RESUMEN
Studies in rodents have shown that astroglial erbB tyrosine kinase receptors are key regulatory elements in neuron-glia communication. Although both astrocytes and deregulation of erbB functions have been implicated in the pathogenesis of many common human brain disorders, erbB signaling in native human brain astrocytes has never been explored. Taking advantage of our ability to perform primary cultures from the cortex and the hypothalamus of human fetuses, we conducted a thorough analysis of erbB signaling in human astrocytes. We showed that human cortical astrocytes express erbB1, erbB2, and erbB3, whereas human hypothalamic astrocytes express erbB1, erbB2, and erbB4 receptors. Ligand-dependent activation of different erbB receptor heterodimeric complexes in these two populations of astrocytes translated into different morphological and proliferative responses. Although morphological plasticity was more pronounced in hypothalamic astrocytes than in cortical astrocytes, the former showed a lower mitogenic potential. Decreasing erbB4 expression via siRNA-mediated gene knockdown revealed that erbB4 constitutively restrains basal proliferative activity in hypothalamic astrocytes. We further show that treatment of human astrocytes with a protein kinase C activator results in rapid tyrosine phosphorylation of erbB receptors that involves cleavage of endogenous membrane bound erbB ligands by metalloproteinases. Together, these results indicate that erbB signaling in primary human brain astrocytes is functional, region-specific, and can be activated in a paracrine and/or autocrine manner. In addition, by revealing that some aspects of astroglial erbB signaling are different between human and rodents, our results provide a molecular framework to explore the potential involvement of astroglial erbB signaling deregulation in human brain disorders.
Asunto(s)
Astrocitos/fisiología , Corteza Cerebral/citología , Receptores ErbB/metabolismo , Hipotálamo/citología , Transducción de Señal/fisiología , Análisis de Varianza , Bromodesoxiuridina , Proliferación Celular , Células Cultivadas , Receptores ErbB/genética , Transportador 1 de Aminoácidos Excitadores/metabolismo , Feto , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunoprecipitación/métodos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neurregulina-1/farmacología , ARN Interferente Pequeño/farmacología , Receptor ErbB-4 , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador alfa/farmacología , Tirosina/metabolismo , Vimentina/metabolismoRESUMEN
The growth factor, transforming growth factor-alpha (TGF-alpha) is strongly expressed in the hypothalamic circadian pacemaker, the suprachiasmatic nucleus (SCN). TGF-alpha is one of several SCN peptides recently suggested to function as a circadian output signal for the regulation of locomotor activity rhythms in nocturnal rodents. When infused in the brain, TGF-alpha suppresses activity. TGF-alpha suppresses other behaviors as well including feeding, resulting in weight loss. Elevated TGF-alpha is correlated with some cancers, and it is possible the TGF-alpha and its receptor, the epidermal growth factor receptor (EGFR), mediate fatigue and weight loss associated with cancer. If true for cancers outside of the brain, then systemic TGF-alpha should also affect behavior. We tested this hypothesis in hamsters with intraperitoneal injections or week-long subcutaneous infusions of TGF-alpha. Both treatments suppressed activity and infusions caused reduced food consumption and weight loss. To identify areas of the brain that might mediate these effects of systemic TGF-alpha, we used immunohistochemistry to localize cells with an activated MAP kinase signaling pathway (phosphorylated ERK1). Cells were activated in two hypothalamic areas, the paraventricular nucleus and a narrow region surrounding the third ventricle. These sites could not only be targets of TGF-alpha produced in the SCN but could also mediate effects of elevated TGF-alpha from tumors both within and outside the central nervous system.
Asunto(s)
Ingestión de Alimentos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Factor de Crecimiento Transformador alfa/fisiología , Pérdida de Peso/efectos de los fármacos , Animales , Cricetinae , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Hipotálamo/citología , Hipotálamo/metabolismo , Inmunohistoquímica , Infusiones Subcutáneas , Inyecciones Intraperitoneales , Masculino , Mesocricetus , Transducción de Señal/efectos de los fármacos , Tercer Ventrículo/citología , Tercer Ventrículo/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador alfa/administración & dosificación , Factor de Crecimiento Transformador alfa/farmacologíaRESUMEN
The activation of transforming growth factor alpha (TGFalpha)-erbB-1 and neuregulin-erbB-4 signaling pathways in hypothalamic astrocytes has been shown to play a key role in the process by which the neuroendocrine brain controls luteinizing hormone-releasing hormone (LHRH) secretion. Earlier studies suggested that tanycytes, an ependymoglial cell type of the median eminence, regulate LHRH release during the estrous cycle by undergoing plastic changes that alternatively allow or prevent direct access of the LHRH nerve terminals to the portal vasculature. Neither the molecules responsible for these plastic changes nor the underlying controlling mechanisms have been identified. Here we show that cultured tanycytes express erbB-1 and erbB-2, two of the four members of the erbB receptor family, and respond to TGFalpha with receptor phosphorylation, release of prostaglandin E2 (PGE2), and a PGE2-dependent increase in the release of TGFbeta1, a growth factor previously implicated in the glial control of LHRH secretion. Blockade of either erbB-1 receptor signal transduction or prostaglandin synthesis prevented the stimulatory effect of TGFalpha on both PGE2 and TGFbeta1 release. Time-lapse studies revealed that TGFalpha and TGFbeta1 have dramatically opposite effects on tanycyte plasticity. Whereas TGFalpha promotes tanycytic outgrowth, TGFbeta1 elicits retraction of tanycytic processes. Blockade of metalloproteinase activity abolished the effect of TGFbeta1, suggesting that TGFbeta1 induces tanycytic retraction by facilitating dissolution of the extracellular matrix. Prolonged (>12 hr) exposure of tanycytes to TGFalpha resulted in focal tanycytic retraction, an effect that was abolished by immunoneutralization of TGFbeta1 action, indicating that the retraction was attributable to TGFalpha-induced TGFbeta1 formation. These in vitro results identify tanycytes as targets of TGFalpha action and demonstrate that activation of erbB-1-mediated signaling in these cells results in plastic changes that, involving PGE2 and TGFbeta1 as downstream effectors, mimic the morphological plasticity displayed by tanycytes during the hours encompassing the preovulatory surge of LHRH.
Asunto(s)
Dinoprostona/biosíntesis , Epéndimo/metabolismo , Receptores ErbB/metabolismo , Eminencia Media/metabolismo , Proteínas del Tejido Nervioso , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Antígenos de Diferenciación/biosíntesis , Astrocitos/citología , Astrocitos/metabolismo , Comunicación Celular/fisiología , Células Cultivadas , Fosfoproteína 32 Regulada por Dopamina y AMPc , Epéndimo/citología , Epéndimo/efectos de los fármacos , Hipotálamo/citología , Metaloproteinasas de la Matriz/metabolismo , Eminencia Media/citología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Fosfoproteínas/biosíntesis , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptor ErbB-2/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor de Crecimiento Transformador alfa/farmacología , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1RESUMEN
Hepatocyte growth factor (HGF) is a potent mitogen for a variety of cells including hepatocytes. While rat oval cells are supposed to be one of hepatic stem cells, biological effects of HGF on oval cells and their relevant signal transduction pathways remain to be determined. We sought to investigate them on OC/CDE22 rat oval cells, which are established from the liver of rats fed a choline-deficient/DL-ethionine-supplemented diet. The oval cells were cultured on fibronectin-coated dishes and stimulated with recombinant HGF, transforming growth factor-alpha (TGF-alpha), and thrombopoietin (TPO) under the serum-free medium condition. HGF treatment enhanced [3H]thymidine incorporation into oval cells in a dose-dependent manner. On the contrary, treatment with TGF-alpha or TPO had no significant effects on [3H]thymidine incorporation into the oval cells. c-Met protein was phosphorylated at the tyrosine residues after the HGF treatment. AKT, extracellular signal-regulated kinase 1/2 (ERK1/2), and p70(s6k) were simultaneously activated after the HGF stimulation, peaking at 30min after the treatment. The activation of AKT, p70(s6k), and ERK1/2 induced by HGF was abolished by pre-treatment with LY294002, a phosphoinositide 3-OH kinase (PI3K) inhibitor, and U0126, a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, respectively. When the cells were pre-treated with LY294002 prior to the HGF stimulation, the proliferative action of HGF was completely abrogated, implying that the PI3K/AKT signaling pathway is responsible for the biological effect of HGF. These in vitro data indicate that HGF exerts a proliferative action on hepatic oval cells via activation of the PI3K/AKT signaling pathway.
Asunto(s)
Factor de Crecimiento de Hepatocito/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Regeneración Hepática/efectos de los fármacos , Regeneración Hepática/fisiología , Complejos Multienzimáticos , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Trombopoyetina/farmacología , Factor de Crecimiento Transformador alfa/farmacologíaRESUMEN
The circadian clock in the suprachiasmatic nucleus (SCN) is thought to drive daily rhythms of behaviour by secreting factors that act locally within the hypothalamus. In a systematic screen, we identified transforming growth factor (TGF)alpha as a likely SCN inhibitor of locomotion. TGFalpha is expressed rhythmically in the SCN, and when infused into the 3rd ventricle it reversibly inhibits locomotor activity and disrupts circadian sleep-wake cycles. These actions are mediated by epidermal growth factor (EGF) receptors, which we identified on neurons in the hypothalamic subparaventricular zone. Mice with a hypomorphic EGF receptor mutation exhibit excessive daytime locomotor activity and fail to suppress activity when exposed to light. These results implicate EGF receptor signalling in the daily control of locomotor activity, and they identify a neural circuit in the hypothalamus that likely mediates the regulation of behaviour both by the SCN and the retina.
Asunto(s)
Ritmo Circadiano/fisiología , Receptores ErbB/fisiología , Actividad Motora/fisiología , Sueño/fisiología , Animales , Ritmo Circadiano/efectos de los fármacos , Cricetinae , Receptores ErbB/genética , Hipotálamo/fisiología , Actividad Motora/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retina/fisiología , Transducción de Señal , Sueño/efectos de los fármacos , Núcleo Supraquiasmático/fisiología , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/farmacología , Factor de Crecimiento Transformador alfa/fisiologíaAsunto(s)
Ritmo Circadiano/fisiología , Receptores ErbB/metabolismo , Hipotálamo/metabolismo , Actividad Motora , Neuronas/metabolismo , Núcleo Supraquiasmático/metabolismo , Factor de Crecimiento Transformador alfa/metabolismo , Animales , Relojes Biológicos/fisiología , Ritmo Circadiano/efectos de los fármacos , Cricetinae , Oscuridad , Receptores ErbB/genética , Luz , Ratones , Actividad Motora/efectos de los fármacos , Mutación , Células Ganglionares de la Retina/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador alfa/farmacologíaRESUMEN
The circadian clock in the suprachiasmatic nucleus (SCN) is thought to drive daily rhythms of behavior by secreting factors that act locally within the hypothalamus. In a systematic screen, we identified transforming growth factor-alpha (TGF-alpha) as a likely SCN inhibitor of locomotion. TGF-alpha is expressed rhythmically in the SCN, and when infused into the third ventricle it reversibly inhibited locomotor activity and disrupted circadian sleep-wake cycles. These actions are mediated by epidermal growth factor (EGF) receptors on neurons in the hypothalamic subparaventricular zone. Mice with a hypomorphic EGF receptor mutation exhibited excessive daytime locomotor activity and failed to suppress activity when exposed to light. These results implicate EGF receptor signaling in the daily control of locomotor activity, and identify a neural circuit in the hypothalamus that likely mediates the regulation of behavior both by the SCN and the retina.
Asunto(s)
Ritmo Circadiano/fisiología , Receptores ErbB/metabolismo , Hipotálamo/metabolismo , Actividad Motora , Sueño/fisiología , Núcleo Supraquiasmático/metabolismo , Animales , Relojes Biológicos/efectos de los fármacos , Relojes Biológicos/fisiología , Temperatura Corporal/efectos de los fármacos , Ventrículos Cerebrales/metabolismo , Ritmo Circadiano/efectos de los fármacos , Cricetinae , Oscuridad , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Femenino , Ligandos , Luz , Masculino , Mesocricetus , Ratones , Actividad Motora/efectos de los fármacos , Vías Nerviosas/fisiología , Neuronas/metabolismo , Mutación Puntual , Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Transducción de Señal , Sueño/efectos de los fármacos , Factor de Crecimiento Transformador alfa/administración & dosificación , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo , Factor de Crecimiento Transformador alfa/farmacologíaRESUMEN
Mutations of the VHL tumor suppressor gene occur in patients with VHL disease and in the majority of sporadic clear cell renal carcinomas (VHL(-/-) RCC). Loss of VHL protein function is associated with constitutive expression of mRNAs encoding hypoxia-inducible proteins, such as vascular endothelial growth factor. Overproduction of angiogenic factors might explain why VHL(-/-) RCC tumors are so highly vascularized, but whether this overproduction is sufficient for oncogenesis still remains unknown. In this report, we examined the activity of transforming growth factor-alpha (TGF-alpha), another VHL-regulated growth factor. We show that TGF-alpha mRNA and protein are hypoxia-inducible in VHL(-/-) RCC cells expressing reintroduced VHL. In addition to its overexpression by VHL(-/-) RCC cells, TGF-alpha can also act as a specific growth-stimulatory factor for VHL(-/-) RCC cells expressing reintroduced wild-type VHL, as well as primary renal proximal tubule epithelial cells, the likely site of origin of RCC. This role is in contrast to those of other growth factors overexpressed by VHL(-/-) RCC cells, such as vascular endothelial growth factor and TGF-beta1, which do not stimulate RCC cell proliferation. A TGF-alpha-specific antisense oligodeoxynucleotide blocked TGF-alpha production in VHL(-/-) RCC cells, which led to the dependence of those cells on exogenous growth factors to sustain growth in culture. Growth of VHL(-/-) RCC cells was also significantly reduced by a drug that specifically inhibits the epidermal growth factor receptor, the receptor through which TGF-alpha stimulates proliferation. These results suggest that the generation of a TGF-alpha autocrine loop as a consequence of VHL inactivation in renal proximal tubule epithelial cells may provide the uncontrolled growth stimulus necessary for the initiation of tumorigenesis.
Asunto(s)
Genes Supresores de Tumor , Ligasas , Proteínas/fisiología , Factor de Crecimiento Transformador alfa/fisiología , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Adenocarcinoma de Células Claras , División Celular/efectos de los fármacos , Hipoxia de la Célula , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Regulación de la Expresión Génica , Sustancias de Crecimiento/farmacología , Humanos , Insulina/farmacología , Neoplasias Renales , Oligodesoxirribonucleótidos Antisentido , Proteínas/genética , Quinazolinas/farmacología , Selenio/farmacología , Transferrina/farmacología , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo , Factor de Crecimiento Transformador alfa/farmacología , Células Tumorales Cultivadas , Tirfostinos/farmacología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Enfermedad de von Hippel-LindauRESUMEN
BACKGROUND: Targeting epigenetic events associated with autonomous growth of advanced prostate cancer (PCA) is a practical approach for its control, prevention, and treatment. Recently we showed that treatment of prostate carcinoma DU145 cells with cancer preventive flavonoid silymarin at 100-200 microM doses inhibits erbB1-Shc mitogenic signaling and modulates cell cycle regulators leading to a G1 arrest and inhibition of cell growth and anchorage-independent colony formation. Here, we asked the question whether these important findings could be extended to other cancer preventive flavonoids and isoflavones such as epigallocatechin 3-gallate (EGCG) and genistein. METHODS: DU145 cells were treated with similar doses (100-200 microM) of silymarin, genistein or EGCG, cell lysates prepared, and levels of activated signaling molecules (erbB1-Shc-ERK1/2) and cell cycle regulators (CDKIs, CDKs, and cyclins) analyzed employing immunoprecipitation and/or immunoblotting techniques. Cell growth studies were done by cell counting during 5 days of treatment with these agents, and cell death was determined by Trypan blue staining. RESULTS: Treatment of cells with silymarin, genistein or EGCG at 100-200 microM resulted in a complete inhibition of TGFalpha-caused activation of erbB1 followed by a moderate to strong inhibition (10-90%) of Shc activation without an alteration in their protein levels. Silymarin and genistein, but not EGCG, also inhibited (10% to complete) ERK1/2 activation suggesting that these agents impair erbB1-Shc-ERK1/2 signaling in DU145 cells. In other studies, silymarin, genistein or EGCG caused a strong induction of Cip1/p21 (up to 2.4-fold) and Kip1/p27 (up to 150-fold), and a strong decrease in CDK4 (40-90%) but had moderate effect on CDK2, and cyclins D1 and E. An enhanced level of CDKIs also led to an increase in their binding to CDK4 and CDK2. Treatment of cells with silymarin, genistein or EGCG also resulted in 50-80% cell growth inhibition at lower doses, and complete inhibition at higher doses. In contrast to silymarin, higher doses of genistein showed cytotoxic effect causing 30-40% cell death. A more profound cytotoxic effect was observed with EGCG accounting for 50% cell death at lower doses and complete loss of viability at higher doses. CONCLUSIONS: These results suggest that similar to silymarin, genistein and EGCG also inhibit mitogenic signaling pathway(s) and alter cell cycle regulators, albeit at different levels, leading to growth inhibition and death of advanced and androgen-independent prostate carcinoma cells. More studies are, therefore, needed with these agents to explore their anti-carcinogenic potential against human prostate cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Anticarcinógenos/farmacología , Catequina/análogos & derivados , Catequina/farmacología , Genisteína/farmacología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/prevención & control , Silimarina/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Muerte Celular/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/fisiología , Receptores ErbB/fisiología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/fisiología , Extractos Vegetales/farmacología , Neoplasias de la Próstata/patología , Unión Proteica , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Proteínas/fisiología , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Factor de Crecimiento Transformador alfa/antagonistas & inhibidores , Factor de Crecimiento Transformador alfa/farmacologíaRESUMEN
Transforming growth factor-alpha (TGF-alpha) contributes to the progression of mammary carcinogenesis in part through synergistic augmentation of estradiol (E2) action. To investigate this further, we sought to determine (1) whether the duration of TGF-alpha treatment might influence the nature of the TGF-alpha/E2 interaction, and (2) whether TGF-alpha would behave in a similar manner when combined with phytoestrogens. To this end, we transfected T47-D breast cancer cells with an estrogen-responsive reporter and then treated the cells (for 4-48 h) with varying concentrations of TGF-alpha, E2, the antiestrogen 4-hydroxy-tamoxifen (HOT), and/or one of three phytoestrogens. Our findings revealed that TGF-alpha has short-term synergistic and long-term inhibitory effects on E2- and phytoestrogen-regulated gene expression. Furthermore, this secondary inhibition of E2 action by TGF-alpha was similar in magnitude to that imposed by HOT. These findings demonstrate a novel role for TGF-alpha and invite reevaluation of current models regarding TGF-alphas interactions with E2 in breast cancer cells. Our results also raise the possibility that phytoestrogens, which interact with TGF-alpha in a manner conceptually identical to that of E2, may subserve a regulatory function in breast cancer cells.
Asunto(s)
Adenocarcinoma/genética , Neoplasias de la Mama/genética , Estrógenos no Esteroides/farmacología , Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Isoflavonas , Factor de Crecimiento Transformador alfa/farmacología , Sinergismo Farmacológico , Antagonistas de Estrógenos/farmacología , Humanos , Luciferasas/metabolismo , Fitoestrógenos , Preparaciones de Plantas , Receptores de Estrógenos/agonistas , Receptores de Estrógenos/efectos de los fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Transfección/genética , Células Tumorales CultivadasRESUMEN
Recent studies in several neuronal lineages suggest that extrinsic factors such as polypeptide growth factors regulate various stages of neuronal development, from initial commitment of multipotent progenitors to induction of specific gene expression that is characteristic of terminal neuronal differentiation. In the present study, immortalized hypothalamic neurons of the GT1-1 lineage were used to analyze proliferative, as well as morphological and molecular differentiation actions of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and insulin-like growth factor-I (IGF-I). These effects were compared with those induced by specific activators of protein kinase A and C pathways, which potently inhibited cell proliferation and gonadotropin-releasing hormone (GnRH) gene expression, but stimulated morphological neuronal maturation as determined by the length and number of neurite outgrowth. bFGF exerted a broad spectrum of stimulatory effects, increasing the rate of proliferation measured both by the incorporation of 3H-thymidine and by cell number, and parameters of terminal differentiation, such as neurite outgrowth and induction of gene expression. bFGF stimulated the expression of the hybrid transgene-containing portions of the rat GnRH promoter. In contrast, EGF, TGF-alpha, and IGF-I inhibited cell proliferation, while having subtle effects on neurite outgrowth. Thus, GT1-1 cells appear to be differentially responsive to distinct neurotrophic factors, providing a model for studying the specific effects of neurotrophic factors on functional differentiation, migration, and connectivity of hypothalamic neurons.
Asunto(s)
Gonadotropinas/metabolismo , Sustancias de Crecimiento/farmacología , Neuritas/efectos de los fármacos , Neuronas/citología , Animales , Carcinógenos/farmacología , Recuento de Células , División Celular/efectos de los fármacos , Línea Celular Transformada , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Expresión Génica/efectos de los fármacos , Hormona Liberadora de Gonadotropina/genética , Hipotálamo/citología , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Neuritas/fisiología , Neuronas/enzimología , Neuronas/ultraestructura , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Timidina/metabolismo , Timidina/farmacología , Factor de Crecimiento Transformador alfa/farmacología , Transgenes/fisiología , TritioRESUMEN
It has been shown in the past that extra hair cells or supernumerary cells can be produced when neonatal cochleae are maintained in vitro. In this report, we investigated the effects of the culture methods, molecules and growth factors that are thought to be involved in cell proliferation. Quantitative studies of supernumerary hair cells were made by measuring the cell density over the entire spiral lamina at two postnatal stages: birth and 3 days after birth. With a standard feeding solution without serum, a difference in cell density was observed between the two methods of culture. Cochlear explants in a standard feeding solution supplemented with serum showed an increase of cell density only when the explantation is made at birth. Retinoic acid added to the standard feeding solution did not increase the hair cell density, while insulin induced an increase, especially at 5 micrograms/ml. Several growth factors were tested. Epidermal growth factor (EGF) presented a dose dependent effect with an increase of up to 30% of hair cell density that was observed in the basal region when the explantation was made at birth. Transforming growth factor-alpha did not induce an increase of cell density, whereas transforming growth factor-beta presented an effect on hair cell density, with a dose dependent effect reaching 37.4% for the basal inner hair cells. Interpretation of these results is limited because of the lack of data concerning the presence of specific membrane receptors. One possibility is that insulin stimulates hair cell differentiation from existing undifferentiated cells. Another hypothesis may be related to the EGF and transforming growth factor-beta, where these molecules might induce transdifferentiation of cells by acting on the transmembrane molecules and the extracellular matrix.
Asunto(s)
Cóclea/citología , Cóclea/fisiología , Células Ciliadas Auditivas Internas/fisiología , Animales , Animales Recién Nacidos , Recuento de Células , Células Cultivadas , Medios de Cultivo , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales , Epitelio/fisiología , Hipoglucemiantes/farmacología , Insulina/farmacología , Regeneración Nerviosa/fisiología , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador alfa/farmacología , Factor de Crecimiento Transformador beta/farmacología , Tretinoina/farmacologíaRESUMEN
Previous studies demonstrated the involvement of transforming growth factor-alpha (TGF alpha), a member of the epidermal growth factor (EGF) family, in the developmental regulation of hypothalamic LHRH release. Although both TGF alpha and EGF stimulate LHRH release, they do not appear to act directly on LHRH neurons, as no EGF/TGF alpha receptors are detected on these cells in vivo. Instead, the stimulatory effect of TGF alpha on LHRH release seems to require a glial intermediacy. The present study identifies one of the glial molecules involved in this process. In vitro exposure of purified hypothalamic astrocytes to TGF alpha or EGF in a defined medium led to activation of the cyclooxygenase-mediated pathway of arachidonic acid metabolism, as indicated by an increase in PGE2 release, but failed to affect lipooxygenase-mediated metabolism, as assessed by the lack of increase in leukotriene C4 production; addition of TGF alpha- (T-CM) or EGF-conditioned medium to cultures of LHRH-producing GT1-1 cells stimulated LHRH release. In contrast, direct exposure of GT1-1 cells to the growth factors was ineffective. Incubation of the cells in medium conditioned by untreated astrocytes (CM) was also ineffective. Blockade of either EGF receptor signal transduction or cyclooxygenase activity in the astrocytic cultures prevented both TGF alpha-induced PGE2 formation in astrocytes and the stimulatory effect of T-CM on LHRH release. Immunoneutralization of PGE2 actions or selective removal of the PG from T-CM also prevented T-CM-induced LHRH release. Addition of exogenous PGE2 restored the effect. Thus, PGE2 is one of the glial molecules involved in mediating the stimulatory effect of TGF alpha on LHRH release. The effectiveness of PGE2 in eliciting LHRH release was, however, greatly reduced when PG was delivered to GT1-1 cells in astrocyte-defined medium instead of CM. Thus, astrocytes appear to produce a yet to be identified substance(s) that facilitates the stimulatory effect of PGE2 on LHRH output. We postulate that the ability of TGF alpha to enhance LHRH release depends on the potentiating interaction of PGE2 with these additional glial-derived molecules.
Asunto(s)
Astrocitos/efectos de los fármacos , Hormona Liberadora de Gonadotropina/metabolismo , Hipotálamo/efectos de los fármacos , Factor de Crecimiento Transformador alfa/farmacología , Animales , Ácido Araquidónico/metabolismo , Astrocitos/metabolismo , Dinoprostona/fisiología , Receptores ErbB/fisiología , Femenino , Hipotálamo/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Transducción de SeñalAsunto(s)
Huesos/enzimología , Dinoprostona/farmacología , Interleucina-1/farmacología , Isoenzimas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador alfa/farmacología , Factor de Crecimiento Transformador beta/farmacología , Células 3T3 , Animales , Ciclooxigenasa 1 , Ciclooxigenasa 2 , ADN Complementario , Dinoprostona/biosíntesis , Sinergismo Farmacológico , Inducción Enzimática , Cinética , Proteínas de la Membrana , Ratones , ARN Mensajero/biosíntesis , Transcripción Genética/inmunologíaRESUMEN
Hepatocyte growth factor (HGF) is a potent mitogen for rat and human hepatocytes in primary culture and appears to be the physiological hepatotrophic factor that triggers or modulates liver regeneration. Regulation of HGF gene expression and the protein production in human skin fibroblasts was examined. Addition of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF) and transforming growth factor-alpha (TGF-alpha) to confluent cultures of the cells markedly stimulated HGF secretion from the cells. The stimulating effect of EGF, PDGF and bFGF was further investigated. The effect of all three growth factors was maximal at 3-30 ng/ml and was accompanied by an increase in HGF mRNA levels. The mRNA levels were not elevated at 5 h but were at 10 h or more after addition of EGF. The levels of HGF mRNA in fibroblasts treated with the optimal doses of EGF, PDGF, bFGF, aFGF and TGF-alpha for 24 h were 6, 4, 5, 4 and 5 times that of control cultures incubated in medium only, respectively. The growth factor-induced HGF mRNA expression and HGF secretion was inhibited by addition of TGF-beta 1 or dexamethasone. Pretreatment with a high dose of phorbol 12-myristate 13-acetate (PMA), which causes down-regulation in protein kinase C (PKC) activity and PMA-induced HGF secretion, did not reduce the effects of the growth factors on HGF mRNA expression and HGF secretion, but rather enhanced them.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Factor 1 de Crecimiento de Fibroblastos/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor de Crecimiento de Hepatocito/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/farmacología , Piel/metabolismo , Northern Blotting , Células Cultivadas , ADN/biosíntesis , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Lactante , Cinética , Proteína Quinasa C/metabolismo , ARN Mensajero/biosíntesis , Proteínas Recombinantes/farmacología , Piel/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Factor de Crecimiento Transformador alfa/farmacologíaRESUMEN
Growth factors have been shown to play a role in intestinal epithelial growth regulation and transformation. Utilizing standard differential cloning techniques, we have isolated a growth factor-inducible gene (RS-2) from rat intestinal epithelial cells that has approximately 95% homology to the mouse mitogen-inducible cyclooxygenase (COX-2) at the amino acid level. This cDNA hybridizes to a approximately 4.5-kb mRNA from transforming growth factor (TGF)-alpha-stimulated rat intestinal epithelial (RIE-1) cells and is constitutively expressed in vivo in adult rat kidney and brain. Nuclear run-on experiments demonstrate that the increase of RS-2 mRNA after TGF-alpha stimulation is in part due to an increased transcription rate of the gene. The coding region for RS-2 was subcloned into a pCMV-2 expression vector, and the RS-2 protein was expressed in COS-1 cells. Microsomal fractions isolated from the COS-1 cells transfected with the RS-2 expression vector contained cyclooxygenase activity. In addition to the production of prostaglandins, the recombinant RS-2 protein also catalyzed the formation of three other eicosanoid products. In summary, we have cloned a mitogen-inducible cyclooxygenase gene from rat intestinal cells that is induced following growth factor stimulation.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Prostaglandina-Endoperóxido Sintasas/biosíntesis , ARN Mensajero/biosíntesis , Factor de Crecimiento Transformador alfa/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Encéfalo/enzimología , Línea Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Clonación Molecular , ADN Complementario/análisis , Inducción Enzimática , Epitelio/efectos de los fármacos , Epitelio/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Biblioteca de Genes , Intestinos , Riñón/enzimología , Ratones , Microsomas/enzimología , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Eleven human endothelial cell lines have been produced by introducing sequences from the DNA tumor virus SV40 into human umbilical vein endothelial cells either by strontium phosphate coprecipitation or electroporation. The resultant lines were confirmed as being endothelial in origin by their production of endothelial-specific von Willebrand factor. The growth characteristics of the different lines in normal and reduced levels of serum was determined, as was their cellular response to endothelial cell growth supplement in combination with heparin, basic fibroblast growth factor, transforming growth factor-alpha, and epidermal growth factor.
Asunto(s)
Endotelio Vascular/citología , Fosfatos/farmacología , Virus 40 de los Simios/genética , Estroncio/farmacología , Antígenos Transformadores de Poliomavirus/genética , División Celular/efectos de los fármacos , Línea Celular , Electroporación , Endotelio Vascular/química , Endotelio Vascular/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Vectores Genéticos , Heparina/farmacología , Humanos , Transfección , Factor de Crecimiento Transformador alfa/farmacología , Factor de von Willebrand/análisis , Factor de von Willebrand/metabolismoRESUMEN
The prognosis of glioblastoma multiforme remains poor despite advances in treatment by surgery, irradiation, and chemotherapy. Many malignant gliomas overexpress growth factor receptors. The possibility of targeting these receptors with selective cytotoxic molecules constructed by fusing deoxyribonucleic acid (DNA)-encoding mutant forms of Pseudomonas exotoxin A (PE) with complementary DNA-encoding growth factors was investigated. Several recombinant toxins have been produced, including those in which transforming growth factor (TGF)-alpha, insulin-like growth factor (IGF)-I, and acidic fibroblast growth factor (FGF) were fused to mutant forms of PE lacking the native cell-binding domain. These recombinant proteins are cytotoxic to cells that express specific cell-surface receptors. The cytotoxic activity of TGF-alpha, IGF-I, and acidic FGF chimeric toxins was tested in vitro against human glioblastoma cell lines. Each recombinant toxin exhibited potent and specific killing of cells. The TGF-alpha-PE40 construct was cytotoxic to seven of the eight cell lines and was active at concentrations as low as 0.5 ng/ml (1.1 x 10(-11) M). The acidic FGF-PE40 toxin was also active on seven of the eight cell lines but was 50-fold less active than the TGF-alpha-PE40. The IGF-I-PE40 construct was active on only two cell lines. To determine the possible therapeutic effect in animals, TGF-alpha-PE40 was administered to nude mice bearing subcutaneous human glioblastoma xenografts. The animals were treated for 7 days via a continuous infusion pump placed in the peritoneal cavity. A constant serum level of TGF-alpha-PE40 was achieved that was nontoxic to the mice yet caused a reduction in tumor volume and retarded growth beyond the treatment period. The overexpression of the epidermal growth factor receptor in glioblastomas multiforme and the potency and specificity of the TGF-alpha-PE40 construct designed to target this receptor suggests that TGF-alpha-PE40 has the potential to be an effective antitumor agent for the adjuvant therapy of these carcinomas.
Asunto(s)
ADP Ribosa Transferasas , Antineoplásicos/farmacología , Toxinas Bacterianas , Exotoxinas/farmacología , Glioblastoma/patología , Sustancias de Crecimiento/farmacología , Proteínas Recombinantes de Fusión/farmacología , Factores de Virulencia , Animales , Estabilidad de Medicamentos , Femenino , Factor 1 de Crecimiento de Fibroblastos/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Dosificación Letal Mediana , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Pseudomonas aeruginosa , Factor de Crecimiento Transformador alfa/farmacología , Trasplante Heterólogo , Células Tumorales Cultivadas , Exotoxina A de Pseudomonas aeruginosaRESUMEN
1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is known to influence cell proliferation/maturation, whereas epidermal growth factor (EGF) is a potent stimulant of proliferation. Recently, hypocalcemia of vitamin D (D) deficiency was shown to significantly perturbe hepatic regeneration, which could be only partly restored by normalizing extracellular calcium, whereas normalization of 1,25-(OH)2D3 fully restored the process. To define the calcium- and/or D3-sensitive mechanisms associated with liver growth, a study of the initial events transduced by EGF was initiated by probing EGF receptor (EGFR) density and affinity, its subsequent autophosphorylation, and the level of its steady state transcript. Studies were carried out in D-depleted rats kept either untreated or supplemented with D3, 1,25-(OH)2D3, or calcium alone. The hepatic EGFR number (picomoles per mg microsomal protein) was significantly affected by hypocalcemic D-depleted (0.82 +/- 0.2), but responded with similar increases to calcium (1.7 +/- 0.09; P < 0.05), D3 (1.6 +/- 0.3; P < 0.05), and 1,25-(OH)2D3 (2.1 +/- 0.3; P < 0.01). The EGFR mRNA level revealed, however, no significant effect of the calcium or D3 status, indicating that posttranscriptional events were playing an important role. Phosphorylation studies showed that EGFR autophosphorylation and tyrosine protein kinase activity paralleled receptor density, with the lowest autophosphorylation values obtained in hypocalcemic D-depleted rats (D-depleted hypocalcemic vs. D3 repleted, P < 0.007). When normalized for receptor number, however, EGFR autophosphorylation increased in D-depleted hypocalcemic rats to a level comparable to that observed in all other groups. To dissociate the effect of the D3 hormone from that of calcium alone on EGFR, D-depleted rats were treated with the nonhypercalcemic 1,25-(OH)2D3 analog 22-OXA-1,25-(OH)2D3 (OCT), with or without calcium supplementation. Hypocalcemic OCT-treated rats did not exhibit any increase in EGFR number (0.6 +/- 0.1) compared to D-depleted hypocalcemic rats, but the addition of dietary calcium to OCT restored extracellular calcium concentrations and EGFR density (1.8 +/- 0.2; P < 0.002) to values comparable to those observed after D3 or 1,25-(OH)2D3 treatment. EGFR autophosphorylation was also decreased in hypocalcemic OCT-treated animals (P < 0.03), but after normalization for receptor density, full restoration of EGFR autophosphorylation was achieved. Our data demonstrate that in normal hepatic tissue, EGFR is highly sensitive to the in vivo prevailing calcium concentration, i.e. hypocalcemia, regardless of the D3 status, leading to a significant decrease in receptor density and its subsequent autophosphorylation.
Asunto(s)
Receptores ErbB/metabolismo , Hipocalcemia/metabolismo , Hígado/metabolismo , Deficiencia de Vitamina D/metabolismo , Animales , Calcitriol/administración & dosificación , Calcitriol/análogos & derivados , Calcitriol/farmacología , Calcio/administración & dosificación , Colecalciferol/administración & dosificación , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Femenino , Hipocalcemia/complicaciones , Masculino , Fosforilación , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador alfa/metabolismo , Factor de Crecimiento Transformador alfa/farmacología , Deficiencia de Vitamina D/complicacionesRESUMEN
Humoral hypercalcemia of malignancy is a multifactorial syndrome caused by the action of tumor-produced factors on target organs of bone, kidney, and intestine to disrupt normal calcium homeostasis. Although parathyroid hormone-related protein (PTHrP) plays an integral role in the syndrome, tumors also produce other hypercalcemic factors, such as transforming growth factor-alpha (TGF-alpha), which may modulate the effects of PTHrP. In order to determine if the effects of PTHrP on calcium homeostasis can be modulated by TGF-alpha, we have used a human squamous cell carcinoma cell line (RWGT2) which produces PTHrP alone and Chinese hamster ovarian (CHO) cells expressing only transfected human TGF-alpha complementary DNA (CHO/TGF-alpha). We studied the effects of these tumors on calcium homeostasis in nude mice bearing both tumors or each tumor alone. Whole blood ionized calcium concentrations (mean +/- SEM in mmol/L) were significantly higher in mice bearing both RWGT2 and CHO/TGF-alpha tumors (3.11 +/- 0.06, P < 0.05) when compared with mice bearing either RWGT2 alone (2.02 +/- 0.06), CHO/TGF-alpha alone (1.42 +/- 0.01), or RWGT2 and nontransfected CHO tumors (1.86 +/- 0.01). This enhanced effect was also observed using continuous PTHrP-(1-34) infusion (2 micrograms/day) in mice bearing CHO/TGF-alpha tumors. In addition, tumor cell conditioned media was tested for bone resorbing activity in organ cultures of fetal rat long bones previously incorporated with 45calcium (45Ca++). Conditioned medium at 0.1% (vol/vol) from either RWGT2 or CHO/TGF-alpha had no bone resorbing activity over control (%45Ca++ release, mean +/- SEM; control 23 +/- 1, RWGT2 19 +/- 1, CHO/TGF-alpha 23 +/- 1). However, the combination of 0.1% conditioned medium from RWGT2 and CHO/TGF-alpha significantly increased bone resorption (53 +/- 2, P < 0.05). These data demonstrate that the hypercalcemic effects of tumor-produced PTHrP are enhanced by TGF-alpha and that this effect may be due to increased bone resorption.