RESUMO
From a fixed number of genes carried in all cells, organisms create considerable diversity in cellular phenotype through differential regulation of gene expression. One prevalent source of transcriptome diversity is alternative pre-mRNA splicing, which is manifested in many different forms. Zebrafish models of splicing dysfunction due to mutated spliceosome components provide opportunity to link biochemical analyses of spliceosome structure and function with whole organism phenotypic outcomes. Drawing from experience with two zebrafish mutants: cephalophonus (a prpf8 mutant, isolated for defects in granulopoiesis) and caliban (a rnpc3 mutant, isolated for defects in digestive organ development), we describe the use of glycerol gradient sedimentation and native gel electrophoresis to resolve components of aberrant splicing complexes. We also describe how RNAseq can be employed to examine relatively rare alternative splicing events including intron retention. Such experimental approaches in zebrafish can promote understanding of how splicing variation and dysfunction contribute to phenotypic diversity and disease pathogenesis.
Assuntos
Processamento Alternativo/genética , Perfilação da Expressão Gênica/métodos , Spliceossomos/genética , Transcriptoma/genética , Animais , Mutação/genética , Fenótipo , Precursores de RNA/genética , Proteínas de Ligação a RNA/genética , Spliceossomos/ultraestrutura , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Uteri of Lif null mice do not support embryo implantation. Since deletion of some genes often prevents the survival of null mice to adulthood, we have used a proven inhibitor of leukaemia inhibitory factor (LIF) signalling to identify the precise window of time during which LIF is required in vivo, and assessed the cellular expression of several LIF-associated targets. On day 4 of pregnancy, mice were injected with hLIF-05 (inhibitor) into the uterine lumen, with corresponding volumes of PBS (vehicle) injected into the contralateral horn. On days 5 and 6, the number of implantation sites was recorded and the uteri processed for immunohistochemistry. Blockade of LIF on day 4 reduced embryo implantation by 50% (PAssuntos
Implantação do Embrião/efeitos dos fármacos
, Fator Inibidor de Leucemia/farmacologia
, Receptores de OSM-LIF/antagonistas & inibidores
, Administração Intravaginal
, Anfirregulina
, Animais
, Ciclo-Oxigenase 2/metabolismo
, Desmina/metabolismo
, Família de Proteínas EGF
, Implantação do Embrião/fisiologia
, Feminino
, Idade Gestacional
, Glicoproteínas/metabolismo
, Imuno-Histoquímica
, Peptídeos e Proteínas de Sinalização Intercelular/metabolismo
, Fator Inibidor de Leucemia/administração & dosagem
, Fator Inibidor de Leucemia/antagonistas & inibidores
, Fator Inibidor de Leucemia/metabolismo
, Fator Inibidor de Leucemia/fisiologia
, Camundongos
, Fosforilação
, Gravidez
, Receptores de OSM-LIF/metabolismo
, Fator de Transcrição STAT3/metabolismo
, Fatores de Tempo
, Útero/anatomia & histologia
, Útero/efeitos dos fármacos
, Útero/metabolismo
RESUMO
We consider a kinetic law of mass action model for Fibroblast Growth Factor (FGF) signaling, focusing on the induction of the RAS-MAP kinase pathway via GRB2 binding. Our biologically simple model suffers a combinatorial explosion in the number of differential equations required to simulate the system. In addition to numerically solving the full model, we show that it can be accurately simplified. This requires combining matched asymptotics, the quasi-steady state hypothesis, and the fact subsets of the equations decouple asymptotically. Both the full and simplified models reproduce the qualitative dynamics observed experimentally and in previous stochastic models. The simplified model also elucidates both the qualitative features of GRB2 binding and the complex relationship between SHP2 levels, the rate SHP2 induces dephosphorylation and levels of bound GRB2. In addition to providing insight into the important and redundant features of FGF signaling, such work further highlights the usefulness of numerous simplification techniques in the study of mass action models of signal transduction, as also illustrated recently by Borisov and co-workers (Borisov et al. in Biophys. J. 89, 951-966, 2005, Biosystems 83, 152-166, 2006; Kiyatkin et al. in J. Biol. Chem. 281, 19925-19938, 2006). These developments will facilitate the construction of tractable models of FGF signaling, incorporating further biological realism, such as spatial effects or realistic binding stoichiometries, despite a more severe combinatorial explosion associated with the latter.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Animais , Proteína Adaptadora GRB2/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Biologia de Sistemas/métodos , Proteínas ras/metabolismoRESUMO
This study addresses the regulation of the interleukin 1 (IL1) system in the murine uterine luminal epithelium (LE) and stroma by leukemia inhibitory factor (LIF). Using RT-PCR we compared expression of Il1a, Il1b, Il1rn, Il1r1, and Il1r2 during the pre- and peri-implantation periods of pregnancy in wild-type (WT) and LIF-null LE and stroma. In WT LE, Il1a transcripts were down-regulated on Day 4 of pregnancy (D4), with renewed expression by the evening of D4 (D4 pm). In Lif(-/-) LE there was a gradual decrease in expression on D2, and expression became undetectable by D6. Il1b and Il1r1 expression were similar in WT and null mice, but Il1rn expression was almost completely lost during the peri-implantation period in Lif(-/-) LE. In the stroma, Il1a was sharply down-regulated on D4 and reappeared on D4 pm but was only expressed from D3 to D5 in the null mice. Stromal Il1r1 and Il1r2 were also misregulated. Il1rn showed constitutive expression in null stroma in contrast to the loss of expression on D4 in the WT mouse. In Lif-deficient mice, immunostaining indicated a reduction of endometrial IL1A at the time of implantation and of IL1B in stroma. LE-stromal coculture revealed that LIF stimulated the apical secretion of both IL1A and PTGES2 by LE cells without affecting basal secretion of IL1A and with only a small effect on basal PTGES2 secretion. We conclude that Il1a and Il1rn in LE and Il1a, Il1rn, and Il1r1 in stroma are regulated by LIF, which stimulates apical secretion of IL1A by LE.
Assuntos
Interleucina-1/genética , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/fisiologia , Transdução de Sinais/genética , Útero/metabolismo , Animais , Células Cultivadas , Implantação do Embrião/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1/metabolismo , Fator Inibidor de Leucemia/metabolismo , Fator Inibidor de Leucemia/farmacologia , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Gravidez , Transdução de Sinais/efeitos dos fármacosRESUMO
Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique (Barone-Nugent et al., 2002). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.
Assuntos
Microscopia Confocal/métodos , Microscopia de Interferência/métodos , Animais , Linhagem Celular Tumoral , Embrião não Mamífero/anatomia & histologia , Fibroblastos , Humanos , Leishmania mexicana , Mastócitos , Camundongos , Células NIH 3T3 , Ratos , Peixe-Zebra/embriologiaRESUMO
Decidualisation of uterine stromal cells is a prerequisite for implantation of the embryo in mice. Here we have used an in vitro culture system in which stromal cells decidualise as indicated by a number of markers, including an increase in alkaline phosphatase (ALP) activity. The latter was used as a quantitative marker of decidualisation in the presence of low (2%) fetal calf serum. Prostaglandin E(2) (PGE(2)), which is known to induce decidualisation, increased ALP activity, and this effect was blocked in a dose-dependent manner by indomethacin. Leukemia inhibitory factor (LIF) was then examined, but it had no effect on PGE(2) secretion. However, LIF suppressed ALP activity in a dose-dependent manner in the presence of 2% serum, while an inhibitor of LIF that competes for binding to its receptor reversed the effect of LIF and increased ALP activity above the control level. In serum-free cultures, stromal cells differentiated rapidly, and no differences were observed between LIF-treated and untreated cultures. Stromal cells produce LIF during in vitro culture, and this peaked at 48 h. Freshly collected stromal cells from both day-2 and -4 pregnant mice expressed mRNA for the LIF receptor, and the transcript level was higher in cells isolated on day 4. However, no differences were observed in the relative levels of transcripts in cells from day 2 and day 4 after culture, nor were there differences between the LIF-treated cultures and controls. Therefore, in this study, we have shown that LIF suppresses decidualisation of murine uterine stromal cells in the presence of serum, this is not due to the regulation of PGE(2) secretion by stromal cells.
Assuntos
Decídua/metabolismo , Chaperonas Moleculares/fisiologia , Proteínas , Útero/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Células Cultivadas , Dinoprostona/metabolismo , Feminino , Interleucina-6 , Fator Inibidor de Leucemia , Subunidade alfa de Receptor de Fator Inibidor de Leucemia , Camundongos , Camundongos Endogâmicos , Gravidez , RNA Mensageiro/análise , Receptores de Citocinas/genética , Receptores de OSM-LIF , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Apoptose , Polaridade Celular , Células Epiteliais/fisiologia , Mucosa Intestinal/citologia , Intestinos/citologia , Animais , Membrana Basal/citologia , Membrana Basal/embriologia , Sobrevivência Celular/efeitos dos fármacos , Quelantes/farmacologia , Meios de Cultura , Meios de Cultura Livres de Soro/farmacologia , Ácido Edético/farmacologia , Células Epiteliais/efeitos dos fármacos , Técnica Indireta de Fluorescência para Anticorpo , Imuno-Histoquímica , Mucosa Intestinal/embriologia , Intestinos/embriologia , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Técnicas de Cultura de Órgãos , Fatores de TempoRESUMO
Sprouty was first identified in Drosophila as a novel antagonist of the fibroblast growth factor signalling pathway. Sprouty proteins comprise a big family, members of which are characterized by a cysteine-rich domain which confers inhibitory activity, whereas differences in the N-terminal region may be responsible for functional divergence. The role of Sprouty in RTK (receptor tyrosine kinase) signalling pathways is still controversial. Sprouty may negatively or positively regulate RTK signalling via differential interaction with different signalling molecules, and hence exert different mechanism of action.
Assuntos
Proteínas de Drosophila/fisiologia , Proteínas de Membrana/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/metabolismoRESUMO
BACKGROUND: Intestinal epithelial cells secrete exosome-like vesicles. The aim of this study was to characterise murine intestinal epithelial exosomes and to analyse their capacity to inform the immune system in vivo in mice. METHODS: Epithelial exosomes were obtained from the murine epithelial cell line MODE K incubated in the presence or absence of interferon gamma (IFN-gamma) together with pepsin/trypsin ovalbumin hydrolysate (hOVA) to mimic luminal digestion. Exosomes isolated from MODE K conditioned media (EXO-hOVA and EXO-hOVA-IFN) were characterised by western blot, peptide mapping, and mass spectrometry. They were injected intraperitoneally to C3H/HeN mice to test their immunocompetence. RESULTS: MODE K epithelial exosomes displayed major histocompatibility complex (MHC) class I and class II (upregulated by IFN-gamma) molecules and tetraspan proteins (CD9, CD81, CD82) potentially involved in the binding to target cells. A33 antigen, an Ig-like molecule highly specific for intestinal epithelial cells, was enriched in exosomes and was also found in mice mesenteric lymph nodes, suggesting exosome migration towards the gut associated lymphoid tissues. Intraperitoneal injection of EXO-hOVA or EXO-hOVA-IFN did not induce humoral or cellular tolerance to OVA in mice. In contrast, exosomes obtained after incubation with IFN-gamma (EXO-hOVA-IFN), bearing abundant MHC class II/OVA complexes, induced a specific humoral immune response. CONCLUSIONS: Epithelial exosomes are antigen presenting vesicles bearing MHC class II/peptide complexes that prime for an immunogenic rather than tolerogenic response in the context of a systemic challenge. In the intestine, both the mucosal microenvironment and local effector cells are probably key players in determining the outcome of the immune response to exosome derived epitopes.
Assuntos
Vesículas Citoplasmáticas/imunologia , Células Epiteliais/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Sistema Imunitário/fisiologia , Mucosa Intestinal/imunologia , Animais , Western Blotting , Linhagem Celular , Feminino , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Interleucina-1/farmacologia , Linfonodos , Mesentério/imunologia , Camundongos , Camundongos Endogâmicos C3HRESUMO
Conditioning injury to adult mammalian sensory neurons enhances their regeneration potential. Here we show that leukemia inhibitory factor (LIF) is a fundamental component of the conditioning response. Conditioning injury in vivo significantly increases the intrinsic growth capacity of sensory neurons in vitro from LIF+/+ mice. This conditioning effect is significantly blunted in sensory neurons from LIF-/- mice. Enhanced growth is rescued in vitro in LIF-/- mice by the addition of exogenous LIF, and the effect blocked by human LIF-05, an LIF receptor antagonist. Furthermore, we demonstrate that LIF promotes elongating but not arborizing neurite outgrowth in vitro and is required for normal regeneration of injured adult sensory neurons in vivo. LIF is also functionally protective to peptidergic sensory neurons after nerve damage in vivo. Our results indicate that the alteration in intrinsic growth status of injured sensory neurons depends, at least in part, on LIF.
Assuntos
Inibidores do Crescimento/metabolismo , Interleucina-6 , Linfocinas/metabolismo , Neurônios Aferentes/metabolismo , Animais , Axotomia , Peptídeo Relacionado com Gene de Calcitonina/biossíntese , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citoproteção/efeitos dos fármacos , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Inibidores do Crescimento/administração & dosagem , Inibidores do Crescimento/genética , Injeções Espinhais , Fator Inibidor de Leucemia , Linfocinas/administração & dosagem , Linfocinas/genética , Masculino , Camundongos , Camundongos Knockout , Fibras Nervosas/metabolismo , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/fisiologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neurônios Aferentes/citologia , Neurônios Aferentes/efeitos dos fármacos , Fenótipo , Ratos , Ratos Wistar , Nervo Isquiático/fisiologia , Nervo Tibial/fisiologiaRESUMO
OBJECTIVE: To determine whether other glycoprotein 130 (gp130) binding cytokines can mimic the effects of oncostatin M (OSM) in acting synergistically with interleukin-1alpha (IL-1alpha) to induce cartilage collagen breakdown and collagenase expression, and to determine which receptors mediate these effects. METHODS: The release of collagen and proteoglycan was assessed in bovine and human cartilage explant cultures. Messenger RNA (mRNA) and protein production from immortalized human chondrocytes (T/C28a4) was analyzed by Northern blotting and specific enzyme-linked immunosorbent assays. Collagenase activity was measured by bioassay. Cell surface receptors were detected by flow cytometry. RESULTS: OSM in combination with IL-1alpha caused a rapid synergistic induction of matrix metalloproteinase 1 mRNA, which was sustained over a 72-hour period. Flow cytometric analyses detected both the OSM-specific receptor and the gp130 receptor at the chondrocyte cell surface, but failed to detect the leukemia inhibitory factor receptor (LIFR). Cartilage degradation assays revealed that, of the gp130 binding cytokines, only OSM and IL-6, in the presence of its soluble receptor (sIL-6R), were able to act synergistically with IL-1alpha to promote collagen release. CONCLUSION: This study demonstrates that IL-6 can mimic OSM in synergizing with IL-1alpha to induce chondrocyte-mediated cartilage collagen breakdown and collagenase production. In order to have this effect, IL-6 requires the presence of its soluble receptor. The apparent absence of LIFR explains why other gp130 binding cytokines do not act in synergy with IL-1alpha. Since OSM, IL-6, and sIL-6R levels have all been shown to be elevated in the rheumatoid joint, our findings suggest that these cytokines may be key mediators of cartilage collagen catabolism in the inflammatory arthritides.
Assuntos
Antígenos CD/metabolismo , Condrócitos/efeitos dos fármacos , Colágeno/metabolismo , Interleucina-1/farmacologia , Interleucina-6/farmacologia , Glicoproteínas de Membrana/metabolismo , Animais , Cartilagem Articular/citologia , Bovinos , Linhagem Celular Transformada , Condrócitos/citologia , Condrócitos/enzimologia , Fator Neurotrófico Ciliar/metabolismo , Fator Neurotrófico Ciliar/farmacologia , Receptor gp130 de Citocina , Citocinas/metabolismo , Citocinas/farmacologia , Sinergismo Farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/farmacologia , Humanos , Interleucina-1/metabolismo , Interleucina-11/metabolismo , Interleucina-11/farmacologia , Interleucina-6/metabolismo , Fator Inibidor de Leucemia , Linfocinas/metabolismo , Linfocinas/farmacologia , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Oncostatina M , Peptídeos/metabolismo , Peptídeos/farmacologia , RNA Mensageiro/análise , Receptores de Interleucina-6/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
The receptor subunit gp130 transduces multiple cell type-specific activities of the leukemia inhibitory factor (LIF)/interleukin (IL)-6 family of cytokines through the signal transducer and activator of transcription (STAT) and src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-2/ras/Erk pathways. To define STAT-dependent physiological responses, we generated mice with a COOH-terminal gp130(DeltaSTAT) "knock-in" mutation which deleted all STAT-binding sites. gp130(DeltaSTAT) mice phenocopyed mice deficient for IL-6 (impaired humoral and mucosal immune and hepatic acute phase responses) and LIF (failure of blastocyst implantation). However, unlike mice with null mutations in any of the components in the gp130 signaling pathway, gp130(DeltaSTAT) mice also displayed gastrointestinal ulceration and a severe joint disease with features of chronic synovitis, cartilaginous metaplasia, and degradation of the articular cartilage. Mitogenic hyperresponsiveness of synovial cells to the LIF/IL-6 family of cyto-kines was caused by sustained gp130-mediated SHP-2/ras/Erk activation due to impaired STAT-mediated induction of suppressor of cytokine signaling (SOCS) proteins which normally limits gp130 signaling. Therefore, the joint pathology in gp130(DeltaSTAT) mice is likely to arise from the disturbance of the otherwise balanced activation of the SHP-2/ras/Erk and STAT signaling cascades emanating from gp130.
Assuntos
Antígenos CD/genética , Antígenos CD/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Proteínas Repressoras , Transativadores/genética , Transativadores/fisiologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Receptor gp130 de Citocina , Primers do DNA/genética , Implantação do Embrião/genética , Implantação do Embrião/fisiologia , Feminino , Artropatias/etiologia , Artropatias/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Úlcera Péptica/etiologia , Úlcera Péptica/patologia , Gravidez , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de CitocinaRESUMO
The expression pattern of the murine A33 antigen has been defined during development using wholemount immunohistochemistry. Two temporally and spatially distinct sites of expression were identified: the inner cell mass of the blastocyst and the endoderm cell layer of the intestinal tract where expression is initiated at E14.5 in the hindgut and subsequently extends throughout the length of the intestine. The onset of mA33 antigen expression in the gut occurs at the beginning of an extensive phase of cell movement involved in the conversion of the endoderm cell layer to a single cell layer of polarized epithelium. Expression of mA33 antigen is then maintained into adulthood, where it is a definitive marker of intestinal epithelium.
Assuntos
Blastocisto/imunologia , Intestinos/embriologia , Intestinos/imunologia , Glicoproteínas de Membrana/metabolismo , Animais , Endoderma/imunologia , Epitélio/embriologia , Epitélio/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos ICRRESUMO
To date, two distinct genes coding for Ras GAP-binding phosphoproteins of 190kDa, p190-A and p190-B, have been cloned from mammalian cells. Rat p190-A of 1513 amino acids shares 50% sequence identity with human p190-B of 1499 amino acids. We have previously demonstrated, using rat p190-A cDNA, that full-length p190-A is a tumor suppressor, reversing v-Ha-Ras-induced malignancy of NIH 3T3 cells through both the N-terminal GTPase (residues 1-251) and the C-terminal Rho GAP (residues 1168-1441) domains. Here we report the cloning of the full-length human p190-A cDNA and its first exon covering more than 80% of this protein, as well as its chromosomal mapping. Human p190-A encodes a protein of 1514 amino acids, and shares overall 97% sequence identity with rat p190-A. Like the p190-B exon, the first exon of p190-A is extremely large (3.7 kb in length), encoding both the GTPase and middle domains (residues 1-1228), but not the remaining GAP domain, suggesting a high conservation of genomic structure between two p190 genes. Using a well characterized monochromosome somatic cell hybrid panel, fluorescent in situ hybridization (FISH) and other complementary approaches, we have mapped the p190-A gene between the markers D19S241E and STD (500 kb region) of human chromosome 19q13.3. Interestingly, this chromosomal region is known to be rearranged in a variety of human solid tumors including pancreatic carcinomas and gliomas. Moreover, at least 40% glioblastoma/astrocytoma cases with breakpoints in this region were previously reported to show loss of the chromosomal region encompassing p190-A, suggesting the possibility that loss or mutations of this gene might be in part responsible for the development of these tumors.
Assuntos
Cromossomos Humanos Par 19/genética , Proteínas de Ligação a DNA , Proteínas de Ligação ao GTP , Genes Supressores de Tumor/genética , Glioma/genética , Proteínas Supressoras de Tumor , Proteínas Ativadoras de ras GTPase/genética , Sequência de Aminoácidos , Sequência de Bases , Bandeamento Cromossômico , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Éxons , Proteínas Ativadoras de GTPase , Deleção de Genes , Genes/genética , Fatores de Troca do Nucleotídeo Guanina , Humanos , Hibridização in Situ Fluorescente , Íntrons , Dados de Sequência Molecular , Mapeamento de Híbridos Radioativos , Proteínas Repressoras , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , ras-GRF1RESUMO
BACKGROUND: The cytokine oncostatin M (OSM) inhibits growth of certain tumour-derived cells, induces proliferation in other cell types (e.g. haemangioblasts) and is a mediator of inflammatory responses. Its mechanism of action is via specific binding to gp130 and either the leukaemia inhibitory factor receptor (LIFR) or oncostatin M receptor (OSMR) systems at the cell surface to form an active signalling complex. RESULTS: We report here the crystal structure of human oncostatin M (hOSM) along with mutagenesis data which map the receptor-binding epitopes of the molecule. The structure was determined to a resolution of 2.2 A and conforms to the haematopoietin cytokine up-up-down-down four-helix bundle topology. The site 2 epitope, responsible for gp130 binding, is centred around Gly120 which forms a 'dimple' on the surface of the molecule located on helices A and C. The site 3 motif, responsible for LIFR and OSMR binding, consists of a protruding Phe160/Lys163 pair located at the start of helix D. CONCLUSIONS: The data presented allow functional dissection of the receptor-binding interfaces to atomic resolution. Modelling suggests that the gp130 residue Phe169 packs into the site 2 dimple in an analogous fashion to structurally equivalent residues at the growth hormone-growth hormone receptor interface, implying that certain key features may underlie recognition across the whole cytokine/receptor superfamily. Conversely, detailed comparison of the available structures suggests that variations on a common theme dictate the specificity of receptor-ligand interactions within the gp130 family of cytokines.
Assuntos
Peptídeos/química , Conformação Proteica , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oncostatina M , Peptídeos/genética , Peptídeos/metabolismo , Receptores de Citocinas/química , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Receptores de Oncostatina M , Transdução de SinaisRESUMO
Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. IL-11 has been shown to induce gp130-dependent signaling through the formation of a high affinity complex with the IL-11 receptor (IL-11R) and gp130. Site-directed mutagenesis studies have identified three distinct receptor binding sites of IL-11, which enable it to form this high affinity receptor complex. Here we present data from immunoprecipitation experiments, using differentially tagged forms of ligand and soluble receptor components, which show that multiple copies of IL-11, IL-11R, and gp130 are present in the receptor complex. Furthermore, it is demonstrated that sites II and III of IL-11 are independent gp130 binding epitopes and that both are essential for gp130 dimerization. We also show that a stable high affinity complex of IL-11, IL-11R, and gp130 can be resolved by nondenaturing polyacrylamide gel electrophoresis, and its composition verified by second dimension denaturing polyacrylamide gel electrophoresis. Results indicate that the three receptor binding sites of IL-11 and the Ig-like domain of gp130 are all essential for this stable receptor complex to be formed. We therefore propose that IL-11 forms a hexameric receptor complex composed of two molecules each of IL-11, IL-11R, and gp130.
Assuntos
Antígenos CD/metabolismo , Interleucina-11/metabolismo , Interleucina-11/farmacologia , Glicoproteínas de Membrana/metabolismo , Receptores de Interleucina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Antígenos CD/química , Antígenos CD/genética , Sítios de Ligação , Linhagem Celular , Receptor gp130 de Citocina , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoglobulinas/química , Interleucina-11/genética , Subunidade alfa de Receptor de Interleucina-11 , Substâncias Macromoleculares , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Mutação/genética , Testes de Precipitina , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina-11 , TermodinâmicaRESUMO
The murine A33 antigen is emerging as a definitive marker of intestinal epithelial cells. Cloning and sequence determination of cDNAs encoding mA33 antigen predict a novel type 1 transmembrane protein of 298 amino acids, comprising an extracellular domain with two immunoglobulin-like domains, a single-span transmembrane domain, and a highly acidic cytoplasmic domain. On the basis of conservation of amino acid sequence and genomic structure, the mA33 antigen is a member of a growing subfamily within the immunoglobulin superfamily, which includes transmembrane proteins CTX/ChT1, CTM/CTH, and CAR. During embryonic development, mA33 antigen expression is first observed in the inner cell mass of blastocysts before implantation. Intestinal expression of mA33 antigen is initiated in the hindgut at E14.5 and increases steadily throughout late embryonic and postnatal life into adulthood. The protein is specifically expressed on the basolateral surfaces of intestinal epithelial cells of all lineages, independent of their position along the rostrocaudal and crypt-villus axes. Thus the mA33 antigen appears to be a novel marker for both proliferating and differentiating intestinal epithelial cells.
Assuntos
Células Epiteliais/química , Mucosa Intestinal/citologia , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , ATPases Associadas a Diversas Atividades Celulares , Animais , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/genética , Sequência de Bases , Biomarcadores , Western Blotting , Carcinoma Embrionário , Moléculas de Adesão Celular/genética , Clonagem Molecular , DNA Complementar , Células Epiteliais/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imunoglobulinas/genética , Mucosa Intestinal/embriologia , Moléculas de Adesão Juncional , Proteínas de Membrana/genética , Metaloendopeptidases , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
Development of the gastrointestinal (GI) tract depends on reciprocal epithelial-mesenchymal cell signaling. Here, we demonstrate a role for platelet-derived growth factor-A (PDGF-A) and its receptor, PDGFR-(alpha), in this process. Mice lacking PDGF-A or PDGFR-(alpha) were found to develop an abnormal GI mucosal lining, including fewer and misshapen villi and loss of pericryptal mesenchyme. Onset of villus morphogenesis correlated with the formation of clusters of PDGFR-(alpha) positive cells, 'villus clusters', which remained located at the tip of the mesenchymal core of the growing villus. Lack of PDGF-A or PDGFR-(alpha) resulted in progressive depletion of PDGFR-(alpha) positive mesenchymal cells, the formation of fewer villus clusters, and premature expression of smooth muscle actin (SMA) in the villus mesenchyme. We found that the villus clusters were postmitotic, expressed BMP-2 and BMP-4, and that their formation correlated with downregulated DNA synthesis in adjacent intestinal epithelium. We propose a model in which villus morphogenesis is initiated as a result of aggregation of PDGFR-(&agr;) positive cells into cell clusters that subsequently function as mesenchymal centers of signaling to the epithelium. The role of PDGF-A seems to be to secure renewal of PDGFR-(alpha) positive cells when they are consumed in the initial rounds of cluster formation.
Assuntos
Intestinos/embriologia , Fator de Crescimento Derivado de Plaquetas/fisiologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/fisiologia , Fator de Crescimento Transformador beta , Animais , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular , Divisão Celular , Sistema Digestório/embriologia , Perfilação da Expressão Gênica , Mucosa Intestinal/metabolismo , Mesoderma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvilosidades , Morfogênese , Músculo Liso/citologia , Fator de Crescimento Derivado de Plaquetas/genéticaRESUMO
Cytokines of the gp130 family exert their diverse biological effects by formation of stable high affinity transmembrane receptor complexes that are characterized by the presence of the shared transmembrane signalling receptor gp130. Different gp130 ligands form signalling complexes that vary in both composition and stoichiometry. Analysis of the three-dimensional structure of selected ligands and receptor elements indicates that ligands display three topologically conserved receptor recognition epitopes that interact with complementary ligand recognition elements. The composition of the signalling complex and downstream biological responses is defined by the relative affinity of different receptor components for these epitopes. The detailed structure of receptor recognition epitopes indicates that the generation of small molecule cytokine mimetics may be a feasible objective.
Assuntos
Antígenos CD/metabolismo , Citocinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Citocinas/metabolismo , Transdução de Sinais/fisiologia , Receptor gp130 de Citocina , Citocinas/química , Dimerização , Desenho de Fármacos , Epitopos/química , Inibidores do Crescimento/química , Hormônio do Crescimento Humano/química , Hormônio do Crescimento Humano/metabolismo , Humanos , Interleucina-6/química , Fator Inibidor de Leucemia , Ligantes , Linfocinas/química , Modelos Moleculares , Família Multigênica , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Receptores da Somatotropina/química , Receptores da Somatotropina/metabolismo , Relação Estrutura-AtividadeRESUMO
Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. A complex of IL-11 and the IL-11 receptor (IL-11R) has been shown to interact with gp130, with high affinity, and to induce gp130- dependent signaling. In this study, we have identified residues crucial for the binding of murine IL-11 (mIL-11) to both the IL-11R and gp130 by examining the activities of mIL-11 mutants in receptor binding and cell proliferation assays. The location of these residues, as predicted from structural studies and a model of IL-11, reveals that mIL-11 has three distinct receptor binding sites. These are structurally and functionally analogous to the previously defined receptor binding sites I, II, and III of interleukin-6 (IL-6). This supports the hypothesis that IL-11 signals via the formation of a hexameric receptor complex and indicates that site III is a generic feature of cytokines that signal via association with gp130.