RESUMEN
Insulin-like growth factor binding protein 2 (IGFBP-2) may represent a critical link between body composition and insulin sensitivity. We investigated the relationship between circulating IGFBP-2 levels, body composition, insulin sensitivity, energy intake and physical activity in children with obesity. Children were recruited via the Weight Management Service at the Royal Children's Hospital, Melbourne, as part of the Childhood Overweight BioRepository of Australia (COBRA). Comprehensive anthropometric, biochemical and environmental data were collected and compared to serum IGFBP-2 levels (measured by enzyme-linked immunosorbent assay). Multiple regression modelling was used to assess the influence of circulating IGFBP-2 levels on anthropometric and biochemical measures. One hundred and ninety-four children were included in this study (46% male). Circulating IGFBP-2 negatively correlated with age, anthropometric measures, blood pressure and insulin concentration. Positive associations were observed between insulin sensitivity index-homeostasis model assessment (ISI-HOMA) and serum IGFBP-2. In multiple regression modelling, IGFBP-2 significantly contributes to variance in systolic blood pressure (-19%, P < 0.05), circulating triglycerides (-16%, P < 0.05) and ISI-HOMA (18%, P < 0.05). No associations were observed between dietary energy intake or physical activity and IGFBP-2 levels. Circulating IGFBP-2 levels in children with obesity correlate inversely with body mass and markers of metabolic dysfunction, and positively with insulin sensitivity. These findings suggest that reduced levels of IGFBP-2 may play an important role in the pathogenesis of obesity complications in early life.
Asunto(s)
Índice de Masa Corporal , Proteínas Portadoras/sangre , Resistencia a la Insulina , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Insulina/metabolismo , Obesidad/complicaciones , Adolescente , Factores de Edad , Australia , Glucemia/metabolismo , Presión Sanguínea , Composición Corporal , Niño , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/deficiencia , Masculino , Obesidad/sangre , Análisis de Regresión , Factores de Riesgo , Triglicéridos/sangreRESUMEN
Brain growth and function are regulated by insulin-like growth factors I and II (IGF-I and IGF-II) but also by IGF-binding proteins (IGFBPs), including IGFBP-2. In addition to modulating IGF activities, IGFBP-2 interacts with a number of components of the extracellular matrix and cell membrane via a Cardin-Weintraub sequence or heparin binding domain (HBD1). The nature and the signalling elicited by these interactions are not fully understood. Here, we examined transgenic mice (H1d-hBP2) overexpressing a mutant human IGFBP-2 that lacks a specific heparin binding domain (HBD1) known as the Cardin-Weintraub sequence. H1d-hBP2 transgenic mice have the genetic background of FVB mice and are characterized by severe deficits in brain growth throughout their lifetime (p<0.05). In tissue lysates from brain hemispheres of 12-21day old male mice, protein levels of the GTPase dynamin-I were significantly reduced (p<0.01). Weight reductions were also found in distinct brain regions in two different age groups (12 and 80weeks). In the younger group, impaired weights were observed in the hippocampus (-34%; p<0.001), cerebellum (-25%; p<0.0001), olfactory bulb (-31%; p<0.05) and prefrontal cortex (-29%; p<0.05). At an age of 12weeks expression of myelin basic protein was reduced (p<0.01) in H1d-BP-2 mice in the cerebellum but not in the hippocampus. At 80weeks of age, weight reductions were similarly present in the cerebellum (-28%; p<0.001) and hippocampus (-31; p<0.05). When mice were challenged in the elevated plus maze, aged but not younger H1d-hBP2 mice displayed significantly less anxiety-like behaviour, which was also observed in a second transgenic mouse model overexpressing mouse IGFBP-2 lacking HBD1 (H1d-mBP2). These in vivo studies provide, for the first time, evidence for a specific role of IGFBP-2 in brain functions associated with anxiety and risk behaviour. These activities of IGFBP-2 could be mediated by the Cardin-Weintraub/HBD1 sequence and are altered in mice expressing IGFBP-2 lacking the HBD1.
Asunto(s)
Ansiedad/prevención & control , Conducta Animal , Biomarcadores/metabolismo , Encéfalo/metabolismo , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/fisiología , Proteína Básica de Mielina/metabolismo , beta-Defensinas/metabolismo , Secuencias de Aminoácidos , Animales , Ansiedad/psicología , Encéfalo/patología , Humanos , Masculino , Ratones , Ratones Transgénicos , Fenotipo , Eliminación de Secuencia , beta-Defensinas/genéticaRESUMEN
The ubiquitous nature of the IGF system, expressed early in embryonic development throughout postnatal and adult life, indicates a key role for this system in human biology. Studies of transgenic mice over-expressing components of the IGF system or mice with disruptions of the same genes have clearly shown that the IGF system plays an important role in vivo. The activity of the IGF ligands, elicited via their receptors and transduced by various intracellular signal pathways, is modulated by the IGFBPs. Among all the IGFBPs, IGFBP-2 has been implicated in the regulation of IGF activity in the nervous system, peripheral tissue and organs. Besides binding to IGFs in the circulation, these IGF-regulatory activities of IGFBP-2 involve interactions with components of the extracellular matrix and cell surface proteoglycans and integrin receptors. In addition to these "local" peri-cellular activities of IGFBP-2, it became evident that IGFBP-2 exerts other key functions within the cell. In the cytoplasm IGFBP-2, most likely in the absence of the IGFs, interacts with regulatory proteins including transcription factors and cytoplasm-nuclear transporters. Within the nucleus IGFBP-2, directly or indirectly, promotes transcriptional activation of specific genes. These intrinsic activities of IGFBP-2 are mediated via specific functional domains. All of these IGFBP-2 activities, intrinsic or dependent on IGFs, contribute to its functional roles in growth/development, metabolism and malignancy as evidenced by studies in IGFBP-2 animal models and also by many in vitro studies. Finally, preclinical studies have demonstrated that IGFBP-2 administration can be beneficial in improving metabolic responses (inhibition of adipogenesis and enhanced insulin sensitivity), while blockade of IGFBP-2 appears to be an effective approach to inhibiting tumor growth and metastasis.
Asunto(s)
Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Neoplasias/metabolismo , Animales , Humanos , Somatomedinas/metabolismoRESUMEN
BACKGROUND/OBJECTIVE: IGF-binding protein (IGFBP)-2 is the principal IGFBP produced by white adipocytes during adipogenesis, and circulating levels are reduced in obesity. Overexpression of IGFBP-2 in transgenic mice prevents obesity, but depot-specific effects of IGFBP-2 on adipo/lipogenesis are unknown. The present study aimed to investigate whether IGFBP-2 affects adipo/lipogenesis in a depot-specific manner and explore potential mechanisms. METHODS: Following adipocyte characterisation, IGFBP-2 levels were measured from human subcutaneous and visceral preadipocytes, and IGFBP-2 dose-responses were then undertaken with exogenous IGFBP-2 in an in vitro IGF-I-free system to examine adipo/lipogenesis. Following this, both types of adipocytes were transfected with human siRNA IGFBP-2 to assess auto-/para-/intra-crine effects, with and without additional add-back IGFBP-2. To elucidate the potential mechanisms, visceral preadipocytes were treated with either wild-type or Heparin Binding Domain (HBD)-mutant IGFBP-2 (which is unable to bind to cell-surface components), and experiments were also undertaken using Echistatin (an integrin receptor blocker). Outcomes included gene expression profiles, protein levels and phosphorylation and lipid staining. RESULTS: Human visceral adipocytes produced significantly more IGFBP-2 than subcutaneous adipocytes. Subsequent dose-responses to IGFBP-2 demonstrated significant reductions in adipo/lipogenesis in visceral, but not subcutaneous, adipocytes in response to increasing IGFBP-2. Silencing IGFBP-2 resulted in exaggerated adipo/lipogenesis in visceral, but not subcutaneous, adipocytes, an effect completely inhibited by add-back IGFBP-2. These effects occurred in the absence of changes in IGF-I levels. HBD-mutant IGFBP-2 had reduced effects compared with wild-type IGFBP-2. Wild-type IGFBP-2 increased phosphorylation of focal adhesion kinase (FAK) and decreased phosphatase and tensin homolog (PTEN) levels, suggestive of integrin-mediated signalling. Blockade of this signalling, using Echistatin, completely negated the effects of IGFBP-2 on visceral adipo/lipogenesis. CONCLUSION: IGFBP-2 inhibits both adipogenesis and lipogenesis in visceral, but not subcutaneous, adipocytes. This depot-specific impairment appears to be independent of IGF-I and involves cell-surface association of IGFBP-2 and activation of integrin signalling pathways.
Asunto(s)
Adipogénesis/efectos de los fármacos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Grasa Intraabdominal/metabolismo , Péptidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Adipocitos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular , Grasa Intraabdominal/efectos de los fármacos , Grasa Intraabdominal/fisiopatología , Lipogénesis/efectos de los fármacos , Ratones , Ratones Transgénicos , Fosforilación/efectos de los fármacosRESUMEN
IGFBP-2 is highly expressed in both the serum and tumor tissues of most cancers, and is considered one of the most significant genes in the signature of major cancers. IGFBP-2 mainly modulates IGF actions in the pericellular space; however, there is considerable evidence to suggest that IGFBP-2 may also act independently of the IGFs. These IGF-independent actions of IGFBP-2 are exerted either via interactions at the cell surface or intracellularly, via interaction with cytoplasmic or nuclear-binding partners. The precise mechanism underlying the intracellular/intranuclear localization of IGFBP-2 remains unclear. In this study, we investigated IGFBP-2 nuclear localization in several common cancer cells with the aim of dissecting the mechanism of its nuclear trafficking. IGFBP-2 is detected in the nuclei of common cancer cells, including breast, prostate and several neuroblastoma cell lines, using cell fractionation and confocal microscopy. Via nuclear import assays, we show that nuclear entry of IGFBP-2 is mediated by the classical nuclear import mechanisms, primarily through importin-α, as demonstrated by the use of blocking, competition and co-immunoprecipitation assays. Bioinformatics analysis of the IGFBP-2 protein sequence with PSORT II identified a classical nuclear localization signal (cNLS) sequence at 179PKKLRPP185, within the IGFBP-2 linker domain, mutagenesis of which abolishes IGFBP-2 nuclear import. Accordingly, the NLSmutIGFBP-2 fails to activate the VEGF promoter, which would otherwise occur in the presence of wild-type IGFBP-2. As a consequence, no activation of angiogenic processes were observed in NLSmutIGFBP-2 expressing SHEP cells when implanted onto our in vivo quail chorio-allantoic membrane model. Taken together, these data show for the first time that IGFBP-2 possesses a functional NLS sequence and that IGFBP-2 actively translocates into the nucleus by a classical nuclear import mechanism, involving formation of IGFBP-2 complexes with importin-α. Nuclear IGFBP-2 is required for the activation of VEGF expression and consequent angiogenesis.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Neoplasias/metabolismo , Señales de Localización Nuclear/genética , Factor A de Crecimiento Endotelial Vascular/biosíntesis , alfa Carioferinas/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Células MCF-7 , Neovascularización Patológica/metabolismo , Regiones Promotoras Genéticas , Alineación de Secuencia , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , beta Carioferinas/metabolismoRESUMEN
We have recently demonstrated that fibroblast growth factor (FGF)-2 promotes neuroblastoma cell differentiation and overrides their mitogenic response to IGF-I. However, the mechanisms involved are unknown. SK-N-MC cells were cultured with FGF-2 (50 ng/ml) and/or IGF-I (100 ng/ml) up to 48 h. Fluorescence-activated cell sorting analysis indicated that FGF-2 promotes G1/G0 cell cycle phase arrest. Gene expression by RT2-PCR and cellular localization showed up-regulation of p21. We then investigated whether FGF-2-induced differentiation of SK-N-MC cells (by GAP43 and NeuroD-6 expression) involves epithelium-mesenchyme transition interconversion. Real-time PCR (RT2-PCR) showed modulation of genes involved in maintenance of the epithelial phenotype and cell-matrix interactions (E-cadherin, Snail-1, MMPs). Zymography confirmed FGF-2 up-regulated MMP2 and induced MMP9, known to contribute to neuronal differentiation and neurite extension. Id1-3 expression was determined by RT2-PCR. FGF-2 induced Id2, while down-regulating Id1 and Id3. FGF-2 induced nuclear accumulation of ID2 protein, while ID1 and ID3 remained cytoplasmic. RNA interference demonstrated that Id3 regulates differentiation and cell cycle (increased Neuro-D6 and p21 mRNA), while d Id2 modulates epithelium-mesenchyme transition-like events (increased E-cadherin mRNA). In conclusion, we have shown for the first time that FGF-2 induces differentiation of neuroblastoma cells via activation of a complex gene expression program enabling modulation of cell cycle, transcription factors, and suppression of the cancer phenotype. The use of RNA interference indicated that Id-3 is a key regulator of these events, thus pointing to a novel therapeutic target for this devastating childhood cancer.
Asunto(s)
Diferenciación Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Fase G1/efectos de los fármacos , Proteína 1 Inhibidora de la Diferenciación/fisiología , Proteína 2 Inhibidora de la Diferenciación/fisiología , Proteínas Inhibidoras de la Diferenciación/fisiología , Proteínas de Neoplasias/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular Tumoral , Matriz Extracelular/fisiología , Regulación Neoplásica de la Expresión Génica , Humanos , Neuroblastoma/genética , Interferencia de ARN , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Estrogen plays a key role in the regulation of growth in both genders, via its stimulation of the pubertal growth spurt and mediation of epiphyseal fusion. Mouse knockout models suggest a differential effect of oestrogen receptor (ER) alpha and beta on the growth plate, with ER beta possibly being more important in regulating epiphyseal fusion. Epiphyseal fusion may also depend on growth plate senescence, which is regulated by oestrogen. While molecular mechanisms for oestrogen's actions remain unclear, local production of oestrogen may be important for growth. Aromatase inhibitors appear to be effective in improving final height outcome in short stature, however long term safety data is lacking particularly in regards to reproductive function. Future studies are required to further understand the mechanisms by which ER alpha and ER beta affect growth plate function, while longer term studies of aromatase inhibitor usage, preferably utilising animal models, are required to verify the safety of these compounds.
Asunto(s)
Estrógenos/fisiología , Crecimiento y Desarrollo/fisiología , Animales , Inhibidores de la Aromatasa/farmacología , Senescencia Celular/fisiología , Estrógenos/deficiencia , Trastornos del Crecimiento/etiología , Placa de Crecimiento/fisiología , Crecimiento y Desarrollo/efectos de los fármacos , Crecimiento y Desarrollo/genética , Antagonistas de Hormonas/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina/fisiología , Modelos Biológicos , Receptores de Estrógenos/genética , Receptores de Estrógenos/fisiología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Transducción de Señal/fisiologíaAsunto(s)
Alérgenos/efectos adversos , Anafilaxia/etiología , Macropodidae , Productos de la Carne/efectos adversos , Adulto , Alérgenos/inmunología , Anafilaxia/inmunología , Animales , Hipersensibilidad a los Alimentos/etiología , Hipersensibilidad a los Alimentos/inmunología , Humanos , Immunoblotting , MasculinoRESUMEN
In recent years, much interest has been devoted to defining the role of the IGF system in the nervous system. The ubiquitous IGFs, their cell membrane receptors, and their carrier binding proteins, the IGFBPs, are expressed early in the development of the nervous system and are therefore considered to play a key role in these processes. In vitro studies have demonstrated that the IGF system promotes differentiation and proliferation and sustains survival, preventing apoptosis of neuronal and brain derived cells. Furthermore, studies of transgenic mice overexpressing components of the IGF system or mice with disruptions of the same genes have clearly shown that the IGF system plays a key role in vivo.
Asunto(s)
Encéfalo/embriología , Encéfalo/fisiología , Factor I del Crecimiento Similar a la Insulina/fisiología , Receptor IGF Tipo 1/fisiología , Animales , HumanosRESUMEN
IGF binding proteins (IGFBPs) modulate IGF cellular bioavailability and may directly regulate tumor growth and invasion. We have previously shown that IGFBP-2 binds and localizes IGF-I to the pericellular matrix and have provided some evidence suggesting that the heparin binding domain (HBD) or the arginine-glycine-aspartic acid (RGD) integrin binding motif may be involved in these interactions. However, the precise mechanisms involved remain to be elucidated. We therefore mutated the HBD or RGD sequence of IGFBP-2 and investigated consequent effects on extracellular matrix (ECM) binding, IGF-induced proliferation, and migration of neuroblastoma cells. IGFBP-2 and its arginine-glycine-glutamic acid (RGE) mutant similarly bound ECM components, whereas binding of mutant HBD-IGFBP-2 to each of the ECM substrates was markedly reduced by 70-80% (P < 0.05). IGF-I (100 ng/ml) increased incorporation of 3H-thymidine in neuroblastoma SK-N-SHEP cells by approximately 30%, an effect blunted by exogenously added native or either mutant IGFBP-2. Overexpression of IGFBP-2 and its RGE mutant potently promoted SHEP cell proliferation (5-fold), whereas SHEP cell proliferation was negligible when HBD-IGFBP-2 was overexpressed. Addition or overexpression of IGFBP-2 and its RGE mutant potently (P < 0.05) enhanced SHEP cell migration/invasion through the ECM. However, overexpression of the HBD-IGFBP-2 mutant potently inhibited (50-60%) SHEP cell invasion through ECM. Thus, IGFBP-2, which binds to the ECM, enhances proliferation and metastatic behavior of neuroblastoma cells, functions that directly or indirectly use the HBD but not the integrin binding sequence. Our novel findings thus point to a key role for the HBD of IGFBP-2 in the control and regulation of neuroblastoma growth and invasion.
Asunto(s)
Matriz Extracelular/metabolismo , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Neuroblastoma/fisiopatología , Secuencia de Bases , División Celular , Línea Celular Tumoral , Movimiento Celular , Cartilla de ADN , Humanos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Cinética , Mutagénesis Sitio-Dirigida , Invasividad Neoplásica , Neuroblastoma/patología , Unión ProteicaRESUMEN
Insulin-like growth factor binding protein-2 (IGFBP-2) as one of the most important IGFBPs has never been assessed in the intracellular compartment in vivo. Since there is evidence for novel intracellular functions of distinct IGFBPs, we investigated the presence of IGFBP-2 inside the cell. In peri/nuclear fractions of various tissues isolated from IGFBP-2 transgenic and non-transgenic mice we were able to show the presence of intact IGFBP-2. In addition, we demonstrate the presence of a highly conserved carboxyl-terminal IGFBP-2 fragment in the peri/nuclear fraction by using different peptide-induced antibodies. In pancreatic sections, confocal microscopy revealed the presence of IGFBP-2 on the nuclear surface but not within the nucleus. Our findings suggest novel functions of intact IGFBP-2 and IGFBP-2 fragments within the cell.
Asunto(s)
Núcleo Celular/metabolismo , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/química , Secuencia de Aminoácidos , Animales , Western Blotting , Centrifugación por Gradiente de Densidad , Inmunoprecipitación , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Ligandos , Ratones , Ratones Transgénicos , Microscopía Confocal , Microscopía Fluorescente , Datos de Secuencia Molecular , Péptidos/química , Propidio/farmacología , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Many factors regulate nervous system development, including complex cross-talk between local neuroendocrine systems. The adipocyte-secreted hormone leptin, mainly known for its key roles in nutrition and reproductive balance, may also be involved in neuroanatomical organization, myelination processes, and neuronal/glia maturation. SK-N-SH-SY5Y neuroblastoma cells were employed as an in vitro model of human neuronal cells to determine whether leptin exerts neuroprotective activities. We show that SH-SY5Y cells express leptin, the long and short isoforms of the leptin receptor (ObRl, ObRs). In SH-SY5Y cells, leptin induced signal transducer and activator of transcription (STAT)-3 phosphorylation and suppressor of cytokine signaling-3 mRNA expression. Leptin dose-dependently increased cell number (up to 200% at 1 microm by 48 h, P < 0.01), and at 24-48 h, leptin at 100 nm increased SH-SY5Y cell number by 30-50%, respectively. SH-SY5Y cell viability was reduced in serum-free conditions at 24 h, and addition of leptin at 100 nm significantly reduced apoptosis by approximately 20% (P < 0.001). Leptin's antiapoptotic activity required Janus kinase/STAT, MAPK, and phosphatidylinositol-3-kinase activation because the antiapoptotic effects of leptin were abolished, and caspase-3 immunoreactivity increased in the presence of the specific blockers AG490, U0126, or LY294002. Gene array demonstrated that leptin inhibits apoptosis via potent down-regulation of caspase-10 and TNF-related apoptosis-inducing ligand. Our data thus demonstrate, for the first time, that leptin stimulates, in a time- and dose-dependent manner, neuroblastoma cell proliferation and that the underlying mechanisms involve suppression of apoptosis via the Janus kinase-STAT, phosphatidylinositol-3 kinase, and MAPK pathways that culminate altogether in the down-regulation of the apoptotic factors caspase-10 and TNF-related apoptosis-inducing ligand.
Asunto(s)
Apoptosis/efectos de los fármacos , Leptina/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Proteínas Sanguíneas/farmacología , Caspasa 10 , Caspasas/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , Técnicas In Vitro , Proteínas Quinasas JNK Activadas por Mitógenos , Leptina/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuroblastoma , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT3 , Ligando Inductor de Apoptosis Relacionado con TNF , Transactivadores/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Glucose is the brain's major energy source; therefore, loss of neuronal cells is a potential consequence of hypoglycaemia. Since apoptosis is a major mechanism of neuronal loss following a range of insults, we explored potent anti-apoptotic systems (IGF-I and bcl-2) as means of enhancing neuronal survival in the face of glucose deprivation. Human neuroblastoma cells (SH-SY5Y, SHEP and SHEP-bcl-2) were exposed to low glucose as a model of glucopenia-induced neuronal damage. Administration of IGF-I and/or over-expression of the survival gene bcl-2 were exploited to attempt to limit neuronal loss. Neuronal survival mechanisms and interactions between these systems were investigated. Low glucose (0.25-2.5 mM) adversely affected cell growth and survival; however, IGF-I ameliorated these outcomes. Over-expression of bcl-2 blunted low glucose-induced apoptosis and up-regulated IGF-I receptor, with the effect of IGF-I addition being negligible on apoptosis, while significantly enhancing mitochondrial activity. In SH-SY5Y cells, IGF-I significantly changed >two-fold mRNA levels of the apoptosis-related genes gadd45, fas, iNOS, NFkB, TRAIL, without further affecting bcl-2 expression. In low glucose, IGF-I acutely enhanced glucose transport and translocation of GLUT1 protein to the cell membrane. GLUT1 mRNA expression was up-regulated by both IGF-I and bcl-2. The potent anti-apoptotic systems IGF-I and bcl-2 are both thus able to enhance cell survival in a glucose-deprived human neuronal model. Although we clearly show evidence of positive cross-talk via bcl-2 modulation of IGF-I receptor, IGF-I also has enhancing effects on mitochondrial function outside the bcl-2 pathway. The common effect of both systems on enhancement of GLUT-1 expression suggests that this is a key mechanism for enhanced survival. These studies also point to the potential use of IGF-I therapy in prevention or amelioration of hypoglycaemic brain injury.
Asunto(s)
Apoptosis , Glucosa/metabolismo , Factor I del Crecimiento Similar a la Insulina/fisiología , Neuronas/fisiología , Análisis de Varianza , Apoptosis/genética , Transporte Biológico , Northern Blotting/métodos , Western Blotting/métodos , Recuento de Células/métodos , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Medio de Cultivo Libre de Suero/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática/métodos , Expresión Génica/efectos de los fármacos , Glucosa/deficiencia , Transportador de Glucosa de Tipo 1 , Humanos , Isótopos de Yodo/farmacocinética , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Monosacáridos/metabolismo , Neuroblastoma , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Translocación Genética/efectos de los fármacosRESUMEN
Using insulin-like growth factor-binding protein-2 (IGFBP-2) transgenic mice (D mice) as a model of elevated IGFBP-2 expression, which is often found in unphysiological conditions, we found association of IGFBP-2 to purified plasma membranes of many organs. To determine whether the RGD (Arg-Gly-Asp) motif of IGFBP-2 mediates cell surface binding in vivo, we mutated the RGD motif of IGFBP-2 into an RGE (Arg-Gly-Glu) sequence and produced transgenic mice (E mice) which express elevated amounts of mutated IGFBP-2. Our data demonstrate that in vivo IGFBP-2 cell surface association is not dependent on the RGD motif and that mutation of this sequence does not alter growth inhibitory effects of IGFBP-2.
Asunto(s)
Peso Corporal/fisiología , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteínas de la Membrana/metabolismo , Oligopéptidos/metabolismo , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/fisiología , Animales , Peso Corporal/genética , Membrana Celular/metabolismo , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/análisis , Factor II del Crecimiento Similar a la Insulina/metabolismo , Proteínas de la Membrana/sangre , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos/crecimiento & desarrollo , Ratones Transgénicos/fisiología , Oligopéptidos/genética , Tamaño de los Órganos/genética , Tamaño de los Órganos/fisiología , Mutación PuntualRESUMEN
Insulin-like growth factor binding protein-6 (IGFBP-6) is an O-linked glycoprotein which specifically inhibits insulin-like growth factor (IGF)-II actions. The effects of O-glycosylation of IGFBP-6 on binding to glycosaminoglycans and proteolysis, both of which reduce the IGF binding affinity of other IGFBPs were studied. Binding of recombinant human nonglycosylated (n-g) IGFBP-6 to a range of glycosaminoglycans in vitro was approximately threefold greater than that of glycosylated (g) IGFBP-6. When bound to glycosaminoglycans, IGFBP-6 had approximately 10-fold reduced binding affinity for IGF-II. Exogenously added n-gIGFBP-6 but not gIGFBP-6 also bound to partially purified rat PC12 phaeochromocytoma membranes. Binding of n-gIGFBP-6 was inhibited by increasing salt concentrations, which is typical of glycosaminoglycan interactions. O-glycosylation also protected human IGFBP-6 from proteolysis by chymotrypsin and trypsin. Proteolysis decreased the binding affinity of IGFBP-6 for IGF-II, even with a relatively small reduction in apparent molecular mass as observed with chymotrypsin. Analysis by ESI-MS of IGFBP-6 following limited chymotryptic digestion showed that a 4.5-kDa C-terminal peptide was removed and peptide bonds involved in the putative high affinity IGF binding site were cleaved. The truncated, multiply cleaved IGFBP-6 remained held together by disulphide bonds. In contrast, trypsin cleaved IGFBP-6 in the mid-region of the molecule, resulting in a 16-kDa C-terminal peptide which did not bind IGF-II. These results indicate that O-glycosylation inhibits binding of IGFBP-6 to glycosaminoglycans and cell membranes and inhibits its proteolysis, thereby maintaining IGFBP-6 in a high-affinity, soluble form and so contributing to its inhibition of IGF-II actions.
Asunto(s)
Glicosaminoglicanos/metabolismo , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Glicosilación , Humanos , Hidrólisis , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/química , Espectrometría de Masas , Datos de Secuencia Molecular , Células PC12 , Unión Proteica , Ratas , Proteínas Recombinantes/metabolismo , Tripsina/metabolismoRESUMEN
Stem cells from the adult forebrain of mice were stimulated to form clones in vitro using fibroblast growth factor-2 (FGF-2). At concentrations above 10 ng/ml of FGF-2, very few clones gave rise to neurons; however, if FGF-2 was removed after 5 days, 20-30% of clones subsequently gave rise to neurons. The number of neuron-containing clones and the number of neurons per clone was significantly enhanced, if insulin-like growth factor (IGF)-1 or heparin were added subsequent to FGF-2 removal. The spontaneous production of neurons after FGF-2 removal was shown to be due to endogenous IGF-1, since antibodies to IGF-1 and an IGF-1 binding protein totally inhibited neuronal production. Similarly, these reagents also abrogated the neuron-promoting effects of heparin. Thus, it appears that endogenous IGF-1 may be a major regulator of stem cell differentiation into neurons. Furthermore, it was found that high levels of IGF-1 or insulin promoted the maturation and affected the neurotransmitter phenotype of the neurons generated.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/fisiología , Neuronas/citología , Células Madre/citología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Senescencia Celular/fisiología , Citocinas/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Heparina/farmacología , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/fisiología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Endogámicos CBA , Factores de Crecimiento Nervioso/farmacología , Neuronas/fisiología , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Insulin-like growth factor (IGF) action in the brain is modulated by IGF-binding proteins (IGFBPs) whose abundance can be altered by other locally expressed growth factors. However, the mechanisms involved are unclear. We here employed the neuroblastoma cell line SK-N-MC as a model to define the mechanisms involved in modulation of IGFBPs in neuronal cells. Western ligand blotting analysis and immunoprecipitation of conditioned media (CM) from SK-N-MC cells showed that in these cells, as in the brain, the most abundantly expressed IGFBP was IGFBP-2. However, IGFBP-2 was barely detectable in CM from cells treated with basic fibroblast growth factor (bFGF) without a change in IGFBP-2 messenger RNA (mRNA) abundance. These CM contained specific IGFBP-2 proteolytic activity, resulting in two IGFBP-2 fragments of 14 and 22 kDa. The activity was inhibited by EDTA/phenylmethylsulfonyl fluoride or aprotinin. Competitive binding studies indicated that IGFBP-2 fragments had reduced binding affinity for IGF-I. bFGF induced IGFBP-3 mRNA and protein. Affinity cross-linking of [125I]IGF-I to neuroblastoma cell membranes followed by immunoprecipitation revealed a approximately 38 kDa [125I]IGF-I/IGFBP-2 complex. Cell surface-associated IGFBP-2 was also susceptible to bFGF-induced proteolysis, with the appearance of a single cross-linked 21-kDa complex with low affinity for IGF-I. These findings indicate that intact IGFBP-2 and the 14-kDa, but not the 22-kDa fragment, bind to the cell surface. Our data suggest that induction of IGFBP-2 proteolysis on neuronal cell surface is a novel mechanism whereby IGF availability is modulated by the local growth factor bFGF.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Neuroblastoma/metabolismo , Péptido Hidrolasas/metabolismo , Sitios de Unión/fisiología , Membrana Celular/metabolismo , Medios de Cultivo/metabolismo , Inducción Enzimática/fisiología , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Neuroblastoma/patología , Fragmentos de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Recombinantes , Células Tumorales CultivadasRESUMEN
Melanocytes, highly differentiated neural crest-derived cells, are located in the basal layer of the epidermis, where they play a role in protecting against UV damage in the skin. Previous studies suggest that both growth hormone (GH) and the insulin-like growth factor I (GH/IGF-I) system may be important for melanocyte growth and function. We have therefore characterized the role of the GH/IGF system in melanocyte growth in vitro and its interaction with the local growth factor basic fibroblast growth factor (bFGF). Analysis of the effects of GH, IGF-I, and bFGF and combinations of these growth factors on melanocyte growth in vitro revealed that 1) GH stimulates the growth of melanocytes when combined with IGF-I, des(1-3)IGF-I [an analog of IGF-I that has a reduced binding affinity for IGF-binding proteins (IGFBPs)], or bFGF, either separately or in combination; 2) in contrast to the lack of effect of GH or bFGF alone, both IGF-I and des(1-3)IGF-I enhance melanocyte growth in a dose-dependent manner; and 3) IGF-I is more efficacious in eliciting a growth response at low concentrations compared to des(1-3)IGF-I. Using Western ligand blotting, affinity cross-linking, immunoprecipitation, RIA, and Northern analysis, we show that cultured human melanocytes synthesize and secrete minimal amounts of IGFBP. IGFBP-4 is the major IGFBP produced by these cells when cultured in complete growth medium or in the presence of either IGF-I or des(1-3)IGF-I alone. In conclusion, these studies provide support for a role for both GH and IGF-I in the growth of human melanocytes in vitro, involving synergy with bFGF. Low levels of melanocyte-derived IGFBP-4 may play a role in enhancing the modulation of IGF action.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/fisiología , Hormona de Crecimiento Humana/fisiología , Factor I del Crecimiento Similar a la Insulina/fisiología , Melanocitos/fisiología , Northern Blotting , Western Blotting , División Celular/efectos de los fármacos , Colorimetría , Reactivos de Enlaces Cruzados , Factor 2 de Crecimiento de Fibroblastos/farmacología , Hormona de Crecimiento Humana/farmacología , Humanos , Técnicas In Vitro , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Melanocitos/efectos de los fármacos , Fragmentos de Péptidos/farmacología , ARN Mensajero/metabolismo , RadioinmunoensayoRESUMEN
Insulin-like growth factor-I (IGF-I) is essential for normal epidermal homeostasis; however, the role of IGF binding proteins (IGFBPs), regulators of IGF action, remains unclear. Here we examine the regulation of human keratinocyte-produced IGFBPs by epidermal growth factor (EGF), transforming growth factor beta 1 (TGFbeta1), and IGF-I, growth factors known to be active in skin. In the absence of added growth factors, IGFBP-3 was the major binding protein secreted into the medium by primary keratinocytes. Addition of EGF or TGFbeta1 to keratinocyte cultures resulted in a significant decrease in IGFBP-3 abundance in conditioned medium when compared with control, untreated cells. Specifically, EGF (50 ng/ml) and TGFbeta1 (50 ng/ml) reduced IGFBP-3 abundance to 15+/-6% and 22+/-9%, respectively. Using Northern blot analysis, we found EGF and TGFbeta1 (50 ng/ml) to reduce IGFBP-3 mRNA levels in keratinocytes to 51+/-12% and 50+/-38%, respectively, when compared with control, untreated cells. Treatment with IGF-I or its analogue des(1-3)IGF-I did not lead to any consistent change in IGFBP-3 abundance. However, both IGF-I and des(1-3)IGF-I at 100 ng/ml led to a modest increase in IGFBP-3 mRNA levels in keratinocytes, suggesting posttranscriptional regulation of IGFBP-3 abundance. We propose that local modulation of IGFBP-3 abundance may represent another level of regulation of growth factor action in the epidermis, where EGF and TGFbeta1 and possibly other local growth factors specifically regulate the availability of IGF-I to its keratinocyte receptors.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Queratinocitos/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Medios de Cultivo Condicionados/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Queratinocitos/efectos de los fármacos , Fragmentos de Péptidos/farmacología , ARN Mensajero/metabolismo , Piel/efectos de los fármacosRESUMEN
Insulin-like growth factor 1 (IGF-1) is induced after hypoxic-ischemic (HI) brain injury, and therapeutic studies suggest that IGF-1 may restrict delayed neuronal and glial cell loss. We have used a well-characterised rat model of HI injury to extend our understanding of the modes of action of the IGF system after injury. The induction of the IGF system by injury was examined by in situ hybridization, immunohistochemistry, Northern blot analysis, RNase protection assay and reverse transcriptase-polymerase chain reaction (RT-PCR). IGF-1 accumulated in blood vessels of the damaged hemisphere within 5 h after a severe injury. By 3 days, IGF-1 mRNA was expressed by reactive microglia in regions of delayed neuronal death, and immunoreactive IGF-1 was associated with these microglia and reactive astrocytes juxtaposed to surviving neurones surrounding the infarct. Total IGF-1 receptor mRNA was unchanged by the injury. IGFBP-2 mRNA was strongly induced in reactive astrocytes throughout the injured hemisphere, and IGFBP-3 and IGFBP-5 mRNA were moderately induced in reactive microglia and neurones of the injured hippocampus, respectively. IGFBP-6 mRNA was induced in the damaged hemisphere by 3 days and increased protein was seen on the choroid plexus, ependyma and reactive glia. In contrast, insulin II was not induced. These results indicate cell type-specific expression for IGF-1, IGFBP-2,3,5 and 6 after injury. Our findings suggest that the IGF-1 produced by microglia after injury is transferred to perineuronal reactive astrocytes expressing IGFBP-2. Thus, modulation of IGF-1 action by IGFBP-2 might represent a key mechanism that restricts neuronal cell loss following HI brain injury.