RESUMEN
The development of a small animal model for hepatitis C virus (HCV) infection is a critical issue for the development of novel anti-HCV drugs. To this aim, we have tried many different approaches for generating mice carrying humanized liver. Main efforts were focused on the transplantation of human hepatocytes into immunocompromised mice (SCID-/-, Bg-/-) carrying a genetic lethal liver disease (Alb-uPA). Survival of homozygotic animals should largely depend on early transplantation with healthy hepatocytes. In parallel to establishing a colony of Alb-uPA/SCID/Bg mice, we developed a microsurgical procedure for intrasplenic xenotransplantation of healthy hepatocytes in 1-week-old mice. So far, we generated several chimeras by xenotransplanting human hepatocytes in Alb-uPA+/+/SCID-/-/Bg-/- mice at 1 week after birth. In a first step, identification of successfully engrafted animals is possible by quantification of human serum albumin and human alpha 1 antitrypsin in mouse sera. Additional preliminary histomorphological analysis of liver sections from chimeric animals was also carried out. One of the mice was transiently infected with HCV, reaching viremia levels of approximately 10(5) genomes/mL. However, the efficiency of this system to generate chimeric mice is still very limited. We are currently exploring the use of more robust models of hepatic disease. Moreover, we have been also exploring novel strategies for the generation of chimeric mice by xenotransplanting human adult stem cells, instead of human hepatocytes, at preimmune stages of development.
Asunto(s)
Hepatitis C/tratamiento farmacológico , Hepatocitos/trasplante , Animales , Anticuerpos Monoclonales/uso terapéutico , Antivirales/uso terapéutico , Carcinoma Hepatocelular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Hepatopatías/cirugía , Neoplasias Hepáticas , Ratones , Ratones SCID , Ratones Transgénicos , Albúmina Sérica/genética , Trasplante Heterólogo , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
We previously demonstrated that high levels of IL-6/sIL-6R complexes are present in sera of patients with systemic juvenile idiopathic arthritis (s-JIA) and that the amount of IL-6 estimated in the IL-6/sIL-6R complexes is markedly higher than that measured by the B9 assay. Here, we show that two additional bioassays, employing human myeloma XG-1 cells and human hepatoma Hep3B cells, detected serum IL-6 levels similar to those measured by the B9 assay and approximately 10-fold lower than the IL-6 levels estimated to be present in the IL-6/sIL-6R complex. Using an assay for the measurement of the amount of circulating IL-6 complexed with the sIL-6R and available for binding to gp130 (gp130 binding activity), we show that the IL-6/gp130 binding activity is similar to that detected by the bioassays and again significantly lower than that estimated to be present in the IL-6/sIL-6R complex. Addition of recombinant human IL-6 (rhIL-6) to sera of patients or controls results in a markedly lower increase in the gp130 binding activity in patients than in controls. Moreover, sera from s-JIA patients inhibited in a dose dependent manner the gp130 binding activity assay. These results show that sera from patients with s-JIA contain a factor, or factors, that inhibit(s) the binding of the IL-6/sIL-6R complex to gp130. This inhibitory activity does not appear to be due to soluble gp130, C-reactive protein or autoantibodies to IL-6.
Asunto(s)
Antígenos CD/sangre , Artritis Juvenil/sangre , Factores Biológicos/sangre , Interleucina-6/sangre , Glicoproteínas de Membrana/sangre , Receptores de Interleucina-6/sangre , Adolescente , Autoanticuerpos/sangre , Unión Competitiva , Bioensayo/métodos , Proteína C-Reactiva/metabolismo , Niño , Preescolar , Receptor gp130 de Citocinas , Humanos , Interleucina-6/inmunología , Unión Proteica , Transducción de Señal , SolubilidadRESUMEN
The transmembrane glycoprotein gp130 is involved in many cytokine-mediated cellular responses and acts therein as the signal transducing receptor subunit. Interleukin-6 (IL-6) and interleukin-11 (IL-11), in complex with their specific alpha-receptors, homodimerize gp130 and, as a consequence, activate the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) signalling pathway in their target cells. So far, it is not clear whether gp130 is bound to these cytokines and their specific alpha-receptor subunits through identical or different epitopes. In order to study the interaction of IL-11 and IL-11R with human gp130 the soluble form of the recently cloned human IL-11R was expressed in baculovirus-infected insect cells. By a coprecipitation binding-assay it is demonstrated that IL-11 and IL-6 compete for binding to gp130. Using deletion and point mutants of gp130 it is shown that IL-11-IL-11R and IL-6-IL-6R recognize overlapping binding motifs on gp130. Moreover, using well-established Jak-deficient cell lines we demonstrate that STAT activation by IL-11 requires Jak1. Taken together, our data support the concept that IL-6 and IL-11 activate gp130 by very similar molecular mechanisms.
Asunto(s)
Antígenos CD/metabolismo , Interleucina-11/farmacología , Interleucina-6/farmacología , Glicoproteínas de Membrana/metabolismo , Transducción de Señal/fisiología , Animales , Antígenos CD/genética , Unión Competitiva/fisiología , División Celular/efectos de los fármacos , Línea Celular , Receptor gp130 de Citocinas , Proteínas de Unión al ADN/metabolismo , Dimerización , Epítopos/inmunología , Humanos , Subunidad alfa del Receptor de Interleucina-11 , Glicoproteínas de Membrana/genética , Mutación/genética , Proteínas Tirosina Quinasas/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-11 , Receptores de Interleucina-6/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Transactivadores/metabolismoRESUMEN
Neutralization of cytokine activity by monoclonal antibodies or receptor antagonists is beneficial in the treatment of immune and neoplastic diseases, but the necessity for continuous parenteral delivery of these anticytokine agents poses considerable practical limitations. A viable alternative is to induce a neutralizing antibody response. Using transgenic mice with high circulating levels of human interleukin-6 (hIL-6), we show that injection of the hIL-6 receptor antagonist Sant1 (an IL-6 variant with seven amino-acid substitutions) induces a strong anti-hIL-6 antibody response. The elicited antibodies bind circulating hIL-6 with very high affinity, totally masking it, and neutralize hIL-6 bioactivity both in vitro and in vivo.
Asunto(s)
Autoanticuerpos/biosíntesis , Interleucina-6/inmunología , Receptores de Interleucina-6/antagonistas & inhibidores , Hidróxido de Aluminio/química , Animales , Reacciones Antígeno-Anticuerpo , Autoanticuerpos/inmunología , Sitios de Unión , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Regulación de la Expresión Génica/inmunología , Ingeniería Genética , Humanos , Inyecciones Intraperitoneales , Interleucina-6/administración & dosificación , Interleucina-6/genética , Ratones , Ratones Transgénicos/inmunología , Proteínas Recombinantes/inmunología , VacunaciónRESUMEN
Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR) and the signal-transducing beta-subunits gp130 and leukemia inhibitory factor receptor-beta (LIFR). CNTFR can function in either membrane-bound or soluble forms. The membrane-bound form mediates the neuronal actions of CNTF, whereas the soluble form serves to confer cytokine responsiveness to non-neuronal cells expressing gp130 and LIFR. The objective of this work was to analyze whether the two receptor isoforms differ in their ability to interact functionally with CNTF and related proteins. Two new types of CNTF variants, characterized by weakened interactions with either CNTFR or both LIFR and gp130, were developed, and the biological activities of these and other mutants were determined in non-neuronal versus neuronal cells, as well as in non-neuronal cells transfected with an expression vector for CNTFR. Membrane anchoring of CNTFR was found to render the CNTF receptor complex relatively insensitive to changes in agonist affinity for either alpha- or beta-receptor subunits and to promote a more efficient interaction with a gp130-depleting antagonistic variant of CNTF. As a result of this phenomenon, which can be rationalized in terms of the multivalent nature of CNTF receptor interaction, CNTF variants display striking changes in receptor selectivity.
Asunto(s)
Antígenos CD/metabolismo , Inhibidores de Crecimiento , Interleucina-6 , Linfocinas , Glicoproteínas de Membrana/metabolismo , Mutación , Proteínas del Tejido Nervioso/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Antígenos CD/química , Bioensayo , Factor Neurotrófico Ciliar , Receptor gp130 de Citocinas , Relación Dosis-Respuesta a Droga , Humanos , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/química , Proteínas de la Membrana , Modelos Químicos , Proteínas del Tejido Nervioso/genética , Unión Proteica , Proteínas Tirosina Quinasas Receptoras/agonistas , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptor de Factor Neurotrófico Ciliar , Receptores de Citocinas/agonistas , Receptores de Citocinas/antagonistas & inhibidores , Receptores de Factor de Crecimiento Nervioso/agonistas , Receptores de Factor de Crecimiento Nervioso/antagonistas & inhibidores , Receptores OSM-LIF , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Transducción de Señal , SolubilidadRESUMEN
Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the tis-11 primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of ob/ob mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in db/db mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus Tipo 2/terapia , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/farmacología , Obesidad/tratamiento farmacológico , Proteínas/farmacología , Receptores de Superficie Celular , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Núcleo Arqueado del Hipotálamo/fisiología , Núcleo Arqueado del Hipotálamo/fisiopatología , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Encéfalo/fisiología , Encéfalo/fisiopatología , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/fisiología , Línea Celular , Factor Neurotrófico Ciliar , Diabetes Mellitus Tipo 2/fisiopatología , Grasas de la Dieta , Aseo Animal/efectos de los fármacos , Humanos , Células Híbridas , Insulina/sangre , Leptina , Masculino , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos C57BL , Ratones Obesos , Actividad Motora/efectos de los fármacos , Neuroblastoma , Neuronas/fisiología , Obesidad/genética , Obesidad/fisiopatología , Mutación Puntual , Proteínas/genética , Proteínas/fisiología , Receptor de Factor Neurotrófico Ciliar , Receptores de Leptina , Receptores de Factor de Crecimiento Nervioso/biosíntesis , Receptores de Factor de Crecimiento Nervioso/fisiología , Proteínas Recombinantes/farmacologíaRESUMEN
The interleukin-6 (IL-6) family of cytokines activates signaling through the formation of either gp130 homodimers, as for IL-6, or gp130-leukemia inhibitory factor receptor (LIFR) heterodimers as for ciliary neurotrophic factor (CNTF), leukemia inhibitory factor, oncostatinM, and cardiotrophin-1. Recent in vitro studies with IL-6 and CNTF have demonstrated that higher order hexameric receptor complexes are assembled in which signaling chain dimerization is accompanied by the dimerization of both the cytokine molecule and its specific receptor alpha subunits (IL-6Ralpha or CNTFRalpha, respectively). IL-11 is a member of the IL-6 family and known to require gp130 but not LIFR for signaling. In this study we investigate the functional and biochemical composition of the IL-11 receptor complex. The human IL-11 receptor alpha-chain was cloned from a human bone marrow cDNA library. IL-11Ralpha was shown to confer IL-11 responsiveness to human hepatoma cells either by cDNA transfection or by adding a soluble form of the receptor (sIL11Ralpha) expressed in the baculovirus system to the culture medium. In vitro immunoprecipitation experiments showed that sIL11Ralpha specifically binds IL-11 and that binding is enhanced by gp130. Similarly to IL-6 and CNTF, gp130 is able to induce dimerization of the IL-11.IL-11Ralpha subcomplex, the result of which is the formation of a pentameric receptor complex. However, in contrast to the other two cytokines, IL-11 was unable to induce either gp130 homodimerization or gp130/LIFR heterodimerization. These results strongly suggest that an as yet unidentified receptor beta-chain is involved in IL-11 signaling.
Asunto(s)
Antígenos CD/química , Glicoproteínas de Membrana/química , Receptores de Interleucina/química , Animales , Clonación Molecular , Receptor gp130 de Citocinas , ADN Complementario/genética , Humanos , Interleucina-11/metabolismo , Subunidad alfa del Receptor de Interleucina-11 , Neoplasias Hepáticas Experimentales , Sustancias Macromoleculares , Unión Proteica , Receptores de Interleucina-11 , Transducción de Señal , Solubilidad , Células Tumorales CultivadasRESUMEN
Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LIFR without affecting binding to CNTFR alpha or gp130. The resulting variants behaved as partial agonists with varying degrees of residual bioactivity in different cell-based assays. Simultaneous alanine substitution of both F152 and K155 totally abolished biological activity. Combining these mutations with amino acid substitutions in the D-helix, which enhance binding affinity for the CNTFR alpha, gave rise to a potent competitive CNTF receptor antagonist. This protein constitutes a new tool for studies of CNTF function in normal physiology and disease.
Asunto(s)
Inhibidores de Crecimiento , Interleucina-6 , Linfocinas , Proteínas del Tejido Nervioso/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Factor de Crecimiento Nervioso/antagonistas & inhibidores , Secuencia de Aminoácidos , Antígenos CD , División Celular/efectos de los fármacos , Células Cultivadas , Colina O-Acetiltransferasa/biosíntesis , Factor Neurotrófico Ciliar , Receptor gp130 de Citocinas , Análisis Mutacional de ADN , Relación Dosis-Respuesta a Droga , Haptoglobinas/biosíntesis , Humanos , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Glicoproteínas de Membrana , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/farmacología , Unión Proteica , Receptor de Factor Neurotrófico Ciliar , Receptores OSM-LIF , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Interleukin-6 (IL-6) is the major growth factor for myeloma cells and is believed to participate in the pathogenesis of chronic autoimmune diseases and postmenopausal osteoporosis. IL-6 has been recently shown to possess three topologically distinct receptor binding sites: site 1 for binding to the subunit specific chain IL-6R alpha and sites 2 and 3 for the interaction with two subunits of the signaling chain gp130. We have generated a set of IL-6 variants that behave as potent cytokine receptor super-antagonists carrying substitutions that abolish interaction with gp130 at either site 2 alone (site 2 antagonist) or at both sites 2 and 3 (site 2 + 3 antagonist). In addition, substitutions have been introduced in site 1 that lead to variable increases in binding for IL-6R alpha up to 70-fold. IL-6 super-antagonists inhibit wild-type cytokine activity with efficacy proportional to the increase in receptor binding on a variety of human call lines of different origin, and the most potent molecules display full antagonism at low molar excess to wild-type IL-6. When tested on a representative set of IL-6-dependent human myeloma cell lines, although site 2 super-antagonists were in general quite effective, only the site 2 + 3 antagonist Sant7 showed antagonism on the full spectrum of cells tested. In conclusion, IL-6 super-antagonists are a useful tool for the study of myeloma in vitro and might constitute, in particular Sant7, effective IL-6 blocking agents in vivo.
Asunto(s)
Antígenos CD/química , Inhibidores de Crecimiento/farmacología , Interleucina-6/análogos & derivados , Interleucina-6/farmacología , Mieloma Múltiple/patología , Fragmentos de Péptidos/farmacología , Receptores de Interleucina/química , Carcinoma Hepatocelular/patología , Inhibidores de Crecimiento/química , Humanos , Interleucina-6/química , Neoplasias Hepáticas/patología , Melanoma/patología , Modelos Moleculares , Morfolinas/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Fragmentos de Péptidos/química , Reacción en Cadena de la Polimerasa , Conformación Proteica , Receptores de Interleucina-6 , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Relación Estructura-Actividad , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Ciliary neurotrophic factor (CNTF) and interleukin-6 (IL-6) potentiate the elevation of serum corticosterone induced by suboptimal doses of interleukin-1 (IL-1). CNTF also potentiates IL-1-induced serum IL-6. Here, we report that four other cytokines (leukemia inhibitory factor [LIF], oncostatin M [OSM], interleukin-11 and cardiotrophin-1) also potentiated the elevation of serum corticosterone and IL-6 levels induced by IL-1. Furthermore, all the six cytokines studied induced the acute-phase protein serum amyloid A when administered alone. Because these cytokines differ both in structure and in function, but share gp130 as a subunit of their receptors, these results indicate that signaling through gp130 mediates potentiation of IL-1 activities. The potentiation of IL-1-induced serum corticosterone levels is not a consequence of the increased serum IL-6 observed after IL-1 administration. In fact, in IL-6 deficient mice, IL-1 increased serum corticosterone to a level comparable to that observed in wild-type mice. Thus, either endogenous IL-6 does not mediate IL-1-induced corticosterone increase, or its role may be fulfilled by other cytokines. To the extent that gp130-dependent cytokines may serve this role, they may be important feedback regulators of inflammation through the activation of the hypothalamus-pituitary-adrenal axis and the potentiation of acute-phase protein synthesis.
Asunto(s)
Antígenos CD/fisiología , Corticosterona/metabolismo , Citocinas/farmacología , Glicoproteínas de Membrana/fisiología , Receptores de Citocinas/química , Transducción de Señal/efectos de los fármacos , Reacción de Fase Aguda , Animales , Antígenos CD/química , Factor Neurotrófico Ciliar , Receptor gp130 de Citocinas , Citocinas/metabolismo , Sinergismo Farmacológico , Inhibidores de Crecimiento/metabolismo , Inhibidores de Crecimiento/farmacología , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/metabolismo , Interleucina-1/metabolismo , Interleucina-1/farmacología , Interleucina-11/metabolismo , Interleucina-11/farmacología , Interleucina-6/metabolismo , Interleucina-6/farmacología , Factor Inhibidor de Leucemia , Hígado/efectos de los fármacos , Hígado/metabolismo , Linfocinas/metabolismo , Linfocinas/farmacología , Masculino , Glicoproteínas de Membrana/química , Ratones , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/farmacología , Oncostatina M , Péptidos/metabolismo , Péptidos/farmacología , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/metabolismo , Receptores de Citocinas/efectos de los fármacos , Receptores de Citocinas/genética , Proteínas Recombinantes/farmacología , Proteína Amiloide A Sérica/metabolismoRESUMEN
Ciliary neurotrophic factor (CNTF) is a cytokine sharing structural and functional similarities with interleukin-6 (IL-6) and other helical cytokines that utilize the common signalling chain gp130. While IL-6 induces gp130 dimerization, CNTF, after the initial interaction with the specific, non-signalling receptor subunit, CNTFR, induces the formation of gp130/LIF-receptor heterodimers. Through immunoprecipitation experiments with tagged soluble receptor molecules, we recently demonstrated that IL-6 drives the formation of a hexameric receptor complex with a defined topology and composed of two IL-6, two IL-6R alpha and two gp130 molecules. Here, we apply the same strategy to study the assembly in vitro of the CNTF receptor complex. We present evidence that both the cytokine and the specific binding chain undergo dimerization in the presence of gp130. Furthermore, although gp130 and LIFR are able to bind independently to the CNTF/CNTFR sub-complex, they never form homodimers but only heterodimers. We propose that CNTF assembles a hexameric receptor complex composed of two CNTF, two CNTFR, one gp130 and one LIFR molecule, and present a model of the reciprocal interaction of these molecules based on similarities with the IL-6 hexameric complex.
Asunto(s)
Interleucina-6/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Factor Neurotrófico Ciliar , ADN Complementario , Glicerofosfatos/metabolismo , Insectos/metabolismo , Datos de Secuencia Molecular , Agregación de Receptores , Receptor de Factor Neurotrófico Ciliar , Receptores de Factor de Crecimiento Nervioso/química , Receptores de Factor de Crecimiento Nervioso/genética , Proteínas Recombinantes/metabolismoRESUMEN
Phage display of proteins can be used to study ligand-receptor interaction and for the affinity-maturation of binding sites in polypeptide hormones and/or cytokines. We have expressed human interleukin-6 (hIL-6) on M13 phage in a monovalent fashion as a fusion protein with the phage coat protein, pIII. Phage-displayed hIL-6 is correctly folded, as judged by its ability to interact with conformation-specific anti-hIL-6 monoclonal antibodies (mAb) and with the hIL-6 receptor complex in vitro. We set up an experimental protocol for the efficient affinity selection of hIL-6 phage using the extracellular portion of the hIL-6 receptor alpha (hIL-6R alpha) fixed on a solid phase. This system was used to affinity-purify from a library of hIL-6 variants, in which four residues in the predicted D-helix of the cytokine were fully randomized, mutants binding hIL-6R alpha with higher efficiency than the wild type. When the best-binder variant Q175I/Q183A was combined with a previously identified superbinder S176R [Savino et al., Proc. Natl. Acad. Sci. 90 (1993) 4067-4071], a triple-substitution mutant Q175I/S176R/Q183A (hIL-6IRA) was obtained with a fivefold increased hIL-6R alpha binding and a 2.5-fold enhanced biological activity.
Asunto(s)
Antígenos CD/metabolismo , Interleucina-6/química , Glicoproteínas de Membrana/metabolismo , Anticuerpos Monoclonales/inmunología , Bacteriófago M13 , Receptor gp130 de Citocinas , Humanos , Interleucina-6/inmunología , Interleucina-6/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Citocinas/metabolismo , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
The helical cytokine interleukin-6 (IL-6) assembles a multiprotein receptor complex. The starting event in the activation of intracellular signaling is the binding of the IL-6/IL-6R alpha subcomplex to two gp130 chains. The homodimerization of gp130 is triggered by two distinct and independent regions of IL-6 called sites 2 and 3. Several IL-6 antagonists have been obtained that affect signaling, but not IL-6 IL-6R alpha subcomplex formation. In this paper, we analyze in detail the impact of these antagonists on gp130 binding and dimerization and show that each signaling variant affects gp130 dimerization in vitro and that biological activity on cells decreases in precise parallel to the decrease in gp130 dimerization in vitro. All IL-6 antagonists can be classified into two groups, mapping at either site 2 or 3 in correspondence to their mode of interaction with gp130. We found that site 3 is a large region, which includes residues at the beginning of helix D spatially flanked by residues in the putative AB loop and located at one extremity of the cytokine 4-helix bundle. Interestingly, in leukemia inhibitory factor, another cytokine that signals through gp130, site 3, is topologically conserved but has evolved to bind leukemia inhibitory factor receptor.
Asunto(s)
Antígenos CD/metabolismo , Interleucina-6/metabolismo , Glicoproteínas de Membrana/metabolismo , Transducción de Señal , Secuencia de Bases , Sitios de Unión , Receptor gp130 de Citocinas , ADN Complementario , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Células Tumorales CultivadasAsunto(s)
Antígenos CD , Interleucina-6/química , Glicoproteínas de Membrana/química , Receptores de Interleucina/antagonistas & inhibidores , Secuencia de Aminoácidos , Receptor gp130 de Citocinas , Humanos , Interleucina-6/metabolismo , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Receptores de Interleucina/química , Receptores de Interleucina/metabolismo , Receptores de Interleucina-6 , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Interleukin-6 (IL-6) triggers the formation of a high affinity receptor complex constituted by the ligand-binding subunit IL-6 receptor alpha (IL-6R alpha) and the signal-transducing beta chain gp130. Since the cytoplasmic region of IL-6R alpha is not required for signal transduction, soluble forms of IL-6R alpha (sIL-6R alpha) show agonistic properties because they are still able to originate IL-6.sIL-6R alpha complexes, which in turn associate with gp130. A three-dimensional model of the human IL-6.IL-6R alpha.gp130 complex has been constructed and verified by site-directed mutagenesis of regions in shIL-6R alpha (where "h" is human) anticipated to contact hgp130, with the final goal of generating receptor variants with antagonistic properties. In good agreement with our structural model, substitutions at Asn-230, His-280, and Asp-281 selectively impaired the capability of shIL-6R alpha to associate with hgp130 both in vitro and on the cell surface, without affecting its affinity for hIL-6. Moreover, the multiple substitution mutant A228D/N230D/H280S/D281V expressed as a soluble protein partially antagonized hIL-6 bioactivity on hepatoma cells.
Asunto(s)
Interleucina-6/antagonistas & inhibidores , Conformación Proteica , Receptores de Interleucina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Carcinoma Hepatocelular/patología , Línea Celular Transformada , Chlorocebus aethiops , Simulación por Computador , Humanos , Neoplasias Hepáticas/patología , Sustancias Macromoleculares , Melanoma/patología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Nucleopoliedrovirus , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina-6 , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Solubilidad , Spodoptera , Células Tumorales CultivadasRESUMEN
The cytokines interleukin 6 (IL-6) and oncostatin M are able to inhibit the growth of cell lines obtained from early but not advanced melanomas. Resistant cell lines have frequently been found to produce IL-6. Acquisition of IL-6 resistance and the relationship between resistance and endogenous IL-6 production are poorly defined phenomena. We have characterized a panel of melanoma cell lines for susceptibility to IL-6 and oncostatin M and have generated lines that acquired resistance to IL-6 by IL-6 cDNA transduction. These lines retained the previous oncostatin M sensitivity, suggesting that the alpha chain of IL-6 receptor (IL-6R alpha) is involved in the acquisition of resistance. In fact transduced cells lost the ability to bind 125I-IL-6 and to release soluble IL-6R alpha in culture. Moreover, addition of soluble recombinant IL-6R alpha were able to restore IL-6 sensitivity in association with IL-6 production. On the contrary, naturally IL-6 resistant melanoma cell lines were not inhibited by treatment with recombinant soluble IL-6R alpha in association with endogenously produced or recombinant IL-6. These results demonstrate that down-regulation of IL-6 receptor is only one of different mechanisms that are responsible of IL-6 resistance in melanoma cells.
Asunto(s)
Regulación hacia Abajo , Interleucina-6/metabolismo , Melanoma/metabolismo , Péptidos/metabolismo , Receptores de Interleucina/metabolismo , División Celular/efectos de los fármacos , Resistencia a Medicamentos/genética , Humanos , Melanoma/genética , Melanoma/patología , Oncostatina M , Receptores de Interleucina-6 , Transfección , Células Tumorales CultivadasRESUMEN
The helical cytokine interleukin (IL) 6 and its specific binding subunit IL-6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution-phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL-6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL-6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL-6 variants complexed with IL-6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL-6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL-6, two IL-6R alpha and two gp130 molecules. We propose here a model representing the IL-6 receptor complex as hexameric, which might be common to other helical cytokines.
Asunto(s)
Antígenos CD , Interleucina-6/metabolismo , Glicoproteínas de Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Receptor gp130 de Citocinas , Variación Genética , Humanos , Interleucina-6/química , Interleucina-6/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-6 , Transducción de SeñalRESUMEN
A nonradioactive receptor binding assay for ciliary neurotrophic factor (CNTF) is described. The assay is based on the interaction between biotinylated human CNTF, soluble gp130, and soluble myc-tagged CNTF receptor captured on a microtiter plate via an antibody against the myc epitope tag. Bound cytokine is revealed by alkaline phosphatase-conjugated avidin. Purified human and rat CNTF competed with biotinylated CNTF for receptor binding, with IC50 values of 29 and 2 nM, respectively. Since the higher affinity of rat vs human CNTF has been previously shown to be conferred by the arginine residue at position 63 of the rat protein, we also tested a human CNTF mutant carrying a Q63R substitution. Secreted forms of wild-type and mutant CNTF were expressed in Escherichia coli, and the amount of cytokines in periplasmic extracts was determined by quantitative Western blotting analysis. The human CNTF mutant (Q63R, N137S) was found to compete with biotinylated CNTF for binding to soluble CNTF receptor with an eightfold higher apparent affinity than wild-type human CNTF. The present method thus faithfully reproduces the relative activities of CNTF analogs determined in other assay systems. The possibility of assaying cytokines in crude bacterial extracts makes the new technique particularly suitable for rapidly determining the receptor binding potencies of genetically engineered CNTF variants.
Asunto(s)
Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factor de Crecimiento Nervioso/análisis , Secuencia de Aminoácidos , Animales , Unión Competitiva , Biotina , Western Blotting/métodos , Factor Neurotrófico Ciliar , Clonación Molecular/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Escherichia coli , Humanos , Cinética , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/farmacología , Ratas , Receptor de Factor Neurotrófico Ciliar , Receptores de Factor de Crecimiento Nervioso/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Oncostatin M is a cytokine that acts as a growth regulator on a wide variety of cells and has diverse biological activities including acute phase protein induction, LDL receptor up-regulation and cell-specific gene expression. In order to gather information about the Onc M structure, we established a protocol for large scale production and single step purification of this functional cytokine from bacterial cells. The cDNA of human Onc M was cloned by RT-PCR from total RNA of PMA induced U937 cells. After the addition of a six histidine tag at the N-terminus, the coding region of mature Onc M was cloned in the pT7.7 expression vector. Histidine tagged Onc M was overexpressed in bacterial cells and purified to homogeneity in one step on a metal chelating column. We found that recombinant 6xHis-OncM remains fully active in a growth inhibition assay. Structural characterization of the purified protein was performed by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry. Thermal and pH stability dependence of Onc M was assessed by circular dichroism spectroscopy; the helical content is about 50%, in agreement with the four helix bundle fold postulated for cytokines that bind haematopoietic receptors of type I.
Asunto(s)
Inhibidores de Crecimiento/biosíntesis , Histidina , Biosíntesis de Péptidos , Péptidos/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Cromatografía Líquida de Alta Presión , Clonación Molecular/métodos , Citocinas/biosíntesis , Escherichia coli , Expresión Génica , Humanos , Modelos Estructurales , Datos de Secuencia Molecular , Oncostatina M , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Espectrometría de Masa Bombardeada por Átomos Veloces , Acetato de Tetradecanoilforbol/farmacología , Transcripción GenéticaRESUMEN
Oncostatin M (OM) and interleukin 6 (IL-6) are functionally related cytokines, which trigger similar biological responses because they share gp130 as a common signal transducing transmembrane receptor. While IL-6 recruits gp130 only upon binding to its specific receptor subunit (IL-6R alpha), reconstitution and cross-linking experiments on cell membranes suggest that OM can directly interact with gp130 and that this interaction is necessary but not sufficient to stimulate cells. However, the issue of the direct binding between gp130 and OM, in the absence of any additional membrane component, remained essentially unclarified. In this paper we show that, uniquely among the family of cytokines that transduce through gp130, OM directly binds in vitro with a 10(-8) M affinity sgp130, a soluble form of gp130. Moreover, titration of sgp130 with OM inhibits the formation of a ternary complex comprising IL-6, sIL-6R alpha, and sgp130. These in vitro properties of OM are consistent with the additional finding that on human hepatoma Hep3B cells, which express gp130 but not functional OM receptors, OM does not mimic IL-6 activity, but rather behaves, at high doses, as an IL-6 antagonist.