RESUMO
Identifying signaling pathways that regulate hematopoietic stem and progenitor cell (HSPC) formation in the embryo will guide efforts to produce and expand HSPCs ex vivo. Here we show that sterile tonic inflammatory signaling regulates embryonic HSPC formation. Expression profiling of progenitors with lymphoid potential and hematopoietic stem cells (HSCs) from aorta/gonad/mesonephros (AGM) regions of midgestation mouse embryos revealed a robust innate immune/inflammatory signature. Mouse embryos lacking interferon γ (IFN-γ) or IFN-α signaling and zebrafish morphants lacking IFN-γ and IFN-Ï activity had significantly fewer AGM HSPCs. Conversely, knockdown of IFN regulatory factor 2 (IRF2), a negative regulator of IFN signaling, increased expression of IFN target genes and HSPC production in zebrafish. Chromatin immunoprecipitation (ChIP) combined with sequencing (ChIP-seq) and expression analyses demonstrated that IRF2-occupied genes identified in human fetal liver CD34(+) HSPCs are actively transcribed in human and mouse HSPCs. Furthermore, we demonstrate that the primitive myeloid population contributes to the local inflammatory response to impact the scale of HSPC production in the AGM region. Thus, sterile inflammatory signaling is an evolutionarily conserved pathway regulating the production of HSPCs during embryonic development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Transdução de Sinais , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Proliferação de Células/genética , Células Cultivadas , Citocinas/imunologia , Embrião de Mamíferos , Embrião não Mamífero , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Inflamação/genética , Inflamação/imunologia , Interferons/genética , Interferons/metabolismo , Camundongos , Peixe-Zebra/embriologiaRESUMO
Cannabinoids (CB) modulate adult hematopoietic stem and progenitor cell (HSPCs) function, however, impact on the production, expansion, or migration of embryonic HSCs is currently uncharacterized. Here, using chemical and genetic approaches targeting CB-signaling in zebrafish, we show that CB receptor (CNR) 2, but not CNR1, regulates embryonic HSC development. During HSC specification in the aorta-gonad-mesonephros (AGM) region, CNR2 stimulation by AM1241 increased runx1;cmyb(+) HSPCs, through heightened proliferation, whereas CNR2 antagonism decreased HSPC number; FACS analysis and absolute HSC counts confirmed and quantified these effects. Epistatic investigations showed AM1241 significantly upregulated PGE2 synthesis in a Ptgs2-dependent manner to increase AGM HSCs. During the phases of HSC production and colonization of secondary niches, AM1241 accelerated migration to the caudal hematopoietic tissue (CHT), the site of embryonic HSC expansion, and the thymus; however these effects occurred independently of PGE2. Using a candidate approach for HSC migration and retention factors, P-selectin was identified as the functional target of CNR2 regulation. Epistatic analyses confirmed migration of HSCs into the CHT and thymus was dependent on CNR2-regulated P-selectin activity. Together, these data suggest CNR2-signaling optimizes the production, expansion, and migration of embryonic HSCs by modulating multiple downstream signaling pathways.
Assuntos
Dinoprostona/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Selectina-P/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Células-Tronco Hematopoéticas/citologia , Transdução de Sinais/fisiologiaRESUMO
Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfRmu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfRmu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1, across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfRmu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.
Assuntos
Barreira Hematoencefálica , Oligonucleotídeos Antissenso , RNA Longo não Codificante , Receptores da Transferrina , Animais , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/administração & dosagem , Barreira Hematoencefálica/metabolismo , Receptores da Transferrina/metabolismo , Humanos , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/genética , Camundongos , Transporte Biológico , Macaca fascicularis , Técnicas de Silenciamento de Genes , Distribuição TecidualRESUMO
Huntington's disease (HD) is caused by an expanded polyglutamine tract in the protein huntingtin (htt). Although HD has historically been viewed as a brain-specific disease, htt is expressed ubiquitously, and recent studies indicate that mutant htt might cause changes to the immune system that could contribute to pathogenesis. Monocytes from HD patients and mouse models are hyperactive in response to stimulation, and increased levels of inflammatory cytokines and chemokines are found in pre-manifest patients that correlate with pathogenesis. In this study, wild-type (WT) bone marrow cells were transplanted into two lethally irradiated transgenic mouse models of HD that ubiquitously express full-length htt (YAC128 and BACHD mice). Bone marrow transplantation partially attenuated hypokinetic and motor deficits in HD mice. Increased levels of synapses in the cortex were found in HD mice that received bone marrow transplants. Importantly, serum levels of interleukin-6, interleukin-10, CXC chemokine ligand 1, and interferon-γ were significantly higher in HD than WT mice but were normalized in mice that received a bone marrow transplant. These results suggest that immune cell dysfunction might be an important modifier of pathogenesis in HD.
Assuntos
Doenças Autoimunes do Sistema Nervoso/terapia , Células da Medula Óssea/imunologia , Transplante de Medula Óssea/métodos , Doença de Huntington/imunologia , Doença de Huntington/terapia , Terapia de Imunossupressão/métodos , Animais , Doenças Autoimunes do Sistema Nervoso/fisiopatologia , Modelos Animais de Doenças , Feminino , Humanos , Doença de Huntington/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Huntington disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin (htt) protein. To uncover candidate therapeutic targets and networks involved in pathogenesis, we integrated gene expression profiling and functional genetic screening to identify genes critical for mutant htt toxicity in yeast. Using mRNA profiling, we have identified genes differentially expressed in wild-type yeast in response to mutant htt toxicity as well as in three toxicity suppressor strains: bna4Δ, mbf1Δ, and ume1Δ. BNA4 encodes the yeast homolog of kynurenine 3-monooxygenase, a promising drug target for HD. Intriguingly, despite playing diverse cellular roles, these three suppressors share common differentially expressed genes involved in stress response, translation elongation, and mitochondrial transport. We then systematically tested the ability of the differentially expressed genes to suppress mutant htt toxicity when overexpressed and have thereby identified 12 novel suppressors, including genes that play a role in stress response, Golgi to endosome transport, and rRNA processing. Integrating the mRNA profiling data and the genetic screening data, we have generated a robust network that shows enrichment in genes involved in rRNA processing and ribosome biogenesis. Strikingly, these observations implicate dysfunction of translation in the pathology of HD. Recent work has shown that regulation of translation is critical for life span extension in Drosophila and that manipulation of this process is protective in Parkinson disease models. In total, these observations suggest that pharmacological manipulation of translation may have therapeutic value in HD.
Assuntos
Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/toxicidade , Proteínas Nucleares/metabolismo , Proteínas Nucleares/toxicidade , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Sequência de Bases , Deleção de Genes , Genômica , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Proteínas Mutantes/biossíntese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/toxicidade , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta/genética , RNA Ribossômico/genética , Sequências Reguladoras de Ácido Nucleico/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/citologia , Transgenes/genéticaRESUMO
Effective delivery of protein therapeutics to the central nervous system (CNS) has been greatly restricted by the blood-brain barrier (BBB). We describe the development of a BBB transport vehicle (TV) comprising an engineered Fc fragment that exploits receptor-mediated transcytosis for CNS delivery of biotherapeutics by binding a highly expressed brain endothelial cell target. TVs were engineered using directed evolution to bind the apical domain of the human transferrin receptor (hTfR) without the use of amino acid insertions, deletions, or unnatural appendages. A crystal structure of the TV-TfR complex revealed the TV binding site to be away from transferrin and FcRn binding sites, which was further confirmed experimentally in vitro and in vivo. Recombinant expression of TVs fused to anti-ß-secretase (BACE1) Fabs yielded antibody transport vehicle (ATV) molecules with native immunoglobulin G (IgG) structure and stability. Peripheral administration of anti-BACE1 ATVs to hTfR-engineered mice and cynomolgus monkeys resulted in substantially improved CNS uptake and sustained pharmacodynamic responses. The TV platform readily accommodates numerous additional configurations, including bispecific antibodies and protein fusions, yielding a highly modular CNS delivery platform.
Assuntos
Secretases da Proteína Precursora do Amiloide , Barreira Hematoencefálica , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Haplorrinos/metabolismo , Fragmentos Fc das Imunoglobulinas , Camundongos , Receptores da Transferrina/metabolismoRESUMO
Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.
Assuntos
Barreira Hematoencefálica , Iduronato Sulfatase , Animais , Encéfalo , Modelos Animais de Doenças , Terapia de Reposição de Enzimas , Lisossomos , CamundongosRESUMO
Clear cell sarcoma is an aggressive malignancy occurring most commonly in the distal extremities of young adults, characterized by t(12;22)(q13;q12) creating the chimeric fusion oncoprotein EWS-ATF1. We assessed growth inhibition and differentiation effects of histone deacetylase inhibitors MS-275 and romidepsin (depsipeptide, FK228) on clear cell sarcoma cells and evaluated drug sensitivity among related translocation-associated sarcomas and other cell models. Three clear cell sarcoma cell lines, seven other sarcomas, six nonsarcoma malignant cell lines, and two nonneoplastic mesenchymal cell models were treated with MS-275 or romidepsin. Growth inhibition was assayed by monolayer 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Induction of cell cycle arrest and apoptosis were assessed by propidium iodide/Annexin V flow cytometry in monolayer and spheroid cultures and by immunoblotting analysis. Expression levels of key genes involved in mesenchymal differentiation and of EWS-ATF1 were measured by quantitative real-time PCR in clear cell sarcoma cells treated with histone deacetylase inhibitors. MS-275 and romidepsin inhibited growth in clear cell sarcoma cells by inducing cell cycle arrest and apoptosis in a time- and dose-dependent manner. Sarcomas showed greater sensitivity than other tumor types, with clear cell sarcomas most sensitive of all, whereas nonmalignant mesenchymal cells were highly resistant. MS-275 at 1 micromol/L and romidepsin at 1 nmol/L induced histone H3 acetylation, cell cycle arrest, apoptosis, and differentiation in clear cell sarcoma cells within 24 hours. Histone deacetylase inhibitors increased expression of SOX9, MYOD1, and PPARG and decreased EWS-ATF1 expression in clear cell sarcoma cells. Histone deacetylase inhibitors show promising preclinical activity in multiple clear cell sarcoma models.
Assuntos
Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Sarcoma de Células Claras/enzimologia , Sarcoma de Células Claras/patologia , Benzamidas/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Depsipeptídeos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sarcoma de Células Claras/genética , Esferoides Celulares/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Translocação Genética/efeitos dos fármacosRESUMO
The core binding factor (CBF) gene RUNX1 is a target of chromosomal translocations in leukemia, including t(8;21) in acute myeloid leukemia (AML). Normal CBF function is essential for activity of AML1-ETO, product of the t(8;21), and for survival of several leukemias lacking RUNX1 mutations. Using virtual screening and optimization, we developed Runt domain inhibitors which bind to the Runt domain and disrupt its interaction with CBFß. On-target activity was demonstrated by the Runt domain inhibitors' ability to depress hematopoietic cell formation in zebrafish embryos, reduce growth and induce apoptosis of t(8;21) AML cell lines, and reduce progenitor activity of mouse and human leukemia cells harboring the t(8;21), but not normal bone marrow cells. Runt domain inhibitors had similar effects on murine and human T cell acute lymphocytic leukemia (T-ALL) cell lines. Our results confirmed that Runt domain inhibitors might prove efficacious in various AMLs and in T-ALL.
Assuntos
Antineoplásicos/metabolismo , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Subunidades alfa de Fatores de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Estrutura Molecular , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Translocação Genética , Peixe-ZebraRESUMO
Zebrafish are one of the preeminent model systems for the study of blood development (hematopoiesis), hematopoietic stem and progenitor cell (HSPC) biology, and hematopathology. The zebrafish hematopoietic system shares strong similarities in functional populations, genetic regulators, and niche interactions with its mammalian counterparts. These evolutionarily conserved characteristics, together with emerging technologies in live imaging, compound screening, and genetic manipulation, have been employed to successfully identify and interrogate novel regulatory mechanisms and molecular pathways that guide hematopoiesis. Significantly, perturbations in many of the key developmental signals controlling hematopoiesis are associated with hematological disorders and disease, including anemia, bone marrow failure syndromes, and leukemia. Thus, understanding the regulatory pathways controlling HSPC production and function has important clinical implications. In this review, we describe how the blood system forms and is maintained in zebrafish, with particular focus on new insights into vertebrate hematological regulation gained using this model. The interplay of factors controlling development and disease in the hematopoietic system combined with the unique attributes of the zebrafish make this a powerful platform to discover novel targets for the treatment of hematological disease.
Assuntos
Neoplasias Hematológicas/patologia , Hematopoese , Peixe-Zebra/fisiologia , Animais , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/citologia , Sistema Hematopoético/embriologia , Peixe-Zebra/embriologiaRESUMO
Hematopoietic stem cells (HSCs) have the ability to both self-renew and differentiate each of the mature blood cell lineages and thereby reconstitute the entire blood system. Therefore, HSCs are therapeutically valuable for treatment of hematological malignances and bone marrow failure. We showed recently that transient glucose elevation elicited dose-dependent effects on HSCs through elevated metabolic activity and subsequent reactive oxygen species-mediated induction of Hypoxia-Inducible Factor 1α (Hif1α). Platelet-Derived Growth Factor B (pdgfb), a Hif1α-target, and its receptor, pdgfrb, were significantly upregulated in response to metabolic stimulation. Although the function of PDGF signaling is well established in vascular development, its role in hematopoiesis is less understood. Exposure to either a pan-PDGF inhibitor or a PDGFRß-selective antagonist in the context of Hif1α stimulation blocked elevations in hematopoietic stem and progenitor cell (HSPC) formation as determined by runx1;cmyb whole-mount in situ hybridization (WISH) and HSPC-reporter flow cytometry analysis. Similar results were observed for morpholino (MO) knockdown of pdgfrb or dominant-negative pdgfrb expression, indicating that PDGFRß signaling is a key downstream mediator of Hif1α-mediated induction of HSPCs. Notably, overexpression of Pdgfb ligand enhanced HSPC numbers in the aorta-gonado-mesonephros (AGM) at 36 hours postfertilization (hpf) and in the caudal hematopoietic tissue at 48 hpf. A survey of known PDGF-B/PDGFRß regulatory targets by expression analysis revealed a significant increase in inflammatory intermediates, including Interleukin 6 (IL-6) and its receptor (IL-6R). MO-mediated knockdown of il6 or chemical inhibition of IL-6R antagonized the effect of Pdgfb overexpression. Furthermore, epistatic analysis of IL-6/IL-6R function confirmed activity downstream of Hif1α. Together, these findings define a Hif1α-regulated signaling axis mediated through PBFGB/PDGFRß and IL-6/IL-6R that acts to control embryonic HSPC production.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia , Interleucina-6/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Proliferação de Células , Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Imuno-Histoquímica , Estabilidade Proteica , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Peixe-ZebraRESUMO
Synovial sarcoma is a soft tissue malignancy with a poor prognosis; many patients will die from this disease within 10 years of diagnosis, despite treatment. Gene expression profiling and immunohistochemistry studies have identified oncogenes that are highly expressed in synovial sarcoma. Included in this group are receptor tyrosine kinases such as epidermal growth factor receptor, insulin-like growth factor receptor 1, fibroblast growth factor receptor 3, KIT, and HER2. Inhibitors of these growth-promoting receptors are likely to inhibit proliferation of synovial sarcoma; however, the effect of receptor tyrosine kinase inhibitors on synovial sarcoma is largely unknown. We assessed the ability of the following receptor tyrosine kinase inhibitors to halt proliferation and induce apoptosis in synovial sarcoma monolayer and three dimensional spheroid in vitro models: gefitinib (Iressa), NVP-AEW541, imatinib mesylate (Gleevec), SU5402, PRO-001, trastuzumab (Herceptin), and 17-allylamino-17-demethoxygeldanamycin (17-AAG). Gefitinib, NVP-AEW541, and imatinib inhibited proliferation only at relatively high concentrations, which are not clinically applicable. 17-AAG, which destabilizes multiple receptor tyrosine kinases and other oncoproteins through heat shock protein 90 inhibition, prevented proliferation and induced apoptosis in synovial sarcoma monolayer models at concentrations achievable in human serum. 17-AAG treatment was also associated with receptor tyrosine kinase degradation and induction of apoptosis in synovial sarcoma spheroid models. 17-AAG was more effective than doxorubicin, particularly in the spheroid models. Here we provide in vitro evidence that 17-AAG, a clinically applicable drug with known pharmacology and limited toxicity, inhibits synovial sarcoma proliferation by inducing apoptosis, and thus has potential as a systemic therapy for this disease.
Assuntos
Apoptose , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Rifabutina/análogos & derivados , Sarcoma Sinovial/tratamento farmacológico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Benzamidas , Benzoquinonas , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Receptores ErbB/metabolismo , Citometria de Fluxo , Gefitinibe , Humanos , Mesilato de Imatinib , Immunoblotting , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Técnicas In Vitro , Concentração Inibidora 50 , Lactamas Macrocíclicas , Piperazinas/farmacologia , Prognóstico , Pirimidinas/farmacologia , Pirróis/farmacologia , Quinazolinas/farmacologia , Rifabutina/farmacologia , TrastuzumabRESUMO
Hematopoietic stem and progenitor cell (HSPC) specification is regulated by numerous defined factors acting locally within the hemogenic niche; however, it is unclear whether production can adapt to fluctuating systemic needs. Here we show that the CNS controls embryonic HSPC numbers via the hypothalamic-pituitary-adrenal/interrenal (HPA/I) stress response axis. Exposure to serotonin or the reuptake inhibitor fluoxetine increased runx1 expression and Flk1(+)/cMyb(+) HSPCs independent of peripheral innervation. Inhibition of neuronal, but not peripheral, tryptophan hydroxlyase (Tph) persistently reduced HSPC number. Consistent with central HPA/I axis induction and glucocorticoid receptor (GR) activation, GR agonists enhanced, whereas GR loss diminished, HSPC formation. Significantly, developmental hypoxia, as indicated by Hif1α function, induced the HPA/I axis and cortisol production. Furthermore, Hif1α-stimulated HSPC enhancement was attenuated by neuronal tph or GR loss. Our data establish that embryonic HSC production responds to physiologic stress via CNS-derived serotonin synthesis and central feedback regulation to control HSC numbers.
Assuntos
Sistema Nervoso Central/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais , Animais , Contagem de Células , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Fluoxetina/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Neurônios Serotoninérgicos/efeitos dos fármacos , Neurônios Serotoninérgicos/metabolismo , Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo , Triptofano Hidroxilase/metabolismo , Peixe-Zebra/embriologiaRESUMO
Vitamin D insufficiency is a worldwide epidemic affecting billions of individuals, including pregnant women and children. Despite its high incidence, the impact of active vitamin D3 (1,25(OH)D3) on embryonic development beyond osteo-regulation remains largely undefined. Here, we demonstrate that 1,25(OH)D3 availability modulates zebrafish hematopoietic stem and progenitor cell (HSPC) production. Loss of Cyp27b1-mediated biosynthesis or vitamin D receptor (VDR) function by gene knockdown resulted in significantly reduced runx1 expression and Flk1+cMyb+ HSPC numbers. Selective modulation in vivo and in vitro in zebrafish indicated that vitamin D3 acts directly on HSPCs, independent of calcium regulation, to increase proliferation. Notably, ex vivo treatment of human HSPCs with 1,25(OH)D3 also enhanced hematopoietic colony numbers, illustrating conservation across species. Finally, gene expression and epistasis analysis indicated that CXCL8(IL-8) was a functional target of vitamin D3-mediated HSPC regulation. Together, these findings highlight the relevance of developmental 1,25(OH)D3 availability for definitive hematopoiesis and suggest potential therapeutic utility in HSPC expansion.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Células-Tronco Hematopoéticas/metabolismo , Interleucina-8/genética , Receptores de Calcitriol/genética , Vitamina D/genética , Proteínas de Peixe-Zebra/genética , Animais , Disponibilidade Biológica , Sinalização do Cálcio/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Humanos , Interleucina-8/metabolismo , Gravidez , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Vitamina D/metabolismo , Deficiência de Vitamina D/genética , Deficiência de Vitamina D/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Hematopoietic stem and progenitor cells (HSPCs) are born from hemogenic endothelium in the dorsal aorta. Specification of this hematopoietic niche is regulated by a signaling axis using Hedgehog (Hh) and Notch, which culminates in expression of Runx1 in the ventral wall of the artery. Here, we demonstrate that the vitamin D precursor cholecalciferol (D3) modulates HSPC production by impairing hemogenic vascular niche formation. Accumulation of D3 through exogenous treatment or inhibition of Cyp2r1, the enzyme required for D3 25-hydroxylation, results in Hh pathway antagonism marked by loss of Gli-reporter activation, defects in vascular niche identity, and reduced HSPCs. Mechanistic studies indicated the effect was specific to D3, and not active 1,25-dihydroxy vitamin D3, acting on the extracellular sterol-binding domain of Smoothened. These findings highlight a direct impact of inefficient vitamin D synthesis on cell fate commitment and maturation in Hh-regulated tissues, which may have implications beyond hemogenic endothelium specification.
Assuntos
Colecalciferol/farmacologia , Proteínas Hedgehog/metabolismo , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Vitaminas/farmacologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Colecalciferol/metabolismo , Colestanotriol 26-Mono-Oxigenase/genética , Colestanotriol 26-Mono-Oxigenase/metabolismo , Deleção de Genes , Células-Tronco Hematopoéticas/citologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened , Vitaminas/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Genetic control of hematopoietic stem and progenitor cell (HSPC) function is increasingly understood; however, less is known about the interactions specifying the embryonic hematopoietic niche. Here, we report that 17ß-estradiol (E2) influences production of runx1+ HSPCs in the AGM region by antagonizing VEGF signaling and subsequent assignment of hemogenic endothelial (HE) identity. Exposure to exogenous E2 during vascular niche development significantly disrupted flk1+ vessel maturation, ephrinB2+ arterial identity, and specification of scl+ HE by decreasing expression of VEGFAa and downstream arterial Notch-pathway components; heat shock induction of VEGFAa/Notch rescued E2-mediated hematovascular defects. Conversely, repression of endogenous E2 activity increased somitic VEGF expression and vascular target regulation, shifting assignment of arterial/venous fate and HE localization; blocking E2 signaling allowed venous production of scl+/runx1+ cells, independent of arterial identity acquisition. Together, these data suggest that yolk-derived E2 sets the ventral boundary of hemogenic vascular niche specification by antagonizing the dorsal-ventral regulatory limits of VEGF.
Assuntos
Antagonistas de Estrogênios/farmacologia , Hemangioblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Compostos Benzidrílicos/farmacologia , Subunidade alfa 2 de Fator de Ligação ao Core/biossíntese , Efrina-B2/antagonistas & inibidores , Estradiol/análogos & derivados , Estradiol/farmacologia , Estrogênios/farmacologia , Etinilestradiol/farmacologia , Fulvestranto , Genisteína/farmacologia , Resposta ao Choque Térmico , Morfolinos/genética , Fenóis/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/biossíntese , Receptores de Estradiol/genética , Receptores Notch/biossíntese , Transdução de Sinais , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidoresRESUMO
In Huntington disease (HD), immune cells are activated before symptoms arise; however, it is unclear how the expression of mutant huntingtin (htt) compromises the normal functions of immune cells. Here we report that primary microglia from early postnatal HD mice were profoundly impaired in their migration to chemotactic stimuli, and expression of a mutant htt fragment in microglial cell lines was sufficient to reproduce these deficits. Microglia expressing mutant htt had a retarded response to a laser-induced brain injury in vivo. Leukocyte recruitment was defective upon induction of peritonitis in HD mice at early disease stages and was normalized upon genetic deletion of mutant htt in immune cells. Migration was also strongly impaired in peripheral immune cells from pre-manifest human HD patients. Defective actin remodeling in immune cells expressing mutant htt likely contributed to their migration deficit. Our results suggest that these functional changes may contribute to immune dysfunction and neurodegeneration in HD, and may have implications for other polyglutamine expansion diseases in which mutant proteins are ubiquitously expressed.
Assuntos
Quimiotaxia , Doença de Huntington/genética , Microglia/fisiologia , Células Mieloides/fisiologia , Proteínas do Tecido Nervoso/genética , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/fisiologia , Animais , Extensões da Superfície Celular/metabolismo , Células Cultivadas , Complemento C5a/fisiologia , Humanos , Proteína Huntingtina , Doença de Huntington/imunologia , Doença de Huntington/patologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/citologia , Monócitos/fisiologia , Mutação , Células Mieloides/citologia , Proteínas do Tecido Nervoso/metabolismo , Peritônio/patologia , Tioglicolatos/farmacologia , Imagem com Lapso de TempoRESUMO
The kynurenine pathway of tryptophan degradation is hypothesized to play an important role in Huntington disease, a neurodegenerative disorder caused by a polyglutamine expansion in the protein huntingtin. Neurotoxic metabolites of the kynurenine pathway, generated in microglia and macrophages, are present at increased levels in the brains of patients and mouse models during early stages of disease, but the mechanism by which kynurenine pathway up-regulation occurs in Huntington disease is unknown. Here we report that expression of a mutant huntingtin fragment was sufficient to induce transcription of the kynurenine pathway in yeast and that this induction was abrogated by impairing the activity of the histone deacetylase Rpd3. Moreover, numerous genetic suppressors of mutant huntingtin toxicity that are functionally unrelated converged unexpectedly on the kynurenine pathway, supporting a critical role for the kynurenine pathway in mediating mutant huntingtin toxicity in yeast. Histone deacetylase-dependent regulation of the kynurenine pathway was also observed in a mouse model of Huntington disease, in which treatment with a neuroprotective histone deacetylase inhibitor blocked activation of the kynurenine pathway in microglia expressing a mutant huntingtin fragment in vitro and in vivo. These findings suggest that a mutant huntingtin fragment can perturb transcriptional programs in microglia, and thus implicate these cells as potential modulators of neurodegeneration in Huntington disease that are worthy of further investigation.