RESUMO
Thrombopoietin (Tpo), which binds to its specific receptor, the Mpl protein, is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Tpo binding to Mpl triggers activation of Janus kinase 2 (Jak2) and phosphorylation of the receptor, as well as activation of several intracellular signalling cascades that mediate cellular responses. Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation required for regulation of major Tpo-stimulated signalling pathways: Mpl-Y565, Mpl-Y599 and Mpl-Y604. Here, we have introduced mutations in the mouse germline and report a consistent physiological requirement for Mpl-Y599, mutation of which resulted in thrombocytopenia, deficient megakaryopoiesis, low hematopoietic stem cell (HSC) number and function, and attenuated responses to myelosuppression. We further show that in models of myeloproliferative neoplasms (MPN), where Mpl is required for pathogenesis, thrombocytosis was dependent on intact Mpl-Y599. In contrast, Mpl-Y565 was required for negative regulation of Tpo responses; mutation of this residue resulted in excess megakaryopoiesis at steady-state and in response to myelosuppression, and exacerbated thrombocytosis associated with MPN.
Assuntos
Hematopoese , Transtornos Mieloproliferativos , Receptores de Trombopoetina , Trombopoetina , Tirosina , Animais , Receptores de Trombopoetina/metabolismo , Receptores de Trombopoetina/genética , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Camundongos , Trombopoetina/metabolismo , Tirosina/metabolismo , Tirosina/genética , Fosforilação , Camundongos Endogâmicos C57BL , Células-Tronco Hematopoéticas/metabolismo , Transdução de Sinais , Mutação , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Trombopoese/genéticaRESUMO
Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-ß induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.
Assuntos
Apoptose , Proteínas Quinases , Humanos , Animais , Camundongos , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Membrana Celular/metabolismo , Mutação , Fatores de Transcrição/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Necroptosis is a pro-inflammatory cell death program executed by the terminal effector, mixed lineage kinase domain-like (MLKL). Previous studies suggested a role for the necroptotic machinery in platelets, where loss of MLKL or its upstream regulator, RIPK3 kinase, impacted thrombosis and haemostasis. However, it remains unknown whether necroptosis operates within megakaryocytes, the progenitors of platelets, and whether necroptotic cell death might contribute to or diminish platelet production. Here, we demonstrate that megakaryocytes possess a functional necroptosis signalling cascade. Necroptosis activation leads to phosphorylation of MLKL, loss of viability and cell swelling. Analyses at steady state and post antibody-mediated thrombocytopenia revealed that platelet production was normal in the absence of MLKL, however, platelet activation and haemostasis were impaired with prolonged tail re-bleeding times. We conclude that MLKL plays a role in regulating platelet function and haemostasis and that necroptosis signalling in megakaryocytes is dispensable for platelet production.
Assuntos
Plaquetas/metabolismo , Morte Celular/fisiologia , Megacariócitos/metabolismo , Necroptose/fisiologia , Animais , Humanos , CamundongosRESUMO
MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Sistema Hematopoético/patologia , Necroptose/genética , Proteínas Quinases/genética , Animais , Animais Recém-Nascidos , Doenças Hereditárias Autoinflamatórias , Humanos , Inflamação/genética , Camundongos , Mutação de Sentido Incorreto , Osteomielite/genética , Proteínas Quinases/metabolismoRESUMO
During haematopoiesis, haematopoietic stem cells differentiate into restricted potential progenitors before maturing into the many lineages required for oxygen transport, wound healing and immune response. We have updated Haemopedia, a database of gene-expression profiles from a broad spectrum of haematopoietic cells, to include RNA-seq gene-expression data from both mice and humans. The Haemopedia RNA-seq data set covers a wide range of lineages and progenitors, with 57 mouse blood cell types (flow sorted populations from healthy mice) and 12 human blood cell types. This data set has been made accessible for exploration and analysis, to researchers and clinicians with limited bioinformatics experience, on our online portal Haemosphere: https://www.haemosphere.org. Haemosphere also includes nine other publicly available high-quality data sets relevant to haematopoiesis. We have added the ability to compare gene expression across data sets and species by curating data sets with shared lineage designations or to view expression gene vs gene, with all plots available for download by the user.
Assuntos
Bases de Dados Genéticas , Expressão Gênica/genética , Hematopoese/genética , Transcriptoma/genética , Animais , Biologia Computacional , Células-Tronco Hematopoéticas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/tendências , Humanos , Camundongos , RNA-Seq , SoftwareRESUMO
The circulating life span of blood platelets is regulated by the prosurvival protein BCL-XL It restrains the activity of BAK and BAX, the essential prodeath mediators of intrinsic apoptosis. Disabling the platelet intrinsic apoptotic pathway in mice by deleting BAK and BAX results in a doubling of platelet life span and concomitant thrombocytosis. Apoptotic platelets expose phosphatidylserine (PS) via a mechanism that is distinct from that driven by classical agonists. Whether there is any role for apoptotic PS in platelet function in vivo, however, is unclear. Apoptosis has also been associated with the platelet storage lesion (PSL), the constellation of biochemical deteriorations that occur during blood bank storage. In this study, we investigated the role of BAK/BAX-mediated apoptosis in hemostasis and thrombosis and in the development of the PSL. We show that although intrinsic apoptosis is rapidly induced during storage at 37°C, it is not detected when platelets are kept at the standard storage temperature of 22°C. Remarkably, loss of BAK and BAX did not prevent the development of the PSL at either temperature. BAK/BAX-deficient mice exhibited increased bleeding times and unstable thrombus formation. This phenotype was not caused by impaired PS exposure, but was associated with a defect in granule release from aged platelets. Strikingly, rejuvenation of BAK/BAX-deficient platelets in vivo completely rescued the observed hemostatic defects. Thus, apoptotic culling of old platelets from the bloodstream is essential to maintain a functional, hemostatically reactive platelet population. Inhibiting intrinsic apoptosis in blood banked platelets is unlikely to yield significant benefit.
Assuntos
Apoptose , Plaquetas/metabolismo , Suscetibilidade a Doenças , Animais , Apoptose/genética , Biomarcadores , Tempo de Sangramento , Contagem de Células Sanguíneas , Coagulação Sanguínea , Caspases/metabolismo , Sobrevivência Celular/genética , Feminino , Genótipo , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.
Assuntos
Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Biologia Computacional , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Análise por Conglomerados , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Humanos , Camundongos , NavegadorRESUMO
Polycomb repressive complex 2 (PRC2) is a chromatin modifier that regulates stem cells in embryonic and adult tissues. Loss-of-function studies of PRC2 components have been complicated by early embryonic dependence on PRC2 activity and the partial functional redundancy of enhancer of zeste homolog 1 (Ezh1) and enhancer of zeste homolog 2 (Ezh2), which encode the enzymatic component of PRC2. Here, we investigated the role of PRC2 in hematopoiesis by conditional deletion of suppressor of zeste 12 protein homolog (Suz12), a core component of PRC2. Complete loss of Suz12 resulted in failure of hematopoiesis, both in the embryo and the adult, with a loss of maintenance of hematopoietic stem cells (HSCs). In contrast, partial loss of PRC2 enhanced HSC self-renewal. Although Suz12 was required for lymphoid development, deletion in individual blood cell lineages revealed that it was dispensable for the development of granulocytic, monocytic, and megakaryocytic cells. Collectively, these data reveal the multifaceted role of PRC2 in hematopoiesis, with divergent dose-dependent effects in HSC and distinct roles in maturing blood cells. Because PRC2 is a potential target for cancer therapy, the significant consequences of modest changes in PRC2 activity, as well as the cell and developmental stage-specific effects, will need to be carefully considered in any therapeutic context.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Linfopoese/genética , Complexo Repressor Polycomb 2/fisiologia , Animais , Proliferação de Células/genética , Células Cultivadas , Feto/imunologia , Feto/fisiologia , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexo Repressor Polycomb 2/genéticaRESUMO
Down syndrome (DS), with trisomy of chromosome 21 (HSA21), is the commonest human aneuploidy. Pre-leukemic myeloproliferative changes in DS foetal livers precede the acquisition of GATA1 mutations, transient myeloproliferative disorder (DS-TMD) and acute megakaryocytic leukemia (DS-AMKL). Trisomy of the Erg gene is required for myeloproliferation in the Ts(1716)65Dn DS mouse model. We demonstrate here that genetic changes specifically attributable to trisomy of Erg lead to lineage priming of primitive and early multipotential progenitor cells in Ts(1716)65Dn mice, excess megakaryocyte-erythroid progenitors, and malignant myeloproliferation. Gene expression changes dependent on trisomy of Erg in Ts(1716)65Dn multilineage progenitor cells were correlated with those associated with trisomy of HSA21 in human DS hematopoietic stem and primitive progenitor cells. These data suggest a role for ERG as a regulator of hematopoietic lineage potential, and that trisomy of ERG in the context of DS foetal liver hemopoiesis drives the pre-leukemic changes that predispose to subsequent DS-TMD and DS-AMKL.
Assuntos
Cromossomos Humanos Par 21/genética , Síndrome de Down/genética , Proteínas Oncogênicas/genética , Células-Tronco/citologia , Transativadores/genética , Fatores de Transcrição/genética , Trissomia , ADP-Ribosil Ciclase 1/metabolismo , Alelos , Animais , Antígenos CD34/metabolismo , Linhagem da Célula , Proliferação de Células , Modelos Animais de Doenças , Células Eritroides/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genótipo , Hematopoese/genética , Sistema Hematopoético/citologia , Sistema Hematopoético/metabolismo , Humanos , Megacariócitos/metabolismo , Camundongos , Camundongos Knockout , Análise em Microsséries , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Análise de Sequência de RNA , Células-Tronco/metabolismo , Regulador Transcricional ERG , TranscriptomaRESUMO
Thrombopoietin (TPO) acting via its receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO-dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (Mpl(PF4cre/PF4cre)). Mpl(PF4cre/PF4cre) mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO overstimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool.
Assuntos
Plaquetas/metabolismo , Megacariócitos/metabolismo , Células Mieloides/citologia , Receptores de Trombopoetina/metabolismo , Trombopoese , Animais , Antígenos CD34/metabolismo , Plaquetas/citologia , Compartimento Celular , Proliferação de Células , Células Clonais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Marcação de Genes , Loci Gênicos/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Integrases/metabolismo , Megacariócitos/citologia , Camundongos , Modelos Biológicos , Células Mieloides/metabolismo , Trombocitose , Trombopoetina/genética , Trombopoetina/metabolismo , Transcrição GênicaRESUMO
Sec1/Munc18 proteins facilitate the formation of trans-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes that mediate fusion of secretory granule (SG) with plasma membrane (PM). The capacity of pancreatic ß-cells to exocytose insulin becomes compromised in diabetes. ß-Cells express three Munc18 isoforms of which the role of Munc18b is unknown. We found that Munc18b depletion in rat islets disabled SNARE complex formation formed by syntaxin (Syn)-2 and Syn-3. Two-photon imaging analysis revealed in Munc18b-depleted ß-cells a 40% reduction in primary exocytosis (SG-PM fusion) and abrogation of almost all sequential SG-SG fusion, together accounting for a 50% reduction in glucose-stimulated insulin secretion (GSIS). In contrast, gain-of-function expression of Munc18b wild-type and, more so, dominant-positive K314L/R315L mutant promoted the assembly of cognate SNARE complexes, which caused potentiation of biphasic GSIS. We found that this was attributed to a more than threefold enhancement of both primary exocytosis and sequential SG-SG fusion, including long-chain fusion (6-8 SGs) not normally (2-3 SG fusion) observed. Thus, Munc18b-mediated exocytosis may be deployed to increase secretory efficiency of SGs in deeper cytosolic layers of ß-cells as well as additional primary exocytosis, which may open new avenues of therapy development for diabetes.
Assuntos
Exocitose/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Munc18/metabolismo , Vesículas Secretórias/metabolismo , Animais , Secreção de Insulina , Masculino , Proteínas Munc18/genética , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Ratos , Ratos Sprague-Dawley , Sintaxina 1/genética , Sintaxina 1/metabolismoRESUMO
Hematopoietic progenitor cells are the progeny of hematopoietic stem cells that coordinate the production of precise numbers of mature blood cells of diverse functional lineages. Identification of cell-surface antigen expression associated with hematopoietic lineage restriction has allowed prospective isolation of progenitor cells with defined hematopoietic potential. To clarify further the cellular origins of megakaryocyte commitment, we assessed the in vitro and in vivo megakaryocyte and platelet potential of defined progenitor populations in the adult mouse bone marrow. We show that megakaryocytes arise from CD150(+) bipotential progenitors that display both platelet- and erythrocyte-producing potential in vivo and that can develop from the Flt3(-) fraction of the pregranulocyte-macrophage population. We define a bipotential erythroid-megakaryocyte progenitor population, the CD150(+)CD9(lo)endoglin(lo) fraction of Lin(-)cKit(+)IL7 receptor alpha(-)FcγRII/III(lo)Sca1(-) cells, which contains the bulk of the megakaryocyte colony-forming capacity of the bone marrow, including bipotential megakaryocyte-erythroid colony-forming capacity, and can generate both erythrocytes and platelets efficiently in vivo. This fraction is distinct from the CD150(+)CD9(hi)endoglin(lo) fraction, which contains bipotential precursors with characteristics of increased megakaryocytic maturation, and the CD150(+)CD9(lo)endoglin(hi) fraction, which contains erythroid lineage-committed cells. Finally, we demonstrate that bipotential erythroid-megakaryocyte progenitor and CD150(+)CD9(hi)endoglin(lo) cells are TPO-responsive and that the latter population specifically expands in the recovery from thrombocytopenia induced by anti-platelet serum.
Assuntos
Células da Medula Óssea/citologia , Eritrócitos/citologia , Megacariócitos/citologia , Células-Tronco/citologia , Trombopoetina/fisiologia , Animais , Antígenos CD/imunologia , Células da Medula Óssea/imunologia , Eritrócitos/imunologia , Megacariócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/imunologiaRESUMO
RATIONALE: The actin cytoskeleton has been implicated in the processing of atherogenic lipoproteins in macrophages. However, the functional role of actin and the regulatory proteins involved are unknown. OBJECTIVE: Coronin-1A (Coro1A) was identified as a differentially expressed transcript in wild-type versus Niemann-Pick type C1 deficient macrophages exposed to acetylated low-density lipoproteins (AcLDL). We investigated whether Coro1A plays a role in the uptake or processing of modified lipoproteins in macrophages and if this is related to its actin regulatory functions. METHODS AND RESULTS: In wild-type primary macrophages, filamentous actin transiently decorated AcLDL containing endosomes that also recruited Coro1A. This dynamic association of F-actin with endosomes was disturbed in Coro1A deficient macrophages. In Coro1A knockout macrophages the uptake of AcLDL was increased, rate of AcLDL delivery to lysosomes enhanced, and lipoprotein-derived cholesteryl ester hydrolysis accelerated. Overexpression of wild-type Coro1A normalized AcLDL uptake in Coro1A knockout macrophages while a Coro1A actin binding mutant did not. Furthermore, the effects of macrophage Coro1A silencing on endosomal actin association and AcLDL delivery to lysosomes resembled those of cofilin silencing. CONCLUSIONS: Coro1A controls actin association with endocytic organelles, thereby negatively regulating endo-lysosomal delivery, degradation of modified lipoproteins and cholesterol deposition in macrophages.
Assuntos
Actinas/metabolismo , Endossomos/metabolismo , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteólise , Animais , Transporte Biológico , Células Cultivadas , Colesterol/metabolismo , Lisossomos/metabolismo , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Modelos Animais , Organelas/metabolismoRESUMO
Diverse mutations in the genes encoding hemoglobin (Hb) have been characterized in human disease. We describe here a mutation in the mouse Hbb-b2 gene, denoted Plt12, that precisely mimics the human hemoglobin Hotel Dieu variant. The mutation results in increased affinity of Hb for oxygen and Plt12 mutant mice exhibited reduced partial pressure of O(2) in the blood, accompanied by erythrocytosis characterized by elevated erythropoietin levels and splenomegaly with excess erythropoiesis. Most homozygous Hbb-b2(Plt12/Plt12) mice succumbed to early lethality associated with emphysema, cardiac abnormalities, and liver degeneration. Survivors displayed a marked thrombocytopenia without significant deficiencies in the numbers of megakaryocytes or megakaryocyte progenitor cells. The lifespan of platelets in the circulation of Hbb-b2(Plt12/Plt12) mice was normal, and splenectomy did not correct the thrombocytopenia, suggesting that increased sequestration was unlikely to be a major contributor. These data, together with the observation that megakaryocytes in Hbb-b2(Plt12/Plt12) mice appeared smaller and deficient in cytoplasm, support a model in which hypoxia causes thrombocytopenia as a consequence of an inability of megakaryocytes, once formed, to properly mature and produce sufficient platelets. The Plt12 mouse is a model of high O(2)-affinity hemoglobinopathy and provides insights into hematopoiesis under conditions of chronic hypoxia.
Assuntos
Hemoglobinas Anormais/genética , Policitemia/genética , Trombocitopenia/genética , Animais , Contagem de Células Sanguíneas , Gasometria , Eritropoese/genética , Eritropoetina/sangue , Meia-Vida , Masculino , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Mutação/genética , Oxigênio/sangue , Policitemia/patologia , Esplenomegalia , Trombocitopenia/patologiaRESUMO
Thrombopoietin (TPO), acting through its receptor Mpl, has two major physiological roles: ensuring production of sufficient platelets via stimulation of megakaryocyte production and maintaining hematopoietic stem cell (HSC) quiescence. Mpl also controls circulating TPO concentration via receptor-mediated internalization and degradation. Here, we demonstrate that the megakaryocytosis and increased platelet mass in mice with mutations in the Myb or p300 genes causes reduced circulating TPO concentration and TPO starvation of the stem-cell compartment, which is exacerbated because these cells additionally exhibit impaired responsiveness to TPO. HSCs from Myb(Plt4/Plt4) mice show altered expression of TPO-responsive genes and, like HSCs from Tpo and Mpl mutant mice, exhibit increased cycling and a decline in the number of HSCs with age. These studies suggest that disorders of platelet number can have profound effects on the HSC compartment via effects on the feedback regulation of circulating TPO concentration.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Animais , Plaquetas/metabolismo , Diferenciação Celular/fisiologia , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Células-Tronco Hematopoéticas/citologia , Megacariócitos/citologia , Megacariócitos/fisiologia , Camundongos , Camundongos Knockout , Análise em Microsséries , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Receptores de Trombopoetina/metabolismo , Trombopoetina/sangueRESUMO
In an N-nitroso-N-ethylurea (ENU) mutagenesis screen using Mpl(-/-) mice, we isolated a semidominant suppressor of thrombocytopenia, termed Plt6. The gene mutated in Plt6 mice encodes the transcriptional coregulator p300, and the mutation, a tyrosine to asparagine substitution at amino acid 630 (Y630N), disrupts the interaction between p300 and c-Myb. Mpl(-/-) p300(Plt6/+) mice displayed elevated platelet counts relative to Mpl(-/-) p300(+/+) controls, whereas mice homozygous for the Plt6 mutation produced supraphysiological levels of circulating platelets. On a wild-type genetic background, mice homozygous for the p300(Plt6) mutation, or recipients of Mpl(+/+) p300(Plt6/Plt6) bone marrow, also exhibited thrombocytosis as well as deficiencies in B-lymphoid cells. Increased platelet numbers in Plt6 mutant mice were accompanied by significant increases in megakaryocyte progenitor cells within the bone marrow and spleen with concomitantly elevated numbers of megakaryocytes. The expansion of megakaryocytopoiesis and suppression of Mpl(-/-) thrombocytopenia in Plt6 mutants is highly reminiscent of that observed in mice with mutations affecting the p300 partner protein c-Myb, suggesting an indispensable repressive role for the c-Myb/p300 transcriptional regulatory complex in megakaryocyte development, the inhibition of which allows substantial thrombopoietin (TPO)-independent platelet production.
Assuntos
Plaquetas/metabolismo , Proteína p300 Associada a E1A/fisiologia , Mutação , Mutação Puntual , Proteínas Proto-Oncogênicas c-myb/fisiologia , Receptores de Trombopoetina/genética , Trombocitopenia/genética , Animais , Sequência de Bases , Proteína p300 Associada a E1A/genética , Homozigoto , Megacariócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-myb/metabolismo , Trombopoetina/metabolismoRESUMO
We identified in a yeast two-hybrid screen the EF-hand Ca(2+)-binding protein Cab45 as an interaction partner of Munc18b. Although the full-length Cab45 resides in Golgi lumen, we characterize a cytosolic splice variant, Cab45b, expressed in pancreatic acini. Cab45b is shown to bind (45)Ca(2+), and, of its three EF-hand motifs, EF-hand 2 is demonstrated to be crucial for the ion binding. Cab45b is shown to interact with Munc18b in an in vitro assay, and this interaction is enhanced in the presence of Ca(2+). In this assay, Cab45b also binds the Munc18a isoform in a Ca(2+)-dependent manner. The endogenous Cab45b in rat acini coimmunoprecipitates with Munc18b, syntaxin 2, and syntaxin 3, soluble N-ethylmaleimide-sensitive factor attachment protein receptors with key roles in the Ca(2+)-triggered zymogen secretion. Furthermore, we show that Munc18b bound to syntaxin 3 recruits Cab45b onto the plasma membrane. Importantly, antibodies against Cab45b are shown to inhibit in a specific and dose-dependent manner the Ca(2+)-induced amylase release from streptolysin-O-permeabilized acini. The present study identifies Cab45b as a novel protein factor involved in the exocytosis of zymogens by pancreatic acini.
Assuntos
Processamento Alternativo/genética , Amilases/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Citosol/metabolismo , Glicoproteínas/metabolismo , Proteínas Munc18/metabolismo , Pâncreas Exócrino/enzimologia , Processamento Alternativo/efeitos dos fármacos , Animais , Anticorpos , Proteínas de Bactérias/farmacologia , Células CHO , Células COS , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Permeabilidade da Membrana Celular/efeitos dos fármacos , Chlorocebus aethiops , Cricetinae , Cricetulus , Citosol/efeitos dos fármacos , Cães , Motivos EF Hand , Perfilação da Expressão Gênica , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Pâncreas Exócrino/efeitos dos fármacos , Pâncreas Exócrino/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Estreptolisinas/farmacologiaRESUMO
An N-ethyl-N-nitrosourea mutagenesis screen in mice was performed to isolate regulators of circulating platelet number. We report here recessive thrombocytopenia and kidney disease in plt1 mice, which is the result of a severe but partial loss-of-function mutation in the gene encoding glycoprotein-N-acetylgalactosamine-3-beta-galactosyltransferase (C1GalT1), an enzyme essential for the synthesis of extended mucin-type O-glycans. Platelet half-life and basic hemostatic parameters were unaffected in plt1/plt1 mice, and the thrombocytopenia and kidney disease were not attenuated on a lymphocyte-deficient rag1-null background. gpIbalpha and podocalyxin were found to be major underglycosylated proteins in plt1/plt1 platelets and the kidney, respectively, implying that these are key targets for C1GalT1, appropriate glycosylation of which is essential for platelet production and kidney function. Compromised C1GalT1 activity has been associated with immune-mediated diseases in humans, most notably Tn syndrome and IgA nephropathy. The disease in plt1/plt1 mice suggests that, in addition to immune-mediated effects, intrinsic C1Gal-T1 deficiency in megakaryocytes and the kidney may contribute to pathology.
Assuntos
Galactosiltransferases/metabolismo , Nefropatias/metabolismo , Trombocitopenia/metabolismo , Animais , Plaquetas/metabolismo , Plaquetas/patologia , Linhagem Celular , Proliferação de Células , Feminino , Galactosiltransferases/genética , Glicosilação , Humanos , Nefropatias/genética , Nefropatias/patologia , Masculino , Camundongos , Mutação/genética , Taxa de Sobrevida , Trombocitopenia/genética , Trombocitopenia/patologiaRESUMO
Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disorder of the childhood caused by mutations in the gene encoding palmitoyl protein thioesterase 1 (PPT1). PPT1 localizes to late endosomes/lysosomes of non-neuronal cells and in neurons also to presynaptic areas. PPT1-deficiency causes massive death of cortical neurons and most tissues show an accumulation of saposins A and D. We have here studied endocytic pathways, saposin localization and processing in PPT1-deficient fibroblasts to elucidate the cellular defects resulting in accumulation of specific saposins. We show that PPT1-deficiency causes a defect in fluid-phase and receptor-mediated endocytosis, whereas marker uptake and recycling endocytosis remain intact. Furthermore, we show that saposins A and D are more abundant and relocalized in PPT-deficient fibroblasts and mouse primary neurons. Metabolic labeling and immunoprecipitation analyses revealed hypersecretion and abnormal processing of prosaposin, implying that the accumulation of saposins may result from endocytic defects. We show for the first time a connection between saposin storage and a defect in the endocytic pathway of INCL cells. These data provide new insights into the metabolism of PPT1-deficient cells and offer a basis for further studies on cellular processes causing neuronal death in INCL and other neurodegenerative diseases.
Assuntos
Endocitose/fisiologia , Saponinas/metabolismo , Tioléster Hidrolases/deficiência , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Dextranos/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Lactente , Lipoproteínas LDL/metabolismo , Macrolídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Saposinas/metabolismo , Soroalbumina Bovina/metabolismo , Tioléster Hidrolases/genética , Transferrina/metabolismoRESUMO
Munc18b is a mammalian Sec1-related protein that is abundant in epithelial cells and regulates vesicle transport to the apical plasma membrane. We constructed a homology model of Munc18b in complex with syntaxin 3 based on the crystal structure of the neuronal Sec1.syntaxin 1A complex. In this model we identified all residues in the interface between the two proteins that contribute directly to the interaction and mutagenized residues in Munc18b to alter its binding to syntaxins 1A, 2, and 3. The syntaxin-binding properties of the mutants were tested using an in vitro assay and by a co-immunoprecipitation approach employing Munc18b expressed in CHO-K1 cells. Three Munc18b variants, W28S, S42K, and E59K, were generated that are defective in binding to all three syntaxins. A fourth mutant protein, S48D, shows abolishment of syntaxin 3 interaction but binds syntaxin 2 at normal and syntaxin 1A at mildly reduced efficiency. Over-expression of Munc18b S48D inhibited transport of influenza hemagglutinin to the apical surface of Madin-Darby canine kidney II cells, which express syntaxin 2 abundantly, but not of Caco-2 cells, in which syntaxin 3 is the major apical target SNARE (soluble NSF (N-ethylmaleimide sensitive factor) attachment protein receptors). This suggests that, although syntaxin 3 is the main target SNARE operating in exocytic transport to the apical plasma membrane in certain epithelial cell types, syntaxin 2 may play an important role in this trafficking route in others.