RESUMEN
There is general agreement that dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens and prefrontal cortex play a key role in drug reinforcement. The activity of these neurons is strongly modulated by the inhibitory and excitatory input they receive. Activation of mu-opioid receptors, located on GABAergic neurons in the VTA, causes hyperpolarization of these GABAergic neurons, thereby causing a disinhibition of VTA dopaminergic neurons. This effect of mu-opioid receptors upon GABA neurotransmission is a likely mechanism for mu-opioid receptor modulation of drug reinforcement. We studied mu-opioid receptor signaling in relation to cocaine reinforcement in wild-type and mu-opioid receptor knockout mice using a cocaine self-administration paradigm and in vitro electrophysiology. Cocaine self-administration was reduced in mu-opioid receptor knockout mice, suggesting a critical role of mu-opioid receptors in cocaine reinforcement. The frequency of spontaneous inhibitory post-synaptic currents onto dopaminergic neurons in the ventral tegmental area was increased in mu-opioid receptor knockout mice compared with wild-type controls, while the frequency of spontaneous excitatory post-synaptic currents was unaltered. The reduced cocaine self-administration and increased GABAergic input to VTA dopaminergic neurons in mu-opioid receptor knockout mice supports the notion that suppression of GABAergic input onto dopaminergic neurons in the VTA contributes to mu-opioid receptor modulation of cocaine reinforcement.
Asunto(s)
Cocaína/farmacología , Neuronas/metabolismo , Receptores Opioides mu/genética , Refuerzo en Psicología , Área Tegmental Ventral/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismo , Vías Aferentes/efectos de los fármacos , Vías Aferentes/metabolismo , Vías Aferentes/fisiopatología , Animales , Trastornos Relacionados con Cocaína/metabolismo , Trastornos Relacionados con Cocaína/fisiopatología , Modelos Animales de Enfermedad , Dopamina/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Noqueados , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas/efectos de los fármacos , Autoadministración , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Área Tegmental Ventral/metabolismo , Área Tegmental Ventral/fisiopatologíaRESUMEN
Insulin-like growth factor binding protein 2 (IGFBP-2) is one member of the family of IGF binding proteins believed to have both endocrine functions elicited by modulating serum IGF half-life and transport as well as autocrine/paracrine functions that result from blocking or enhancing the availability of IGFs to bind cell surface receptors. To clarify the in vivo role of IGFBP-2, we have used gene targeting to introduce a null IGFBP-2 allele into the mouse genome. Animals homozygous for the altered allele are viable and fertile, contain no IGFBP-2 mRNA, and have no detectable IGFBP-2 in the adult circulation. Heterozygous and homozygous animals showed no significant differences in prenatal or postnatal body growth. Analyses of organ weights in adult males, however, revealed that spleen weight was reduced and liver weight was increased in the absence of IGFBP-2. In addition, ligand blot analyses of sera from adult IGFBP-2 null males showed that IGFBP-1, IGFBP-3, and IGFBP-4 levels were increased relative to wild-type mice. These results demonstrate that up-regulation of multiple IGFBPs accompanies the absence of IGFBP-2 and that IGFBP-2 has a critical role, either directly or indirectly, in modulating spleen and liver size.
Asunto(s)
Crecimiento/genética , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Eliminación de Secuencia , Animales , Peso Corporal , Fertilidad/genética , Corazón/anatomía & histología , Heterocigoto , Homocigoto , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , 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/deficiencia , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Riñón/anatomía & histología , Hígado/anatomía & histología , Pulmón/anatomía & histología , Masculino , Ratones , Ratones Noqueados , Tamaño de los Órganos , ARN Mensajero/genética , Mapeo Restrictivo , Bazo/anatomía & histologíaRESUMEN
The insulin-like growth factor (IGF) system is an important regulator of fetal growth and differentiation. IGF bioavailability is modulated by IGF binding proteins (IGFBPs). We have generated six different antisera, directed to synthetic peptide fragments of mouse IGFBP-1 through -6. The specificity of the produced antisera was demonstrated by enzyme-linked immunosorbent assay, Western blotting, and by immunohistochemistry on sections of mouse embryos of 13.5 days post coitum. Specificity for the IGFBP-2 through -6 antisera also was confirmed immunohistochemically in liver and lung of corresponding gene deletion (knock-out) mutant mice and wild-type litter mates. Immunohistochemistry and messenger RNA (mRNA) in situ hybridization on sections of mouse embryos of 13.5 days post coitum revealed tissue-specific expression patterns for the six IGFBPs. The only site of IGFBP-1 protein and mRNA production was the liver. IGFBP-2, -4, and -5 protein and mRNA were detected in various organs and tissues. IGFBP-3 and -6 protein and mRNA levels were low. In several tissues, such as lung, liver, kidney, and tongue, more than one IGFBP (protein and mRNA) could be detected. Differences between mRNA and protein localization were extensive for IGFBP-3, -5, and -6, suggesting that these IGFBPs are secreted and transported. These results confirm the different spatial localization of the IGFBPs, on the mRNA and protein level. The overlapping mRNA and protein localization for IGFBP-2 and -4, on the other hand, may indicate that these IGFBPs also function in an auto- or paracrine manner.
Asunto(s)
Embrión de Mamíferos/química , Sueros Inmunes/biosíntesis , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , ARN Mensajero/análisis , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Inmunohistoquímica , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/química , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/inmunología , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/química , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/inmunología , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/química , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/inmunología , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/química , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/inmunología , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/química , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/inmunología , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/química , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/inmunología , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/inmunología , Leucemia Eritroblástica Aguda , Ratones , Datos de Secuencia Molecular , Células Tumorales CultivadasRESUMEN
Non-islet-cell tumour-induced hypoglycaemia (NICTH) is, in most cases, attributable to tumour production of insulin-like growth factor II (IGF-II). Tumour-derived IGF-II has a higher than normal molecular weight (big 'IGF-II') and an impaired ability to form the normal ternary 150 kD complex with IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). Consequently, tumoral IGF-II circulates mainly in smaller binary complexes which have a higher bioavailability than the ternary complex. We had the opportunity to analyze IGFs and IGF-related factors in both pre- and post-operative blood, tumour tissue and tumour cyst fluid from a patient with a disseminated haemangiopericytoma and severe hypoglycaemia. In addition, the effect of serum and tumour cyst fluid on autophosphorylation of the insulin receptor was examined. Patient serum contained low levels of IGF-I, IGFBP-3 and ALS, while the concentrations of IGFBP-2 and IGFBP-6 were markedly elevated. The total level of circulating IGF-II was within the normal range, but Biogel P-60 gel filtration of patient serum revealed that 77% of the IGF-II was present in high molecular weight forms (normal: 10-15%), which decreased to 53% after partial removal of the tumour. Most of the IGF-II immunoreactivity in pre- and post-operative patient serum was associated with 50-60 kD complexes with only a minimal contribution (<10%) from the 150 kD complex. Tumour cyst fluid contained excessive amounts of both big IGF-II and IGFBP-6. Northern blot analysis of total mRNA isolated from the tumour demonstrated high expression of the IGF-II gene and abundant 1.1 kb IGFBP-6 transcript, while the genes encoding IGFBP-3, -4 and -5 were only weakly expressed and mRNA of IGFBP-1, -2 and IGF-I could not be detected. mRNAs for the IGF type II receptor could be easily demonstrated, whereas those for the insulin- and IGF type I receptor were hardly detectable. In contrast to patient serum tumour cyst fluid strongly stimulated the insulin receptor in vitro. The present study suggests an important role of the simultaneous production of IGF-II and IGFBP-6 in the pathophysiology of tumour-induced hypoglycaemia.
Asunto(s)
Hemangiopericitoma/metabolismo , Hipoglucemia/metabolismo , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Northern Blotting , Western Blotting , Cromatografía en Gel , Hemangiopericitoma/complicaciones , Humanos , Hipoglucemia/etiología , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/genética , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana Edad , ARN Mensajero/análisis , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/metabolismo , Receptor de Insulina/metabolismoRESUMEN
Expression of IGF-I, IGF-II, the Type-I IGF receptor and six IGF binding proteins were examined in three different T-ag-driven mouse tumors. Unlike the widespread expression of IGF-II in pancreatic beta-cell tumors, IGF-II was not widely expressed in the two different pituitary tumors examined indicating that a mechanism independent of focal IGF-II expression can also drive T-antigen tumorigenesis. In addition, multiple IGF binding proteins were expressed in all three tumor types. This expression, however, was generally heterogeneous with no specific changes to indicate a required role for any IGF binding protein in T-antigen tumorigenesis.
Asunto(s)
Antígenos Virales de Tumores/genética , Factor II del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Neoplasias Hipofisarias/genética , Receptor IGF Tipo 1/genética , Animales , ADN de Neoplasias/análisis , Regulación Neoplásica de la Expresión Génica , Hibridación in Situ , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Ratones , Estadificación de Neoplasias , Hipófisis/química , Hipófisis/patología , Hipófisis/fisiología , Neoplasias Hipofisarias/patología , ARN Mensajero/análisisRESUMEN
Morphine produces analgesia by activating mu opioid receptors encoded by the MOR-1 gene. Although morphine-6 beta-glucuronide (M6G), heroin and 6-acetylmorphine also are considered mu opioids, recent evidence suggests that they act through a distinct receptor mechanism. We examined this question in knockout mice containing disruptions of either the first or second coding exon of MOR-1. Mice homozygous for either MOR-1 mutation were insensitive to morphine. Heroin, 6-acetylmorphine and M6G still elicited analgesia in the exon-1 MOR-1 mutant, which also showed specific M6G binding, whereas M6G and 6-acetylmorphine were inactive in the exon-2 MOR-1 mutant. These results provide genetic evidence for a unique receptor site for M6G and heroin analgesia.
Asunto(s)
Analgésicos Opioides/farmacología , Exones/genética , Heroína/farmacología , Derivados de la Morfina/farmacología , Receptores Opioides mu/genética , Animales , Resistencia a Medicamentos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/genética , Ratones Noqueados/fisiología , Transcripción Genética/fisiologíaRESUMEN
Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.
Asunto(s)
Analgesia , Tolerancia a Medicamentos , Morfina , Receptores Opioides delta/genética , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/metabolismo , Animales , Encefalina D-Penicilamina (2,5)/administración & dosificación , Encefalina D-Penicilamina (2,5)/metabolismo , Exones , Eliminación de Gen , Marcación de Gen , Inyecciones Intraventriculares , Inyecciones Espinales , Ratones , Ratones Noqueados , Oligopéptidos/administración & dosificación , Oligopéptidos/metabolismo , Receptores Opioides delta/fisiología , Médula Espinal/efectos de los fármacos , TritioRESUMEN
The mitogenic and metabolic activities of insulin-like growth factors (IGF) are modulated by a family of six high affinity IGF binding proteins (IGFBPs). This study describes the expression of the mouse IGFBP-6 which is unique among IGFBPs in its preferential binding of IGF II, in insect cells using the baculovirus system. The purified, O-glycosylated IGFBP-6 was functional as shown by IGF binding and by inhibition of IGF II-stimulated DNA synthesis in human fibroblasts. Specific antibodies generated in chicken against the recombinant IGFBP-6 were used for Western blotting analysis and immunohistochemistry. Strong immunoreactivity was found in ossifying bones of the cranial base, in cell clusters of the pancreas anlage, in the trigeminal ganglion, on myoblasts, on motoneurons of the spinal cord of embryonic mice. In tissues of adult mouse, strong IGFBP-6 immunostaining was present in epidermal and peridermal layers of the skin, in meningeal layers, in long-striated skeletal muscle, and in the Langerhans' islets of the pancreas. No immunopositive staining was observed in lung and liver indicating that sites of synthesis and IGFBP action are different.
Asunto(s)
Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/aislamiento & purificación , Animales , Especificidad de Anticuerpos , Baculoviridae/genética , Pollos , Humanos , Sueros Inmunes/biosíntesis , Inmunohistoquímica , Insectos , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/inmunología , Ratones , Peso Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Dietary potassium (K) depletion is known to reduce body weight gain and organ growth, except for kidney which increases in weight. This renal hypertrophy is preceded by increased renal IGF-I levels. In the present study, we investigated IGF-I and -II, type I IGF receptor and IGF-binding protein (IGFBP) mRNA expression in liver and kidney of K-depleted and normal rats infused with vehicle or recombinant human IGF-I. Body weight gain was almost completely arrested in K-depleted rats without any stimulatory effect of IGF-I infusion. Both absolute and relative kidney weight (kidney weight/body weight) were significantly increased in K-depleted rats and this was further enhanced by IGF-I infusion. In contrast, relative liver weight was comparable in the different groups and unaffected by IGF-I infusion. IGF-I mRNA expression was significantly lower in kidney and liver of K-depleted animals whereas type I IGF receptor levels were unchanged. In contrast, in kidney, K depletion increased IGFBP-1 and -2 mRNA expression with no additional effect of IGF-I infusion. In liver of K-depleted animals, IGFBP-1 mRNA expression was increased whereas increased IGFBP-1 and -2 mRNA expression was observed when these animals were infused with IGF-I. These observations may point towards a differential mode of action of the IGFBPs. In kidney increased IGFBP-1 and -2 mRNA expression may enhance IGF-I bioavailability with subsequent kidney growth. In liver, with clearly detectable type I IGF receptor mRNA expression, increased IGFBP levels may protect from IGF-I-induced organ growth by decreasing IGF-I bioavailability.
Asunto(s)
Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Riñón/metabolismo , Hígado/metabolismo , Deficiencia de Potasio/genética , Receptor IGF Tipo 1/genética , Animales , Glucemia/análisis , Femenino , Expresión Génica , Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Factor I del Crecimiento Similar a la Insulina/metabolismo , Riñón/patología , Hígado/patología , Tamaño de los Órganos , Deficiencia de Potasio/sangre , ARN Mensajero/genética , Ratas , Ratas Wistar , Tibia/anatomía & histología , Aumento de PesoRESUMEN
It has recently been demonstrated in various clinical experiments that native somatostatin and its long-acting analogues increase circulating levels of insulin-like growth factor-binding protein-1 (IGFBP-1) within 1-2 h, independent of effects on circulating insulin or glucose levels. Using human hepatoma cells in vitro the somatostatin analogue, octreotide, has been shown to increase IGFBP-1 mRNA within 24 h indicative of a direct stimulatory effect of octreotide on IGFBP-1 synthesis. In order to ascertain whether octreotide acutely stimulates IGFBP-1 mRNA in vivo, placebo or two doses of octreotide were injected subcutaneously into three groups of rats. One hour after saline or octreotide administration, liver, kidney and serum were obtained for the measurement of IGFBPs-1 to -6 mRNA in tissue and IGFBPs and IGF-I in serum. Octreotide increased liver IGFBP-1 (562%) and IGFBP-3 (23%) mRNA expression with a concomitant rise in the circulating 30 kDa (106%) and 38-42 kDa (23%) IGFBPs. No detectable changes were seen in other liver IGFBP transcripts, other circulating IGFBPs or in any of the kidney IGFBP transcripts. Serum IGF-I increased by 37% in the animals receiving the high octreotide dose. No concomitant changes were observed in glucose or insulin levels. These data show that octreotide acutely stimulates hepatic IGFBP-1 and -3 mRNA in vivo in rats. The stimulating effect on IGFBP-3 presents a possible hitherto unknown form of regulation of IGFBP-3 whilst the effect on IGFBP-1 indicates that the stimulatory effect of octreotide on circulating IGFBP-1 described in clinical trials may be due to increased hepatic production. The present findings may be of importance in the clinical use of octreotide.
Asunto(s)
Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Hígado/metabolismo , Octreótido/farmacología , Animales , Femenino , Expresión Génica , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Riñón/metabolismo , Hígado/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Estimulación QuímicaRESUMEN
Insulin-like growth factor II (IGF-II)-overexpressing NIH 3T3 cells were used to examine regulation of insulin-like growth factor binding protein (IGFBP) and mannose 6-phosphate (M6P)/IGF-II receptor expression. Ligand blot analysis of conditioned media indicated a predominant IGFBP of 26-28 kilodaltons the abundance of which is 3- to 10-fold higher in media of IGF-II-overexpressing cells. The IGFBP level in control cell medium was increased by incubation in the presence of IGF-II, IGF-I, and mutant IGF-II forms with reduced affinities for IGF-I or M6P/IGF-II receptors. Further proof that IGF-II regulated the IGFBP was obtained by incubation of IGF-II overexpressing cells in the presence of antisense IGF-II oligomers or anti-IGF-II antibodies, which resulted in significant reduction of the IGFBP in conditioned medium. Mouse IGFBP-6 mRNA expression was increased in IGF-II-overexpressing or IGF-II-treated control cells. The IGFBP contained O-linked carbohydrate residues and was recognized by an antiserum to rat IGFBP-6. To determine whether IGFs were influencing proteolytic degradation of IGFBPs, cell-free conditioned media were incubated at 37 C with recombinant human IGFBPs. At neutral pH proteolysis of IGFBP-5 occurred during incubation in conditioned media from control and IGF-II-overexpressing cells. Upon acidification of the medium samples, only the degradation of IGFBP-6 was prevented in IGF-II-overexpressing cell-conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Receptor IGF Tipo 2/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Medios de Cultivo Condicionados , Regulación de la Expresión Génica , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , RatasRESUMEN
The IGFs are important mitogens involved in lung growth and development. The regulation of IGF action depends not only on the expression of IGFs and IGF receptors, but also on the modulation of IGF activity by IGF-binding proteins (IGFBPs). In this study, we describe the mRNA expression of IGF-I, IGF-II, type I IGF receptor, IGFBP-2, IGFBP-4 and IGFBP-5 during mouse lung development as studied by in situ hybridization techniques. The IGF, type I IGF receptor and IGFBP-2, -4 and -5 genes were expressed in developing lung as early as embryonal day 12.5. Expression of IGFBPs-1, -3 and -6 was below detection. IGF and IGFBP-2 mRNAs were expressed both in mesenchymal and epithelial cells. Type I IGF receptor transcripts were also observed throughout the developing lung, with the exception of the epithelial cells of the bronchi after embryonal day 15. Furthermore, mRNA expression of IGFBPs-4 and -5 was noted in neighbouring cell types, and after embryonal day 15, co-expression of the type I IGF receptor and IGFBP-4 transcripts was detected. The observed expression patterns imply that the IGFBP-2, -4 and -5 genes are differentially regulated during embryonic development and suggest that each may have a discrete function. A possible role for IGFBPs-2, -4 and -5 is to participate in the regulation of cell-specific IGF responses during mouse lung development.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Fetales/genética , Regulación del Desarrollo de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/genética , Pulmón/embriología , ARN Mensajero/biosíntesis , Receptor IGF Tipo 1/genética , Animales , Bronquios/embriología , Bronquios/metabolismo , Proteínas Portadoras/biosíntesis , Epitelio/embriología , Epitelio/metabolismo , Proteínas Fetales/biosíntesis , Hibridación in Situ , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Factor II del Crecimiento Similar a la Insulina/genética , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , Alveolos Pulmonares/embriología , Alveolos Pulmonares/metabolismo , ARN Mensajero/genética , Receptor IGF Tipo 1/biosíntesisRESUMEN
We investigated the spatio-temporal changes in RNA and protein expression of growth factors and their receptors by in situ hybridization and immunocytochemistry during regeneration after acute injury of mouse urothelium in vivo. These data were correlated with changes in morphology and proliferation during regeneration. Except for an enhanced muscular transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta type II receptor expression, changes in expression patterns of growth factors or receptors were confined to the urothelium. Increased mucosal RNA expression of insulin-like growth factor-II (IGF-II) and particularly of type I IGF receptor, as well as fibroblast growth factor-1 (FGF-1) but not of FGF-2, coincided with re-epithelialization and urothelial proliferation. Both high levels of urothelial TGF-beta 1 RNA and protein expression were associated with re-epithelialization and differentiation. In addition, TGF beta type II receptor protein expression was similarly enhanced in the same urothelial cells. Platelet-derived growth factor-A (PDGF-A) RNA was expressed constitutively in the mucosa but decreased in the reepithelialization phase. The data are consistent with the notion that urothelial regeneration can be achieved by paracrine or autocrine acting, urothelium-derived growth factors. Since analogous growth factor RNA expression patterns in regenerating skin epidermis have been found, a more general growth factor-regulated mechanism for epithelial regeneration may be suggested.
Asunto(s)
Sustancias de Crecimiento/biosíntesis , Receptores de Factores de Crecimiento/biosíntesis , Regeneración , Vejiga Urinaria/fisiología , Animales , Diferenciación Celular , División Celular , ADN/biosíntesis , Epitelio/química , Epitelio/patología , Epitelio/fisiología , Femenino , Sustancias de Crecimiento/genética , Técnicas para Inmunoenzimas , Hibridación in Situ , Ratones , ARN Mensajero/análisis , Vejiga Urinaria/química , Vejiga Urinaria/patología , Cicatrización de Heridas/fisiologíaRESUMEN
The insulin-like growth factor binding proteins (IGFBPs) comprise a family of six distinct proteins which modulate insulin-like growth factor action. We have isolated cDNAs encoding the six mouse IGFBPs (mIGFBPs). In addition, we studied the mRNA expression of the six mIGFBPs during development and in various adult tissues. Our results show that each of the six mIGFBPs is highly homologous to their human and rat counterparts, whereas only the N and C terminal ends are conserved between the six mIGFBPs. Northern blotting revealed that mIGFBP-2, -3, -4 and -5 genes are already expressed at gestational day 11.5, suggesting a role for these mIGFBPs in embryonal development. In liver, a peak of mIGFBP-1 mRNA expression was found around birth, suggesting a function for mIGFBP-1 in the newborn mouse. Finally, tissue-specific expression of the six mouse IGFBP genes was observed in adult tissues suggesting different roles or modes of actions in adult life.
Asunto(s)
Proteínas Portadoras/genética , ADN Complementario/genética , ARN Mensajero/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Complementario/análisis , Femenino , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Riñón/química , Hígado/química , Pulmón/química , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , ARN Mensajero/análisis , Ratas , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Somatomedinas/genética , Bazo/químicaRESUMEN
In order to investigate potential changes in insulin-like growth factor binding proteins (IGFBPs) during human follicle maturation, we examined the IGFBP profiles in follicular fluid from follicles in different stages of maturation. Samples were obtained from ovaries of women with regular menstrual cycles and of subjects with cycle abnormalities and polycystic ovaries (diagnosed as polycystic ovary syndrome (PCOS)) and analyzed by Western ligand blotting. IGFBPs of 43 kDa, 37 kDa, 31 kDa, a doublet around 28 kDa and a minor band of 24 kDa were detected in follicle fluid of normal non-dominant (size < 10 mm) and atretic (androstenedione/estradiol ratio > 4) follicles of both regularly menstruating women and PCOS patients. The 43 and 37 kDa IGFBPs could be identified as IGFBP-3 and the 31 kDa IGFBP as IGFBP-2, whereas the 28 kDa IGFBP could not be identified as IGFBP-1, all by immunoblotting techniques. A dramatic decrease in IGFBP-2, the 28 kDa and 24 kDa IGFBPs was observed in follicular fluid of dominant follicles (size > 10 mm) of both regular menstruating individuals and one PCOS patient as compared with follicular fluid of normal non-dominant or atretic follicles. These observations indicate that the PCOS follicle may not be different from normal with respect to IGFBP profiles. Furthermore, these results suggest that at least one of these IGFBPs might be involved in human folliculogenesis.
Asunto(s)
Proteínas Portadoras/metabolismo , Líquido Folicular/metabolismo , Ciclo Menstrual/metabolismo , Síndrome del Ovario Poliquístico/metabolismo , Adulto , Western Blotting , Proteínas Portadoras/análisis , Electroforesis en Gel de Poliacrilamida , Femenino , Hormona Folículo Estimulante/sangre , Líquido Folicular/química , Humanos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina , Hormona Luteinizante/sangre , Peso Molecular , Síndrome del Ovario Poliquístico/sangre , Valores de ReferenciaRESUMEN
The insulin-like growth factor-binding proteins (IGFBPs) comprise at least six distinct species that may modulate the action of IGFs. IGFs are important regulators of fetal growth and differentiation. To define sites of IGFBP mRNA synthesis, we have used in situ hybridization techniques in mouse conceptuses of different gestational ages (11-18 days). Expression of mouse (m) IGFBP-1 was detected in mouse conceptuses after day 12 of gestation and was restricted to the liver. Transcripts for mIGFBP-2, -4, and -5 were detected in various tissues and were found in all stages tested. In contrast, expression of mIGFBP-3 and -6 could be only weakly detected in late gestational conceptuses. Comparison of the expression patterns of mIGFBP-2, -4, and -5, which were found widely distributed in mouse conceptuses, revealed that mIGFBP-2 was expressed in the mesoderm-derived part of the tongue (day 13.5), but mainly in the ectodermal layer. Transcripts for mIGFBP-4, however, were detected only in the mesodermal part, whereas expression of mIGFBP-5 was restricted to the ectodermal layer. A similar distribution pattern was observed in the lung (day 18). In general, expression of mIGFBP-2 and -5 was detected in the same cells, whereas mIGFBP-4 and -5 were expressed mainly in different cell types. These data suggest that the different mIGFBPs might play distinct roles in mouse embryonal and fetal life.
Asunto(s)
Proteínas Portadoras/genética , Embrión de Mamíferos/fisiología , Expresión Génica , Ratones/embriología , Animales , Autorradiografía , Proteínas Portadoras/metabolismo , Embrión de Mamíferos/metabolismo , Edad Gestacional , Hibridación in Situ , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Ratones/genética , Somatomedinas/metabolismo , Distribución TisularRESUMEN
In order to identify regions in insulin-like growth factor binding protein-1 involved in the binding of IGFs, we tested three monoclonal antibodies, designated MAb A, B, and C on their interference with IGF-binding. Monoclonal A interfered with the binding of IGF to IGFBP-1 as determined by immunoprecipitation whereas monoclonal B and C did not. Furthermore MAb A was found to abolish IGFBP-1 inhibition of IGF stimulation in an in vitro proliferation assay. The epitopes of all three monoclonal antibodies were found to be located within the C-terminal part of IGFBP-1. The regions surrounding residue 188-196 and 222-227 are especially important for antibody recognition. These results indicate that MAb A functionally interferes with the binding of IGF to IGFBP-1. Furthermore, we suggest that part of the epitope of MAb A is located at or sterically near the IGF binding domain of IGFBP-1.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas Portadoras/metabolismo , Epítopos/análisis , Somatomedinas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Línea Celular , Epítopos/inmunología , Humanos , Hibridomas , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina , Ratones , Datos de Secuencia Molecular , Mutación Puntual , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Somatomedinas/metabolismoRESUMEN
The IGF binding proteins (IGFBPs) comprise at least six distinct species which may modulate the action of IGFs. IGFs are important regulators of fetal growth and differentiation. We have studied the mRNA expression of the six IGFBPs during post-implantation embryogenesis (day 11-18) by in situ hybridization techniques. Expression of IGFBP-1 was detected in mouse conceptuses after day 12 of gestation and seemed restricted to the liver. Transcripts for IGFBP-2, -4 and -5 were detected in various tissues and were found in all stages tested. In contrast, expression of IGFBP-3 and -6 could be detected only weakly in late gestational embryos. Comparison of the expression pattern of IGFBP-2, -4 and -5, which were found widely distributed in mouse conceptuses, revealed that IGFBP-2 was expressed mainly in the ectodermal layer and also in the mesoderm derived part of the tongue (day 13.5). Transcripts for IGFBP-4 however, only were detected in the mesoderm derived tissues, whereas expression of IGFBP-5 was restricted to the ectodermal layer. A similar distribution pattern was observed in the lung. In general, expression of IGFBP-2 and -5 was detected in the same cells, whereas IGFBP-4 and -5 were expressed mainly in different cell types. In rodents as in the human there is widespread expression of the genes coding IGFs, the IGFBPs and the receptors during pre- and postimplantation embryogenesis. These data support the assumption that the IGFs play an important role during embryogenesis.
Asunto(s)
Proteínas Portadoras/biosíntesis , Feto/metabolismo , Expresión Génica , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Receptor IGF Tipo 1/biosíntesis , Receptor IGF Tipo 2/biosíntesis , Animales , Blastocisto/metabolismo , Desarrollo Embrionario y Fetal , Feto/fisiología , Edad Gestacional , Humanos , Hibridación in Situ , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Ratones , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/metabolismoRESUMEN
To date six IGF binding proteins (IGFBP) have been characterized. Analysis of the amino acid sequence reveals that the IGFBPs are clearly distinct but are sharing regions with strong homology. Specifically the hydrophobic cysteine rich N-terminal region and to a lesser extend the C-terminal part are preserved. The alignment of the cysteine molecules is strongly conserved across the 6 IGFBPs. The middle one-third region, where no cysteines are present (except for IGFBP-4) is most divergent. IGFBP-3 and -4 are glycosylated, whereas IGFBP-1 and -2 contain an Arg-Gly-Asp sequence near the carboxyl terminus. Determination of the number of free-SH groups of IGFBP-1 and -3 has revealed that most likely all cysteine residues are involved in disulfide bond formation. All members of the IGFBP family bind IGF-I and IGF-II with about equal affinity. Studies involving deletion mutation and site-directed mutagenesis of IGFBP-1 and -3 have suggested that the three-dimensional structure of the protein plays an important role in IGF binding. However at present it is unclear whether the IGFBPs share one or more specific IGF binding domain. The predominant function of the IGFBPs is to allocate IGF in the various body fluid compartments and tissues and to modulate IGF binding to receptors. For this purpose there exists a very sophisticated control of the routing of circulating IGF both from and to the cell. There is mounting evidence that the structure of the IGFBP proteins plays a key role in the regulation of IGF bioavailability, by modulating its molecular size, capillary membrane permeability, target tissue specificity, cell membrane adherence and IGF affinity.
Asunto(s)
Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/fisiología , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Datos de Secuencia Molecular , Estructura Molecular , Homología de Secuencia de AminoácidoRESUMEN
To define domains involved in IGF binding 60 N-terminal amino acid residues of IGFBP-1 were deleted. This deletion resulted in loss of IGF binding suggesting that the N-terminus may enclose an IGF binding domain. However, most point mutations introduced in this region did not affect IGF binding. In contrast to Cys-34, only substitution of Cys-38 for a tyrosine residue abolished IGF binding. With the determination that all 18 cysteine residues are involved in disulphide bond formation our data suggest that, although not all cysteines contribute to the same extent, the ligand binding site may be spatially organized.