RESUMEN
The functional consequence of N6-methyladenosine (m6A) RNA modification is mediated by "reader" proteins of the YTH family. YTH domain-containing 2 (YTHDC2) is essential for mammalian fertility, but its molecular function is poorly understood. Here, we identify U-rich motifs as binding sites of YTHDC2 on 3' UTRs of mouse testicular RNA targets. Although its YTH domain is an m6A-binder in vitro, the YTH point mutant mice are fertile. Significantly, the loss of its 3'â5' RNA helicase activity causes mouse infertility, with the catalytic-dead mutation being dominant negative. Biochemical studies reveal that the weak helicase activity of YTHDC2 is enhanced by its interaction with the 5'â3' exoribonuclease XRN1. Single-cell transcriptomics indicate that Ythdc2 mutant mitotic germ cells transition into meiosis but accumulate a transcriptome with mixed mitotic/meiotic identity that fail to progress further into meiosis. Finally, our demonstration that ythdc2 mutant zebrafish are infertile highlights its conserved role in animal germ cell development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Exorribonucleasas/metabolismo , ARN Helicasas , Pez Cebra , Animales , Fertilidad/genética , Mamíferos/metabolismo , Meiosis , Ratones , ARN/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , Pez Cebra/genéticaRESUMEN
Whole-exome and targeted sequencing of 13 individuals from 10 unrelated families with overlapping clinical manifestations identified loss-of-function and missense variants in KIAA1109 allowing delineation of an autosomal-recessive multi-system syndrome, which we suggest to name Alkuraya-Kucinskas syndrome (MIM 617822). Shared phenotypic features representing the cardinal characteristics of this syndrome combine brain atrophy with clubfoot and arthrogryposis. Affected individuals present with cerebral parenchymal underdevelopment, ranging from major cerebral parenchymal thinning with lissencephalic aspect to moderate parenchymal rarefaction, severe to mild ventriculomegaly, cerebellar hypoplasia with brainstem dysgenesis, and cardiac and ophthalmologic anomalies, such as microphthalmia and cataract. Severe loss-of-function cases were incompatible with life, whereas those individuals with milder missense variants presented with severe global developmental delay, syndactyly of 2nd and 3rd toes, and severe muscle hypotonia resulting in incapacity to stand without support. Consistent with a causative role for KIAA1109 loss-of-function/hypomorphic variants in this syndrome, knockdowns of the zebrafish orthologous gene resulted in embryos with hydrocephaly and abnormally curved notochords and overall body shape, whereas published knockouts of the fruit fly and mouse orthologous genes resulted in lethality or severe neurological defects reminiscent of the probands' features.
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Artrogriposis/genética , Encéfalo/embriología , Mutación/genética , Proteínas/genética , Adolescente , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Niño , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Lactante , Recién Nacido , Imagen por Resonancia Magnética , Masculino , Linaje , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent Tg(itga2b:EGFP) thrombocytes measured. The fga-/- larvae failed to support occlusive venous thrombosis and showed reduced thrombocyte binding and aggregation at injury sites. The fga+/- larvae were largely unaffected. When genome editing zebrafish to produce fibrinogen Aα R28C, equivalent to the human Aα R35C dysfibrinogenemia mutation, we detected in-frame skipping of exon 2 in the fga mRNA, thereby encoding AαΔ19-56. This mutation is similar to Fibrinogen Montpellier II which causes hypodysfibrinogenemia. Aα+/Δ19-56 fish had prolonged TTO and reduced thrombocyte activity, a dominant effect of the mutation. Finally, we used transgenic expression of fga R28C cDNA in fga knock-down or fga-/- mutants to model thrombosis in dysfibrinogenemia. Aα R28C expression had similar effects on TTO and thrombocyte activity as Aα+/Δ19-56. We conclude that thrombosis assays in larval zebrafish can distinguish between quantitative and qualitative fibrinogen disorder models and may assist in anticipating a thrombotic phenotype of novel fibrinogen mutations.
Asunto(s)
Biomarcadores , Plaquetas/metabolismo , Fibrinógeno/metabolismo , Trombosis de la Vena/sangre , Trombosis de la Vena/etiología , Animales , Secuencia de Bases , Coagulación Sanguínea , Modelos Animales de Enfermedad , Exones , Fibrinógeno/química , Fibrinógeno/genética , Edición Génica , Expresión Génica , Plásmidos/genética , Activación Plaquetaria , ARN Guía de Kinetoplastida , Eliminación de Secuencia , Trombosis de la Vena/diagnóstico , Pez CebraRESUMEN
Fibrinogen is an abundant protein synthesized in the liver, present in human blood plasma at concentrations ranging from 1.5-4 g/L in healthy individuals with a normal half-life of 3-5 days. With fibrin, produced by thrombin-mediated cleavage, fibrinogen plays important roles in many physiological processes. Indeed, the formation of a stable blood clot, containing polymerized and cross-linked fibrin, is crucial to prevent blood loss and drive wound healing upon vascular injury. A balance between clotting, notably the conversion of fibrinogen to fibrin, and fibrinolysis, the proteolytic degradation of the fibrin mesh, is essential. Disruption of this equilibrium can cause disease in distinct manners. While some pathological conditions are the consequence of altered levels of fibrinogen, others are related to structural properties of the molecule. The source of fibrinogen expression and the localization of fibrin(ogen) protein also have clinical implications. Low levels of fibrinogen expression have been detected in extra-hepatic tissues, including carcinomas, potentially contributing to disease. Fibrin(ogen) deposits at aberrant sites including the central nervous system or kidney, can also be pathological. In this review, we discuss disorders in which fibrinogen and fibrin are implicated, highlighting mechanisms that may contribute to disease.
Asunto(s)
Fibrina , Fibrinógeno , Humanos , TrombinaRESUMEN
Circulating microRNA (miRNA) expression profiles correlate with platelet reactivity. MiR-126 is a promising candidates in this regard. We generated a transgenic zebrafish line with thrombocyte-specific overexpression of miR-126. Laser injury of the posterior cardinal vein of 5 day-old larvae was performed with or without antithrombotic pre-treatment. Platelet-like structures (PLS) derived from human megakaryocytes transfected with miR-126 were also evaluated for procoagulant activity. Finally, we studied the correlation between miR-126 level and thrombin generation markers in a cohort of stable cardiovascular patients. Control zebrafish developed small thrombocyte-rich thrombi at the site of vessel injury, without vessel occlusion. The miR-126 transgenic line developed an occluding thrombus in 75% (95% CI: 51-91%) of larvae. Pre-treatment with the direct thrombin inhibitor argatroban, but not aspirin, prevented vessel occlusion in the transgenic line (0% occlusion, 95%CI: 0-18%). Upon activation, human PLS showed an increased procoagulant profile after miR-126 transfection compared to control. Finally, the plasma levels of miR-126, but not a control platelet-derived miRNA, correlated with markers of in vivo thrombin generation in a cohort of 185 cardiovascular patients. Our results from three complementary approaches support a key role for miR-126 in platelet-supported thrombin generation and open new avenues in the tailoring of antithrombotic treatment.
Asunto(s)
Plaquetas/metabolismo , MicroARNs/sangre , Trombina/metabolismo , Animales , Humanos , MicroARNs/genética , Trombina/genética , Pez CebraRESUMEN
GNB5 encodes the G protein ß subunit 5 and is involved in inhibitory G protein signaling. Here, we report mutations in GNB5 that are associated with heart-rate disturbance, eye disease, intellectual disability, gastric problems, hypotonia, and seizures in nine individuals from six families. We observed an association between the nature of the variants and clinical severity; individuals with loss-of-function alleles had more severe symptoms, including substantial developmental delay, speech defects, severe hypotonia, pathological gastro-esophageal reflux, retinal disease, and sinus-node dysfunction, whereas related heterozygotes harboring missense variants presented with a clinically milder phenotype. Zebrafish gnb5 knockouts recapitulated the phenotypic spectrum of affected individuals, including cardiac, neurological, and ophthalmological abnormalities, supporting a direct role of GNB5 in the control of heart rate, hypotonia, and vision.
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Bradicardia/genética , Bradicardia/fisiopatología , Discapacidades del Desarrollo/genética , Subunidades beta de la Proteína de Unión al GTP/genética , Genes Recesivos/genética , Mutación/genética , Nodo Sinoatrial/fisiopatología , Adolescente , Animales , Niño , Discapacidades del Desarrollo/fisiopatología , Femenino , Subunidades beta de la Proteína de Unión al GTP/deficiencia , Reflujo Gastroesofágico/genética , Reflujo Gastroesofágico/fisiopatología , Eliminación de Gen , Frecuencia Cardíaca/genética , Heterocigoto , Humanos , Masculino , Hipotonía Muscular/genética , Mutación Missense/genética , Linaje , Fenotipo , Enfermedades de la Retina/genética , Enfermedades de la Retina/fisiopatología , Convulsiones/genética , Síndrome , Adulto Joven , Pez Cebra/genética , Pez Cebra/fisiología , Proteínas de Pez CebraRESUMEN
We report five individuals with loss-of-function of the X-linked AMMECR1: a girl with a balanced X-autosome translocation and inactivation of the normal X-chromosome; two boys with maternally inherited and de novo nonsense variants; and two half-brothers with maternally inherited microdeletion variants. They present with short stature, cardiac and skeletal abnormalities, and hearing loss. Variants of unknown significance in AMMECR1 in four male patients from two families with partially overlapping phenotypes were previously reported. AMMECR1 is coexpressed with genes implicated in cell cycle regulation, five of which were previously associated with growth and bone alterations. Our knockdown of the zebrafish orthologous gene resulted in phenotypes reminiscent of patients' features. The increased transcript and encoded protein levels of AMMECR1L, an AMMECR1 paralog, in the t(X;9) patient's cells indicate a possible partial compensatory mechanism. AMMECR1 and AMMECR1L proteins dimerize and localize to the nucleus as suggested by their nucleic acid-binding RAGNYA folds. Our results suggest that AMMECR1 is potentially involved in cell cycle control and linked to a new syndrome with growth, bone, heart, and kidney alterations with or without elliptocytosis.
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Huesos/fisiología , Corazón/fisiología , Proteínas/genética , Animales , Western Blotting , Huesos/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular , Exoma/genética , Femenino , Células HeLa , Humanos , Masculino , Secuenciación Completa del Genoma , Pez CebraRESUMEN
In mammals, embryonic hematopoiesis occurs in successive waves, culminating with the emergence of hematopoietic stem cells (HSCs) in the aorta. HSCs first migrate to the fetal liver (FL), where they expand, before they seed the bone marrow niche, where they will sustain hematopoiesis throughout adulthood. In zebrafish, HSCs emerge from the dorsal aorta and colonize the caudal hematopoietic tissue (CHT). Recent studies showed that they interact with endothelial cells (ECs), where they expand, before they reach their ultimate niche, the kidney marrow. We identified tfec, a transcription factor from the mitf family, which is highly enriched in caudal endothelial cells (cECs) at the time of HSC colonization in the CHT. Gain-of-function assays indicate that tfec is capable of expanding HSC-derived hematopoiesis in a non-cell-autonomous fashion. Furthermore, tfec mutants (generated by CRISPR/Cas9) showed reduced hematopoiesis in the CHT, leading to anemia. Tfec mediates these changes by increasing the expression of several cytokines in cECs from the CHT niche. Among these, we found kitlgb, which could rescue the loss of HSCs observed in tfec mutants. We conclude that tfec plays an important role in the niche to expand hematopoietic progenitors through the modulation of several cytokines. The full comprehension of the mechanisms induced by tfec will represent an important milestone toward the expansion of HSCs for regenerative purposes.
Asunto(s)
Animales Modificados Genéticamente/embriología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Embrión no Mamífero/citología , Desarrollo Embrionario/fisiología , Células Madre Hematopoyéticas/citología , Nicho de Células Madre/fisiología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Citocinas/genética , Citocinas/metabolismo , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Mutations in the human fibrinogen genes can lead to the absence of circulating fibrinogen and cause congenital afibrinogenemia. This rare bleeding disorder is associated with a variable phenotype, which may be influenced by environment and genotype. Here, we present a zebrafish model of afibrinogenemia. We introduced targeted mutations into the zebrafish fga gene using zinc finger nuclease technology. Animals carrying 3 distinct frameshift mutations in fga were raised and bred to produce homozygous mutants. Using a panel of anti-zebrafish fibrinogen antibodies, fibrinogen was undetectable in plasma preparations from homozygous mutant fish. We observed hemorrhaging in fga mutants and reduced survival compared with control animals. This model will now serve in the search for afibrinogenemia modifying genes or agents and, to our knowledge, is the first transmissible zebrafish model of a defined human bleeding disorder.
Asunto(s)
Afibrinogenemia/congénito , Modelos Animales de Enfermedad , Fibrinógeno/genética , Mutagénesis Sitio-Dirigida , Pez Cebra/genética , Afibrinogenemia/genética , Afibrinogenemia/patología , Animales , Animales Modificados Genéticamente , Hemorragia/genética , Humanos , Larva , FenotipoRESUMEN
The expression of plasma proteins changes dramatically as a result of cytokine induction, particularly interleukin-6, and their levels are used as clinical markers of inflammation. miRNAs are important regulators of gene expression and play significant roles in many inflammatory diseases and processes. The interactions between miRNAs and the genes that they regulate during the acute phase response have not been investigated. We examined the effects of IL-6 stimulation on the transcriptome and miRNome of human and mouse primary hepatocytes and the HepG2 cell line. Using an integrated analysis, we identified differentially expressed miRNAs whose seed sequences are significantly enriched in the 3' untranslated regions of differentially expressed genes, many of which are involved in inflammation-related pathways. Our finding that certain miRNAs may de-repress critical acute phase proteins within acute timeframes has important biological and clinical implications.
Asunto(s)
Hepatocitos/metabolismo , Interleucina-6/farmacología , MicroARNs/metabolismo , ARN Mensajero/metabolismo , Proteínas de Fase Aguda/biosíntesis , Proteínas de Fase Aguda/genética , Animales , Células Cultivadas , Células Hep G2 , Hepatocitos/efectos de los fármacos , Humanos , Ratones , Transcriptoma/efectos de los fármacosRESUMEN
The plasma protein fibrinogen is encoded by 3 structural genes (FGA, FGB, and FGG) that are transcribed to mRNA, spliced, and translated to 3 polypeptide chains (Aα, Bß, and γ, respectively). These chains are targeted for secretion, decorated with post-translational modifications, and assembled into a hexameric "dimer of trimers" (AαBßγ)2. Fully assembled fibrinogen is secreted into the blood as a 340 kDa glycoprotein. Fibrinogen is one of the most prevalent coagulation proteins in blood, and its expression is induced by inflammatory cytokines, wherein circulating fibrinogen levels may increase up to 3-fold during acute inflammatory events. Abnormal levels of circulating fibrinogen are associated with bleeding and thrombotic disorders, as well as several inflammatory diseases. Notably, therapeutic strategies to modulate fibrinogen levels have shown promise in experimental models of disease. Herein, we review pathways mediating fibrinogen synthesis, from gene expression to secretion. Knowledge of these mechanisms may lead to the identification of biomarkers and new therapeutic targets to modulate fibrinogen in health and disease.
RESUMEN
BACKGROUND: Blood clots are primarily composed of red blood cells (RBCs), platelets/thrombocytes, and fibrin. Despite the similarities observed between mammals and zebrafish, the composition of fish thrombi is not as well known. OBJECTIVES: To analyze the formation of zebrafish blood clots ex vivo and arterial and venous thrombi in vivo. METHODS: Transgenic zebrafish lines and laser-mediated endothelial injury were used to determine the relative ratio of RBCs and thrombocytes in clots. Scanning electron and confocal microscopy provided high-resolution images of the structure of adult and larval clots. Adult and larval thrombocyte spreading on fibrinogen was evaluated ex vivo. RESULTS: RBCs were present in arterial and venous thrombi, making up the majority of cells in both circulations. However, bloodless mutant fish demonstrated that fibrin clots can form in vivo in the absence of blood cells. Scanning electron and confocal microscopy showed that larval and adult zebrafish thrombi and mammalian thrombi look surprisingly similar externally and internally, even though the former have nucleated RBCs and thrombocytes. Although adult thrombocytes spread on fibrinogen, we found that larval cells do not fully activate without the addition of plasma from adult fish, suggesting a developmental deficiency of a plasma activating factor. Finally, mutants lacking αIIbß3 demonstrated that this integrin mediates thrombocyte spreading on fibrinogen. CONCLUSION: Our data showed strong conservation of arterial and venous and clot/thrombus formation across species, including developmental regulation of thrombocyte function. This correlation supports the possibility that mammals also do not absolutely require circulating cells to form fibrin clots in vivo.
Asunto(s)
Hemostáticos , Tromboembolia , Trombosis , Animales , Pez Cebra , Trombosis/genética , Plaquetas , Fibrina/química , Fibrinógeno/genética , MamíferosRESUMEN
The plasma concentration of fibrinogen varies in the healthy human population between 1.5 and 3.5 g/L. Understanding the basis of this variability has clinical importance because elevated fibrinogen levels are associated with increased cardiovascular disease risk. To identify novel regulatory elements involved in the control of fibrinogen expression, we used sequence conservation and in silico-predicted regulatory potential to select 14 conserved noncoding sequences (CNCs) within the conserved block of synteny containing the fibrinogen locus. The regulatory potential of each CNC was tested in vitro using a luciferase reporter gene assay in fibrinogen-expressing hepatoma cell lines (HuH7 and HepG2). 4 potential enhancers were tested for their ability to direct enhanced green fluorescent protein expression in zebrafish embryos. CNC12, a sequence equidistant from the human fibrinogen alpha and beta chain genes, activates strong liver enhanced green fluorescent protein expression in injected embryos and their transgenic progeny. A transgenic assay in embryonic day 14.5 mouse embryos confirmed the ability of CNC12 to activate transcription in the liver. While additional experiments are necessary to prove the role of CNC12 in the regulation of fibrinogen, our study reveals a novel regulatory element in the fibrinogen locus that is active in the liver and may contribute to variable fibrinogen expression in humans.
Asunto(s)
Carcinoma Hepatocelular/genética , Elementos de Facilitación Genéticos/genética , Fibrinógeno/genética , Neoplasias Hepáticas/genética , Familia de Multigenes , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Animales Modificados Genéticamente , Carcinoma Hepatocelular/metabolismo , Células Cultivadas , Secuencia Conservada , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Hibridación in Situ , Riñón/citología , Riñón/metabolismo , Neoplasias Hepáticas/metabolismo , Ratones , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
Elevated levels of fibrinogen are associated with increased risk of cardiovascular disease, whereas low fibrinogen can lead to a bleeding disorder. We investigated whether microRNAs (miRNAs), known to act as post-transcriptional regulators of gene expression, regulate fibrinogen production. Using transfection of a library of 470 annotated human miRNA precursor molecules in HuH7 hepatoma cells and quantitative measurements of fibrinogen production, we identified 23 miRNAs with down-regulating (up to 64% decrease) and 4 with up-regulating effects (up to 129% increase) on fibrinogen production. Among the down-regulating miRNAs, we investigated the mechanism of action of 3 hsa-miR-29 family members and hsa-miR-409-3p. Overexpression of hsa-miR-29 members led to decreased steady-state levels of all fibrinogen gene (FGA, FGB, and FGG) transcripts in HuH7 cells. Luciferase reporter gene assays demonstrated that this was independent of miRNA-fibrinogen 3'-untranslated region interactions. In contrast, overexpression of hsa-miR-409-3p specifically lowered fibrinogen Bß mRNA levels, and this effect was dependent on a target site in the fibrinogen Bß mRNA 3'-untranslated region. This study adds to the known mechanisms that control fibrinogen production, points toward a potential cause of variable circulating fibrinogen levels, and demonstrates that a screening approach can identify miRNAs that regulate clinically important proteins.
Asunto(s)
Fibrinógeno/genética , Regulación de la Expresión Génica , MicroARNs/genética , Regiones no Traducidas 3' , Línea Celular , Línea Celular Tumoral , Fibrinógeno/metabolismo , Genes Reporteros , HumanosRESUMEN
SerpinB2 (plasminogen activator inhibitor-2) is widely described as an inhibitor of urokinase plasminogen activator; however, SerpinB2(-/-) mice show no detectable increase in urokinase plasminogen activator activity. In this study, we describe an unexpected immune phenotype in SerpinB2(-/-) mice. After immunization with OVA in CFA, SerpinB2(-/-) mice made approximately 6-fold more IgG2c and generated approximately 2.5-fold more OVA-specific IFN-gamma-secreting T cells than SerpinB2(+/+) littermate controls. In SerpinB2(+/+) mice, high inducible SerpinB2 expression was seen at the injection site and in macrophages low levels in draining lymph nodes and conventional dendritic cells, and no expression was seen in plasmacytoid dendritic, B, T, or NK cells. SerpinB2(-/-) macrophages promoted greater IFN-gamma secretion from wild-type T cells in vivo and in vitro and, when stimulated with anti-CD40/IFN-gamma or cultured with wild-type T cells in vitro, secreted more Th1-promoting cytokines than macrophages from littermate controls. Draining lymph node SerpinB2(-/-) myeloid APCs similarly secreted more Th1-promoting cytokines when cocultured with wild-type T cells. Regulation of Th1 responses thus appears to be a physiological function of inflammation-associated SerpinB2; an observation that may shed light on human inflammatory diseases like pre-eclampsia, lupus, asthma, scleroderma, and periodontitis, which are associated with SerpinB2 polymorphisms or dysregulated SerpinB2 expression.
Asunto(s)
Inmunidad Adaptativa/inmunología , Inflamación/inmunología , Inhibidor 2 de Activador Plasminogénico/fisiología , Células TH1/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Western Blotting , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Citometría de Flujo , Inmunización , Inmunoglobulina G/inmunología , Inflamación/fisiopatología , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidor 2 de Activador Plasminogénico/genética , Inhibidor 2 de Activador Plasminogénico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células TH1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Many malignant tissues, including human papilloma virus (HPV)-associated cancers, express SerpinB2, also known as plasminogen activator inhibitor type-2 (PAI-2). Whether SerpinB2 is expressed by the HPV-transformed cancer cells, and if so, whether SerpinB2 is mutated or behaves aberrantly remains unclear. Here we show that HPV-transformed CaSki cells express high levels of constitutive wild-type SerpinB2, with cellular distribution, glycosylation, secretion, cleavage, induction and urokinase binding similar to that reported for primary cells. Neutralization of secreted SerpinB2 failed to affect CaSki cell migration or growth. Lentivirus-based over-expression of SerpinB2 also had no effect on growth, and we were unable to confirm a role for SerpinB2 in binding or regulating expression of the retinoblastoma protein. CaSki cells thus emerge as a useful tool for studying SerpinB2, with the physiological function of SerpinB2 expression by tumor cells remaining controversial. Using CaSki cells as a source of endogenous SerpinB2, we confirmed that SerpinB2 efficiently binds the proteasomal subunit member ß1.
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Neoplasias/metabolismo , Papillomaviridae , Inhibidor 2 de Activador Plasminogénico/análisis , Línea Celular Transformada , Movimiento Celular , Proliferación Celular , Transformación Celular Viral , Humanos , Proteínas de Neoplasias , Neoplasias/patología , Neoplasias/virología , Inhibidor 2 de Activador Plasminogénico/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteína de Retinoblastoma/metabolismoRESUMEN
Many patients with anemia fail to respond to treatment with erythropoietin (Epo), a commonly used hormone that stimulates erythroid progenitor production and maturation by human BM or by murine spleen. The protein product of growth arrest-specific gene 6 (Gas6) is important for cell survival across several cell types, but its precise physiological role remains largely enigmatic. Here, we report that murine erythroblasts released Gas6 in response to Epo and that Gas6 enhanced Epo receptor signaling by activating the serine-threonine kinase Akt in these cells. In the absence of Gas6, erythroid progenitors and erythroblasts were hyporesponsive to the survival activity of Epo and failed to restore hematocrit levels in response to anemia. In addition, Gas6 may influence erythropoiesis via paracrine erythroblast-independent mechanisms involving macrophages. When mice with acute anemia were treated with Gas6, the protein normalized hematocrit levels without causing undesired erythrocytosis. In a transgenic mouse model of chronic anemia caused by insufficient Epo production, Gas6 synergized with Epo in restoring hematocrit levels. These findings may have implications for the treatment of patients with anemia who fail to adequately respond to Epo.
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Anemia/tratamiento farmacológico , Anemia/genética , Eritropoyesis/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/uso terapéutico , Animales , Adhesión Celular/genética , Supervivencia Celular , Modelos Animales de Enfermedad , Resistencia a Medicamentos , Eritroblastos/efectos de los fármacos , Eritroblastos/metabolismo , Eritropoyetina/genética , Eritropoyetina/farmacología , Eritropoyetina/uso terapéutico , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Mutantes , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Eritropoyetina/agonistas , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor AxlRESUMEN
Thrombosis is a leading cause of morbidity and mortality. Fibrinogen, the soluble substrate for fibrin-based clotting, has a central role in haemostasis and thrombosis and its plasma concentration correlates with cardiovascular disease event risk and a prothrombotic state in experimental models. We aimed to identify chemical entities capable of changing fibrinogen production and test their impact on experimental thrombosis. A total of 1,280 bioactive compounds were screened for their ability to alter fibrinogen production by hepatocyte-derived cancer cells and a selected panel was tested in zebrafish larvae. Anthralin and all-trans retinoic acid (RA) were identified as fibrinogen-lowering and fibrinogen-increasing moieties, respectively. In zebrafish larvae, anthralin prolonged laser-induced venous- occlusion times and reduced thrombocyte accumulation at injury sites. RA had opposite effects. Treatment with RA, a nuclear receptor ligand, increased fibrinogen mRNA levels. Using an antisense morpholino oligonucleotide to deplete zebrafish fibrinogen, we correlated a shortening of laser-induced venous thrombosis times with RA treatment and fibrinogen protein levels. Anthralin had little effect on fibrinogen mRNA in zebrafish larvae, despite leading to lower detectable fibrinogen. Therefore, we made a proteomic scan of anthralin-treated cells and larvae. A reduced representation of proteins linked to the canonical secretory pathway was detected, suggesting that anthralin affects protein secretion. In summary, we found that chemical modulation of fibrinogen levels correlates with measured effects on experimental venous thrombosis and could be investigated as a therapeutic avenue for thrombosis prevention.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Fibrinógeno/metabolismo , Fibrinolíticos/farmacología , Trombosis de la Vena/tratamiento farmacológico , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Antralina/farmacología , Modelos Animales de Enfermedad , Fibrinógeno/genética , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Integrina alfa2/genética , Integrina alfa2/metabolismo , Morfolinos/farmacología , Mutación , Oligonucleótidos Antisentido/farmacología , Proteómica , Bibliotecas de Moléculas Pequeñas , Tretinoina/farmacología , Trombosis de la Vena/genética , Trombosis de la Vena/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
MicroRNAs (miRNAs) are small noncoding RNAs modulating protein production. They are key players in regulation of cell function and are considered as biomarkers in several diseases. The identification of the proteins they regulate, and their impact on cell physiology, may delineate their role as diagnostic or prognostic markers and identify new therapeutic strategies. During the last 3 decades, development of a large panel of techniques has given rise to multiple models dedicated to the study of miRNAs. Since plasma samples are easily accessible, circulating miRNAs can be studied in clinical trials. To quantify miRNAs in numerous plasma samples, the choice of extraction and purification techniques, as well as normalization procedures, are important for comparisons of miRNA levels in populations and over time. Recent advances in bioinformatics provide tools to identify putative miRNAs targets that can then be validated with dedicated assays. In vitro and in vivo approaches aim to functionally validate candidate miRNAs from correlations and to understand their impact on cellular processes. This review describes the advantages and pitfalls of the available techniques for translational research to study miRNAs with a focus on their role in regulating platelet reactivity.