RESUMO
UNLABELLED: The incidence of asthma and allergy has increased throughout the developed world over the past decades. During the same period of time, the use of industrial chemicals such as phthalates, commonly used as plasticizers in polyvinylchloride (PVC) flooring material, has increased. The aim of this study was to investigate whether PVC flooring in the home of children in the age of 1-5 years is associated with the development of asthma in 5- and 10-year follow-up investigations (n = 3228). Dampness in Buildings and Health Study (DBH Study) commenced in 2000 in Värmland, Sweden. The current analyses included subjects who answered all baseline and follow-up questionnaires. Logistic regression analyses were applied to questionnaire results. Children who had PVC floorings in the bedroom at baseline were more likely to develop doctor-diagnosed asthma during the following 10-year period when compared with children living without. There were indications that PVC flooring in the parents' bedrooms was strongly associated with the new cases of doctor-diagnosed asthma when compared with child's bedroom. Our results suggest that PVC flooring exposure during pregnancy could be a critical period in the development of asthma in children at a later time; prenatal exposure and measurements of phthalate metabolites should be included in the future. PRACTICAL IMPLICATIONS: This study has found that PVC flooring material in early life was related to incidence of asthma during the following 10 years when compared with other flooring materials and especially when comparing with wood flooring type.The study has further indicated that PVC flooring in the parents' bedroom (proxy for prenatal exposure) was more associated with the development of asthma than PVC in the child's bedroom was. Our results suggest that PVC flooring exposure during pregnancy could be a critical period in the development of asthma in children at a later time. In future prospective cohort study, prenatal exposure and measurements of phthalate metabolites should be included.
Assuntos
Asma/etiologia , Ácidos Ftálicos/efeitos adversos , Asma/epidemiologia , Pré-Escolar , Estudos de Coortes , Feminino , Pisos e Cobertura de Pisos , Seguimentos , Humanos , Incidência , Lactente , Modelos Logísticos , Masculino , Inquéritos e Questionários , Suécia/epidemiologiaRESUMO
During the last decades more than 100 000 new chemicals have been introduced to the environment. Many of these new chemicals and many common consumer products that include these have been shown to be toxic in animal studies and an increasing body of evidence suggests that they are also impacting human health. Among the suspect chemicals, the endocrine disrupting chemicals (EDCs) are of particular concern. One such chemical group is the phthalates, used in soft poly vinyl chloride (PVC) material and in a huge number of consumer products. During the same period of time that the prevalence of these modern chemicals has increased, there has been a remarkable increase in several chronic illnesses, including asthma and allergy in children. In this article we outline the scientific knowledge on phthalate exposure for asthma and airway diseases in children by examining epidemiological and experimental peer review data for potential explanatory mechanisms. Epidemiological data point to a possible correlation between phthalate exposure and asthma and airway diseases in children. Experimental studies present support for an adjuvant effect on basic mechanisms in allergic sensitization by several phthalates. Despite variations in the experimental design and reported result in the individual studies, a majority of published reports have identified adjuvant effects on Th2 differentiation, production of Th2 cytokines and enhanced levels of Th2 promoted immunoglobulins (mainly IgG1 but also IgE) in mice. A limited amount of data do also suggest phthalate-induced enhancement of mast cell degranulation and eosinophilic infiltration which are important parts in the early inflammation phase. Thus, some of the early key mechanisms in the pathology of allergic asthma could possibly be targeted by phthalate exposure. But the important questions of clinical relevance of real life exposure and identification of molecular targets that can explain interactions largely remain to be answered.
Assuntos
Asma/imunologia , Disruptores Endócrinos/toxicidade , Poluentes Ambientais/toxicidade , Hipersensibilidade/imunologia , Ácidos Ftálicos/toxicidade , Adolescente , Animais , Asma/epidemiologia , Criança , Dietilexilftalato/toxicidade , Exposição Ambiental , Feminino , Humanos , Inflamação/fisiopatologia , Masculino , Camundongos , Células Th1/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologiaRESUMO
Phosphoinositide 3OH-kinases (PI3K) are a family of lipid kinases that activates signalling pathways important for migration, cytoskeletal rearrangements, and cell survival. These processes are important hallmarks in transformation. We have evaluated the functional role of PI3K for development of a transformed morphology and migratory responses of murine fibroblasts (NIH/sis and COL1A1/NIH3T3 cell lines) stimulated in an autocrine fashion by constitutive expression of platelet-derived growth factor-BB (PDGF-BB). We show that prolonged treatment with the specific PI3K inhibitor LY294002, induced a reversion of the transformed morphology, and prevented density-independent growth and focus formation. Functional PI3K was also required for development of the transformed morphology of NIH/sis and COL1A1/NIH3T3. Furthermore, treatment with LY294002 completely perturbed random migration of the cells. In addition our data show that, in the signalling pathways downstream of PI3K, activation of the small GTPase Rac was a prerequisite for the transformation signal. Our data also indicate the presence of a suramin-insensitive PI3K activity. Most likely this was due to the presence of a suramin-insensitive intracellular PDGFR pool that allowed activation of PI3K located in intracellular compartments. In conclusion these data show that intact PI3K activity was required for the morphological alterations and the enhanced migratory response that are hallmarks for PDGF induced autocrine transformation.
Assuntos
Comunicação Autócrina/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Serina-Treonina Quinases , Animais , Becaplermina , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Extensões da Superfície Celular/efeitos dos fármacos , Cromonas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Morfolinas/farmacologia , Fenótipo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-sis , Pseudópodes/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas/metabolismo , Suramina/farmacologia , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
The human neuroblastoma cell line SH-SY5Y can differentiate into a functional sympathetic neuronal phenotype when treated with low concentrations of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of serum or defined growth factors. When TrkA is introduced into the cells, NGF also induces differentiation. In both cases, protein kinase C (PKC) is pivotal for induction and maintenance of the differentiated phenotype. We have recently shown that PKC activity is needed to enable the MAPK ERK to accumulate in the nucleus of SH-SY5Y cells and hence activate transcription. To find out whether this could be one reason for the PKC dependency in the differentiation process we have investigated the role of ERK during neuronal differentiation of these cells. The results show that ERK was needed for full upregulation of the neuronal marker genes NPY and GAP-43. However, ERK activity was not necessary for TPA-induced neurite formation. Neither was activation of ERK sufficient to promote neurite outgrowth. The results clearly show that there was no correlation between nuclear ERK activity, measured as SRE transactivation, and neurite formation in TPA-differentiated SH-SY5Y neuroblastoma cells.
Assuntos
Expressão Gênica , MAP Quinase Quinase Quinase 1 , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Neuritos/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Proteína GAP-43/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitógenos/farmacologia , Neuroblastoma , Neuropeptídeo Y/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Receptor trkA/genética , Fator de Resposta Sérica , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Proteínas ras/metabolismoRESUMO
The human neuroblastoma cell line SH-SY5Y/TrkA differentiates in vitro and acquires a sympathetic phenotype in response to phorbolester (activator of protein kinase C, PKC) in the presence of serum or growth factors, or nerve growth factor (NGF). We have now investigated to what extent phorbolester and NGF cause activation of Ras and Raf-1 and the involvement of PKC in this response in differentiating SH-SY5Y/TrkA cells. NGF stimulated increased accumulation of Ras-GTP and a threefold activation of Raf-1. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect on the amount of Ras-GTP but led to a smaller activation of Raf-1. NGF caused a limited increase in phosphorylation of Raf-1 compared with TPA, and NGF-induced Raf activity was independent of PKC. Analysis of phosphorylation of the endogenous PKC substrate myristoylated alanine-rich C-kinase substrate (MARCKS), and of subcellular distribution of PKC-alpha, -delta, and -epsilon revealed that NGF only caused a very small activation of PKC in SH-SY5Y/TrkA cells. The results identify Raf-1 as a target for both TPA- and NGF-induced signals in differentiating SH-SY5Y/TrkA cells and demonstrate that signalling to Raf-1 was mediated via distinct mechanisms.
Assuntos
Carcinógenos , Fator de Crescimento Neural/farmacologia , Neuroblastoma/enzimologia , Ésteres de Forbol , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/metabolismo , Western Blotting , Diferenciação Celular , Ativação Enzimática , Humanos , Modelos Genéticos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ácidos Mirísticos/metabolismo , Fenótipo , Fosforilação , Testes de Precipitina , Isoformas de Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-raf/química , Transdução de Sinais , Frações Subcelulares/metabolismo , Acetato de Tetradecanoilforbol , Fatores de Tempo , Distribuição Tecidual , Células Tumorais Cultivadas , Regulação para CimaRESUMO
The human neuroblastoma cell line SH-SY5Y is a well characterized model for sympathetic neuronal differentiation in vitro. Several differentiation protocols exist, one of which, the addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of serum, has been thoroughly studied. Wild-type SH-SY5Y cells are unresponsive to nerve growth factor (NGF), but cells transfected with the high-affinity NGF receptor TrkA (SH-SY5Y/TrkA) differentiate in response to NGF. In the present study, we have addressed the existence of a differentiation-specific mode of activation and subcellular distribution of the extracellular signal-regulated kinases ERK1 and ERK2 in SH-SY5Y/wt and SH-SY5Y/TrkA. Both TPA and NGF induced a sustained activation and nuclear accumulation of ERK that was accompanied by transactivation of a serum response element (SRE)-driven reporter and of the c-fos gene. However, activation and nuclear accumulation of ERK were not sufficient to induce neuronal differentiation in SH-SY5Y, as demonstrated by the response to TPA in serum-free cultures. Nuclear accumulation but not activation of ERK was demonstrated to require active protein kinase C (PKC). The effect of specific PKC inhibitors on subcellular distribution of ERK and ERK-dependent transcription suggests a functional role for PKC in the regulation of nuclear ERK activity in SH-SY5Y neuroblastoma cells.
Assuntos
Diferenciação Celular , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Becaplermina , Núcleo Celular/enzimologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Fator de Crescimento Neural/farmacologia , Neuroblastoma , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Testes de Precipitina , Proteína Quinase C/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-sis , Frações Subcelulares/enzimologia , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The cell signaling docking protein p130cas became tyrosine-phosphorylated in SH-SY5Y human neuroblastoma cells during induced differentiation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum or a combination of basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The differentiating cells develop a neuronal phenotype with neurites and growth cones and sustained activation of protein kinase C (PKC) and pp60c-src. The TPA-induced p130cas phosphorylation increased within 5 min of stimulation and persisted for at least 4 days, whereas bFGF/IGF-I-induced p130cas phosphorylation was biphasic. However, the increase in tyrosine phosphorylation of p130cas was not restricted to differentiation inducing stimuli. The phosphorylation was blocked by the specific PKC inhibitor GF 109203X, and transient transfection with active PKC-epsilon induced p130cas tyrosine phosphorylation. pp60c-src, known to directly phosphorylate p130cas in other cell systems, was not activated after stimulation with TPA or bFGF/IGF-I for up to 30 min, and the initial p130cas phosphorylation was resistant to the Src family kinase inhibitor herbimycin A. However, in long term stimulated cells, herbimycin A blocked the induced phosphorylation of p130cas. Also, overexpression of src induced phosphorylation of p130cas. p130cas protein and phosphorylated p130cas were present in growth cones isolated from differentiated SH-SY5Y cells. Inhibition of PKC activity in differentiating cells with GF 109203X leads to a rapid retraction of growth cone filopodia, and p130cas phosphorylation decreased transiently (within minutes). Growth cones isolated from these cells were virtually devoid of phosphorylated p130cas. These data suggest a function for p130cas as a PKC downstream target in SH-SY5Y cells and possibly also in their growth cones.
Assuntos
Neuroblastoma/patologia , Fosfoproteínas/metabolismo , Proteína Quinase C/metabolismo , Proteínas , Quinases da Família src/metabolismo , Bucladesina/farmacologia , Diferenciação Celular , Proteína Substrato Associada a Crk , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/farmacologia , Isoenzimas/metabolismo , Maleimidas/farmacologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C-épsilon , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Proteína p130 Retinoblastoma-Like , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
A combination of basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) or 16 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum induces human SH-SY5Y neuroblastoma cells to undergo differentiation and acquire a neuronal phenotype. Nerve growth factor (NGF) added to SH-SY5Y cells stably transfected with the NGF-receptor TRK-A (SH-SY5Y/trk) induces a similar differentiated phenotype. SH-SY5Y cells express protein kinase C (PKC)-alpha, PKC-beta I, PKC-epsilon, and PKC-zeta protein, and phorbol ester- or growth factor-induced differentiation results in a sustained activation of PKC. The specific PKC inhibitor GF 109203X blocked TPA- and bFGF-IGF-I-induced neurite outgrowth in wild-type SH-SY5Y cells and NGF-induced neurite outgrowth in SH-SY5Y/trk cells. When added to differentiated cells, GF 109203X caused rapid retraction of growth cone filopodia. In TPA- and bFGF-IGF-I-treated cells, addition of GF 109203X also blocked induced expression of growth associated protein-43 and neuropeptide tyrosine while the increase in expression of these two genes was only slightly affected by the inhibitor in NGF-treated SH-SY5Y/trk cells. Thus, a portion of the NGF-induced phenotypic changes appears not to be mediated via PKC-dependent signaling. A high concentration of TPA (1.6 microM) down regulated PKC-alpha and PKC-beta I almost completely and PKC-epsilon partially in wild-type SH-SY5Y and SH-SY5Y/trk cells. Cells with down-regulated PKC-alpha and PKC-beta I after 1.6 microM TPA treatment still differentiated with growth factors. In these cells, the PKC-epsilon level was restored, and the PKC-epsilon protein was enriched in the growth cones. The 1.6 microM TPA-induced down-regulation of PKC-epsilon was counteracted by bFGF and NGF but not by platelet-derived growth factor or IGF-I. These data indicate that PKC activity is vital for neurite formation, and that the cells can differentiate under conditions when PKC-alpha and PKC-beta I are extensively down regulated. The close correlation between differentiation and presence of PKC-epsilon protein suggests an important function for this isoform during this process.
Assuntos
Isoenzimas/fisiologia , Neuritos/efeitos dos fármacos , Neurônios/enzimologia , Proteína Quinase C/fisiologia , Diferenciação Celular/fisiologia , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Substâncias de Crescimento/farmacologia , Humanos , Indóis/farmacologia , Isoenzimas/antagonistas & inibidores , Maleimidas/farmacologia , Neuroblastoma , Neurônios/citologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C-alfa , Proteína Quinase C-épsilon , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
SH-SY5Y cells differentiate into neuronal-like cells and express marker proteins like growth-associated protein (GAP-43) and neuropeptide tyrosine when treated with a low concentration (16 nM) of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of growth factors or serum. Both control and differentiated cells expressed protein kinase C-alpha (PKC-alpha), PKC-epsilon, and PKC-zeta as revealed by Western blot analyses, but the subcellular distribution of the three isoforms was not uniform, indicating specific localized functions of the enzymes. In growth cones prepared from differentiating cells PKC-alpha and PKC-epsilon were enriched. In contrast, PKC-zeta was more evenly distributed within the differentiating cell. Cells treated with a high concentration of TPA (1.6 microM) differentiate poorly and continue to proliferate. In those cells, PKC-alpha and PKC-epsilon were found to be down-regulated while PKC-zeta remained present. Thus, down-regulation of PKC-alpha and PKC-epsilon appears to be incompatible with neuronal differentiation of SH-SY5Y cells. These cells also differentiate when treated with a combination of basic fibroblast growth factor and insulin-like growth factor I. Growth cones isolated from such cells are also enriched in PKC-alpha and PKC-epsilon, but not in PKC-zeta. Based on the subcellular distribution of PKC-alpha and epsilon, and that PKC substrates like GAP-43 and pp60c-src are enriched in SH-SY5Y growth cones, a role during neurite growth is suggested.
Assuntos
Diferenciação Celular , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/metabolismo , Proteína Quinase C/genética , Northern Blotting , Células Cultivadas , Proteína GAP-43 , Humanos , Imuno-Histoquímica , RNA Mensageiro/biossínteseRESUMO
Human neuroblastoma cell lines frequently express the TRK-A proto-oncogene and bind nerve growth factor (NGF) but do not differentiate when exposed to NGF. Transient transfection of an exogenous TRK-A gene into SH-SY5Y and LA-N-5 neuroblastoma cells restored the ability of these tumor cells to differentiate with NGF. Stable TRK-A-transfected SH-SY5Y cell clones were isolated, and they responded to NGF by autophosphorylation of p140trk-A, c-fos induction, morphological differentiation, and increased expression of two neuronal marker genes, neuropeptide tyrosine and GAP-43. In phorbol ester-induced differentiated wild-type cells, TRK-A expression was increased with no change in NGF responsiveness. Thus, the restoration of the NGF-induced differentiation pathway by exogenous TRK-A presents a system of NGF-responsive human cultured cells and focuses attention on the trk-A protein and its function or malfunction in neuroblastoma.
Assuntos
Fatores de Crescimento Neural/farmacologia , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator de Crescimento Neural/genética , Diferenciação Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/metabolismo , Receptor trkA , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Phosphorylated tyrosine residues in receptor tyrosine kinases serve as binding sites for signal transduction molecules. We have identified two autophosphorylation sites, Tyr-988 and Tyr-1018, in the platelet-derived growth factor (PDGF) alpha-receptor carboxyl-terminal tail, which are involved in binding of phospholipase C-gamma (PLC-gamma). The capacities of the Y988F and Y1018F mutant PDGF alpha-receptors, expressed in porcine aortic endothelial cells, to bind PLC-gamma are 60 and 5% of that of the wild-type receptor, respectively. Phosphorylated but not unphosphorylated peptides containing Tyr-1018 are able to compete with the intact receptor for binding to immobilized PLC-gamma SH2 domains; a phosphorylated Tyr-988 peptide competes 10 times less efficiently. The complex between PLC-gamma and the PDGF alpha-receptor is more stable than that of PLC-gamma and the PDGF beta-receptor. However, PDGF stimulation results in a smaller fraction of tyrosine-phosphorylated PLC-gamma and a smaller accumulation of inositol trisphosphate in cells expressing the alpha-receptor as compared with cells expressing the beta-receptor. We conclude that phosphorylated Tyr-988 and Tyr-1018 in the PDGF alpha-receptor carboxyl-terminal tail bind PLC-gamma, but this association leads to only a relatively low level of tyrosine phosphorylation and activation of PLC-gamma.
Assuntos
Endotélio Vascular/metabolismo , Isoenzimas/metabolismo , Fosfolipases/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Sequência de Aminoácidos , Animais , Aorta , Sequência de Bases , Becaplermina , Ligação Competitiva , Divisão Celular , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fosfopeptídeos/farmacologia , Fosforilação , Fosfotirosina , Fator de Crescimento Derivado de Plaquetas/farmacologia , Mutação Puntual , Proteínas Proto-Oncogênicas c-sis , Receptores Proteína Tirosina Quinases/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Proteínas Recombinantes/farmacologia , Homologia de Sequência de Aminoácidos , Suínos , Timidina/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Human neuroblastoma cell lines are established from high-stage, highly malignant tumours. Despite this and the fact that these tumours are arrested at an early, immature stage, many cell lines have the capacity to undergo neuronal differentiation under proper growth conditions. One such cell line is the noradrenergic SH-SY5Y cell line. These cells can be induced to mature by a variety of modalities, resulting in different mature phenotypes. The use of this cell system as a model to study the stem cell character of neuroblastoma is reviewed and discussed. In particular, we focus on growth factor dependencies in the SH-SY5Y system, and compare that to the normal situation, i.e. growth factor control of sympathetic neuronal and neuroendocrine differentiation during human and rat embryogenesis.
Assuntos
Substâncias de Crescimento/fisiologia , Neuroblastoma/metabolismo , Diferenciação Celular/efeitos dos fármacos , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/farmacologia , Células-Tronco Neoplásicas/patologia , Neuroblastoma/patologia , Fenótipo , Proteína Quinase C/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Divisão Celular , Células Cultivadas , Análise Mutacional de DNA , Proteína Adaptadora GRB2 , Guanosina Trifosfato/metabolismo , Humanos , Ligantes , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Receptores Proteína Tirosina Quinases/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Análise de Sequência , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Phorbolester-triggered differentiation of SH-SY5Y neuroblastoma cells requires serum and a prolonged activation of protein kinase C (PKC). Under serum-free conditions development of a mature phenotype requires phorbolester in combination with a member of either the insulin-like growth factor (IGF) or the platelet-derived growth factor family. Here we report that basic and acidic fibroblast growth factor (FGF) and epidermal growth factor, but not nerve growth factor, synergistically potentiate phorbolester-induced differentiation. Alone these factors induced a mitogenic response which varied in magnitude, with basic FGF and IGF-I being the two most potent mitogens. However, a combination of basic FGF and IGF-I induced differentiation as judged by morphology and the increase in growth associated protein (GAP-43) and neuropeptide tyrosine mRNA levels. In contrast to the phenotype obtained in the presence of phorbolester, bFGF and IGF-I-treated SH-SY5Y cells retained their capacity to proliferate. Finally, in these cells, the phosphorylation of the endogenous PKC substrate, myristoylated alanine-rich C-kinase substrate (MARCKS), was slightly increased during several days, suggesting an involvement of PKC in the bFGF and IGF-I-induced differentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Northern Blotting , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Fator 1 de Crescimento de Fibroblastos/farmacologia , Proteína GAP-43 , Substâncias de Crescimento/biossíntese , Humanos , Insulina/farmacologia , Fator de Crescimento Insulin-Like II/farmacologia , Glicoproteínas de Membrana/biossíntese , Substrato Quinase C Rico em Alanina Miristoilada , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/biossíntese , Neuroblastoma , Neuropeptídeos/biossíntese , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína Quinase C/metabolismo , Proteínas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
In the present study an attempt was made to further elucidate the molecular mechanisms whereby protein kinase C (PKC) modulates the beta-cell stimulus-secretion coupling. Regulation of Ca2+ channel activity, [Ca2+]i, and insulin release were investigated in both normal pancreatic mouse beta-cells and in similar beta-cells deprived of PKC activity. [Ca2+]i was measured with the intracellular fluorescent Ca2+ indicator fura-2 and the Ca2+ channel activity was estimated by the whole cell configuration of the patch-clamp technique. To reveal the various isoenzymes of PKC present in the mouse beta-cell, proteins were separated by one-dimensional gel electrophoresis and Western blotting was performed. The production of inositol phosphates was measured by ion-exchange chromatography and insulin release was measured radioimmunologically. Acute stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate resulted in suppression of both the carbamylcholine-induced increase in [Ca2+]i and production of inositol 1,4,5-trisphosphate. Under these conditions the increase in [Ca2+]i in response to glucose was similar to that found in control cells. When beta-cells were deprived of PKC, by exposure to 200 nM 12-O-tetradecanoylphorbol-13-acetate for 24-48 h, there was an enhanced response to carbamylcholine. This response constituted increases in both the [Ca2+]i signal and production of inositol 1,4,5-trisphosphate. Interestingly, cells with down-regulated PKC activity responded more slowly to glucose stimulation, when comparing the initial increase in [Ca2+]i, than control cells. On the other hand, the maximal increase in [Ca2+]i was similar whether or not PKC was present. Moreover, PKC down-regulated cells exhibited a significant reduction of maximal whole cell Ca2+ currents, a finding that may explain the altered kinetics with regard to the [Ca2+]i increase in response to the sugar. Both the alpha and beta 1 forms of the PKC isoenzymes were present in the mouse beta-cell and were also subjected to PKC down-regulation. Hence, either of these isoenzymes or both may be involved in the modulation of phospholipase C and Ca2+ channel activity. Since insulin release under physiological conditions is critically dependent on Ca(2+)-influx through the voltage-gated L-type Ca2+ channels, the kinetics of hormone release was expected to demonstrate a similar delay as that of the [Ca2+]i increase. Although not as pronounced, such a delay was indeed also observed in the onset of insulin release. There was, however, no effect on the total amounts of hormone released.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Canais de Cálcio/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Proteína Quinase C/metabolismo , Animais , Canais de Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Células Cultivadas , Fura-2 , Galopamil/farmacologia , Glucose/farmacologia , Homeostase , Immunoblotting , Inositol 1,4,5-Trifosfato/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Obesos , Proteína Quinase C/isolamento & purificação , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The potent mitogen platelet-derived growth factor (PDGF) induced a rapid increase in Ras.GTP in permeabilized human and murine fibroblasts. The effect was initiated by both PDGF-AA acting exclusively through PDGF alpha-receptors, and by PDGF-BB interacting with both alpha- and beta-type receptors. The dose-response curves suggest that both receptor types mediate the response. PDGF-dependent Ras activation, measured as increased formation of Ras.GTP, was rapid and reversible. At 37 degrees C the effect had a duration of around 10 min. The PDGF-dependent increase in Ras.GTP was followed by a simultaneous increase in Ras.GDP. Under no experimental condition could a relative increase in Ras.GTP be detected. 0.5 microM GDP and 0.5 microM GTP were equally potent competing for the formation of Ras.[alpha-32P]GTP upon PDGF stimulation. Furthermore, when the basal nucleotide exchange rate on Ras was elevated by omission of Mg2+ from the medium, PDGF had no further effect on the formation of Ras.GTP. We therefore conclude that PDGF activates Ras through a mechanism leading to an increased nucleotide exchange on Ras.
Assuntos
Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células 3T3 , Animais , Bombesina/farmacologia , Permeabilidade da Membrana Celular , Células Cultivadas , Replicação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Humanos , Cinética , Magnésio/farmacologia , Masculino , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/isolamento & purificação , Pele/metabolismo , Tionucleotídeos/farmacologia , Timidina/metabolismo , Fatores de TempoRESUMO
Activation of protein kinase C results in phosphorylation of a 19-kDa protein termed 19K. Isolation and sequence analysis of a cDNA encoding the 19K protein revealed that this protein has been studied in other systems under different names. The name oncoprotein 18 (Op18) has been proposed on the basis of a postulated up-regulation in neoplastic cells. In the present report we adopt the designation Op18 for the 19K protein, and quantify this phosphoprotein in a series of leukemia/lymphoma cell lines, a panel of non-transformed cells and some terminally differentiated cell types. For this purpose we have developed reagents allowing quantitative Western-blot analysis, and quantification of Op18 on the single cell level by flow cytometric analysis. The data demonstrates a pronounced up-regulation of the Op18 protein in most leukemia/lymphoma cell lines. The HPB-ALL cell line provided the most extreme case and expressed 7 x 10(6) Op18 molecules/cell, which compares with 0.65 x 10(6) Op18 molecules/cell in non-transformed lymphoblastoid cells. The expression of Op18 appears to be restricted to cell types with proliferative potential, but it is clear from our results that up-regulation of Op18 is uncoupled from cellular proliferation. Moreover, by employing an Epstein-Barr virus based shuttle vector, we expressed Op18 cDNA in lymphoblastoid cells. This resulted in a three to fourfold up-regulation of Op18 that did not have any detectable consequences for cell-surface phenotype or cell size. However, increased expression of Op18 resulted in a partial inhibition of cell proliferation. Taken altogether, the results suggest that up-regulation Op18 levels in leukemia/lymphoma cells are strongly associated with, but not a direct cause of tumour progression.
Assuntos
Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Leucemia/metabolismo , Linfoma/metabolismo , Proteínas dos Microtúbulos , Fosfoproteínas/biossíntese , Western Blotting , Diferenciação Celular , Divisão Celular , Linhagem Celular , Transformação Celular Neoplásica , Citometria de Fluxo , Humanos , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosforilação , Proteína Quinase C/biossíntese , Estatmina , Transfecção , Células Tumorais CultivadasRESUMO
SH-SY5Y human neuroblastoma cells can be induced to differentiate into a neuronal phenotype by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In other cell systems, TPA treatment frequently leads to down-regulation of protein kinase C (PKC). However, we now report that TPA-treated and non-treated SH-SY5Y cells express PKC-alpha, but not PKC-beta and PKC-gamma, mRNA. Furthermore, only a slight down-regulation of the PKC-alpha protein could be seen during prolonged treatment with 16 nM TPA, the concentration giving optimal differentiation. In contrast, a higher concentration of TPA (1.6 microM) results in a poor neuronal differentiation and a complete down-regulation of PKC-alpha. PKC-alpha was rapidly translocated to the particulate fraction and remained membrane bound for at least 4 days during treatment with 16 nM TPA. In such cells a sustained increased level of the phosphorylated form of a 80,000 Dalton PKC-substrate was found. In addition to this sustained augmented phosphorylation, administration of fresh TPA at day 4 caused a small but reproducible further increased level of phosphorylated substrate. When the PKC activity was measured by the histone phosphorylation assay a substantial fraction of the initial enzyme activity could still be detected after 4 days of TPA treatment. Taken together, the data demonstrate that PKC remains functionally active during TPA induced differentiation of SH-SY5Y cells, which may suggest a continuous role for the enzyme during the differentiation process.
Assuntos
Neurônios/enzimologia , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Northern Blotting , Diferenciação Celular , Membrana Celular/enzimologia , Citosol/enzimologia , Humanos , Neuroblastoma , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fosforilação , Células Tumorais CultivadasRESUMO
To elucidate the transmembrane signalling processes initiated by fibroblast growth factor (FGF), we have studied the effect of recombinant basic FGF (bFGF) on various early events associated with mitogenesis in Swiss 3T3 fibroblasts. bFGF, at mitogenic concentrations, neither induced Ca2+ mobilization from intracellular stores nor increased the accumulation of inositol phosphates. In contrast, bFGF stimulated the phosphorylation of the Mr 80,000 (80K) cellular protein which is a major substrate of protein kinase C. This effect was potentiated by the diacylglycerol kinase inhibitor R59022. Two-dimensional polyacrylamide gel electrophoresis and phosphopeptide mapping showed that the 80K phosphoproteins generated in response to bFGF, bombesin, and phorbol 12,13-dibutyrate were indistinguishable. Down-regulation of protein kinase C prevented bFGF stimulation of 80K phosphorylation. Other protein kinase C-dependent early events such as transmodulation of the epidermal growth factor receptor, cytoplasmic alkalinization, inhibition of vasopressin induced increase in cytosolic [Ca2+], and enhancement of cAMP accumulation in response to forskolin were also induced by bFGF. Similar results were obtained when bFGF was added to quiescent cultures of tertiary mouse embryo fibroblasts. We conclude that bFGF stimulates protein kinase C through a signal transduction pathway distinct from inositol phospholipid turnover and Ca2+ mobilization.
Assuntos
Cálcio/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Fosfatos de Inositol/metabolismo , Proteína Quinase C/metabolismo , Animais , Bombesina/farmacologia , Linhagem Celular , AMP Cíclico/fisiologia , DNA/biossíntese , Diacilglicerol Quinase , Eletroforese em Gel Bidimensional , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Concentração de Íons de Hidrogênio , Camundongos , Fosfoproteínas/metabolismo , Fosfotransferases/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/farmacologia , Pirimidinonas/farmacologia , Transdução de Sinais , Tiazóis/farmacologiaRESUMO
Platelet-derived growth factor (PDGF) occurs as three dimeric isoforms, AA, BB, and AB. Two distinct receptor subunits, alpha and beta, have been identified which bind either all three isoforms of PDGF (alpha) or PDGF-BB only (beta). Here, we have compared the effect of purified PDGF homodimers on the early intracellular signaling events and mitogenesis in Swiss 3T3 cells, which possess equivalent numbers of the alpha and beta subunits. Both PDGF-AA and PDGF-BB stimulated receptor phosphorylation, inositol phosphate formation, activation of protein kinase C, calcium mobilization, EGF receptor transmodulation, sodium uptake, arachidonic acid release, cyclic AMP accumulation, and c-fos induction in a comparable, dose-dependent manner (half-maximal values for all these response were in the 2-10 ng/ml range for both homodimers). At high concentrations of PDGF (greater than 10 ng/ml), the BB homodimer effect on early membrane and cytosolic signals was 20-30% greater than PDGF-AA, reflecting the greater number of available binding sites for PDGF-BB. DNA synthesis studies indicated that PDGF-AA and PDGF-BB were potent mitogens for Swiss 3T3 cells, displaying identical dose-response effects. Moreover, the mitogenic activities of both homodimers were equally potentiated in the presence of insulin. These results indicate that both PDGF-AA and PDGF-BB stimulate the full complement of molecular responses required for the synergistic interactions mediating long-term mitogenesis. We conclude that alpha and beta receptor subunits do not differ in their ability to transduce PDGF-mediated signals leading to DNA synthesis in Swiss 3T3 cells.