RESUMO
The intestine-specific caudal-related homeobox gene-2 (CDX2) homeobox gene, while being a tumor suppressor in the gut, is ectopically expressed in a large proportion of acute leukemia and is associated with poor prognosis. Here, we report that turning on human CDX2 expression in the hematopoietic lineage of mice induces acute monoblastic leukemia, characterized by the decrease in erythroid and lymphoid cells at the benefit of immature monocytic and granulocytic cells. One of the highly stimulated genes in leukemic bone marrow cells was BMP and activin membrane-bound inhibitor (Bambi), an inhibitor of transforming growth factor-ß (TGF-ß) signaling. The CDX2 protein was shown to bind to and activate the transcription of the human BAMBI promoter. Moreover, in a leukemic cell line established from CDX2-expressing mice, reducing the levels of CDX2 or Bambi stimulated the TGF-ß-dependent expression of Cd11b, a marker of monocyte maturation. Taken together, this work demonstrates the strong oncogenic potential of the homeobox gene CDX2 in the hematopoietic lineage, in contrast with its physiological tumor suppressor activity exerted in the gut. It also reveals, through BAMBI and TGF-ß signaling, the involvement of CDX2 in the perturbation of the interactions between leukemia cells and their microenvironment.
Assuntos
Fator de Transcrição CDX2/genética , Leucemia Monocítica Aguda/genética , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Antígeno CD11b/genética , Linhagem da Célula , Humanos , Leucemia Monocítica Aguda/patologia , Proteínas de Membrana/genética , Camundongos , Transdução de Sinais , Microambiente TumoralRESUMO
BACKGROUND: Autosomal dominant gain-of-function mutations in human stimulator of interferon genes (STING) lead to a severe autoinflammatory disease called STING-associated vasculopathy with onset in infancy that is associated with enhanced expression of interferon-stimulated gene transcripts. OBJECTIVE: The goal of this study was to analyze the phenotype of a new mouse model of STING hyperactivation and the role of type I interferons in this system. METHODS: We generated a knock-in model carrying an amino acid substitution (V154M) in mouse STING, corresponding to a recurrent mutation seen in human patients with STING-associated vasculopathy with onset in infancy. Hematopoietic development and tissue histology were analyzed. Lymphocyte activation and proliferation were assessed in vitro. STING V154M/wild-type (WT) mice were crossed to IFN-α/ß receptor (IFNAR) knockout mice to evaluate the type I interferon dependence of the mutant Sting phenotype recorded. RESULTS: In STING V154M/WT mice we detected variable expression of inflammatory infiltrates in the lungs and kidneys. These mice showed a marked decrease in survival and developed a severe combined immunodeficiency disease (SCID) affecting B, T, and natural killer cells, with an almost complete lack of antibodies and a significant expansion of monocytes and granulocytes. The blockade in B- and T-cell development was present from early immature stages in bone marrow and thymus. In addition, in vitro experiments revealed an intrinsic proliferative defect of mature T cells. Although the V154M/WT mutant demonstrated increased expression of interferon-stimulated genes, the SCID phenotype was not reversed in STING V154M/WT IFNAR knockout mice. However, the antiproliferative defect in T cells was rescued partially by IFNAR deficiency. CONCLUSIONS: STING gain-of-function mice developed an interferon-independent SCID phenotype with a T-cell, B-cell, and natural killer cell developmental defect and hypogammaglobulinemia that is associated with signs of inflammation in lungs and kidneys. Only the intrinsic proliferative defect of T cells was partially interferon dependent.
Assuntos
Linfócitos B/fisiologia , Inflamação/genética , Células Matadoras Naturais/imunologia , Proteínas de Membrana/genética , Mutação/genética , Imunodeficiência Combinada Severa/genética , Linfócitos T/fisiologia , Agamaglobulinemia , Animais , Diferenciação Celular/genética , Modelos Animais de Doenças , Humanos , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/genéticaRESUMO
BACKGROUND AND PURPOSE: Valvular heart disease (VHD) is highly prevalent in industrialized countries. Chronic use of anorexigens, amphetamine or ergot derivatives targeting the 5-HT system is associated with VHD. Here, we investigated the contribution of 5-HT receptors in a model of valve degeneration induced by nordexfenfluramine, the main metabolite of the anorexigens, dexfenfluramine and benfluorex. EXPERIMENTAL APPROACH: Nordexfenfluramine was infused chronically (28 days) in mice ((WT and transgenic Htr2B -/- , Htr2A -/- , and Htr2B/2A -/- ) to induce mitral valve lesions. Bone marrow transplantation was also carried out. Haemodynamics were measured with echocardiography; tissues and cells were analysed by histology, immunocytochemistry, flow cytometry and RT -qPCR. Samples of human prolapsed mitral valves were also analysed. KEY RESULTS: Chronic treatment of mice with nordexfenfluramine activated 5-HT2B receptors and increased valve thickness and cell density in a thick extracellular matrix, mimicking early steps of mitral valve remodelling. Lesions were prevented by 5-HT2A or 5-HT2B receptor antagonists and in transgenic Htr2B -/- or Htr2A/2B -/- mice. Surprisingly, valve lesions were mainly formed by numerous non-proliferative CD34+ endothelial progenitors. These progenitors originated from bone marrow (BM) as revealed by BM transplantation. The initial steps of mitral valve remodelling involved mobilization of BM-derived CD34+ CD31+ cells by 5-HT2B receptor stimulation. Analysis of human prolapsed mitral valves showing spontaneous degenerative lesions, demonstrated the presence of non-proliferating CD34+ /CD309+ /NOS3+ endothelial progenitors expressing 5-HT2B receptors. CONCLUSIONS AND IMPLICATIONS: BM-derived endothelial progenitor cells make a crucial contribution to the remodelling of mitral valve tissue. Our data describe a new and important mechanism underlying human VHD.
Assuntos
Células Progenitoras Endoteliais , Doenças das Valvas Cardíacas/metabolismo , Valva Mitral/metabolismo , Receptor 5-HT2B de Serotonina/metabolismo , Animais , Transplante de Medula Óssea , Células Progenitoras Endoteliais/metabolismo , Doenças das Valvas Cardíacas/patologia , Masculino , Camundongos Transgênicos , Valva Mitral/efeitos dos fármacos , Valva Mitral/patologia , Norfenfluramina/farmacologia , Receptor 5-HT2A de Serotonina/genética , Receptor 5-HT2B de Serotonina/genética , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologiaRESUMO
The Notch signaling pathway is activated in many cell types, but its effects are cell type- and stage-specific. In the immune system, Notch activity is required for the differentiation of T cell progenitors, but it is reduced in more mature thymocytes, in which Notch is oncogenic. Studies based on single-gene models have suggested that the tumor suppressor protein Ikaros plays an important role in repressing the transcription of Notch target genes. We used genome-wide analyses, including chromatin immunoprecipitation sequencing, to identify genes controlled by Notch and Ikaros in gain- and loss-of-function experiments. We found that Ikaros bound to and directly repressed the expression of most genes that are activated by Notch. Specific deletion of Ikaros in thymocytes led to the persistent expression of Notch target genes that are essential for T cell maturation, as well as the rapid development of T cell leukemias in mice. Expression of Notch target genes that are normally silent in T cells, but are activated by Notch in other cell types, occurred in T cells of mice genetically deficient in Ikaros. We propose that Ikaros shapes the timing and repertoire of the Notch transcriptional response in T cells through widespread targeting of elements adjacent to Notch regulatory sequences. These results provide a molecular framework for understanding the regulation of tissue-specific and tumor-related Notch responses.
Assuntos
Genes Supressores de Tumor , Fator de Transcrição Ikaros/fisiologia , Receptores Notch/metabolismo , Linfócitos T/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica , Humanos , Fator de Transcrição Ikaros/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Transcrição GênicaRESUMO
Tubular aggregates are regular arrays of membrane tubules accumulating in muscle with age. They are found as secondary features in several muscle disorders, including alcohol- and drug-induced myopathies, exercise-induced cramps, and inherited myasthenia, but also exist as a pure genetic form characterized by slowly progressive muscle weakness. We identified dominant STIM1 mutations as a genetic cause of tubular-aggregate myopathy (TAM). Stromal interaction molecule 1 (STIM1) is the main Ca(2+) sensor in the endoplasmic reticulum, and all mutations were found in the highly conserved intraluminal Ca(2+)-binding EF hands. Ca(2+) stores are refilled through a process called store-operated Ca(2+) entry (SOCE). Upon Ca(2+)-store depletion, wild-type STIM1 oligomerizes and thereby triggers extracellular Ca(2+) entry. In contrast, the missense mutations found in our four TAM-affected families induced constitutive STIM1 clustering, indicating that Ca(2+) sensing was impaired. By monitoring the calcium response of TAM myoblasts to SOCE, we found a significantly higher basal Ca(2+) level in TAM cells and a dysregulation of intracellular Ca(2+) homeostasis. Because recessive STIM1 loss-of-function mutations were associated with immunodeficiency, we conclude that the tissue-specific impact of STIM1 loss or constitutive activation is different and that a tight regulation of STIM1-dependent SOCE is fundamental for normal skeletal-muscle structure and function.
Assuntos
Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Miopatias Congênitas Estruturais/patologia , Proteínas de Neoplasias/metabolismo , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Criança , Feminino , Homeostase , Humanos , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Músculos/patologia , Músculos/ultraestrutura , Mutação/genética , Mioblastos/metabolismo , Mioblastos/patologia , Miopatias Congênitas Estruturais/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Linhagem , Fenótipo , Molécula 1 de Interação Estromal , Adulto JovemRESUMO
Several epidemiological studies suggest that a diet rich in fruits and vegetables, which contain high levels of polyphenols, is associated with a reduced risk of cancer. The aim of the present study was to determine whether a red wine polyphenolic extract (RWPs, a rich source of polyphenols; 2.9g/L) affects the proliferation of human lymphoblastic leukaemia cells (Jurkat cells) and, if so, to determine the underlying mechanism. Cell proliferation and viability were determined by the MTS and trypan blue exclusion assays, respectively. Cell cycle analysis, apoptosis activity and oxidative stress levels were assessed by flow cytometry, and the expression of p73, UHRF1 and active caspase-3 by Western blot analysis. RWPs inhibited the proliferation of Jurkat cells and induced G0/G1 cell cycle arrest in a concentration-dependent manner. Moreover, RWPs triggered apoptosis, which is associated with an increased expression level of the pro-apoptotic protein p73 and the active caspase-3. RWPs induced apoptosis confirmed by DNA fragmentation analysis, and this effect was associated with down-regulation of the antiapoptotic protein UHRF1. Furthermore RWPs significantly increased the formation of reactive oxygen species (ROS). Intracellular scavengers of superoxide anions (MnTMPyP, MnTBAP, PEG-SOD) prevented the RWPs-induced formation of ROS and apoptosis, while native extracellular superoxide dismutase (SOD) was without effect. In addition, the effect of RWPs on the expression levels of p73, active caspase-3 and UHRF1 was also prevented by MnTMPyP. Thus, these findings indicate that RWPs induce apoptosis in Jurkat cells by a redox-sensitive mechanism involving the intracellular formation of superoxide anions and consequently the up-regulation of p73 and down-regulation of UHRF1.
Assuntos
Apoptose , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Flavonoides/farmacologia , Proteínas Nucleares/metabolismo , Fenóis/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Vinho , Caspase 3/biossíntese , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Humanos , Células Jurkat/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Oxirredução , Polifenóis , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Proteína Tumoral p73 , Ubiquitina-Proteína Ligases , Regulação para Cima/efeitos dos fármacosRESUMO
Arthritis in the K/BxN mouse model is provoked by pathogenic antibodies (Abs) directed against a ubiquitously expressed protein, glucose-6-phosphate isomerase (GPI). To begin dissecting the repertoire of arthritogenic immunoglobulins (Igs) in the K/BxN model, and to provide a basis for comparison with RA patients we have generated anti-GPI monoclonal Abs (mAbs) from spontaneously activated B cells in the lymphoid organs of arthritic mice. B cell clones with anti-GPI specificities were present at extraordinarily high frequencies in the spleen, and less frequently in other lymphoid organs and in the synovial fluid. None of the anti-GPI mAbs induced arthritis when injected individually into healthy recipients, but most were effective when combined in pairs or larger pools. Arthritogenic combinations depended on mAbs of the IgG1 isotype, which bound to GPI with Kd in the 10(-9) M range, with no indication of cooperative binding between complementing pairs. Pathogenicity was not associated with recognition of a particular epitope, but the ability to form mAb/GPI multimers by simultaneous recognition of different epitopes was clearly required, consistent with the known role of complement and FcRs in this model. Sequence analysis revealed structural similarities amongst the mAbs, indicating that a particular subset of B cells may evade tolerance in K/BxN mice, and that affinity maturation by somatic mutation likely takes place. These results confirm that GPI itself, rather than a cross-reactive molecule, is the target of pathogenic Igs.