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1.
J Vet Intern Med ; 30(4): 1129-37, 2016 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-27306920

RESUMEN

BACKGROUND: Limited options are available for the treatment of pain in cats. Monoclonal antibodies (mAbs) that neutralize nerve growth factor (NGF) have demonstrated analgesic capacity in rodent models, people with osteoarthritis, and dogs with degenerative joint disease. HYPOTHESIS/OBJECTIVES: This study describes the design and characterization of a fully felinized anti-NGF monoclonal antibody. In vitro potency, pharmacokinetics, and the ability of the antibody to treat pain in a self-resolving, acute inflammation model were investigated in cats. ANIMALS: Thirty-eight cats at a research colony at Charles River Laboratories, Ireland. METHODS: Felinized anti-NGF mAb, NV-02, was produced using a complementary DNA (cDNA)-based method (PETization). Purified NV-02 was tested for affinity, potency, and immunoreactivity in vitro, then for safety and plasma pharmacokinetic distribution in vivo, and analgesic efficacy in a model of kaolin-induced inflammatory pain. RESULTS: Anti-NGF mAb, NV-02 neutralized NGF with high affinity and potency and did not bind complement. NV-02-administered SC had a plasma half-life of 7-15 days and was well tolerated at dosages up to 28 mg/kg. A dosage of 2 mg/kg NV-02 SC significantly decreased signs of lameness on day 2 (P = .0027), day 3 (P = .016), day 4, (P = .0063), day 5 (P = .0085), day 6 (P = .0014), and day 7 (P = .0034) after induction of inflammation. CONCLUSIONS AND CLINICAL IMPORTANCE: The high affinity, long plasma half-life, safety, and analgesic efficacy of felinized anti-NGF mAb (NV-02) support further investigation of the analgesic potential of this antibody in the cat.


Asunto(s)
Analgésicos no Narcóticos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Enfermedades de los Gatos/terapia , Factor de Crecimiento Nervioso/inmunología , Osteoartritis/veterinaria , Dolor Intratable/veterinaria , Analgésicos no Narcóticos/farmacocinética , Animales , Anticuerpos Monoclonales/farmacocinética , Gatos , Femenino , Cojera Animal/terapia , Masculino , Osteoartritis/terapia , Manejo del Dolor/veterinaria , Dolor Intratable/terapia , Especificidad de la Especie , Resultado del Tratamiento
2.
J Vet Intern Med ; 30(4): 1138-48, 2016 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-27334504

RESUMEN

BACKGROUND: Neutralizing antibodies against nerve growth factor (NGF) are analgesic in rodent models, naturally occurring degenerative joint disease (DJD) pain in dogs, and chronic pain in humans. OBJECTIVES: To evaluate the efficacy of a fully felinized anti-NGF antibody (NV-02) for the treatment of DJD pain and mobility impairment in cats. ANIMALS: Thirty-four client-owned cats with DJD-associated pain and mobility impairment. METHODS: In a placebo-controlled, pilot, masked clinical study, cats were randomized to a single treatment with NV-02 (0.4 mg/kg SC [n = 11] or 0.8 mg/kg SC [n = 12]) or placebo (saline, SC [n = 11]). Activity was measured objectively. Additionally, owners completed clinical metrology instruments (client-specific outcome measures [CSOM] and feline musculoskeletal pain index [FMPI]) on days 0 (screening), 14 (baseline), 35, 56, and 77. A repeated-measures model was used to evaluate the objective activity data. RESULTS: NV-02 significantly increased objectively measured activity overall (P = .017) and at 2 (P = .035), 3 (P = .007), 4 (P = .006), 5 (P = .007), and 6 (P = .017) weeks after treatment. CSOM scores (P = .035) and pain (P = .024) showed a significant effect of treatment 3 weeks after administration. In the treatment group, 83% of the owners correctly identified the treatment administered compared with 45% of owners in the placebo group (P = .013). No treatment-related adverse effects were identified. CONCLUSIONS: These pilot data demonstrate a 6-week duration positive analgesic effect of this fully felinized anti-NGF antibody in cats suffering from DJD-associated pain.


Asunto(s)
Analgésicos no Narcóticos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Enfermedades de los Gatos/terapia , Factor de Crecimiento Nervioso/inmunología , Osteoporosis/veterinaria , Dolor Intratable/veterinaria , Animales , Gatos , Método Doble Ciego , Femenino , Inyecciones Subcutáneas/veterinaria , Cojera Animal/terapia , Masculino , Osteoporosis/terapia , Dolor Intratable/terapia , Proyectos Piloto , Especificidad de la Especie , Resultado del Tratamiento
3.
Nature ; 410(6827): 483-7, 2001 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-11260717

RESUMEN

Bone morphogenetic proteins (BMPs), including the fly homologue Decapentaplegic (DPP), are important regulators of early vertebrate and invertebrate dorsal-ventral development. An evolutionarily conserved BMP regulatory mechanism operates from fly to fish, frog and mouse to control the dorsal-ventral axis determination. Several secreted factors, including the BMP antagonist chordin/Short gastrulation (SOG), modulate the activity of BMPs. In Drosophila, Twisted gastrulation (TSG) is also involved in dorsal-ventral patterning, yet the mechanism of its function is unclear. Here we report the characterization of the vertebrate Tsg homologues. We show that Tsg can block BMP function in Xenopus embryonic explants and inhibits several ventral markers in whole-frog embryos. Tsg binds directly to BMPs and forms a ternary complex with chordin and BMPs. Coexpression of Tsg with chordin leads to a more efficient inhibition of the BMP activity in ectodermal explants. Unlike other known BMP antagonists, however, Tsg also reduces several anterior markers at late developmental stages. Our data suggest that Tsg can function as a BMP inhibitor in Xenopus; furthermore, Tsg may have additional functions during frog embryogenesis.


Asunto(s)
Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas de Drosophila , Gástrula/metabolismo , Proteínas/fisiología , Animales , Biomarcadores , Proteínas Morfogenéticas Óseas/metabolismo , Clonación Molecular , Embrión no Mamífero , Péptidos y Proteínas de Señalización Intercelular , Péptidos/metabolismo , Unión Proteica , Proteínas/química , Proteínas/metabolismo , ARN , Transducción de Señal , Xenopus
4.
Genomics ; 66(2): 213-6, 2000 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-10860666

RESUMEN

Members of the IL-1 family of cytokines are important in mediating inflammatory responses. The genes encoding IL-1alpha, IL-beta, and the IL-1 receptor antagonist (IL-1Ra) are clustered within 450 kb on human chromosome 2q. By searching the EST databases and sequencing this region of chromosome 2, we have identified three novel genes that show homology to the IL-1 family, which we have named IL-1-related protein 1, 2, and 3 (IL-1RP1, IL-1RP2, and IL-1RP3). All three genes contain a signature motif common to the IL-1 family and appear to be more closely related to IL-1Ra. Similar to the intracellular form of IL-1Ra, these genes lack conventional hydrophobic signal sequences. The expression of these genes appears to be highly restricted to various epithelial cell populations. Our results demonstrate the existence of additional IL-1 gene family members within the previously defined IL-1 cluster and point to this region of chromosome 2 as an evolutionary hotspot for IL-1 gene duplication. These genes may prove to have an important role in inflammatory responses.


Asunto(s)
Cromosomas Humanos Par 2 , Interleucina-1/genética , Secuencia de Aminoácidos , Humanos , Interleucina-1/química , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido
5.
Gene ; 238(2): 301-13, 1999 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-10570958

RESUMEN

Wnt proteins influence many aspects of embryonic development, and their activity is regulated by several secreted antagonists, including the Xenopus Dickkopf-1 (xDkk-1) protein. xDkk-1 inhibits Wnt activities in Xenopus embryos and may play a role in induction of head structures. Here, we characterize a family of human Dkk-related genes composed of Dkk-1, Dkk-2, Dkk-3, and Dkk-4, together with a unique Dkk-3 related protein termed Soggy (Sgy). hDkks 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Sgy is a novel secreted protein related to Dkk-3 but which lacks the cysteine-rich domains. Members of the Dkk-related family display unique patterns of mRNA expression in human and mouse tissues, and are secreted when expressed in 293T cells. Furthermore, secreted hDkk-2 and hDkk-4 undergo proteolytic processing which results in cleavage of the second cysteine-rich domain from the full-length protein. Members of the human Dkk-related family differ not only in their structures and expression patterns, but also in their abilities to inhibit Wnt signaling. hDkk-1 and hDkk-4, but not hDkk-2, hDkk-3 or Sgy, suppress Wnt-induced secondary axis induction in Xenopus embryos. hDkk-1 and hDkk-4 do not block axis induction triggered either by Xenopus Dishevelled (Xdsh) or Xenopus Frizzled-8 (Xfz8), both of which function to transduce signals from Wnt ligands. Thus, hDkks 1 and 4 may inhibit Wnt activity by a mechanism upstream of Frizzled. Our findings highlight the structural and functional heterogeneity of human Dkk-related proteins.


Asunto(s)
Familia de Multigenes , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Xenopus/embriología , Proteínas de Xenopus
6.
Mol Cell Biol ; 17(7): 4007-14, 1997 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-9199335

RESUMEN

Members of the epidermal growth factor family of receptors have long been implicated in the pathogenesis of various tumors, and more recently, apparent roles in the developing heart and nervous system have been described. Numerous ligands that activate these receptors have been isolated. We report here on the cloning and initial characterization of a second ligand for the erbB family of receptors. This factor, which we have termed Don-1 (divergent of neuregulin 1), has structural similarity with the neuregulins. We have isolated four splice variants, two each from human and mouse, and have shown that they are capable of inducing tyrosine phosphorylation of erbB3, erbB4, and erbB2. In contrast to those of neuregulin, high levels of expression of Don-1 are restricted to the cerebellum and dentate gyrus in the adult brain and to fetal tissues.


Asunto(s)
Cerebelo/fisiología , Glicoproteínas/fisiología , Hipocampo/fisiología , Factores de Crecimiento Nervioso/genética , Secuencia de Aminoácidos , Animales , División Celular , Mapeo Cromosómico , Cromosomas Humanos Par 5 , Clonación Molecular , Expresión Génica , Ligamiento Genético , Humanos , Hibridación in Situ , Ligandos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neurregulinas , Fosfotirosina/metabolismo , Receptores de Factores de Crecimiento/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Distribución Tisular
7.
Blood ; 89(8): 2706-16, 1997 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-9108388

RESUMEN

The adhesion molecule BEN/SC1/DM-GRASP (BEN) is a marker in the developing chicken nervous system that is also expressed on the surface of embryonic and adult hematopoietic cells such as immature thymocytes, myeloid progenitors, and erythroid progenitors. F84.1 and KG-CAM, two monoclonal antibodies to rat neuronal glycoproteins with similarity to BEN, cross-react with an antigen on rat hematopoietic progenitors, but F84.1 only also recognizes human blood cell progenitors. We have defined the antigen recognized by F84.1 as the hematopoietic cell antigen (HCA). HCA expression was detected on 40% to 70% of CD34+ fetal and adult bone marrow cells and mobilized peripheral blood cells. Precursor cell activity for long-term in vitro bone marrow cell culture was confined to the subset of CD34+ cells that coexpress HCA. HCA is expressed by the most primitive subsets of CD34+ cells, including all rhodamine 123(lo), Thy-1+, and CD38(-/lo) CD34+ adult bone marrow cells. HCA was also detected on myeloid progenitors but not on early B-cell progenitors. We also describe here the cloning and characterization of cDNAs encoding two variants of the human HCA antigen (huHCA-1 and huHCA-2) and of a cDNA clone encoding rat HCA (raHCA). The deduced amino acid sequences of huHCA and raHCA are homologous to that of chicken BEN. Recombinant proteins produced from either human or rat HCA cDNAs were recognized by F84.1, whereas rat HCA but not human HCA was recognized by antirat KG-CAM. Expression of either form of huHCA in CHO cells conferred homophilic adhesion that could be competed with soluble recombinant huHCA-Fc. The molecular cloning of HCA and the availability of recombinant HCA should permit further evaluation of its role in human and rodent hematopoiesis.


Asunto(s)
Antígenos de Superficie/aislamiento & purificación , Células de la Médula Ósea , Moléculas de Adhesión Celular Neuronal , Células Madre Hematopoyéticas/inmunología , Adulto , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD , Antígenos CD34/análisis , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/genética , Médula Ósea/embriología , Pollos , Clonación Molecular , Secuencia de Consenso , ADN Complementario/genética , Proteínas Fetales/biosíntesis , Proteínas Fetales/genética , Proteínas Fetales/aislamiento & purificación , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/aislamiento & purificación , Neuronas/inmunología , Neuronas/metabolismo , Especificidad de Órganos , Ratas , Proteínas Recombinantes de Fusión/inmunología , Homología de Secuencia de Aminoácido , Especificidad de la Especie
8.
Blood ; 87(11): 4618-28, 1996 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-8639830

RESUMEN

Attempts to maintain or expand primitive hematopoietic stem cells in vitro without the concomitant loss of their differentiative and proliferative potential in vivo have largely been unsuccessful. To investigate this problem, we compared the ability of three cloned bone marrow (BM) stromal cell lines to support the growth of primitive Thy-1lo Sca-1+H-2Khi cells isolated by fluorescence-activated cell sorting from the BM of Ly-5.2 mice treated 1 day previously with 5-fluo- rouracil. Sorted cells were highly enriched in cobblestone area-forming cells (CAFC), but their frequency was dependent on the stromal cell lines used in this assay (1 per 45 cells on SyS-1; 1 per 97 cells on PA6). In the presence of recombinant leukemia inhibitory factor (LIF), CAFC cloning efficiency was increased to 1 per 8 cells on SyS-1 and 1 per 11 cells on PA6, thus showing the high clonogenicity of this primitive stem cell population. More primitive stem cells with competitive repopulating potential were measured by injecting the sorted cells into lethally irradiated Ly-5.1 mice together with 10(5) radioprotective Ly-5.1 BM cells whose long-term repopulating ability has been "compromised" by two previous cycles of marrow transplantation and regeneration. Donor-derived lymphocytes and granulocytes were detected in 66% of animals injected with 50 sorted cells. To quantitate the maintenance of competitive repopulating units (CRU) by stromal cells, sorted cells were transplanted at limiting dilution before and after being cultured for 2 weeks on adherent layers of SyS-1, PA6, or S17 cells. CRU represented 1 per 55 freshly sorted cells. CRU could be recovered from cocultures supported by all three stromal cell lines, but their numbers were approximately-sevenfold less than on day 0. In contrast, the addition of LIF to stromal cultures improved CRU survival by 2.5-fold on S17 and PA6 cells (approximately two-fold to threefold decline), and enabled their maintenance on SyS-1. LIF appeared to act indirectly, because alone it did not support the proliferation of Thy-1lo Sca-1+H-2Khi cells in stroma-free cultures. Polymerase chain reaction (RT-PCR) analysis revealed that Interleukin-1beta (IL-1 beta) IL-2, IL-6, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, transforming growth factors, LIF, and Steel Factor (SLF) mRNAs were upregulated in SyS-1 within 1 to 6 hours of LIF-stimulation. To determine if increased expression of SLF by LIF-stimulated SyS-1 cells could account for their capacity to support stem cells, sorted calls were cocultured on simian CV-E cells that were transfected with an expression vector encoding membrane-bound SLF, or supplemented with soluble SLF. In both cases, SLF synergized with IL-6 produced endogenously by CV-E cells enabling CAFC growth equivalent to that on LIF-stimulated SyS-1. CAFC development on LIF-stimulated SyS-1 could also be completely abrogated by an anti-SLF antibody. These data provide evidence for a role of LIF in the support of long-term repopulating stem cells by indirectly promoting cytokine expression by BM stroma. Furthermore, we have used quantitative assays to show a maintenance of CRU numbers, with retention of in vivo function following ex vivo culture.


Asunto(s)
Tejido Conectivo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-6 , Linfocinas/farmacología , Animales , Antígenos de Diferenciación/análisis , Secuencia de Bases , Células de la Médula Ósea , Trasplante de Médula Ósea , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Células del Tejido Conectivo , Fluorouracilo/toxicidad , Supervivencia de Injerto , Antígenos H-2/genética , Hematopoyesis , Células Madre Hematopoyéticas/citología , Factor Inhibidor de Leucemia , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Antígenos Thy-1/genética
9.
Eur J Neurosci ; 8(2): 291-9, 1996 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-8714700

RESUMEN

Previously we have shown that leukaemia inhibitory factor (LIF) potentiates the development of murine spinal cord neurons in vitro, suggesting that it, or related factors, may play an important regulatory role in neuronal development. We have further investigated this role and show here that the generation of neurons in cultures of embryonic day 10 spinal cord cells is inhibited by antibodies to the beta subunit of the LIF receptor. Since there are more undifferentiated precursors in antibody-treated cultures than in control and LIF-treated cultures, it is concluded that the primary action of LIF, or related molecules, is to promote neuronal differentiation, not precursor survival. In addition, the failure of LIF to support neuronal survival in the period immediately following differentiation suggests that the increased numbers of neurons generated with LIF are not attributable to its neurotrophic action. By selecting neuronal precursors on the basis of their inability to express class 1 major histocompatibility complex molecules, it was shown that LIF acted directly upon these cells and not via an intermediary cell. LIF also appears to be involved in regulating the differentiation of astrocytes, since it increases the number of glial fibrillary protein (GFAP)-positive cells present in the cultures and since the spontaneous production of GFAP-positive cells is blocked by antibodies to the LIF beta receptor. These findings suggest that LIF or related factors promote the differentiation of neural precursors in the spinal cord, but that they are not involved in preferentially promoting precursors down a specific differentiation pathway.


Asunto(s)
Astrocitos/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Interleucina-6 , Linfocinas/farmacología , Neuronas/efectos de los fármacos , Médula Espinal/embriología , Células Madre/efectos de los fármacos , Animales , Anticuerpos Monoclonales/farmacología , Astrocitos/citología , Factores Biológicos/farmacología , Células CHO , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Linaje de la Célula , Células Cultivadas , Técnicas de Cocultivo , Cricetinae , Medios de Cultivo/farmacología , Compensación de Dosificación (Genética) , Fibroblastos/fisiología , Fibronectinas/farmacología , Genes Reporteros , Laminina/farmacología , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Ratones Transgénicos , Neuronas/citología , Ornitina/farmacología , Receptores de Citocinas/efectos de los fármacos , Receptores de Citocinas/inmunología , Receptores OSM-LIF , Médula Espinal/citología , Células Madre/citología
10.
J Biol Chem ; 270(40): 23254-7, 1995 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-7559477

RESUMEN

Transient transfection of expression vectors for various members of the hematopoietin receptor family and STAT proteins into COS-1 cells indicated that each receptor was capable of stimulating the DNA binding activity of STAT1, STAT3, and STAT5B. However, gp130 preferentially activated STAT1 and STAT3. Activation of STAT5B differed from that of the other two in that the box 3 sequence motif in the cytoplasmic domain of gp130 was not required. Moreover, STAT5B and STAT3 enhanced gene transcription via separate regulatory elements. This study has identified two potential signal transduction pathways by which hematopoietin receptors, including the interleukin-6 receptor, control transcription of acute phase plasma protein genes in hepatic cells.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Crecimiento de Célula Hematopoyética/metabolismo , Proteínas de la Leche , Receptores de Factores de Crecimiento/metabolismo , Transactivadores/metabolismo , Proteínas de Fase Aguda/genética , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Línea Celular , Receptor gp130 de Citocinas , Citocinas/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Receptores de Factores de Crecimiento/genética , Factor de Transcripción STAT3 , Factor de Transcripción STAT5 , Transducción de Señal , Transactivadores/genética , Transcripción Genética , Transfección
11.
Blood ; 86(5): 1671-9, 1995 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-7654999

RESUMEN

To determine the specificity of signal transducer and activator of transcription (STAT) protein activation by box 3 motif-deficient hematopoietin receptors, expression vectors encoding the receptors for growth hormone, interleukin-3 (IL-3), and IL-4 were transiently transfected into COS-1 cells, together with expression vectors for Janus kinases (JAKs) and STAT proteins. Each receptor mediated a dose-dependent activation of STAT1 and STAT3, and for IL-3R and GHR this process was enhanced by JAK2. The data suggest that a box 3 motif in the cytoplasmic domain of the signal-transducing receptor to the JAK/STAT pathway. Transfection of the receptors, in combination with STAT3, into HepG2 cells reconstituted a cytokine-dependent stimulation of gene transcription through IL-6 response elements, providing evidence for a functional role of STAT3 in controlling gene expression.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Interleucina-6/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Receptores de Interleucina-3/fisiología , Receptores de Somatotropina/fisiología , Transactivadores/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Carcinoma Hepatocelular , Línea Celular , Chlorocebus aethiops , Citocinas/farmacología , Activación Enzimática , Expresión Génica/efectos de los fármacos , Humanos , Janus Quinasa 2 , Cinética , Neoplasias Hepáticas , Receptores del Factor Estimulante de Colonias/fisiología , Receptores de Interleucina/efectos de los fármacos , Receptores de Interleucina/fisiología , Receptores de Interleucina-3/biosíntesis , Receptores de Interleucina-3/efectos de los fármacos , Receptores de Interleucina-4 , Receptores de Somatotropina/biosíntesis , Receptores de Somatotropina/efectos de los fármacos , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT3 , Transducción de Señal , Transfección , Células Tumorales Cultivadas
12.
J Biol Chem ; 270(25): 14847-50, 1995 Jun 23.
Artículo en Inglés | MEDLINE | ID: mdl-7797460

RESUMEN

The cytoplasmic receptor sequences required for the transcriptional control via the IL-6 response element (IL-6RE) and the hematopoietin receptor response element (HRRE) in hepatoma cells were defined by transient expression of wild-type and mutant granulocyte-colony stimulating factor receptor-gp130 chimeric receptors. gp130 generated two separate transcriptional signals, one of which was directed to IL-6RE and required an intact box 3 motif, and another, which was directed to HRRE and was box 3-independent. The activation of DNA-binding of STAT3 required the same gp130 domains as the IL-6RE response. A box 3-independent activation of STAT proteins was achieved by overexpression of the kinases JAK2 or TYK2. The increase in the DNA-binding activity of STAT proteins, however, did not result in a corresponding increase in transcription via either IL-6RE or HRRE. The data indicate that activation of the DNA-binding potential of STAT proteins via gp130 is not sufficient to achieve transcriptional up-regulation of specific target genes.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Interleucina-6/farmacología , Transducción de Señal , Animales , Cloranfenicol O-Acetiltransferasa/biosíntesis , Chlorocebus aethiops , Riñón , Neoplasias Hepáticas Experimentales , Ratas , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética , Transfección , Células Tumorales Cultivadas
13.
Proc Natl Acad Sci U S A ; 92(12): 5361-5, 1995 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-7777512

RESUMEN

We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose- and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor alpha-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokine-stimulated expression of a reporter gene under the control of the beta-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism of heterologous receptor regulation of LIFR signaling occurring at Ser-1044.


Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Inhibidores de Crecimiento/metabolismo , Interleucina-6 , Linfocinas/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Citocinas/metabolismo , Células 3T3 , Animales , Humanos , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Ratas , Receptores OSM-LIF , Células Tumorales Cultivadas
14.
Oncogene ; 10(10): 2007-16, 1995 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-7761101

RESUMEN

By means of a combined in vitro and in vivo analysis we provide evidence that IL-1 beta and PDGF-B, but not OSM (oncostatin M) or IL-6, are major mitogens for the spindle cells of Kaposi's sarcoma (KS) in vivo. PDGF-B and IL-1 beta stimulated proliferation of cultivated KS spindle cells in vitro. Analysis of gene expression in vivo revealed that both factors as well as the PDGF beta-receptor are present in KS lesions. By contrast, IL-6 had no effect and OSM inhibited proliferation of cultivated KS spindle cells. Again, the effect of these factors on cultivated KS spindle cells in vitro was reflected by the gene expression observed in KS lesions in vivo. Neither the expression of IL-6 receptor nor of OSM could be detected in KS lesions by in situ hybridization. Moreover, in situ hybridization revealed an identical pattern of gene expression in cultivated KS spindle cells and KS spindle cells in vivo with respect to the above-mentioned cytokines [PDGF-B, IL-1 beta, IL-1 alpha, IL-6, OSM] and their receptors [PDGF beta-receptor, gp130, IL-6 receptor, leukemia inhibitory factor (LIF) receptor]. This further supported the suitability of cultivated KS spindle cells as an in vitro model in order to determine which cytokines may activate proliferation of KS spindle cells in vivo.


Asunto(s)
Interleucina-1/análisis , Interleucina-6/análisis , Péptidos/análisis , Factor de Crecimiento Derivado de Plaquetas/análisis , Proteínas Proto-Oncogénicas/análisis , Sarcoma de Kaposi/patología , División Celular/efectos de los fármacos , Humanos , Hibridación in Situ , Interleucina-1/farmacología , Masculino , Oncostatina M , Péptidos/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas/farmacología , Proteínas Proto-Oncogénicas c-sis , Receptores de Citocinas/análisis , Receptores de Interleucina/análisis , Receptores de Interleucina-6 , Receptores de Oncostatina M , Receptores del Factor de Crecimiento Derivado de Plaquetas/análisis , Sarcoma de Kaposi/química
15.
J Biol Chem ; 270(14): 8298-310, 1995 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-7713938

RESUMEN

The gene regulatory functions of the human IL-2 receptor (IL-2R) were reconstituted in transiently transfected hepatoma cells. The combination of IL-2R beta and -gamma mediated a strong stimulation via the cytokine response element of the alpha 1-acid glycoprotein gene and the hematopoietin receptor response element, but none via the IL-6 response element or the sis-inducible element. IL-2R alpha enhanced 10-fold the sensitivity of the IL-2R beta.gamma complex to respond to IL-2 or IL-15, but did not modify the specificity or the magnitude of maximal gene regulation. A homodimerizing chimeric receptor G-CSFR-IL-2R beta could mimic the IL-2R action. The IL-2R-mediated gene regulation was similar to that seen with receptors for IL-4 and IL-7, but differed from that for IL-6 type cytokines, thrombopoietin, erythropoietin, and growth hormone. The activation of STAT proteins by the IL-2R was assessed in transfected L-cells and COS-1 cells. Although IL-2R subunits were highly expressed in these cells, no STAT protein activation was detectable. Transient overexpression of JAK3 was unable to change the signaling specificity of the hematopoietin receptors in rat hepatoma, L-, and COS cells, but established a prominent activation of the IL-6 response elements by the IL-2R and IL-4R in HepG2 cells. The data support the model that the IL-2R and related hematopoietin receptors produce at least two separate signals which control gene expression.


Asunto(s)
Hígado/metabolismo , Receptores del Factor Estimulante de Colonias/metabolismo , Receptores de Interleucina-2/metabolismo , Transducción de Señal , Animales , Secuencia de Bases , Línea Celular , Citoplasma/metabolismo , Cartilla de ADN , Humanos , Interleucina-6/metabolismo , Hígado/citología , Ratones , Datos de Secuencia Molecular , Ratas , Receptores de Interleucina-2/química , Transcripción Genética , Células Tumorales Cultivadas
16.
Development ; 120(11): 3145-53, 1994 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-7720558

RESUMEN

The pleiotropic cytokine leukemia inhibitory factor (LIF) is able to promote the growth of mouse primordial germ cells (PGCs) in culture. It is unclear whether LIF acts directly on PGCs or indirectly via feeder cells or embryonic somatic cells. To understand the role of LIF in PGC growth, we have carried out molecular and cell culture analyses to investigate the role of both the LIF ligand and its receptor in PGC development. LIF is able to stimulate PGC growth independently of the presence of feeder cells supporting the hypothesis that LIF acts directly on PGCs to promote their growth. We show here that transcripts for the low-affinity LIF receptor (LIFR), an integral component of the functional LIF receptor complex, are expressed in the developing gonad. Fluorescence-activated cell sorter (FACS) analysis, using an anti-LIFR antiserum, demonstrates that LIFR is present on the surface of PGCs, suggesting that PGCs are likely to be a direct target of LIF action in culture. Signalling via LIFR is essential for PGC growth in culture since the anti-LIFR antiserum, which blocks LIF binding to its receptor, abolishes PGC survival in culture. Two LIF-related cytokines, namely oncostatin M and ciliary neurotrophic factor, can also promote PGC growth in culture in addition to LIF. Thus one or more of these LIFR-dependent cytokines may play an important role in PGC development in mice.


Asunto(s)
Células Germinativas/crecimiento & desarrollo , Inhibidores de Crecimiento/fisiología , Linfocinas/fisiología , Receptores de Citocinas/fisiología , Animales , Células Cultivadas , Factor Neurotrófico Ciliar , Citocinas/fisiología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Células Germinativas/citología , Interleucina-6/fisiología , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Ratones , Ratones Endogámicos , Proteínas del Tejido Nervioso/fisiología , Oncostatina M , Péptidos/fisiología , Receptores OSM-LIF
17.
EMBO J ; 13(7): 1574-84, 1994 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-8156996

RESUMEN

The role played by the Src-related tyrosine kinase, Hck, in embryonic stem (ES) cell differentiation was investigated by replacing a conserved C-terminally located tyrosine with phenylalanine by gene targeting. Targeted ES cells display a 7- to 9-fold elevation in constitutive Hck kinase activity and require approximately 15 times less leukaemia inhibitory factor (LIF) than parental ES cells to maintain their stem cell character in vitro. We also demonstrate a rapid and transient increase in Hck tyrosine kinase activity in parental ES cells stimulated by LIF and, finally, show that Hck is physically associated with gp130, an affinity converter and signal transducing component of the LIF receptor. Thus, these results provide biological and biochemical evidence that Hck participates in signal transduction from the LIF receptor.


Asunto(s)
Inhibidores de Crecimiento , Interleucina-6 , Linfocinas , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Interleucina/metabolismo , Transducción de Señal , Células Madre/metabolismo , Animales , Secuencia de Bases , Factor Inhibidor de Leucemia , Datos de Secuencia Molecular , Mutagénesis , Fosforilación , Proteínas Tirosina Quinasas/análisis , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-hck , Receptores de Interleucina-6 , Receptores OSM-LIF , Recombinación Genética
18.
J Biol Chem ; 269(8): 6215-22, 1994 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-8119965

RESUMEN

Leukemia inhibitory factor (LIF) and oncostatin M (OSM) both bind to the same receptor with high affinity and thus mediate an overlapping spectrum of biological activities, the signal transduction mechanisms for which are unclear. We show that mitogen-activated protein kinases are involved in both the LIF and OSM signal transduction pathways. However, we found that OSM is a much more potent inducer of both mitogen-activated protein kinase activity and biological response, both of which correlate with the expression of a second OSM receptor that does not bind LIF. In addition, different patterns of tyrosine-phosphorylated proteins were stimulated by OSM and LIF. We therefore suggest that the two receptors for OSM can be coupled to different signal transduction events.


Asunto(s)
Inhibidores de Crecimiento/metabolismo , Interleucina-6 , Linfocinas/metabolismo , Péptidos/metabolismo , Receptores de Citocinas/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Línea Celular , Activación Enzimática , Humanos , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Proteína Quinasa 1 Activada por Mitógenos , Datos de Secuencia Molecular , Oncostatina M , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores OSM-LIF , Receptores de Oncostatina M , Células Tumorales Cultivadas , Tirosina/metabolismo
19.
Proc Natl Acad Sci U S A ; 91(3): 1119-23, 1994 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-8302840

RESUMEN

Specific low-affinity receptors for leukemia inhibitory factor (LIF), oncostatin M (OSM; gp130), and ciliary neurotrophic factor (CNTF; receptor alpha, CNTFR alpha) may be utilized in various combinations to generate high-affinity binding sites and signal transduction. We have tested the ability of combinations of these receptors to transduce a proliferative signal in BAF-B03 cells. Coexpression of the LIF receptor and gp130 in these cells conferred high-affinity LIF and OSM binding and responsiveness to LIF and OSM. These cells also responded to CNTF in the absence of detectable binding. The further addition of CNTFR alpha conferred high-affinity CNTF binding and enhanced responsiveness to CNTF but did not modify responses to LIF or OSM. Coexpression of LIF receptor and CNTFR alpha resulted in a nonfunctional high-affinity binding site. These data are consistent with a role for the CNTFR alpha in enhancing CNTF action but the CNTFR alpha is not absolutely required for CNTF action and suggest a wider range of targets for CNTF.


Asunto(s)
Inhibidores de Crecimiento , Células Madre Hematopoyéticas/metabolismo , Interleucina-6 , Linfocinas , Receptores de Citocinas/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Animales , Sitios de Unión , División Celular , Línea Celular , Células Madre Hematopoyéticas/citología , Humanos , Cinética , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Ratones , Receptor de Factor Neurotrófico Ciliar , Receptores de Citocinas/química , Receptores de Citocinas/genética , Receptores de Factores de Crecimiento/química , Receptores de Factores de Crecimiento/genética , Receptores OSM-LIF , Receptores de Oncostatina M , Transducción de Señal , Transfección
20.
Mol Cell Biol ; 14(1): 138-46, 1994 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-8264582

RESUMEN

The receptor for leukemia inhibitory factor (LIFR), in combination with the signal-transducing subunit for interleukin-6-type cytokine receptors, gp130, and LIF, activates transcription of acute-phase plasma protein genes in human and rat hepatoma cells and the vasoactive intestinal peptide gene in a human neuroblastoma cell line. To identify the regions within the cytoplasmic domain of LIFR that initiate signal transduction independently of gp130, we constructed a chimeric receptor by linking the extracellular domain of the granulocyte colony-stimulating factor receptor (G-CSFR) to the transmembrane and cytoplasmic domain of human LIFR. The function of the chimeric receptor protein in transcriptional activation was assessed by G-CSF-mediated stimulation of cotransfected cytokine-responsive reporter gene constructs in hepatoma and neuroblastoma cells. By using the full-length cytoplasmic domain and mutants with progressive carboxy-terminal deletions, internal deletions, or point mutations, we identified the first 150 amino acid residues of LIFR as the minimal region necessary for signaling. The signaling reaction appears to involve a cooperativity between the first 70-amino-acid region containing the two sequence motifs conserved among hematopoietin receptors (box 1 and box 2) and a critical sequence between residues 141 and 150 (box 3). Analogous analyses of the cytoplasmic domains of G-CSFR and gp130 indicated similar arrangements of functional domains in these receptor subunits and the requirement of a box 3-related motif for signaling.


Asunto(s)
Antígenos CD , Inhibidores de Crecimiento , Interleucina-6 , Linfocinas , Glicoproteínas de Membrana/metabolismo , Receptores de Citocinas/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Receptor gp130 de Citocinas , Citoplasma/metabolismo , ADN/genética , Humanos , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Hígado/metabolismo , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Mutagénesis , Neuronas/metabolismo , Ratas , Receptores de Citocinas/genética , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Receptores OSM-LIF , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal/genética , Activación Transcripcional
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