RESUMO
OBJECTIVE: Interleukin-6 (IL-6) signaling plays an important proinflammatory role, but this role is restricted by regulatory mechanisms that, for example, reduce the cell surface availability of the signal-transducing chain of the IL-6 receptor, gp130. The aim of this study was to determine whether the inflammatory environment in arthritic joints has an impact on monocytic gp130 surface expression and the extent to which regulatory processes in the synovial fluid (SF) can be reproduced in an in vitro model. METHODS: Flow cytometry and live cell imaging were used to measure the cell surface expression and internalization of gp130. STAT-3 phosphorylation was monitored by flow cytometry and Western blotting. RESULTS: In patients with juvenile idiopathic arthritis (JIA), levels of cell surface gp130 expression in SF monocytes were reduced compared to those in peripheral blood (PB) monocytes. These reduced levels were reproduced when PB monocytes from healthy donors were stimulated with SF, and this reduction was dependent on p38 MAPK. The induction of p38 by IL-1ß in PB monocytes interfered with IL-6 signaling due to the reduced cell surface expression of gp130. CONCLUSION: These results suggest that p38-mediated proinflammatory stimuli induce the down-regulation of gp130 on monocytes and thus restrict gp130-mediated signal transduction. This regulatory mechanism could be of relevance to processes in the inflamed joints of patients with JIA.
Assuntos
Artrite Juvenil/fisiopatologia , Receptor gp130 de Citocina/fisiologia , Regulação para Baixo/fisiologia , Interleucina-6/fisiologia , Transdução de Sinais/fisiologia , Líquido Sinovial/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Adolescente , Artrite Juvenil/patologia , Células Cultivadas , Criança , Citometria de Fluxo , Humanos , Técnicas In Vitro , Interleucina-1beta/fisiologia , Monócitos/patologia , Monócitos/fisiologia , Fosforilação/fisiologia , Fator de Transcrição STAT3/fisiologia , Serina/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Líquido Sinovial/citologiaRESUMO
BACKGROUND: Mutations in the IGF-I receptor (IGF1R) gene can be responsible for intrauterine and postnatal growth disorders. OBJECTIVE: Here we report on a novel mutation in the IGF1R gene in a female patient. The aim of our study was to analyze the functional impact of this mutation. PATIENT: At birth, the girl's length was 47 cm [-1.82 sd score (SDS)], and her weight was 2250 g (-2.26 SDS). Clinical examination revealed microcephaly and retarded cognitive development. She showed no postnatal catch-up growth but had relatively high IGF-I levels (+1.83 to +2.17 SDS). RESULTS: Denaturing HPLC screening and direct DNA sequencing disclosed a heterozygous missense mutation resulting in an amino acid exchange from valine to glutamic acid at position 599 (V599E-IGF1R). Using various cell systems, we found that the V599E-IGF1R mutant was not tyrosine phosphorylated and had an impaired downstream signaling in the presence of IGF-I. Flow cytometry and live cell confocal laser scanning microscopy revealed a lack of cell surface expression due to an extensive retention of V599E-IGF1R proteins within the endoplasmic reticulum. CONCLUSION: The V599E-IGF1R mutation interferes with the receptor's trafficking path, thereby abrogating proreceptor processing and plasma membrane localization. Diminished cell surface receptor density solely expressed from the patient's wild-type allele is supposed to lead to insufficient IGF-I signaling. We hypothesize that this mechanism results in intrauterine and postnatal growth retardation of the affected patient. The reported retention of the nascent IGF1R in the endoplasmic reticulum presents a novel mechanism of IGF-I resistance.
Assuntos
Retículo Endoplasmático/metabolismo , Retardo do Crescimento Fetal/genética , Transtornos do Crescimento/genética , Heterozigoto , Mutação de Sentido Incorreto , Receptor IGF Tipo 1/genética , Substituição de Aminoácidos , Peso ao Nascer , Tamanho Corporal , Transtornos Cognitivos/genética , Éxons/genética , Feminino , Ácido Glutâmico/genética , Humanos , Recém-Nascido , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Deficiência Intelectual/genética , Microcefalia/genética , Reação em Cadeia da Polimerase , Receptor IGF Tipo 1/metabolismo , Valina/genéticaRESUMO
The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on multiple levels, for instance by regulating the mRNA stability of targeted cytokines through activation of the p38-MAPK pathway. In our study we discovered a new mechanism that answers the long-standing question how pro-inflammatory cytokines and environmental stress restrict immediate signalling of interleukin (IL)-6-type cytokines. We show that p38, activated by IL-1beta, TNFalpha or environmental stress, impairs IL-6-induced JAK/STAT signalling through phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalisation and degradation. We identify MK2 as the kinase that phosphorylates serine 782 in the cytoplasmic part of gp130. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalisation motif blocks receptor depletion and restores IL-6-dependent STAT activation as well as gene induction. Hence, a novel negative crosstalk mechanism for cytokine signalling is described, where cytokine receptor turnover is regulated in trans by pro-inflammatory cytokines and stress stimuli to coordinate the inflammatory response.
Assuntos
Receptor gp130 de Citocina/metabolismo , Endocitose , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1beta/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Neuroblastoma cell lines are commonly used as a model to study neuronal differentiation as they retain the capacity to differentiate into a neuronal-like phenotype. It is of great medical interest to understand the signalling pathways biasing differentiation versus proliferation. Neuroblastoma cells differentiate in response to serum reduction or addition of the cholesterol synthesis inhibitor mevastatin. The responsible pathways are not well characterized. In Neuro2a neuroblastoma cells, we found that mevastatin and serum withdrawal triggered the production of nitric oxide (NO). In addition, the differentiation of Neuro2a cells and the activation of Akt/PKB triggered by serum withdrawal could be blocked by addition of the NO synthetase (NOS) inhibitor l-NAME. Moreover, mevastatin and serum withdrawal rapidly increased the expression of the neuronal NOS isoform nNOS. However, addition of an NO donor SNP per se did not trigger neurite outgrowth. Taken together, we report for the first time a role of NO in neurite outgrowth of neuroblastoma cells triggered by mevastatin or serum reduction.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lovastatina/análogos & derivados , Neuritos/fisiologia , Óxido Nítrico/fisiologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro , Ativação Enzimática , Lovastatina/farmacologia , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Neuritos/efeitos dos fármacos , Neuroblastoma , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Óxido Nítrico/biossíntese , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/biossíntese , Nitroprussiato/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Dysregulated cytokine production contributes to inflammatory and proliferative diseases. Therefore, inhibition of proinflammatory mediators such as TNF, IL-1, and IL-6 is of great clinical relevance. Actual strategies are aimed at preventing receptor activation through sequestration of the ligand. Here we describe the development of an inhibitor of murine IL-6 based on fused receptor fragments. Molecular modeling-guided analysis of the murine IL-6Ralpha revealed that mutations in the Ig-like domain D1 severely affect protein function, although D1 is not directly involved in the ligand-binding interface. The resulting single chain IL-6 inhibitor (mIL-6-RFP) consisting of domains D1-D3 of mgp130, a flexible linker, and domains D1-D3 of mIL-6Ralpha is a highly potent and specific IL-6 inhibitor. mIL-6-RFP will permit further characterization of the role of IL-6 in various disease models and could ultimately lead to anti-IL-6 therapy.
Assuntos
Desenho de Fármacos , Interleucina-6/antagonistas & inibidores , Engenharia de Proteínas/métodos , Receptores de Interleucina-6/genética , Animais , Inflamação/tratamento farmacológico , Ligantes , Camundongos , MutaçãoRESUMO
The Janus kinases, Jaks, constitutively associate with the cytoplasmic region of cytokine receptors and play an important role in a multitude of biological processes. Jak2 dysfunction has been implicated in myeloproliferative diseases and leukemia. Although Jaks were studied extensively for many years, the molecular mechanism of Jak activation upon cytokine stimulation of cells is still incompletely understood. In this study, we investigated the importance of an unusual insertion located within the kinase domain in Jak2. We found that the deletion of this insertion, which we named the Jak-specific insertion (JSI), totally abrogates Jak2 autophosphorylation. We further point mutated four residues within the JSI that are conserved in all Jak family members. Three of these mutants showed abrogated or reduced autophosphorylation, whereas the fourth displayed increased autophosphorylation. We found that the phosphorylation state of these mutants is not influenced by other domains of the kinase. Our data further suggest that the JSI is not required for the negative regulation of kinase activity by the suppressor of cytokine signaling proteins, SOCS. Most importantly, we show that mutations in this region differentially affect IFN-gamma and erythropoietin signal transduction. Taken together, the dramatic effects on the phosphorylation status of Jak2 as well as the differential effects on the signaling via different cytokines highlight the importance of this unusual region for the catalytic activity of Jaks.
Assuntos
Citocinas/fisiologia , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Mutagênese Insercional , Sequência de Aminoácidos , Animais , Domínio Catalítico/genética , Domínio Catalítico/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Simulação por Computador , Citocinas/biossíntese , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Humanos , Janus Quinase 2/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação PuntualRESUMO
Haematopoietic stem cell transplantation is the treatment of choice for severe primary immunodeficiencies, but only has moderate prognosis in Omenn syndrome as it is complicated by highly activated Omenn T-cells resulting in delayed T-cell engraftment and a high rate of graft failure. A 6 1/2 months old patient with a previously unknown compound heterozygous defect within the RAG1 gene (R474C; R975W) underwent 8/10 HLA-matched cord blood transplantation after myeloablative conditioning. Immune reconstitution was impressive with T-, B- and NK-cells reaching the median of age-dependent reference values within twelve, four and two months respectively. With a continuous decrease of activated Omenn T-cells there was a steady increase of naive, probably thymus-derived T-cells. Polyclonal B-cell activation and hypergammaglobulinaemia disappeared with B-cell engraftment. This case emphasizes that, despite their naive status and HLA-barriers, cord blood T-cells were apparently able to achieve T-effector function resulting in the elimination of all activated Omenn T-cells.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Dermatite Esfoliativa/terapia , Imunodeficiência Combinada Severa/terapia , Dermatite Esfoliativa/imunologia , Feminino , Citometria de Fluxo , Humanos , Lactente , Contagem de Leucócitos , Imunodeficiência Combinada Severa/imunologia , Condicionamento Pré-Transplante , Transplante HomólogoRESUMO
Some membrane-permeable antagonists restore cell surface expression of misfolded receptors retained in the endoplasmic reticulum (ER) and are therefore termed pharmacochaperones. Whether pharmacochaperones increase protein stability, thereby preventing rapid degradation, or assist folding via direct receptor interactions or interfere with quality control components remains elusive. We now show that the cell surface expression and function (binding of the agonist) of the mainly ER-retained wild-type murine vasopressin V2 receptor GFP fusion protein (mV2R.GFP) is restored by the vasopressin receptor antagonists SR49059 and SR121463B with EC50 values similar to their KD values. This effect was preserved when protein synthesis was abolished. In addition, SR121463B rescued eight mutant human V2Rs (hV2Rs, three are responsible for nephrogenic diabetes insipidus) characterized by amino acid exchanges at the C-terminal end of transmembrane helix TM I and TM VII. In contrast, mutants with amino acid exchanges at the interface of TM II and IV were not rescued by either antagonist. The mechanisms involved in successful rescue of cell surface delivery are explained in a three-dimensional homology model of the antagonist-bound hV2R.
Assuntos
Membrana Celular/metabolismo , Chaperonas Moleculares/química , Processamento de Proteína Pós-Traducional , Receptores de Vasopressinas/química , Sequência de Aminoácidos , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Transporte Biológico , Biotinilação , Linhagem Celular , Retículo Endoplasmático/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Cinética , Lasers , Camundongos , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Morfolinas/farmacologia , Mutação , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Compostos de Espiro/farmacologia , Fatores de Tempo , TransfecçãoRESUMO
Growth deficit associated with stimulants in children who have attention deficit hyperactivity disorder (ADHD) has long been the subject of scientific discussion. More recent studies describe the effects on the final height of children with ADHD who are treated with methylphenidate (MPH) as only slight. There are only a handful of reports of severe growth deficits attributed to MPH that are associated with gastrointestinal side effects. We describe a 10-year-old boy with ADHD and chronic asthma who underwent corticosteroid therapy and developed an almost complete growth arrest during MPH treatment. Growth hormone (GH) stimulation tests and measurement of GH-dependent growth factors point to the influence of MPH on GH secretion with subsequent impaired growth. One may conclude that some children are at risk of serious growth decrement when treated with MPH. The growth of children should thus be monitored carefully, even if there are no alarming gastrointestinal side effects from MPH. We found that the determination of growth velocity was a sensitive marker for the evaluation of growth impairment in our patient.