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1.
Mol Cell ; 83(12): 2003-2019.e6, 2023 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-37257451

RESUMO

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase , Neoplasias de Cabeça e Pescoço , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Carcinogênese
2.
Nat Immunol ; 16(8): 810-8, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26147687

RESUMO

Foxm1 is known as a typical proliferation-associated transcription factor. Here we found that Foxm1 was essential for maintenance of the quiescence and self-renewal capacity of hematopoietic stem cells (HSCs) in vivo in mice. Reducing expression of FOXM1 also decreased the quiescence of human CD34(+) HSCs and progenitor cells, and its downregulation was associated with a subset of myelodysplastic syndrome (MDS). Mechanistically, Foxm1 directly bound to the promoter region of the gene encoding the receptor Nurr1 (Nr4a2; called 'Nurr1' here), inducing transcription, while forced expression of Nurr1 reversed the loss of quiescence observed in Foxm1-deficient cells in vivo. Thus, our studies reveal a previously unrecognized role for Foxm1 as a critical regulator of the quiescence and self-renewal of HSCs mediated at least in part by control of Nurr1 expression.


Assuntos
Proliferação de Células/genética , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Animais , Células Cultivadas , Citometria de Fluxo , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Nat Immunol ; 15(3): 239-47, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24487321

RESUMO

Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas Interatuantes com Canais de Kv/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/biossíntese , Fatores Estimuladores Upstream/metabolismo , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Animais , Imunoprecipitação da Cromatina , Cisteína Endopeptidases , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/imunologia , Immunoblotting , Inflamação/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Ubiquitina-Proteína Ligases/genética
4.
J Biol Chem ; 298(9): 102385, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35985424

RESUMO

Asparagine synthetase (ASNS) catalyzes synthesis of asparagine (Asn) and Glu from Asp and Gln in an ATP-dependent reaction. Asparagine synthetase deficiency (ASNSD) results from biallelic mutations in the ASNS gene. Affected children exhibit congenital microcephaly, continued brain atrophy, seizures, and often premature mortality. However, the underlying mechanisms are unclear. This report describes a compound heterozygotic ASNSD child with two novel mutations in the ASNS gene, c.1118G>T (paternal) and c.1556G>A (maternal), that lead to G373V or R519H ASNS variants. Structural mapping suggested that neither variant participates directly in catalysis. Growth of cultured fibroblasts from either parent was unaffected in Asn-free medium, whereas growth of the child's cells was suppressed by about 50%. Analysis of Asn levels unexpectedly revealed that extracellular rather than intracellular Asn correlated with the reduced proliferation during incubation of the child's cells in Asn-free medium. Our attempts to ectopically express the G373V variant in either HEK293T or JRS cells resulted in minimal protein production, suggesting instability. Protein expression and purification from HEK293T cells revealed reduced activity for the R519H variant relative to WT ASNS. Expression of WT ASNS in ASNS-null JRS cells resulted in nearly complete rescue of growth in Asn-free medium, whereas we observed no proliferation for the cells expressing either the G373V or R519H variant. These results support the conclusion that the coexpression of the G373V and R519H ASNS variants leads to significantly reduced Asn synthesis, which negatively impacts cellular growth. These observations are consistent with the ASNSD phenotype.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Aspartato-Amônia Ligase , Deficiência Intelectual , Microcefalia , Doenças Neurodegenerativas , Trifosfato de Adenosina , Asparagina/genética , Aspartato-Amônia Ligase/química , Atrofia , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Criança , Células HEK293 , Humanos , Deficiência Intelectual/genética , Microcefalia/genética , Mutação
5.
Blood ; 137(5): 610-623, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33538795

RESUMO

This study was conducted to determine the dosage effect of c-Myc on hematopoiesis and its distinct role in mediating the Wnt/ß-catenin pathway in hematopoietic stem cell (HSC) and bone marrow niche cells. c-Myc haploinsufficiency led to ineffective hematopoiesis by inhibiting HSC self-renewal and quiescence and by promoting apoptosis. We have identified Nr4a1, Nr4a2, and Jmjd3, which are critical for the maintenance of HSC functions, as previously unrecognized downstream targets of c-Myc in HSCs. c-Myc directly binds to the promoter regions of Nr4a1, Nr4a2, and Jmjd3 and regulates their expression. Our results revealed that Nr4a1 and Nr4a2 mediates the function of c-Myc in regulating HSC quiescence, whereas all 3 genes contribute to the function of c-Myc in the maintenance of HSC survival. Adenomatous polyposis coli (Apc) is a negative regulator of the Wnt/ß-catenin pathway. We have provided the first evidence that Apc haploinsufficiency induces a blockage of erythroid lineage differentiation through promoting secretion of IL6 in bone marrow endothelial cells. We found that c-Myc haploinsufficiency failed to rescue defective function of Apc-deficient HSCs in vivo but it was sufficient to prevent the development of severe anemia in Apc-heterozygous mice and to significantly prolong the survival of those mice. Furthermore, we showed that c-Myc-mediated Apc loss induced IL6 secretion in endothelial cells, and c-Myc haploinsufficiency reversed the negative effect of Apc-deficient endothelial cells on erythroid cell differentiation. Our studies indicate that c-Myc has a context-dependent role in mediating the function of Apc in hematopoiesis.


Assuntos
Genes myc , Hematopoese/fisiologia , Proteínas Proto-Oncogênicas c-myb/fisiologia , Proteína da Polipose Adenomatosa do Colo/fisiologia , Anemia/genética , Anemia/prevenção & controle , Animais , Apoptose/fisiologia , Transplante de Medula Óssea , Autorrenovação Celular/fisiologia , Ensaio de Unidades Formadoras de Colônias , Células Endoteliais/patologia , Células Eritroides/patologia , Deleção de Genes , Genes APC , Haploinsuficiência , Hematopoese/genética , Células-Tronco Hematopoéticas , Interleucina-6/fisiologia , Histona Desmetilases com o Domínio Jumonji/fisiologia , Camundongos Mutantes , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , Poli I-C/farmacologia , Quimera por Radiação , Via de Sinalização Wnt/fisiologia
6.
Blood ; 138(26): 2838-2852, 2021 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-34255814

RESUMO

YTHDC1 has distinct functions as a nuclear N6-methyladenosine (m6A) reader in regulating RNA metabolism. Here we show that YTHDC1 is overexpressed in acute myeloid leukemia (AML) and that it is required for the proliferation and survival of human AML cells. Genetic deletion of Ythdc1 markedly blocks AML development and maintenance as well as self-renewal of leukemia stem cells (LSCs) in vivo in mice. We found that Ythdc1 is also required for normal hematopoiesis and hematopoietic stem and progenitor cell (HSPC) maintenance in vivo. Notably, Ythdc1 haploinsufficiency reduces self-renewal of LSCs but not HSPCs in vivo. YTHDC1 knockdown has a strong inhibitory effect on proliferation of primary AML cells. Mechanistically, YTHDC1 regulates leukemogenesis through MCM4, which is a critical regulator of DNA replication. Our study provides compelling evidence that shows an oncogenic role and a distinct mechanism of YTHDC1 in AML.


Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Proteínas de Manutenção de Minicromossomo/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Adenosina/análogos & derivados , Adenosina/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Replicação do DNA , Humanos , Camundongos Transgênicos , Componente 4 do Complexo de Manutenção de Minicromossomo/genética , Regulação para Cima
7.
Nucleic Acids Res ; 49(10): 5779-5797, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34048572

RESUMO

Faithful genome integrity maintenance plays an essential role in cell survival. Here, we identify the RNA demethylase ALKBH5 as a key regulator that protects cells from DNA damage and apoptosis during reactive oxygen species (ROS)-induced stress. We find that ROS significantly induces global mRNA N6-methyladenosine (m6A) levels by modulating ALKBH5 post-translational modifications (PTMs), leading to the rapid and efficient induction of thousands of genes involved in a variety of biological processes including DNA damage repair. Mechanistically, ROS promotes ALKBH5 SUMOylation through activating ERK/JNK signaling, leading to inhibition of ALKBH5 m6A demethylase activity by blocking substrate accessibility. Moreover, ERK/JNK/ALKBH5-PTMs/m6A axis is activated by ROS in hematopoietic stem/progenitor cells (HSPCs) in vivo in mice, suggesting a physiological role of this molecular pathway in the maintenance of genome stability in HSPCs. Together, our study uncovers a molecular mechanism involving ALKBH5 PTMs and increased mRNA m6A levels that protect genomic integrity of cells in response to ROS.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Dano ao DNA , Reparo do DNA , Espécies Reativas de Oxigênio/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Desmetilação/efeitos dos fármacos , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metilação/efeitos dos fármacos , Camundongos , Fosforilação , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA-Seq , Sumoilação/efeitos dos fármacos , Espectrometria de Massas em Tandem , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo
9.
Brief Bioinform ; 19(4): 627-635, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28203711

RESUMO

Long noncoding RNAs (lncRNAs) are a large family of noncoding RNAs that play a critical role in various normal bioprocesses as well as tumorigenesis. However, the expression patterns and biological functions of lncRNAs in acute leukemia have not been well studied. Here, we performed transcriptome-wide lncRNA expression profiling of acute myeloid leukemia (AML) patient samples, along with non-leukemia control hematopoietic samples. We found that lncRNAs were differentially expressed in AML samples relative to control samples. Notably, we identified that lncRNAs upregulated in AML (relative to the control samples) are associated with a lower degree of DNA methylation and a higher ratio of being bound by transcription factors such as SP1, STAT4, ATF-2 and ELK-1 compared with those downregulated in AML. Moreover, an enrichment of H3K4me3 and a depletion of H3K27me3 were observed in upregulated lncRNAs in AML. Expression patterns of three types of lncRNAs (antisense, enhancer and intergenic lncRNAs) have previously been characterized. Of the identified lncRNAs, we found that high expression level lncRNA LOC285758 is associated with the poor prognosis in AML patients. Furthermore, we found that LOC285758 regulates proliferation of AML cell lines by enhancing the expression of HDAC2, a key factor in carcinogenesis. Collectively, our study depicts a landscape of important lncRNAs in AML and provides novel potential therapeutic targets and prognostic markers for AML treatment.


Assuntos
Regulação Neoplásica da Expressão Gênica , Histona Desacetilase 2/metabolismo , Leucemia Mieloide Aguda/genética , RNA Longo não Codificante/genética , Transcriptoma , Estudos de Casos e Controles , Histona Desacetilase 2/genética , Humanos , Células Tumorais Cultivadas
10.
Circ Res ; 123(1): 43-56, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29794022

RESUMO

RATIONALE: Microvascular inflammation and endothelial dysfunction secondary to unchecked activation of endothelium play a critical role in the pathophysiology of sepsis and organ failure. The intrinsic signaling mechanisms responsible for dampening excessive activation of endothelial cells are not completely understood. OBJECTIVE: To determine the central role of YAP (Yes-associated protein), the major transcriptional coactivator of the Hippo pathway, in modulating the strength and magnitude of endothelial activation and vascular inflammation. METHODS AND RESULTS: Endothelial-specific YAP knockout mice showed increased basal expression of E-selectin and ICAM (intercellular adhesion molecule)-1 in endothelial cells, a greater number of adherent neutrophils in postcapillary venules and increased neutrophil counts in bronchoalveolar lavage fluid. Lipopolysaccharide challenge of these mice augmented NF-κB (nuclear factor-κB) activation, expression of endothelial adhesion proteins, neutrophil and monocyte adhesion to cremaster muscle venules, transendothelial neutrophil migration, and lung inflammatory injury. Deletion of YAP in endothelial cells also markedly augmented the inflammatory response and cardiovascular dysfunction in a polymicrobial sepsis model induced by cecal ligation and puncture. YAP functioned by interacting with the E3 ubiquitin-protein ligase TLR (Toll-like receptor) signaling adaptor TRAF6 (tumor necrosis factor receptor-associated factor 6) to ubiquitinate TRAF6, and thus promoted TRAF6 degradation and modification resulting in inhibition of NF-κB activation. TRAF6 depletion in endothelial cells rescued the augmented inflammatory phenotype in mice with endothelial cell-specific deletion of YAP. CONCLUSIONS: YAP modulates the activation of endothelial cells and suppresses vascular inflammation through preventing TRAF6-mediated NF-κB activation and is hence essential for limiting the severity of sepsis-induced inflammation and organ failure.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Células Endoteliais/fisiologia , Endotélio Vascular/fisiopatologia , Fosfoproteínas/fisiologia , Fator 6 Associado a Receptor de TNF/metabolismo , Vasculite/etiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Permeabilidade Capilar , Adesão Celular , Proteínas de Ciclo Celular , Selectina E/metabolismo , Células Endoteliais/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Molécula 1 de Adesão Intercelular/metabolismo , Contagem de Leucócitos , Camundongos , Camundongos Knockout , Microvasos , Monócitos/fisiologia , NF-kappa B/metabolismo , Neutrófilos/citologia , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Sepse/complicações , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Vênulas/citologia , Proteínas de Sinalização YAP
11.
Mol Cancer ; 18(1): 30, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30813931

RESUMO

Ectopic Viral Integration site 1 (EVI1) upregulation is implicated in 10-25% of pediatric acute myeloid leukemia (AML) and has an inferior outcome with current chemotherapy regimens. Here we report that EVI1 upregulation is associated with methylation of the miR-9 promoter and correlated with downregulation of miR-9 in human AML cell lines and bone marrow (BM) cells from pediatric patients. Reactivation of miR-9 by hypomethylating agents and forced expression of miR-9 in EVI1high leukemia cell lines and primary leukemia cells results in apoptosis and decreased proliferation of EVI1high leukemia cells. Furthermore, re-expression of miR-9 delays disease progression in EVI1high leukemia-xenograft mice. Our results suggest that EVI1-induced hypermethylation and downregulation of the miR-9 plays an important role in leukemogenesis in EVI-1high pediatric AML, indicating that hypomethylating agents may be a potential therapeutic strategy for EVI1high pediatric AML.


Assuntos
Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Proteína do Locus do Complexo MDS1 e EVI1/genética , MicroRNAs/genética , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células , Criança , Metilação de DNA/efeitos dos fármacos , Decitabina/farmacologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Proteína do Locus do Complexo MDS1 e EVI1/metabolismo , Camundongos , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Stem Cells ; 36(7): 1097-1108, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29575305

RESUMO

Multiple functions have been proposed for transcription factor FoxM1, including the regulation of cell proliferation, differentiation, senescence, apoptosis, and tissue homeostasis. However, the role of FoxM1 in muscle satellite cells (SCs) remains unclear. In the present study, we demonstrated that FoxM1 was essential for the proliferation and survival of SCs. Crucially, we found that long noncoding RNAs (lncRNAs) Snhg8 and Gm26917 significantly regulated the proliferation and apoptosis of SCs, respectively, and these lncRNAs were directly regulated by FoxM1 in SCs. Mechanistically, Snhg8 sustained SCs proliferation by promoting the transcription of ribosomal proteins, while Gm26917 acted as a competing endogenous RNA for microRNA-29b, which accelerated apoptosis of SCs. In mice, conditional knockout of FoxM1 in skeletal muscle resulted in decreased proliferation and increased apoptosis of SCs. Thus, our studies revealed a previously unrecognized role of FoxM1 in SCs and uncovered two lncRNAs, Snhg8 and Gm26917, which function as novel targets of FoxM1 in the regulation of SCs proliferation and survival. Stem Cells 2018;36:1097-1108.


Assuntos
Proteína Forkhead Box M1/metabolismo , RNA Longo não Codificante/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Animais , Apoptose , Proliferação de Células , Sobrevivência Celular , Humanos , Camundongos
13.
Am J Respir Crit Care Med ; 198(6): 788-802, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29664678

RESUMO

RATIONALE: Angioproliferative vasculopathy is a hallmark of pulmonary arterial hypertension (PAH). However, little is known about how endothelial cell (EC) and smooth muscle cell (SMC) crosstalk regulates the angioproliferative vascular remodeling. OBJECTIVES: To investigate the role of EC and SMC interaction and underlying signaling pathways in pulmonary hypertension (PH) development. METHODS: SMC-specific Foxm1 (forkhead box M1) or Cxcr4 knockout mice, EC-specific Foxm1 or Egln1 knockout mice, and EC-specific Egln1/Cxcl12 double knockout mice were used to assess the role of FoxM1 on SMC proliferation and PH. Lung tissues and cells from patients with PAH were used to validate clinical relevance. FoxM1 inhibitor thiostrepton was used in Sugen 5416/hypoxia- and monocrotaline-challenged rats. MEASUREMENTS AND MAIN RESULTS: FoxM1 expression was markedly upregulated in lungs and pulmonary arterial SMCs of patients with idiopathic PAH and four discrete PH rodent models. Mice with SMC- (but not EC-) specific deletion of Foxm1 were protected from hypoxia- or Sugen 5416/hypoxia-induced PH. The upregulation of FoxM1 in SMCs induced by multiple EC-derived factors (PDGF-B, CXCL12, ET-1, and MIF) mediated SMC proliferation. Genetic deletion of endothelial Cxcl12 in Egln1Tie2Cre mice or loss of its cognate receptor Cxcr4 in SMCs in hypoxia-treated mice inhibited FoxM1 expression, SMC proliferation, and PH. Accordingly, pharmacologic inhibition of FoxM1 inhibited severe PH in both Sugen 5416/hypoxia and monocrotaline-challenged rats. CONCLUSIONS: Multiple factors derived from dysfunctional ECs induced FoxM1 expression in SMCs and activated FoxM1-dependent SMC proliferation, which contributes to pulmonary vascular remodeling and PH. Thus, targeting FoxM1 signaling represents a novel strategy for treatment of idiopathic PAH.


Assuntos
Endotélio Vascular/fisiopatologia , Proteína Forkhead Box M1/fisiologia , Hipertensão Pulmonar/patologia , Músculo Liso Vascular/fisiopatologia , Remodelação Vascular , Animais , Endotélio Vascular/metabolismo , Proteína Forkhead Box M1/metabolismo , Humanos , Hipertensão Pulmonar/metabolismo , Camundongos , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Transdução de Sinais
15.
Proc Natl Acad Sci U S A ; 110(14): 5594-9, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23509296

RESUMO

MicroRNA-9 (miR-9) is emerging as a critical regulator of organ development and neurogenesis. It is also deregulated in several types of solid tumors; however, its role in hematopoiesis and leukemogenesis is not yet known. Here we show that miR-9 is detected in hematopoietic stem cells and hematopoietic progenitor cells, and that its expression increases during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis and promotes apoptosis in vitro and in vivo. Conversely, in hematopoietic progenitor cells, the inhibition of miR-9 with a miRNA sponge blocks myelopoiesis. Ecotropic viral integration site 1 (EVI1), required for normal embryogenesis, is considered an oncogene because its inappropriate up-regulation induces malignant transformation in solid and hematopoietic cancers. Here we show that EVI1 binds to the promoter of miR-9-3, leading to DNA hypermethylation of the promoter and repression of miR-9. Moreover, miR-9 expression reverses a myeloid differentiation block that is induced by EVI1. Our findings indicate that EVI1, when inappropriately expressed, delays or blocks myeloid differentiation at least in part by DNA hypermethylation and down-regulation of miR-9. It was reported that Forkhead box class O genes (FoxOs) inhibit myeloid differentiation and prevent differentiation of leukemia-initiating cells. Here we identify both FoxO1 and FoxO3 as direct targets of miR-9 in hematopoietic cells and find that up-regulation of FoxO3 inhibits miR-9-induced myelopoiesis. These results reveal a unique role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms and provide insights into the epigenetic regulation of miR9 in tumorigenesis.


Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/metabolismo , Mielopoese/fisiologia , Fatores de Transcrição/metabolismo , Animais , Imunoprecipitação da Cromatina , Ensaio de Unidades Formadoras de Colônias , Primers do DNA/genética , Citometria de Fluxo , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Células HEK293 , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Mielopoese/genética , Células NIH 3T3 , Proto-Oncogenes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA
16.
Blood ; 121(20): 4063-72, 2013 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-23547052

RESUMO

Emerging evidence suggests that adenomatous polyposis coli (Apc) plays a critical role in the maintenance of hematopoietic stem/progenitor cells (HSCs/HPCs). The molecular pathways responsible for the function of Apc in HSCs/HPCs remain unclear. By genetic approach, we demonstrated that inactivation of ß-catenin rescued the exhaustion of Apc-deficient HSCs/HPCs, thereby preventing bone marrow failure in Apc-deficient mice. ß-catenin loss inhibited the excessive proliferation and apoptosis of Apc-deficient HSCs/HPCs, as well as their defects in myeloid and erythroid differentiation. In addition, loss of ß-catenin reversed the down-regulation of Cdkn1a, Cdkn1b, and Mcl1 induced by Apc ablation in Lin(-)Sca(+)c-Kit(+). In assays of long-term stem cell function, the HSCs with deficiency of both Apc and ß-catenin displayed a significantly enhanced self-renewal capacity compared with ß-catenin-deficient and control HSCs. Our findings suggest that Apc regulates the survival, proliferation, and differentiation of HSCs/HPCs largely through a ß-catenin-mediated pathway. They also indicate that multiple downstream targets of Apc including ß-catenin may coordinately regulate HSC self-renewal.


Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Células-Tronco Hematopoéticas/fisiologia , beta Catenina/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Deleção de Genes , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , beta Catenina/genética , beta Catenina/metabolismo
17.
Stem Cells ; 32(7): 1855-64, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24578354

RESUMO

Adult stem cell treatment is a potential novel therapeutic approach for acute respiratory distress syndrome. Given the extremely low rate of cell engraftment, it is believed that these cells exert their beneficial effects via paracrine mechanisms. However, the endogenous mediator(s) in the pulmonary vasculature remains unclear. Using the mouse model with endothelial cell (EC)-restricted disruption of FoxM1 (FoxM1 CKO), here we show that endothelial expression of the reparative transcriptional factor FoxM1 is required for the protective effects of bone marrow progenitor cells (BMPC) against LPS-induced inflammatory lung injury and mortality. BMPC treatment resulted in rapid induction of FoxM1 expression in wild type (WT) but not FoxM1 CKO lungs. BMPC-induced inhibition of lung vascular injury, resolution of lung inflammation, and survival, as seen in WT mice, were abrogated in FoxM1 CKO mice following LPS challenge. Mechanistically, BMPC treatment failed to induce lung EC proliferation in FoxM1 CKO mice, which was associated with impaired expression of FoxM1 target genes essential for cell cycle progression. We also observed that BMPC treatment enhanced endothelial barrier function in WT but not in FoxM1-deficient EC monolayers. Restoration of ß-catenin expression in FoxM1-deficient ECs normalized endothelial barrier enhancement in response to BMPC treatment. These data demonstrate the requisite role of endothelial FoxM1 in the mechanism of BMPC-induced vascular repair to restore vascular integrity and accelerate resolution of inflammation, thereby promoting survival following inflammatory lung injury.


Assuntos
Fatores de Transcrição Forkhead/fisiologia , Pulmão/irrigação sanguínea , Células-Tronco Mesenquimais/fisiologia , Síndrome do Desconforto Respiratório/terapia , Animais , Permeabilidade Capilar , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Proteína Forkhead Box M1 , Lipopolissacarídeos/farmacologia , Pulmão/imunologia , Pulmão/patologia , Masculino , Transplante de Células-Tronco Mesenquimais , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regeneração , Ativação Transcricional
18.
Haematologica ; 99(6): 1032-40, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24532040

RESUMO

The control of mRNA stability plays a central role in orchestrating gene-regulatory networks in hematopoietic cell growth, differentiation and tumorigenesis. HNRNPA0, which encodes an RNA-binding protein shown to regulate transcript stability via binding to the AU-rich elements of mRNAs, is located within the commonly deleted segment of 5q31.2 in myeloid neoplasms with a del(5q), and is expressed at haploinsufficient levels in these patients. We show that HNRNPA0 is normally highly expressed in hematopoietic stem cells and exhibits dynamic changes in expression during the course of differentiation. To model HNRNPA0 haploinsufficiency, we used RNAi interference in primary murine cells and an experimental cell system, and found that reduced Hnrnpa0 expression leads to a shift from monocytic towards granulocytic differentiation. Microarray-based global expression profiling revealed that Hnrnpa0 knockdown disproportionally impacts AU-rich containing transcripts and alters expression of myeloid specification genes. In therapy-related myeloid neoplasms with a del(5q), AU-rich containing mRNAs are enriched in transcripts that encode proteins associated with increased growth and proliferation. Our findings implicate haploinsufficiency of HNRNPA0 as one of the key initiating mutations in the pathogenesis of myeloid neoplasms with a del(5q), and suggest that therapies that target AU-rich elements warrant consideration in efforts to develop new mechanism-based treatment strategies.


Assuntos
Sequência Rica em At , Deleção Cromossômica , Cromossomos Humanos Par 5 , Ribonucleoproteínas Nucleares Heterogêneas/genética , Células Mieloides/metabolismo , Transcrição Gênica , Animais , Linhagem Celular , Transdiferenciação Celular/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Granulócitos/citologia , Granulócitos/metabolismo , Hematopoese/genética , Humanos , Leucemia Mieloide/genética , Camundongos , Camundongos Knockout , Monócitos/citologia , Monócitos/metabolismo , Segunda Neoplasia Primária/genética
19.
STAR Protoc ; 5(1): 102855, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38300798

RESUMO

RNA-binding proteins (RBPs) regulate gene expression both co-transcriptionally and post-transcriptionally. Here, we provide a protocol for photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation followed by next-generation sequencing (PAR-CLIP-seq). PAR-CLIP-seq is a transcriptome-scale technique for identifying in vivo binding sites of RBPs at the single-nucleotide level. We detail procedures for the establishment of FLAG-RBM33 stable cell line, the sequencing library preparation, and the data analysis.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Ligação a RNA , Humanos , Células HEK293 , Sítios de Ligação , Proteínas de Ligação a RNA/metabolismo , Transcriptoma
20.
Genes Dis ; 11(1): 382-396, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37588203

RESUMO

As the most common internal modification of mRNA, N6-methyladenosine (m6A) and its regulators modulate gene expression and play critical roles in various biological and pathological processes including tumorigenesis. It was reported previously that m6A methyltransferase (writer), methyltransferase-like 3 (METTL3) adds m6A in primary microRNAs (pri-miRNAs) and facilitates its processing into precursor miRNAs (pre-miRNAs). However, it is unknown whether m6A modification also plays a role in the maturation process of pre-miRNAs and (if so) whether such a function contributes to tumorigenesis. Here, we found that YTHDF2 is aberrantly overexpressed in acute myeloid leukemia (AML) patients, especially in relapsed patients, and plays an oncogenic role in AML. Moreover, YTHDF2 promotes expression of miR-126-3p (also known as miR-126, as it is the main product of precursor miR-126 (pre-miR-126)), a miRNA that was reported as an oncomiRNA in AML, through facilitating the processing of pre-miR-126 into mature miR-126. Mechanistically, YTHDF2 recognizes m6A modification in pre-miR-126 and recruits AGO2, a regulator of pre-miRNA processing, to promote the maturation of pre-miR-126. YTHDF2 positively and negatively correlates with miR-126 and miR-126's downstream target genes, respectively, in AML patients, and forced expression of miR-126 could largely rescue YTHDF2/Ythdf2 depletion-mediated suppression on AML cell growth/proliferation and leukemogenesis, indicating that miR-126 is a functionally important target of YTHDF2 in AML. Overall, our studies not only reveal a previously unappreciated YTHDF2/miR-126 axis in AML and highlight the therapeutic potential of targeting this axis for AML treatment, but also suggest that m6A plays a role in pre-miRNA processing that contributes to tumorigenesis.

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