RESUMO
Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-ß. In vivo, this precipitates an elevation in IFN-ß levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.
Assuntos
Apoptose , Caspases/metabolismo , Interferon Tipo I/metabolismo , Transdução de Sinais , Animais , Caspase 9/genética , Caspase 9/metabolismo , Caspases/classificação , Cruzamentos Genéticos , DNA Mitocondrial/metabolismo , Feminino , Células-Tronco Hematopoéticas/metabolismo , Interferon Tipo I/imunologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BLRESUMO
cGAS-STING signalling is induced by detection of foreign or mislocalised host double-stranded (ds)DNA within the cytosol. STING acts as the major signalling hub, where it controls production of type I interferons and inflammatory cytokines. Basally, STING resides on the ER membrane. Following activation STING traffics to the Golgi to initiate downstream signalling and subsequently to endolysosomal compartments for degradation and termination of signalling. While STING is known to be degraded within lysosomes, the mechanisms controlling its delivery remain poorly defined. Here we utilised a proteomics-based approach to assess phosphorylation changes in primary murine macrophages following STING activation. This identified numerous phosphorylation events in proteins involved in intracellular and vesicular transport. We utilised high-temporal microscopy to track STING vesicular transport in live macrophages. We subsequently identified that the endosomal complexes required for transport (ESCRT) pathway detects ubiquitinated STING on vesicles, which facilitates the degradation of STING in murine macrophages. Disruption of ESCRT functionality greatly enhanced STING signalling and cytokine production, thus characterising a mechanism controlling effective termination of STING signalling.
Assuntos
Imunidade Inata , Proteínas de Membrana , Camundongos , Animais , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Macrófagos/metabolismo , Nucleotidiltransferases/metabolismo , DNA , Complexos Endossomais de Distribuição Requeridos para Transporte/genéticaRESUMO
Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.
Assuntos
Apoptose/genética , Plaquetas/citologia , Linfócitos T/citologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Linhagem Celular , Sobrevivência Celular/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genéticaRESUMO
Dysregulated platelet functions contribute to the development and progression of ischemic stroke. Utilizing mice with a platelet-specific deletion of cyclophilin D (CypD), a mediator of necrosis, we found that platelet necrosis regulates tissue damage and outcomes during ischemic stroke in vivo. Mice with loss of CypD in platelets (CypDplt-/-mice) exhibited significantly enhanced cerebral blood flow, improved neurological and motor functions, and reduced ischemic stroke infarct volume after cerebral ischemia-reperfusion injury. These effects were attributable, at least in part, to platelet-neutrophil interactions. Twenty-four hours after stroke, significantly more circulating platelet-neutrophil aggregates (PNAs) were found in CypDplt+/+ mice. Underscoring the role of platelet necrosis in PNA formation, we observed a significant number of phosphatidylserine (PS)+ platelets in PNAs in CypDplt+/+ mice. In contrast, significantly fewer platelets in PNAs were PS+ in CypDplt-/- counterparts. Accordingly, mice with CypD-deficient platelets had fewer neutrophils and PNAs recruited to their brain following stroke relative to wild-type counterparts. Neutrophil depletion in wild-type mice conferred protection from ischemic stroke to a similar degree as observed in mice with CypD-deficient platelets. Neutrophil depletion in CypDplt-/- mice did not further reduce infarct size. Transmission electron microscopy of ex vivo-formed PNAs revealed a propensity of necrotic platelets to interact with neutrophils. These results suggest that necrotic platelets interact with neutrophils to exacerbate brain injury during ischemic stroke. Because inhibiting platelet necrosis does not compromise hemostasis, targeting platelet CypD may be a potential therapeutic strategy to limit brain damage following ischemic stroke.
Assuntos
Plaquetas/patologia , Encéfalo/patologia , Infarto da Artéria Cerebral Média/patologia , Animais , Encéfalo/irrigação sanguínea , Peptidil-Prolil Isomerase F/genética , Feminino , Deleção de Genes , Infarto da Artéria Cerebral Média/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Necrose/genética , Necrose/patologia , Neutrófilos/patologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologiaRESUMO
Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.
Assuntos
Transformação Celular Neoplásica , Transição Epitelial-Mesenquimal/genética , Histona Desmetilases/metabolismo , Leucemia Mieloide Aguda/patologia , Fatores de Transcrição da Família Snail/fisiologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células HEK293 , Células HL-60 , Histona Desmetilases/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Transgênicos , Ligação Proteica , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismoRESUMO
OBJECTIVE: People with diabetes are at a significantly higher risk of cardiovascular disease, in part, due to accelerated atherosclerosis. Diabetic subjects have increased number of platelets that are activated, more reactive, and respond suboptimally to antiplatelet therapies. We hypothesized that reducing platelet numbers by inducing their premature apoptotic death would decrease atherosclerosis. Approach and Results: This was achieved by targeting the antiapoptotic protein Bcl-xL (B-cell lymphoma-extra large; which is essential for platelet viability) via distinct genetic and pharmacological approaches. In the former, we transplanted bone marrow from mice carrying the Tyr15 to Cys loss of function allele of Bcl-x (known as Bcl-xPlt20) or wild-type littermate controls into atherosclerotic-prone Ldlr+/- mice made diabetic with streptozotocin and fed a Western diet. Reduced Bcl-xL function in hematopoietic cells significantly decreased platelet numbers, exclusive of other hematologic changes. This led to a significant reduction in atherosclerotic lesion formation in Bcl-xPlt20 bone marrow transplanted Ldlr+/- mice. To assess the potential therapeutic relevance of reducing platelets in atherosclerosis, we next targeted Bcl-xL with a pharmacological strategy. This was achieved by low-dose administration of the BH3 (B-cell lymphoma-2 homology domain 3) mimetic, ABT-737 triweekly, in diabetic Apoe-/- mice for the final 6 weeks of a 12-week study. ABT-737 normalized platelet numbers along with platelet and leukocyte activation to that of nondiabetic controls, significantly reducing atherosclerosis while promoting a more stable plaque phenotype. CONCLUSIONS: These studies suggest that selectively reducing circulating platelets, by targeting Bcl-xL to promote platelet apoptosis, can reduce atherosclerosis and lower cardiovascular disease risk in diabetes. Graphic Abstract: A graphic abstract is available for this article.
Assuntos
Aterosclerose/sangue , Aterosclerose/complicações , Plaquetas/patologia , Angiopatias Diabéticas/sangue , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Aterosclerose/prevenção & controle , Compostos de Bifenilo/administração & dosagem , Plaquetas/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/complicações , Feminino , Humanos , Leucócitos/patologia , Leucócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrofenóis/administração & dosagem , Piperazinas/administração & dosagem , Contagem de Plaquetas , Receptores de LDL/deficiência , Receptores de LDL/genética , Fatores de Risco , Sulfonamidas/administração & dosagemRESUMO
The stem cell leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1, and Runx1, mutations of Scl have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, lymphoblastic leukemia 1 (Lyl1) might explain this observation. To determine whether Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking 1 or both factors in megakaryocytes. Conditional Scl knockout (KO) mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 (Pf4) promoter generated megakaryocytes with markedly reduced but not absent Scl These Pf4Sclc-KO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, Pf4Sclc-KO mice generated on an Lyl1-null background (double knockout [DKO] mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation, and markedly impaired platelet aggregation. DKO megakaryocytes, but not single-knockout megakaryocytes, had reduced expression of Gata1, Fli1, Nfe2, and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared Scl and Lyl1 E-box binding sites that were also enriched for Gata1, Ets, and Runx1 motifs. Thus, Scl and Lyl1 share functional roles in platelet production by regulating expression of partner proteins including Gata1. We propose that this functional redundancy provides one explanation for the absence of Scl and Lyl1 mutations in inherited thrombocytopenia.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Plaquetas/fisiologia , Proteínas de Neoplasias/fisiologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T/fisiologia , Trombopoese/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica , Megacariócitos/patologia , Megacariócitos/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Trombocitopenia/sangue , Trombocitopenia/genéticaRESUMO
Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.
Assuntos
Apoptose/fisiologia , Bibliotecas de Moléculas Pequenas/metabolismo , Canal de Ânion 2 Dependente de Voltagem/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Animais , Camundongos , Ligação Proteica , Canal de Ânion 2 Dependente de Voltagem/metabolismoRESUMO
Cytopenias are key prognostic indicators of life-threatening infection, contributing to immunosuppression and mortality. Here we define a role for Caspase-1-dependent death, known as pyroptosis, in infection-induced cytopenias by studying inflammasome activation in hematopoietic progenitor cells. The NLRP1a inflammasome is expressed in hematopoietic progenitor cells and its activation triggers their pyroptotic death. Active NLRP1a induced a lethal systemic inflammatory disease that was driven by Caspase-1 and IL-1ß but was independent of apoptosis-associated speck-like protein containing a CARD (ASC) and ameliorated by IL-18. Surprisingly, in the absence of IL-1ß-driven inflammation, active NLRP1a triggered pyroptosis of hematopoietic progenitor cells resulting in leukopenia at steady state. During periods of hematopoietic stress induced by chemotherapy or lymphocytic choriomeningitis virus (LCMV) infection, active NLRP1a caused prolonged cytopenia, bone marrow hypoplasia, and immunosuppression. Conversely, NLRP1-deficient mice showed enhanced recovery from chemotherapy and LCMV infection, demonstrating that NLRP1 acts as a cellular sentinel to alert Caspase-1 to hematopoietic and infectious stress.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Células-Tronco Hematopoéticas/metabolismo , Inflamassomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Adaptadoras de Sinalização CARD , Caspase 1/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Dermatite/imunologia , Dermatite/metabolismo , Fluoruracila/farmacologia , Hematopoese/efeitos dos fármacos , Hematopoese/imunologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/virologia , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interferon gama/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Camundongos Knockout , Mutação , Pancitopenia/imunologia , Pancitopenia/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The immune system must distinguish viable cells from cells damaged by physical and infective processes. The damaged cell-recognition molecule Clec9A is expressed on the surface of the mouse and human dendritic cell subsets specialized for the uptake and processing of material from dead cells. Clec9A recognizes a conserved component within nucleated and nonnucleated cells, exposed when cell membranes are damaged. We have identified this Clec9A ligand as a filamentous form of actin in association with particular actin-binding domains of cytoskeletal proteins. We have determined the crystal structure of the human CLEC9A C-type lectin domain and propose a functional dimeric structure with conserved tryptophans in the ligand recognition site. Mutation of these residues ablated CLEC9A binding to damaged cells and to the isolated ligand complexes. We propose that Clec9A provides targeted recruitment of the adaptive immune system during infection and can also be utilized to enhance immune responses generated by vaccines.
Assuntos
Citoesqueleto de Actina/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Receptores Imunológicos/metabolismo , Receptores Mitogênicos/metabolismo , Actinas/metabolismo , Imunidade Adaptativa , Animais , Sítios de Ligação , Linhagem Celular , Membrana Celular/metabolismo , Células Dendríticas/citologia , Feminino , Humanos , Lectinas Tipo C/química , Lectinas Tipo C/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Secundária de Proteína , Receptores Imunológicos/genética , Receptores Mitogênicos/química , Receptores Mitogênicos/genética , Espectrina/metabolismoRESUMO
Ets-related gene (ERG), which encodes a member of the Ets family of transcription factors, is a potent oncogene. Chromosomal rearrangements involving ERG are found in acute myeloid leukemia, acute lymphoblastic leukemia, Ewing's sarcoma and more than half of all prostate cancers; however, the normal physiological function of Erg is unknown. We did a sensitized genetic screen of the mouse for regulators of hematopoietic stem cell function and report here a germline mutation of Erg. We show that Erg is required for definitive hematopoiesis, adult hematopoietic stem cell function and the maintenance of normal peripheral blood platelet numbers.
Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Transativadores/genética , Transativadores/metabolismo , Animais , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Mutantes , Mutação , Transcrição Gênica , Regulador Transcricional ERGRESUMO
Despite their profoundly different cellular composition, size, and function, megakaryocytes and platelets both depend on restraint of the intrinsic (or "mitochondrial") apoptosis pathway by BCL-2 family prosurvival proteins for their development and viability. Activation of the pathway contributes to the clearance of megakaryocytes following platelet shedding and constrains platelet lifespan in the circulation. Important questions remain as to how apoptosis is initiated in these cells at steady state and in response to pathophysiological insults.
Assuntos
Apoptose/fisiologia , Plaquetas/fisiologia , Linhagem da Célula/fisiologia , Megacariócitos/fisiologia , Animais , Morte Celular , Diferenciação Celular , Humanos , Fagocitose/fisiologiaRESUMO
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
Although many genes are known to be critical for early hematopoiesis in the embryo, it remains unclear whether distinct regulatory pathways exist to control hematopoietic specification versus hematopoietic stem cell (HSC) emergence and function. Due to their interaction with key regulators of hematopoietic commitment, particular interest has focused on the role of the ETS family of transcription factors; of these, ERG is predicted to play an important role in the initiation of hematopoiesis, yet we do not know if or when ERG is required. Using in vitro and in vivo models of hematopoiesis and HSC development, we provide strong evidence that ERG is at the center of a distinct regulatory program that is not required for hematopoietic specification or differentiation but is critical for HSC maintenance during embryonic development. We show that, from the fetal period, ERG acts as a direct upstream regulator of Gata2 and Runx1 gene activity. Without ERG, physiological HSC maintenance fails, leading to the rapid exhaustion of definitive hematopoiesis.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Proteínas Oncogênicas/metabolismo , Animais , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Fator de Transcrição GATA2/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas/genética , Fatores de Transcrição , Regulador Transcricional ERGRESUMO
Human platelets contain microRNAs (miRNAs) and miRNA processing machinery, but their contribution to platelet function remains incompletely understood. Here, we show that murine megakaryocyte (MK)-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduces the level of the majority of miRNAs in platelets. This leads to altered platelet messenger RNA (mRNA) expression profiles and mild thrombocytopenia. Fibrinogen receptor subunits Itga2b (αIIb) and Itgb3 (ß3) mRNAs were among the differentially expressed transcripts that are increased in platelets lacking Dicer1. Argonaute 2 (Ago2), a member of the miRNA silencing complex, co-immunoprecipitated with αIIband ß3mRNAs in wild-type platelets. Furthermore, co-immunoprecipitation experiments suggested reduced αIIb/ß3/Ago2 complexes in miRNA-deficient platelets. These results suggested that miRNAs regulate both integrin subunits. Subsequent 3' untranslated region luciferase reporter assays confirmed that the translation of both αIIband ß3mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion ofDicer1resulted in increased surface expression of integrins αIIband ß3, and enhanced platelet binding to fibrinogen in vivo and in vitro. Heightened platelet reactivity, shortened tail-bleeding time, and reduced survival following collagen/epinephrine-induced pulmonary embolism were also observed in Dicer1-deficient animals. CombinedPf4-cre-mediated deletion of Drosha and Dicer1 did not significantly exacerbate phenotypes observed in single Dicer1 knockout mice. In summary, these findings indicate that Dicer1-dependent generation of mature miRNAs in late-stage MKs and platelets modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets.
Assuntos
Plaquetas/metabolismo , RNA Helicases DEAD-box/metabolismo , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mensageiro/metabolismo , Ribonuclease III/metabolismo , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , RNA Helicases DEAD-box/genética , Humanos , Integrina alfa2/biossíntese , Integrina alfa2/genética , Integrina beta3/biossíntese , Integrina beta3/genética , Camundongos , Camundongos Knockout , MicroRNAs/genética , Embolia Pulmonar/induzido quimicamente , Embolia Pulmonar/genética , Embolia Pulmonar/metabolismo , RNA Mensageiro/genética , Ribonuclease III/genéticaRESUMO
MYH9-related disorders (MYH9-RDs) caused by mutation of the MYH9 gene which encodes non-muscle myosin heavy-chain-IIA (NMMHC-IIA), an important motor protein in hemopoietic cells, are the most commonly encountered cause of inherited macrothrombocytopenia. Despite distinguishing features including an autosomal dominant mode of inheritance, giant platelets on the peripheral blood film accompanied by leucocytes with cytoplasmic inclusion bodies (döhle-like bodies), these disorders remain generally under-recognized and often misdiagnosed as immune thrombocytopenia (ITP). This may result in inappropriate treatment with corticosteroids, immunosupressants and in some cases, splenectomy. We explored the efficacy of next generation sequencing (NGS) with a candidate gene panel to establish the aetiology of thrombocytopenia for individuals who had been referred to our center from hematologists in the Australasian region in whom the cause of thrombocytopenia was suspected to be secondary to an inherited condition but which remained uncharacterized despite phenotypic investigations. Pathogenic MYH9 variants were detected in 15 (15/121, 12.4%) individuals and the pathogenecity of a novel variant of uncertain significance was confirmed in a further two related individuals following immunofluorescence (IF) staining performed in our laboratory. Concerningly, only one (1/17) individual diagnosed with MYH9-RD had been referred with this as a presumptive diagnosis, in all other cases (16/17, 94.1%), a diagnosis was not suspected by referring clinicians, indicating a lack of awareness or a failing of our diagnostic approach to these conditions. We examined the mean platelet diameter (MPD) measurements as a means to better identify and quantify platelet size. MPDs in cases with MYH9-RDs were significantly larger than controls (p < 0.001) and in 91% were greater than a previously suggested threshold for platelets in cases of ITP. In addition, we undertook IF staining in a proportion of cases and confirm that this test and/or NGS are satisfactory diagnostic tests. We propose that fewer cases of MYH9-RDs would be missed if diagnostic algorithms prioritized IF and/or NGS in cases of thrombocytopenia associated with giant platelets, even if döhle-like bodies are not appreciated on the peripheral blood film. Finally, our report describes the long-term use of a thrombopoietin agonist in a case of MYH9-RD that had previously been diagnosed as ITP, and demonstrates that treatment with these agents may be possible, and is well tolerated, in this group of patients.
Assuntos
Plaquetas/metabolismo , Perda Auditiva Neurossensorial/genética , Mutação , Cadeias Pesadas de Miosina/genética , Púrpura Trombocitopênica Idiopática/genética , Receptores Fc/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Trombocitopenia/congênito , Trombopoetina/uso terapêutico , Adulto , Australásia , Plaquetas/efeitos dos fármacos , Plaquetas/patologia , Tamanho Celular , Estudos de Coortes , Diagnóstico Diferencial , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Genes Dominantes , Perda Auditiva Neurossensorial/sangue , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/tratamento farmacológico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Corpos de Inclusão/efeitos dos fármacos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Masculino , Volume Plaquetário Médio , Pessoa de Meia-Idade , Cadeias Pesadas de Miosina/sangue , Púrpura Trombocitopênica Idiopática/sangue , Púrpura Trombocitopênica Idiopática/diagnóstico , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Trombocitopenia/sangue , Trombocitopenia/diagnóstico , Trombocitopenia/tratamento farmacológico , Trombocitopenia/genéticaRESUMO
The Piwi pathway is deeply conserved amongst animals because one of its essential functions is to repress transposons. However, many Piwi-interacting RNAs (piRNAs) do not base-pair to transposons and remain mysterious in their targeting function. The sheer number of piRNA cluster (piC) loci in animal genomes and infrequent piRNA sequence conservation also present challenges in determining which piC loci are most important for development. To address this question, we determined the piRNA expression patterns of piC loci across a wide phylogenetic spectrum of animals, and reveal that most genic and intergenic piC loci evolve rapidly in their capacity to generate piRNAs, regardless of known transposon silencing function. Surprisingly, we also uncovered a distinct set of piC loci with piRNA expression conserved deeply in Eutherian mammals. We name these loci Eutherian-Conserved piRNA cluster (ECpiC) loci. Supporting the hypothesis that conservation of piRNA expression across ~100 million years of Eutherian evolution implies function, we determined that one ECpiC locus generates abundant piRNAs antisense to the STOX1 transcript, a gene clinically associated with preeclampsia. Furthermore, we confirmed reduced piRNAs in existing mouse mutations at ECpiC-Asb1 and -Cbl, which also display spermatogenic defects. The Asb1 mutant testes with strongly reduced Asb1 piRNAs also exhibit up-regulated gene expression profiles. These data indicate ECpiC loci may be specially adapted to support Eutherian reproduction.
Assuntos
Mamíferos/genética , Família Multigênica , RNA Interferente Pequeno/genética , Animais , Evolução Molecular , Mamíferos/classificaçãoRESUMO
Harlequin ichthyosis (HI) is a severe skin disease which leads to neonatal death in â¼50% of cases. It is the result of mutations in ABCA12, a protein that transports lipids required to establish the protective skin barrier needed after birth. To better understand the life-threatening newborn HI phenotype, we analysed the developing epidermis for consequences of lipid dysregulation in mouse models. We observed a pro-inflammatory signature which was characterized by chemokine upregulation in embryonic skin which is distinct from that seen in other types of ichthyosis. Inflammation also persisted in grafted HI skin. To examine the contribution of inflammation to disease development, we overexpressed interleukin-37b to globally suppress fetal inflammation, observing considerable improvements in keratinocyte differentiation. These studies highlight inflammation as an unexpected contributor to HI disease development in utero, and suggest that inhibiting inflammation may reduce disease severity.
Assuntos
Ictiose Lamelar/embriologia , Ictiose Lamelar/imunologia , Animais , Diferenciação Celular , Quimiocinas/genética , Quimiocinas/imunologia , Modelos Animais de Doenças , Epiderme/embriologia , Epiderme/imunologia , Feminino , Humanos , Ictiose Lamelar/genética , Ictiose Lamelar/fisiopatologia , Interleucina-1/genética , Interleucina-1/imunologia , Queratinócitos/citologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Pele/embriologia , Pele/imunologiaRESUMO
In a dominant mouse ethylnitrosurea mutagenesis screen for genes regulating erythropoiesis, we identified a pedigree with a novel microcytic hypochromia caused by a V235G missense mutation in Dynamin 2 (Dnm2). Mutations in Dnm2, a GTPase, are highly disease-specific and have been implicated in four forms of human diseases: centronuclear myopathy, Charcot-Marie Tooth neuropathy and, more recently, T-cell leukaemia and Hereditary Spastic Paraplegia, but red cell abnormalities have not been reported to date. The V235G mutation lies within a crucial GTP nucleotide-binding pocket of Dnm2, and resulted in defective GTPase activity and incompatibility with life in the homozygous state. Dnm2 is an essential mediator of clathrin-mediated endocytosis, which is required for the uptake of transferrin (Tf) into red cells for incorporation of haem. Accordingly, we observed significantly reduced Tf uptake by Dnm2+/V235G cells, which led to impaired endosome formation. Despite these deficiencies, surprisingly all iron studies were unchanged, suggesting an unexplained alternative mechanism underlies microcytic anaemia in Dnm2+/V235G mice. This study provides the first in vivo evidence for the requirements of Dnm2 in normal erythropoiesis.
Assuntos
Anemia Hipocrômica/genética , Dinamina II/genética , Mutação de Sentido Incorreto , Anemia Hipocrômica/sangue , Animais , Mapeamento Cromossômico/métodos , Modelos Animais de Doenças , Dinamina II/deficiência , Dinamina II/fisiologia , Endocitose/genética , Endocitose/fisiologia , Eritrócitos/metabolismo , Eritrócitos/patologia , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos Knockout , Transferrina/metabolismoRESUMO
Apoptosis is required to maintain tissue homeostasis in multicellular organisms. Platelets, the anucleate cells that are essential for blood clotting, are a prime example. Their brief life span in the circulation is regulated by the intrinsic apoptosis pathway. Pro-survival BCL-XL (also termed BCL2L1) is essential for platelet viability. It functions to restrain the pro-apoptotic BCL-2 family members BAK (also termed BAK1) and BAX, the essential mediators of intrinsic apoptosis. Genetic deletion or pharmacological inhibition of BCL-XL results in thrombocytopenia. Conversely, deletion of BAK in platelets doubles their circulating life span. However, what triggers platelet apoptosis in vivo remains unclear. The pro-apoptotic BH3-only proteins are essential for initiating apoptosis in nucleated cells, and there is some evidence to suggest they also play a role in platelet biology. We investigated whether PUMA (also termed BBC3), a potent BH3-only protein that can inhibit all pro-survival BCL-2 family members as well as directly activate BAX, regulates the death of platelets. Surprisingly, loss of PUMA had no impact on the loss of platelets caused by loss of BCL-XL. It therefore remains to be established whether other BH3-only proteins play a critical role in induction of apoptosis in platelets or whether their death is controlled solely by the interactions between BCL-XL with BAK and BAX.