sustained by a reservoir of self-renewing cells, termed tumor-initiating cells or cancer stem cells. Osteosarcomas are high-grade sarcomas derived from osteoblast progenitor cells and are the most common pediatric bone malignancy. In this report we show that the stem cell transcription factor Sox2 is highly expressed in human and murine osteosarcoma (mOS) cell lines as well as in the tumor samples. Osteosarcoma cells have increased ability to grow in suspension as osteospheres, that are greatly enriched in expression of Sox2 and the stem cell marker, Sca-1. Depletion of Sox2 by short-hairpin RNAs in independent mOS-derived cells drastically reduces their transformed properties in vitro and their ability to form tumors. Sox2-depleted osteosarcoma cells can no longer form osteospheres and differentiate into mature osteoblasts. Concomitantly, they exhibit decreased Sca-1 expression and upregulation of the Wnt signaling pathway. Thus, despite other mutations, these cells maintain a requirement for Sox2 for tumorigenicity. Our data indicate that Sox2 is required for osteosarcoma cell self renewal, and that Sox2 antagonizes the pro-differentiation Wnt pathway that can in turn reduce Sox2 expression. These studies define Sox2 as a survival factor and a novel biomarker of self renewal in osteosarcomas, and support a tumor suppressive role for the Wnt pathway in tumors of mesenchymal origin. Our findings could provide the basis for novel therapeutic strategies based on inhibiting Sox2 or enhancing Wnt signaling for the treatment of osteosarcomas.

Asunto(s)

Neoplasias Óseas/genética , Proliferación Celular , Células Madre Neoplásicas , Osteosarcoma/genética , Factores de Transcripción SOXB1/genética , Animales , Antígenos Ly/genética , Antígenos Ly/metabolismo , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Neoplasias Óseas/patología , Diferenciación Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Osteosarcoma/patología , Transducción de Señal , Vía de Señalización Wnt

RESUMEN

The development and maintenance of most tissues and organs require the presence of multipotent and unipotent stem cells that have the ability of self-renewal as well as of generating committed, further differentiated cell types. The transcription factor Sox2 is essential for embryonic development and maintains pluripotency and self-renewal in embryonic stem cells. It is expressed in immature osteoblasts/osteoprogenitors in vitro and in vivo and is induced by fibroblast growth factor signaling, which stimulates osteoblast proliferation and inhibits differentiation. Sox2 overexpression can by itself inhibit osteoblast differentiation. To elucidate its function in the osteoblastic lineage, we generated mice with an osteoblast-specific, Cre-mediated knockout of Sox2. These mice are small and osteopenic, and mosaic for Sox2 inactivation. However, culturing calvarial osteoblasts from the mutant mice for 2-3 passages failed to yield any Sox2-null cells. Inactivation of the Sox2 gene by Cre-mediated excision in cultured osteoblasts showed that Sox2-null cells could not survive repeated passage in culture, could not form colonies, and arrested their growth with a senescent phenotype. In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability. Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression. These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

Asunto(s)

Osteoblastos/citología , Factores de Transcripción SOXB1/metabolismo , Factor de Transcripción Activador 2/metabolismo , Animales , Diferenciación Celular , Línea Celular , Linaje de la Célula , Desarrollo Embrionario , Ratones , Ratones Noqueados , Interferencia de ARN , Factores de Transcripción SOXB1/genética , Transducción de Señal

RESUMEN

This study explores the psychometric properties of the Spanish adaptation of the Mood Spectrum Self-Report (MOODS-SR), an instrument designed to assess a broad range of manifestations of mood psychopathology. A total of 71 Spanish subjects participated: 49 outpatients who met criteria for a mood disorder or generalized anxiety disorder, and 22 normal controls. The instrument proved to have good internal consistency and test-retest reliability. Significant positive correlations were found between the depressive subdomains of the questionnaire and the Beck Depression Inventory, as well as between the manic-hypomanic subdomains and the Clinician-Administered Rating Scale for Mania. Clinical subjects displayed higher mean scores than normal subjects in all domains, and patients with bipolar disorder displayed higher scores than patients with unipolar disorder in the Manic component, particularly in the Energy and the Cognition subdomains. Differences between patients with generalized anxiety and mood disorders were small. The former, however, did not differ from normal controls in several subdomains, whereas patients with mood disorders did.

Asunto(s)

Trastornos de Ansiedad/diagnóstico , Trastornos del Humor/diagnóstico , Psicometría/métodos , Encuestas y Cuestionarios , Adulto , Femenino , Humanos , Lenguaje , Masculino , Persona de Mediana Edad , Inventario de Personalidad , Reproducibilidad de los Resultados , Traducción

RESUMEN

BACKGROUND: Expression of fibroblast growth factors (FGFs) is increased in a substantial fraction of human prostate cancers in vivo and in prostate cancer cell lines. Altered FGF signaling can potentially have a variety of effects, including stimulating cell proliferation and inhibiting cell death. To determine the biologic significance of altered FGF signaling in human prostate cancer, we disrupted signaling by expression of a dominant-negative (DN) FGF receptor in prostate cancer cell lines. METHODS: PC-3, LNCaP, and DU145 prostate cancer cells were stably transfected with DN FGFR constructs, and LNCaP and DU145 cells were infected with a recombinant adenovirus expressing DN FGFR-1. The effect of DN FGFR-1 expression was assessed by colony-formation assays, cell proliferation assays, flow cytometry, and cytogenetic analysis. Key regulators involved in the G(2)-to-M cell cycle transition were assessed by western blotting to examine cyclin B1 expression and by in vitro kinase assay to assess cdc2 kinase activity. RESULTS: Stable transfection of the DN FGFR-1 construct inhibited colony formation by more than 99% in all three cell lines. Infection of LNCaP and DU145 prostate cancer cells with adenovirus expressing DN FGFR-1 led to extensive cell death within 48 hours. Flow cytometry and cytogenetic analysis revealed that the DN FGFR-1 receptor led to arrest in the G(2) phase of the cell cycle before cell death. Cyclin B1 accumulated in DN FGFR-1-infected LNCaP cells, but cdc2 kinase activity was decreased. CONCLUSIONS: These findings reveal an unexpected dependence of prostate cancer cells on FGF receptor signal transduction to traverse the G(2)/M checkpoint. The mechanism for the G(2) arrest is not clear. Our results raise the possibility that FGF-signaling antagonists might enhance the cell death induced by other prostate cancer therapies.

Asunto(s)

Neoplasias de la Próstata/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Transducción de Señal , Adenoviridae/genética , Western Blotting , Proteína Quinasa CDC2/metabolismo , Ciclo Celular , Muerte Celular , División Celular , Supervivencia Celular , Cromosomas , Ciclina B/biosíntesis , Ciclina B1 , Citogenética , Citometría de Flujo , Genes Dominantes , Humanos , Operón Lac , Masculino , Pruebas de Precipitina , Factores de Tiempo , Transfección , Células Tumorales Cultivadas

RESUMEN

Fibroblast growth factors (FGFs) are known to play an important role in the growth of normal prostatic epithelial cells. In addition to their effects on proliferation, FGFs can promote cell motility, increase tumor angiogenesis, and inhibit apoptosis, all of which play an important role in tumor progression. To determine whether FGFs are overexpressed in human prostate cancers, we analyzed 26 prostate cancer RNAs by reverse transcription-PCR for expression of FGF3, FGF4, and FGF6, which cannot be detected in normal prostate tissue by this technique. Fourteen of 26 prostate cancers expressed FGF6 mRNA. No expression of FGF3 or FGF4 was detected. An ELISA of tissue extracts of normal prostate, high-grade prostatic intraepithelial neoplasia (PIN), and prostate cancer for FGF6 showed that this growth factor was undetectable in normal prostate but was present at elevated levels in 4 of 9 PIN lesions and in 15 of 24 prostate cancers. Immunohistochemical analysis with anti-FGF6 antibody revealed weak staining of prostatic basal cells in normal prostate that was markedly elevated in PIN. In the prostate cancers, the majority of cases revealed expression of FGF6 by the prostate cancer cells themselves. In two cases, expression was present in prostatic stromal cells. Exogenous FGF6 was able to stimulate proliferation of primary prostatic epithelial and stromal cells, immortalized prostatic epithelial cells, and prostate cancer cell lines in tissue culture. FGF receptor 4, which is the most potent FGF receptor for FGF6, is expressed in the human prostate in vivo and in all of the cultured cell lines. Thus, FGF6 is increased in PIN and prostate cancer and can promote the proliferation of the transformed prostatic epithelial cells via paracrine and autocrine mechanisms.

Asunto(s)

Factores de Crecimiento de Fibroblastos/biosíntesis , Neoplasia Intraepitelial Prostática/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , División Celular/efectos de los fármacos , División Celular/fisiología , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Factor 6 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/farmacología , Factores de Crecimiento de Fibroblastos/fisiología , Humanos , Inmunohistoquímica , Masculino , Próstata/citología , Próstata/efectos de los fármacos , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas/farmacología , Proteínas Proto-Oncogénicas/fisiología , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/biosíntesis , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células Tumorales Cultivadas

RESUMEN

Fibroblast growth factors (FGF) play a critical role in bone growth and development affecting both chondrogenesis and osteogenesis. During the process of intramembranous ossification, which leads to the formation of the flat bones of the skull, unregulated FGF signaling can produce premature suture closure or craniosynostosis and other craniofacial deformities. Indeed, many human craniosynostosis disorders have been linked to activating mutations in FGF receptors (FGFR) 1 and 2, but the precise effects of FGF on the proliferation, maturation and differentiation of the target osteoblastic cells are still unclear. In this report, we studied the effects of FGF treatment on primary murine calvarial osteoblast, and on OB1, a newly established osteoblastic cell line. We show that FGF signaling has a dual effect on osteoblast proliferation and differentiation. FGFs activate the endogenous FGFRs leading to the formation of a Grb2/FRS2/Shp2 complex and activation of MAP kinase. However, immature osteoblasts respond to FGF treatment with increased proliferation, whereas in differentiating cells FGF does not induce DNA synthesis but causes apoptosis. When either primary or OB1 osteoblasts are induced to differentiate, FGF signaling inhibits expression of alkaline phosphatase, and blocks mineralization. To study the effect of craniosynostosis-linked mutations in osteoblasts, we introduced FGFR2 carrying either the C342Y (Crouzon syndrome) or the S252W (Apert syndrome) mutation in OB1 cells. Both mutations inhibited differentiation, while dramatically inducing apoptosis. Furthermore, we could also show that overexpression of FGF2 in transgenic mice leads to increased apoptosis in their calvaria. These data provide the first biochemical analysis of FGF signaling in osteoblasts, and show that FGF can act as a cell death inducer with distinct effects in proliferating and differentiating osteoblasts.

Asunto(s)

Apoptosis/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Osteoblastos/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento de Fibroblastos/genética , Transducción de Señal/efectos de los fármacos , Acrocefalosindactilia/genética , Fosfatasa Alcalina/metabolismo , Animales , Bromodesoxiuridina/metabolismo , Calcificación Fisiológica/genética , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Disostosis Craneofacial/genética , Fragmentación del ADN/genética , Factor 1 de Crecimiento de Fibroblastos , Histocitoquímica , Ratones , Ratones Transgénicos , Mutación Puntual , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transfección

RESUMEN

Several genetic forms of human dwarfism have been linked to activating mutations in FGF receptor 3, indicating that FGF signaling has a critical role in chondrocyte maturation and skeletal development. However, the mechanisms through which FGFs affect chondrocyte proliferation and differentiation remain poorly understood. We show here that activation of FGF signaling inhibits chondrocyte proliferation both in a rat chondrosarcoma (RCS) cell line and in primary murine chondrocytes. FGF treatment of RCS cells induces phosphorylation of STAT-1, its translocation to the nucleus, and an increase in the expression of the cell-cycle inhibitor p21WAF1/CIP1. We have used primary chondrocytes from STAT-1 knock-out mice to provide genetic evidence that STAT-1 function is required for the FGF mediated growth inhibition. Furthermore, FGF treatment of metatarsal rudiments from wild-type and STAT-1(-/-) murine embryos produces a drastic impairment of chondrocyte proliferation and bone development in wild-type, but not in STAT-1(-/-) rudiments. We propose that STAT-1 mediated down regulation of chondrocyte proliferation by FGF signaling is an homeostatic mechanism which ensures harmonious bone development and morphogenesis.

Asunto(s)

Desarrollo Óseo , Condrocitos/citología , Proteínas de Unión al ADN/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Células 3T3 , Animales , Desarrollo Óseo/efectos de los fármacos , Huesos/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Proteínas de Unión al ADN/genética , Factor 1 de Crecimiento de Fibroblastos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Huesos Metatarsianos/efectos de los fármacos , Huesos Metatarsianos/crecimiento & desarrollo , Ratones , Ratones Noqueados , Técnicas de Cultivo de Órganos , Fosforilación , Ratas , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Transactivadores/genética , Células Tumorales Cultivadas

RESUMEN

Normal breast tissue as well as most breast tumors are dependent on estrogen for growth. Breast tumors often progress to a hormone-independent state which is associated with poor prognosis. It has been proposed that activation of growth factor signaling pathways in the tumor cells may free them from hormonal control. Certain growth factors can mimic estrogen responses by activating the estrogen receptor via its phosphorylation by mitogen-activated protein (MAP) kinase. In this report, however, we show that fibroblast growth factor (FGF), despite activating MAP kinase, is growth-inhibitory for estrogen-dependent MCF-7 breast cancer cells. MCF-7 cells treated with FGFs exhibit slower growth than controls in both the presence and absence of estrogen, with a concomitant increase in the number of cells in G0/G1. Expression of a constitutively activated FGF receptor in these cells further decreases their growth rate, which is no longer influenced by FGF treatment. Activation of the FGF signaling pathway also reduces the induction of an estrogen-responsive CAT reporter plasmid by estrogen, an effect which appears to be independent of serine 118 in the estrogen receptor, a MAP kinase target site. The inhibitory effects of FGF are probably mediated through the sustained induction of the cyclin kinase inhibitor p21/WAF1/CIP1, which is upregulated at the mRNA and protein level by FGF. FGF treatment also results in the phosphorylation of STAT1. This upregulation of p21 and phosphorylation of STAT1 is not detectable in T47D breast cancer cells upon which FGF has no inhibitory effect.

Asunto(s)

Neoplasias de la Mama/metabolismo , Ciclinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Estrógenos/farmacología , Factores de Crecimiento de Fibroblastos/farmacología , Neoplasias Hormono-Dependientes/metabolismo , Transactivadores/metabolismo , Neoplasias de la Mama/patología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Regulación hacia Abajo , Inhibidores Enzimáticos/metabolismo , Regulación de la Expresión Génica , Humanos , Neoplasias Hormono-Dependientes/patología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Factor de Transcripción STAT1 , Transducción de Señal , Células Tumorales Cultivadas

RESUMEN

FGF signaling is clearly important for proper bone development, and several autosomally dominant forms of genetic bone disorders have been mapped to FGF receptors 1, 2, and 3. We have studied the biological effects of the most commonly mutated cysteine residue in FGFR-2 which is detected in individuals with Crouzon syndrome, an autosomally dominant trait which causes premature fusion of the skull bones (craniosynostosis). This Crouzon mutation replaces the cysteine at position 342 with tyrosine, thus disrupting the formation of the third immunoglobulin (Ig)-like loop in the extracellular portion of the receptor. By transfecting mutated and wild-type receptors into a variety of cell lines, we have shown that the C342Y mutation in FGFR-2 produces a receptor which is constitutively activated and capable of transforming NIH3T3 cells and preventing the differentiation of C2 myoblasts in the absence of ligand. Constitutive activation appears to result from the ability of this receptor to form stable interreceptor dimers which involve disulfide bonds between the remaining free cysteine in the mutant receptor. The altered conformation of the third Ig-like domain in the mutated receptor also results in a drastically reduced ability to bind FGF-1 or FGF-2 and in a reduced level of receptor glycosylation. Thus it appears that Crouzon syndrome results from constitutive activation of FGFR-2 and that uncontrolled FGF signaling produces alterations of intramembranous bone development and premature closing of cranial sutures.

Asunto(s)

Craneosinostosis/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Células 3T3 , Animales , Células COS , Diferenciación Celular , Craneosinostosis/metabolismo , Dimerización , Disulfuros , Genes Dominantes , Glicosilación , Ligandos , Ratones , Mutación , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas Receptoras/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/genética

RESUMEN

Signaling through FGF receptors, which constitute a family of membrane-spanning tyrosine kinases, can stimulate cell proliferation, induce or inhibit cell differentiation and plays an important role in development. Recently, mutations in FGF receptors have been shown to be associated with a number of genetically dominant human skeletal disorders. A remarkably conserved mutation (Gly 380-->Arg) in the transmembrane region of FGFR-3 has been shown to be responsible for achondroplasia (ACH) but it was not clear whether such mutations result in loss of receptor function or constitutive activation. We have therefore made mutations in the transmembrane regions of murine FGFR-2 and FGFR-3 and studied their effect on receptor activity. We show here that the ACH mutation in FGFR-3 as well as two similar mutations in FGFR-2 result in constitutive activation of these receptors. This is manifested in their ability to become autophosphorylated in the absence of ligand in L6 cells, transforming activity on NIH3T3 fibroblasts, and the ability to inhibit myogenic differentiation in the absence of growth factor. Thus the transmembrane region of FGFR-2 and FGFR-3 plays a regulatory role in receptor function and the ACH mutation produces a dominant oversignaling receptor which is no longer regulated by FGF binding. These findings also support the newly identified role of FGF signaling as a negative regulator of bone growth.

Asunto(s)

Proteínas Tirosina Quinasas , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Células 3T3 , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Transformación Celular Neoplásica , Humanos , Proteínas de la Membrana/química , Ratones , Datos de Secuencia Molecular , Músculos/citología , Fosforilación , Proteínas Tirosina Quinasas Receptoras/química , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Relación Estructura-Actividad , Transfección

RESUMEN

PURPOSE: To evaluate the expression of fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in human prostate. MATERIALS AND METHODS: RNA was extracted from surgically excised human prostate glands and from primary cultures of human prostatic epithelial and stromal cells. Expression of FGFs and FGF receptors was evaluated by Northern blotting and reverse-transcriptase polymerase chain reaction (RT-PCR) techniques. RESULTS: FGF7 (KGF) is the major FGF mRNA expressed in the human prostate, with smaller amounts of FGF2 and extremely small amounts of FGF1. For all three FGFs, the prostatic stromal cells were the primary site of expression. Prostatic epithelial cells express primarily the FGFR-3 IIIc isoform, which preferentially binds FGF1 over FGF2, with smaller amounts of the FGFR-2 IIIb (FGF7 binding) isoform. Prostatic stromal cells express primarily the FGFR-3 IIIc isoform and smaller amounts of FGFR-1 IIIc and FGFR-2 IIIc isoforms, which bind both FGF1 and FGF2. CONCLUSION: The pattern of expression of FGFs and FGF receptors in the prostate is consistent with a paracrine stimulation of epithelial growth by stromal-derived FGFs and potential autocrine stimulation of stromal cell proliferation by stromal FGFs.

Asunto(s)

Factores de Crecimiento de Fibroblastos/biosíntesis , Próstata/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/biosíntesis , Factores de Crecimiento de Fibroblastos/genética , Humanos , Masculino , Próstata/química , ARN/análisis , Receptores de Factores de Crecimiento de Fibroblastos/genética

RESUMEN

Mutant cDNAs coding for dominant-negative forms of the fibroblast growth factor receptors 1 (FGFR-1) and 2 (FGFR-2) that lack tyrosine kinase activity were ligated to a 2.2 kb DNA fragment containing the bovine rhodopsin promoter and used to generate transgenic mice. Six independent lines were generated with the FGFR-1 construct, and five were generated with the FGFR-2 construct. Five of the six FGFR-1 mutant lines and all five FGFR-2 mutant lines showed transgene expression in the retina by reverse transcription-PCR. By both in situ hybridization and immunohistochemistry, mutant FGFRs were found to be expressed specifically in photoreceptors of transgene-positive FGFR-1 and FGFR-2 mice. Lines expressing the FGFR-2 mutant showed progressive photoreceptor degeneration; the retinas showed minimal or no abnormalities at 1 month, but by 2 months they showed focal areas of thinning of the outer nuclear layer and disruption of photoreceptors. By 2-4 months, areas of complete loss of photoreceptors were seen. These abnormalities were not seen in control littermates not expressing the transgene. Mice from two FGFR-1 mutant lines showed focal areas of thinning of the outer nuclear layer and numerous photoreceptors with fragmented chromatin, whereas the other FGFR-1 lines showed minimal or no abnormalities. These data indicate that perturbation of FGF signaling in photoreceptors is associated with progressive photoreceptor degeneration, suggesting that one or more of the FGFs may act as a survival factor for photoreceptor cells.

Asunto(s)

Genes Dominantes , Ratones Transgénicos/genética , Células Fotorreceptoras/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Degeneración Retiniana/genética , Animales , Secuencia de Bases , Bovinos , Ratones , Sondas Moleculares/genética , Datos de Secuencia Molecular , Mutación , Conejos , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología

RESUMEN

We have studied the expression of two of the receptors for fibroblast growth factors, FGFR-1 and FGFR-2, in response to ligand binding and in embryonal carcinoma (EC cells). Exposure of mouse fibroblasts to FGF-4 or FGF-2 results in a drastic downregulation of the mRNA levels for FGFR-2, while expression of FGFR-1 mRNA appears unaffected. Furthermore, FGF-4 transformed cells display low levels of FGFR-2 mRNA and these levels are significantly increased by treatment with anti FGF-4 neutralizing antibodies. In undifferentiated F9 EC cells, the levels of FGFR-2 mRNA are very low and increase substantially upon induction of differentiation. The levels of mRNA for FGFR-1 are again unaffected. To gain information on the regulation of expression of the gene encoding FGFR-2 (bek) we have cloned the FGFR-2 promoter region and used it to drive the expression of plasmids encoding the bacterial CAT enzyme. Transfection of these plasmids into FGF treated and untreated cells did not produce significant variation in CAT activity, suggesting that FGFR-2 downregulation in response to ligand binding occurs mainly by a post-transcriptional mechanism. In contrast, plasmids containing as little as 140 nt of the FGFR-2 promoter region were regulated in F9 cells, showing substantially higher expression in differentiated than in undifferentiated cells. It appears therefore that FGFR-2 expression in fibroblasts and EC cells is regulated by somewhat different mechanisms. In contrast, FGFR-1 expression does not vary substantially under the conditions shown to affect FGFR-2 expression. The implications of these findings are discussed.

Asunto(s)

Carcinoma Embrionario/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Proteínas Proto-Oncogénicas/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Secuencia de Bases , Carcinoma Embrionario/patología , Diferenciación Celular , Línea Celular , Regulación hacia Abajo , Factor 4 de Crecimiento de Fibroblastos , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética

RESUMEN

Ligand-induced dimerization and transphosphorylation are thought to be important events by which receptor tyrosine kinases generate cellular signals. We have investigated the ability of signalling-defective, truncated fibroblast growth factor (FGF) receptors (FGFR-1 and FGFR-2) to block the FGF response in cells that express both types of endogenous FGF receptors. When these dominant negative receptors are expressed in NIH 3T3 cells transformed by the secreted FGF-4, the transformed properties of the cells can be reverted to various degrees, with better reversion phenotype correlating with higher levels of truncated receptor expression. Furthermore, truncated FGFR-2 is significantly more efficient at producing reversion than FGFR-1, indicating that FGF-4 preferentially utilizes the FGFR-2 signalling pathway. NIH 3T3 clones expressing these truncated receptors are more resistant to FGF-induced mitogenesis and also exhibit reduced tyrosine phosphorylation upon treatment with FGF. The block in FGF-signalling, however, can be overcome by the addition of excess growth factor. The truncated receptors have binding affinities that are four- to eightfold lower than those of wild-type receptors, as measured by Scatchard analysis. We also observed a partial specificity in the responses of truncated-receptor-expressing clones to FGF-2 or FGF-4. Our results suggest that the block to signal transduction produced by kinase-negative FGF receptors is achieved through a combination of dominant negative effects and competition for growth factor binding with functional receptors.

Asunto(s)

Transformación Celular Neoplásica/metabolismo , Factores de Crecimiento de Fibroblastos/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Células 3T3 , Animales , Sitios de Unión , Células CHO , Transformación Celular Neoplásica/genética , Cricetinae , Factores de Crecimiento de Fibroblastos/genética , Ratones , Mutagénesis , Fenotipo , Fosforilación , Receptores de Factores de Crecimiento de Fibroblastos/genética , Transducción de Señal , Transfección

RESUMEN

The role of heparin or heparan sulfates in the interaction of basic fibroblast growth factor (bFGF) with its high affinity receptor were investigated using purified extracellular ligand-binding region of FGF receptor-1 (FGFR-1) and intact receptors expressed in a myeloid cell line (32D) that does not express detectable levels of heparan sulfate proteoglycans or in Chinese hamster ovary (CHO) cell mutants defective in heparan sulfate synthesis. The purified extracellular domain of FGFR-1 formed complexes with 125I-bFGF both in the presence or absence of heparin. Intact FGFR-1 expressed in 32D cells also bound the same amount of 125I-bFGF in the presence or absence of heparin when saturating concentrations of bFGF were used. Varying the concentration of 125I-bFGF showed that heparin increased the amount of 125I-bFGF bound at low bFGF concentrations and increased the affinity of bFGF for its receptor by about 3-fold. To eliminate the possibility of alteration of bFGF properties through the chemical modification reactions, bFGF was labeled biosynthetically. The binding of biosynthetically labeled bFGF to FGFR-1 also did not require heparin. When FGFR-1 or FGFR-2 were expressed in mutant CHO cells deficient in heparan sulfate synthesis, the cells also bound 125I-bFGF in the absence of heparin, and the addition of heparin increased the affinity of bFGF for its receptors 2-3-fold. Thus, heparin or heparan sulfate is not required for the binding of bFGF to its receptors but increases the binding affinity to a moderate degree. Finally, the requirement for heparin in signal transduction through the receptor was investigated. Expression of c-fos mRNA was induced by bFGF in 32D cells expressing FGFR-1 to the same extent in the presence or absence of heparin.

Asunto(s)

Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparina/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Condroitinasas y Condroitín Liasas/farmacología , Expresión Génica/efectos de los fármacos , Genes fos , Liasa de Heparina , Humanos , Ratones , Polisacárido Liasas/farmacología , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Transfección

RESUMEN

The bek gene encodes a member of the high-affinity fibroblast growth factor receptor family. The BEK/FGFR-2 receptor is a membrane-spanning tyrosine kinase with the typical features of FGF receptors. We have cloned a murine bek cDNA and expressed it in receptor-negative Chinese hamster ovary cells and in 32D myeloid cells. The BEK receptor expressed in Chinese hamster ovary cells binds acidic FGF, basic FGF, and Kaposi FGF equally well but does not bind keratinocyte growth factor or FGF-5 appreciably. Upon treatment with basic FGF or Kaposi FGF, the BEK receptor is phosphorylated and a mitogenic response is achieved. Heparan sulfate proteoglycans have been shown to play an obligate role in basic FGF binding to the high-affinity FLG receptor. Unlike the BEK-expressing Chinese hamster ovary cells, 32D cells expressing the BEK receptor require the addition of exogenous heparin in order to grow in the presence of basic FGF or Kaposi FGF. We show that the addition of heparin greatly enhances the binding of radio-labeled basic FGF to the receptor. Thus the BEK receptor, like FLG, also requires an interaction with heparan sulfate proteoglycans to facilitate binding to its ligands.

Asunto(s)

Factores de Crecimiento de Fibroblastos/metabolismo , Heparina/farmacología , Proteínas Tirosina Quinasas/genética , Receptores de Superficie Celular/genética , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células CHO , División Celular/efectos de los fármacos , Línea Celular , Clonación Molecular , Cricetinae , ADN/genética , ADN/aislamiento & purificación , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Factor 1 de Crecimiento de Fibroblastos/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factores de Crecimiento de Fibroblastos/farmacología , Biblioteca de Genes , Cinética , Masculino , Datos de Secuencia Molecular , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos , Transfección

RESUMEN

We have cloned a murine cDNA on the basis of homology to the tyrosine kinase domain of the bek fibroblast growth factor receptor. This cDNA encodes a putative tyrosine kinase receptor with a unique structural pattern in its extracellular domain. It is a new member of the immunoglobulin superfamily with two immunoglobulin-like domains. It also contains two fibronectin type III domains which are found on diverse proteins such as receptor tyrosine phosphatases and neural cell adhesion molecules. This protein tyrosine kinase called ark (adhesion-related kinase) is likely to represent a new class of receptor tyrosine kinase. Ark mRNA appears to be expressed in most cell lines and adult tissues examined except those of hematopoietic lineage. It is undetectable in undifferentiated teratocarcinoma cells, F9 and N Tera 2.

Asunto(s)

Proteínas Oncogénicas , Proteínas Tirosina Quinasas , Proteínas/genética , Proteínas Tirosina Quinasas Receptoras , Secuencia de Aminoácidos , Sondas de ADN , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sistemas de Lectura Abierta , Proteínas Proto-Oncogénicas , Tirosina Quinasa del Receptor Axl

RESUMEN

We have cloned a murine cDNA encoding a tyrosine kinase receptor with about 90% similarity to the chicken fibroblast growth factor (FGF) receptor and the human fms-like gene (FLG) tyrosine kinase. This mouse receptor lacks 88 amino acids in the extracellular portion, leaving only two immunoglobulin-like domains compared to three in the chicken FGF receptor. The cDNA was cloned into an expression vector and transfected into receptor-negative CHO cells. We show that cells expressing the receptor can bind both basic FGF and Kaposi FGF. Although the receptor binds basic FGF with a 15- to 20-fold higher affinity, Kaposi FGF is able to induce down-regulation of the receptor to the same extent as basic FGF. The receptor is phosphorylated upon stimulation with both FGFs, DNA synthesis is stimulated, and a proliferative response is produced in cells expressing the receptor, whereas cells expressing the cDNA in the antisense orientation show none of these responses to basic FGF or Kaposi FGF. Thus this receptor can functionally interact with two growth factors of the FGF family.

Asunto(s)

Factores de Crecimiento de Fibroblastos/fisiología , Proteínas Tirosina Quinasas/fisiología , Receptores de Superficie Celular/fisiología , Secuencia de Aminoácidos , Animales , Western Blotting , División Celular , Línea Celular , Clonación Molecular , Cricetinae , Cricetulus , ADN/genética , Regulación hacia Abajo , Proteínas Filagrina , Ratones , Datos de Secuencia Molecular , Fosforilación , Receptores de Factores de Crecimiento de Fibroblastos , Sarcoma de Kaposi/fisiopatología

RESUMEN

Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen responsible for considerable morbidity in the general population. The results presented herein establish the basic fibroblast growth factor (FGF) receptor as a means of entry of HSV-1 into vertebrate cells. Inhibitors of basic FGF binding to its receptor and competitive polypeptide antagonists of basic FGF prevented HSV-1 uptake. Chinese hamster ovary (CHO) cells that do not express FGF receptors are resistant to HSV-1 entry; however, HSV-1 uptake is dramatically increased in CHO cells transfected with a complementary DNA encoding a basic FGF receptor. The distribution of this integral membrane protein in vivo may explain the tissue and cell tropism of HSV-1.

Asunto(s)

Receptores de Superficie Celular/fisiología , Simplexvirus/fisiología , Adsorción , Secuencia de Aminoácidos , Animales , Unión Competitiva , Línea Celular , Membrana Celular/microbiología , Cricetinae , ADN/genética , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Heparitina Sulfato/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/farmacología , Receptores de Superficie Celular/genética , Receptores de Factores de Crecimiento de Fibroblastos , Transfección