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1.
Mol Cell Biol ; 39(22)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31501274

RESUMO

GATA factors GATA1 and GATA2 and ETS factor PU.1 are known to function antagonistically during hematopoietic development. In mouse mast cells, however, these factors are coexpressed and activate the expression of the Ms4a2 gene encoding the ß chain of the high-affinity IgE receptor (FcεRI). The present study showed that these factors cooperatively regulate Ms4a2 gene expression through distinct mechanisms. Although GATA2 and PU.1 contributed almost equally to Ms4a2 gene expression, gene ablation experiments revealed that simultaneous knockdown of both factors showed neither a synergistic nor an additive effect. A chromatin immunoprecipitation analysis showed that they shared DNA binding to the +10.4-kbp region downstream of the Ms4a2 gene with chromatin looping factor LDB1, whereas the proximal -60-bp region was exclusively bound by GATA2 in a mast cell-specific manner. Ablation of PU.1 significantly reduced the level of GATA2 binding to both the +10.4-kbp and -60-bp regions. Surprisingly, the deletion of the +10.4-kbp region by genome editing completely abolished the Ms4a2 gene expression as well as the cell surface expression of FcεRI. These results suggest that PU.1 and LDB1 play central roles in the formation of active chromatin structure whereas GATA2 directly activates the Ms4a2 promoter.


Assuntos
Fator de Transcrição GATA2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de IgE/genética , Transativadores/metabolismo , Animais , Células da Medula Óssea/citologia , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição GATA2/genética , Regulação da Expressão Gênica , Proteínas com Domínio LIM/metabolismo , Mastócitos/metabolismo , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Receptores de IgE/metabolismo , Transativadores/genética
2.
Int J Mol Sci ; 20(18)2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31533351

RESUMO

Mast cell tryptases have crucial roles in allergic and inflammatory diseases. The mouse tryptase genes represent a cluster of loci on chromosome 16p3.3. While their functional studies have been extensively performed, transcriptional regulation of tryptase genes is poorly understood. In this study, we examined the molecular basis of the tryptase gene expression in bone marrow-derived mast cells (BMMCs) of C57BL/6 mice and in MEDMC-BRC6 mast cells. The expression of the Tpsb2 and Tpsg1 genes, which reside at the 3'-end of the tryptase locus, is significantly decreased by the reduction of the GATA transcription factors GATA1 or GATA2. Chromatin immunoprecipitation assays have shown that the GATA factors bind at multiple regions within the locus, including 1.0 and 72.8 kb upstream of the Tpsb2 gene, and that GATA1 and GATA2 facilitate each other's DNA binding activity to these regions. Deletion of the -72.8 kb region by genome editing significantly reduced the Tpsb2 and Tpsg1 mRNA levels in MEDMC-BRC6 cells. Furthermore, binding of CTCF and the cohesin subunit Rad21 was found upstream of the -72.8 kb region and was significantly reduced in the absence of GATA1. These results suggest that mouse tryptase gene expression is coordinately regulated by GATA1 and GATA2 in BMMCs.


Assuntos
Células da Medula Óssea/metabolismo , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica , Mastócitos/metabolismo , Triptases/genética , Animais , Técnicas de Inativação de Genes , Loci Gênicos , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno/genética , Triptases/metabolismo
3.
Tumour Biol ; 41(7): 1010428318824815, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31296150

RESUMO

GATA2 is a pioneering transcription factor governing androgen receptor expression and signaling in prostate cells. To understand the prognostic potential of GATA2 assessment in prostate cancer, we analyzed nuclear GATA2 expression on an annotated tissue microarray with 12,427 prostate cancer samples. Normal prostate glands were negative to weakly positive. GATA2 staining was found in almost all prostate cancers (95%). Strong GATA2 staining was linked to advanced tumor stage, high classical and quantitative Gleason grade (p < 0.0001 each), positive nodal stage (p = 0.0116), and early biochemical recurrence (p < 0.0001). GATA2 was linked to ERG-fusion-type cancers, with strong GATA2 staining in 29% of ERG-negative and 53% of ERG-positive cancers (p < 0.0001). Separate calculations in 3854 cancers with and 4768 cancers without TMPRSS2:ERG fusion revealed that these associations with tumor phenotype and patient outcome were largely driven by the subset of ERG-negative tumors. GATA2 expression was further linked to androgen receptor expression: Only 8% of androgen receptor-negative, but 56% of strongly androgen receptor expressing cancers had strong GATA2 expression (p < 0.0001). In conclusion, the results of our study demonstrate that increasing GATA2 levels are linked to prostate cancer progression and aggressiveness. The prognostic value of GATA2 is remarkable in ERG-negative cancers. However, the upregulation of GATA2 in ERG-positive cancers makes it unsuitable as a prognostic marker in this patient subset.


Assuntos
Fator de Transcrição GATA2/metabolismo , Neoplasias da Próstata/metabolismo , Adulto , Idoso , Progressão da Doença , Intervalo Livre de Doença , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Invasividade Neoplásica , Recidiva Local de Neoplasia , Fenótipo , Prognóstico , Prostatectomia , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Análise Serial de Tecidos , Regulador Transcricional ERG/metabolismo , Resultado do Tratamento
4.
Exp Hematol ; 75: 53-63.e11, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31260717

RESUMO

Currently, there is a growing need for culturing hematopoietic stem/progenitor cells (HSPCs) in vitro for various clinical applications including gene therapy. Compared with cord blood (CB) CD34+ HSPCs, it is more challenging to maintain or expand CD34+ peripheral blood mobilized stem/progenitor cells (PBSCs) ex vivo. To fill this knowledge gap, we have systematically surveyed 466 small-molecule drug compounds for their potential in cytokine-dependent expansion of human CD34+CD90+ HSPCs. We found that epigenetic modifiers, especially histone deacetylase inhibitors (HDACis), could preferentially maintain and expand these cells. In particular, treatment of CD34+ PBSCs with a single dose of HDACi trichostatin A (TSA) at a concentration of 50 nmol/L ex vivo yielded the greatest expansion (11.7-fold) of CD34+CD90+ cells when compared with the control (dimethyl sulfoxide [DMSO] plus cytokines) group. Additionally, TSA-treated PBSC CD34+ cells had a statistically significant higher engraftment rate than the control-treated group in xenotransplantation experiments. Mechanistically, TSA treatment was associated with increased expression of HSPC-related genes such as GATA2 and SALL4. Furthermore, TSA-mediated CD34+CD90+ expansion was reduced by downregulation of SALL4 but not GATA2. Overall, we have developed a robust, short-term (5-day), PBSC ex vivo maintenance/expansion culture technique and found that the HDACi-TSA/SALL4 axis is important for the biological process.


Assuntos
Técnicas de Cultura de Células , Sobrevivência de Enxerto/efeitos dos fármacos , Mobilização de Células-Tronco Hematopoéticas , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Transplante de Células-Tronco de Sangue Periférico , Células-Tronco de Sangue Periférico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Animais , Fator de Transcrição GATA2/metabolismo , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Células-Tronco de Sangue Periférico/citologia
5.
J Exp Clin Cancer Res ; 38(1): 192, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088566

RESUMO

BACKGROUND: Modulation of cell surface expression of MHC class I chain-related protein A/B (MICA/B) has been proven to be one of the mechanisms by which tumor cells escape from NK cell-mediated killing. Abnormal metabolic condition, such as high glucose, may create a cellular stress milieu to induce immune dysfunction. Hyperglycemia is frequently presented in the majority of pancreatic cancer patients and is associated with poor prognosis. In this study, we aimed to detect the effects of high glucose on NK cell-mediated killing on pancreatic cancer cells through reduction of MICA/B expression. METHODS: The lysis of NK cells on pancreatic cancer cells were compared at different glucose concentrations through lactate dehydrogenase release assay. Then, qPCR, Western Blot, Flow cytometry and Immunofluorescence were used to identify the effect of high glucose on expression of MICA/B, Bmi1, GATA2, phosphorylated AMPK to explore the underlying mechanisms in the process. Moreover, an animal model with diabetes mellitus was established to explore the role of high glucose on NK cell-mediated cytotoxicity on pancreatic cancer in vivo. RESULTS: In our study, high glucose protects pancreatic cancer from NK cell-mediated killing through suppressing MICA/B expression. Bmi1, a polycomb group (PcG) protein, was found to be up-regulated by high glucose, and mediated the inhibition of MICA/B expression through promoting GATA2 in pancreatic cancer. Moreover, high glucose inhibited AMP-activated protein kinase signaling, leading to high expression of Bmi1. CONCLUSION: Our findings identify that high glucose may promote the immune escape of pancreatic cancer cells under hyperglycemic tumor microenvironment. In this process, constitutive activation of AMPK-Bmi1-GATA2 axis could mediate MICA/B inhibition, which may serve as a therapeutic target for further intervention of pancreatic cancer immune evasion.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fator de Transcrição GATA2/metabolismo , Glucose/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Evasão Tumoral/imunologia , Animais , Glicemia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Citotoxicidade Imunológica , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Masculino , Camundongos , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/genética , Transdução de Sinais , Microambiente Tumoral
6.
Blood Cancer J ; 9(3): 33, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850577

RESUMO

The inv(16) acute myeloid leukemia-associated CBFß-MYH11 fusion is proposed to block normal myeloid differentiation, but whether this subtype of leukemia cells is poised for a unique cell lineage remains unclear. Here, we surveyed the functional consequences of CBFß-MYH11 in primary inv(16) patient blasts, upon expression during hematopoietic differentiation in vitro and upon knockdown in cell lines by multi-omics profiling. Our results reveal that primary inv(16) AML cells share common transcriptomic signatures and epigenetic determiners with megakaryocytes and erythrocytes. Using in vitro differentiation systems, we reveal that CBFß-MYH11 knockdown interferes with normal megakaryocyte maturation. Two pivotal regulators, GATA2 and KLF1, are identified to complementally occupy RUNX1-binding sites upon fusion protein knockdown, and overexpression of GATA2 partly induces a gene program involved in megakaryocyte-directed differentiation. Together, our findings suggest that in inv(16) leukemia, the CBFß-MYH11 fusion inhibits primed megakaryopoiesis by attenuating expression of GATA2/KLF1 and interfering with a balanced transcriptional program involving these two factors.


Assuntos
Fator de Transcrição GATA2/metabolismo , Regulação Leucêmica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Megacariócitos/metabolismo , Proteínas de Fusão Oncogênica/genética , Sítios de Ligação , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Epigênese Genética , Células Eritroides/citologia , Células Eritroides/metabolismo , Eritropoese/genética , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Megacariócitos/citologia , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica , Trombopoese , Transcrição Genética
7.
Development ; 146(4)2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30760481

RESUMO

Specification of neurons in the spinal cord relies on extrinsic and intrinsic signals, which in turn are interpreted by expression of transcription factors. V2 interneurons develop from the ventral aspects of the spinal cord. We report here a novel neuronal V2 subtype, named V2s, in zebrafish embryos. Formation of these neurons depends on the transcription factors sox1a and sox1b. They develop from common gata2a- and gata3-dependent precursors co-expressing markers of V2b and V2s interneurons. Chemical blockage of Notch signalling causes a decrease in V2s and an increase in V2b cells. Our results are consistent with the existence of at least two types of precursor arranged in a hierarchical manner in the V2 domain. V2s neurons grow long ipsilateral descending axonal projections with a short branch at the ventral midline. They acquire a glycinergic neurotransmitter type during the second day of development. Unilateral ablation of V2s interneurons causes a delay in touch-provoked escape behaviour, suggesting that V2s interneurons are involved in fast motor responses.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Interneurônios/metabolismo , Neurônios Motores/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Medula Espinal/metabolismo , Peixe-Zebra/embriologia , Animais , Comportamento Animal , Fator de Transcrição GATA2/metabolismo , Genótipo , Glicina/química , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Receptores Notch/metabolismo , Transdução de Sinais , Especificidade da Espécie , Medula Espinal/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
8.
Cancer Sci ; 110(4): 1183-1193, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30710465

RESUMO

The transcription factor GATA2 regulates normal hematopoiesis, particularly in- stem cell maintenance and myeloid differentiation. Various heteroallelic GATA2 gene mutations are associated with a variety of hematological neoplasms, including myelodysplastic syndromes and leukemias. Here, we report that impaired GATA2 expression induces myelodysplastic and myeloproliferative neoplasm development in elderly animals, and this neoplasm resembles chronic myelomonocytic leukemia in humans. GATA2 hypomorphic mutant (G2f GN / fGN ) mice that were generated by the germline insertion of a neocassette into the Gata2 gene locus avoided the early embryonic lethality observed in Gata2-null mice. However, adult G2f GN / fGN mice suffered from exacerbated leukocytosis concomitant with progressive anemia and thrombocytopenia and eventually developed massive granulomonocytosis accompanied by trilineage dysplasia. The reconstitution activity of G2f GN / fGN mouse stem cells was impaired. Furthermore, G2f GN / fGN progenitors showed myeloid lineage-biased proliferation and differentiation. Myeloid progenitor accumulation started at a younger age in G2f GN / fGN mice and appeared to worsen with age. G2f GN / fGN mice showed increased expression of transcripts encoding cytokine receptors, such as macrophage colony-stimulating factor receptor and interleukin-6 receptor, in granulocyte-monocyte progenitors. This increased expression could be correlated with the hypersensitive granulomonocytic proliferation reaction when the mice were exposed to lipopolysaccharide. Taken together, these observations indicate that GATA2 hypomorphism leads to a hyperreactive defense response to infections, and this reaction is attributed to a unique intrinsic cell defect in the regulation of myeloid expansion that increases the risk of hematological neoplasm transformation.


Assuntos
Fator de Transcrição GATA2/genética , Predisposição Genética para Doença , Leucemia Mielomonocítica Crônica/genética , Polimorfismo Genético , Fatores Etários , Animais , Biomarcadores , Modelos Animais de Doenças , Fator de Transcrição GATA2/metabolismo , Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mielomonocítica Crônica/metabolismo , Leucemia Mielomonocítica Crônica/patologia , Contagem de Leucócitos , Leucocitose/genética , Leucocitose/metabolismo , Leucocitose/patologia , Camundongos , Camundongos Knockout , Monócitos , RNA Mensageiro
9.
Cells ; 8(2)2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30736420

RESUMO

NFATc1, which is ubiquitous in many cell types, is the master regulator of osteoclastogenesis. However, the molecular mechanisms by which NFATc1 drives its transcriptional program to produce osteoclasts from macrophages (M) remains poorly understood. We performed quantitative PCR (QPCR) arrays and bioinformatic analyses to discover new direct and indirect NFATc1 targets. The results revealed that NFATc1 significantly modified the expression of 55 genes in untransfected cells and 31 genes after NFATc1-knockdown (≥2). Among them, we focused on 19 common genes that showed changes in the PCR arrays between the two groups of cells. Gene Ontology (GO) demonstrated that genes related to cell differentiation and the development process were significantly (p > 0.05) affected by NFATc1-knockdown. Among all the genes analyzed, we focused on GATA2, which was up-regulated in NFATc1-knockdown cells, while its expression was reduced after NFATc1 rescue. Thus, we suggest GATA2 as a new target of NFATc1. Ingenuity Pathway Analysis (IPA) identified up-regulated GATA2 and the STAT family members as principal nodes involved in cell differentiation. Mechanistically, we demonstrated that STAT6 was activated in parallel with GATA2 in NFATc1-knockdown cells. We suggest an alternative pathway for macrophage differentiation in the absence of NFATc1 due to the GATA2 transcription factor.


Assuntos
Diferenciação Celular/genética , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Macrófagos/citologia , Macrófagos/metabolismo , Fatores de Transcrição NFATC/metabolismo , Animais , Fator de Transcrição GATA2/metabolismo , Ontologia Genética , Camundongos , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese , Mapas de Interação de Proteínas , Células RAW 264.7
10.
J Clin Invest ; 129(3): 1180-1192, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30620726

RESUMO

The development and function of stem and progenitor cells that produce blood cells are vital in physiology. GATA-binding protein 2 (GATA2) mutations cause GATA-2 deficiency syndrome involving immunodeficiency, myelodysplastic syndrome, and acute myeloid leukemia. GATA-2 physiological activities necessitate that it be strictly regulated, and cell type-specific enhancers fulfill this role. The +9.5 intronic enhancer harbors multiple conserved cis-elements, and germline mutations of these cis-elements are pathogenic in humans. Since mechanisms underlying how GATA2 enhancer disease mutations impact hematopoiesis and pathology are unclear, we generated mouse models of the enhancer mutations. While a multi-motif mutant was embryonically lethal, a single-nucleotide Ets motif mutant was viable, and steady-state hematopoiesis was normal. However, the Ets motif mutation abrogated stem/progenitor cell regeneration following stress. These results reveal a new mechanism in human genetics, in which a disease predisposition mutation inactivates enhancer regenerative activity, while sparing developmental activity. Mutational sensitization to stress that instigates hematopoietic failure constitutes a paradigm for GATA-2 deficiency syndrome and other contexts of GATA-2-dependent pathogenesis.


Assuntos
Elementos Facilitadores Genéticos , Deficiência de GATA2 , Fator de Transcrição GATA2 , Mutação em Linhagem Germinativa , Hematopoese/genética , Motivos de Nucleotídeos , Regeneração/genética , Animais , Deficiência de GATA2/genética , Deficiência de GATA2/metabolismo , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Humanos , Camundongos , Camundongos Mutantes
11.
Mol Cell Endocrinol ; 483: 39-49, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30615908

RESUMO

The transcription factor GATA2 is an anti-adipogenic factor whose expression is downregulated during adipocyte differentiation. The present study attempted to clarify the molecular mechanism underlying the GATA2 repression and found that the repression is dependent on the activation of the glucocorticoid receptor (GR) during 3T3-L1 preadipocyte differentiation. Although several recognition sequences for GR were found in both the proximal and distal regions of the Gata2 locus, the promoter activity was not affected by the GR activation in the reporter assays, and the CRISPR-Cas9-mediated deletion of the two distal regions of the Gata2 locus was not involved in the GR-mediated Gata2 repression. Notably, the level of histone acetylation was markedly reduced at the Gata2 locus during 3T3-L1 differentiation, and the GR-mediated Gata2 repression was significantly relieved by histone deacetylase inhibition. These results suggest that GR regulates the Gata2 gene by reducing histone acetylation in the early phase of adipogenesis.


Assuntos
Adipócitos/citologia , Fator de Transcrição GATA2/genética , Histonas/metabolismo , Receptores de Glucocorticoides/metabolismo , Células 3T3-L1 , Acetilação , Adipócitos/metabolismo , Animais , Diferenciação Celular , Regulação para Baixo , Epigênese Genética , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica , Camundongos , Regiões Promotoras Genéticas , Ativação Transcricional
12.
Leukemia ; 33(3): 638-652, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30291337

RESUMO

The consequences of immune dysfunction in B-chronic lymphocytic leukemia (CLL) likely relate to the incidence of serious recurrent infections and second malignancies that plague CLL patients. The well-described immune abnormalities are not able to consistently explain these complications. Here, we report bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. Numbers of CD34+ BM hematopoietic progenitors responsive in standard colony-forming unit (CFU) assays, including CFU-GM/GEMM and CFU-E, were significantly reduced. Flow cytometry revealed corresponding reductions in frequencies of all hematopoietic stem and progenitor cell (HSPC) subsets assessed in CLL patient marrow. Consistent with the reduction in HSPCs, BM resident monocytes and natural killer cells were reduced, a deficiency recapitulated in blood. Finally, we report increases in protein levels of the transcriptional regulators HIF-1α, GATA-1, PU.1, and GATA-2 in CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Importantly, PU.1 and GATA-2 were rapidly increased when healthy HSPCs were exposed in vitro to TNFα, a cytokine constitutively produced by CLL B cells. Together, these findings reveal BM hematopoietic dysfunction in untreated CLL patients that provides new insight into the etiology of the complex immunodeficiency state in CLL.


Assuntos
Células da Medula Óssea/patologia , Medula Óssea/patologia , Leucemia Linfocítica Crônica de Células B/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD34/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Feminino , Citometria de Fluxo/métodos , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
13.
Immunol Cell Biol ; 97(2): 142-151, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30221399

RESUMO

Myeloid-derived suppressor cells (MDSCs) represent a group of immature myeloid cells composed of myeloid progenitor cells and immature myeloid cells that can negatively regulate immune responses by inhibiting T-cell function. In mice, MDSCs are broadly defined by the expression of CD11b and Gr1. We and others have shown that injection of a lethal or sublethal dose of lipopolysaccharide (LPS) into mice could result in the expansion of MDSCs in the bone marrow (BM), spleen and blood. Until now, the molecular mechanisms responsible for this expansion are poorly studied; specifically, the roles of the individual microRNAs (miRNAs) which may be involved remain largely unknown. We performed microarray analysis to compare the miRNA expression profiles of CD11b+ Gr1+ cells sorted from the BM of LPS-injected and phosphate-buffered saline-injected mice. We identified let-7e, which was highly upregulated in the LPS-treated group, as a potent regulator of LPS-induced MDSC expansion. Furthermore, let-7e overexpression in BM chimeric mice led to a noticeable increase in the population of CD11b+ Gr1+ cells, which resulted from reduced cellular apoptosis. Further studies showed that let-7e could directly target caspase-3 to inhibit cell apoptosis, and upregulation of let-7e in LPS-stimulated MDSCs could be due to the relieved repression of let-7e transcription exerted by downregulated GATA2. Our findings suggest that LPS expands MDSCs by inhibiting apoptosis through the regulation of the GATA2/let-7e axis.


Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Fator de Transcrição GATA2/imunologia , Fator de Transcrição GATA2/metabolismo , MicroRNAs/imunologia , MicroRNAs/metabolismo , Células Supressoras Mieloides/metabolismo , Animais , Apoptose/efeitos dos fármacos , Medula Óssea/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Células Supressoras Mieloides/imunologia
14.
Stem Cells ; 37(3): 430-440, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30537419

RESUMO

Previously, we reported that although the HSPC frequency in bone marrow cells (BMC) was comparable between ß2-/- and ß2+/+ mice, transplantation of ß2-/- BMC into lethally irradiated CD45.1 recipient resulted in more myeloid cell production than ß2+/+ BMC. The objective of this study is to address if integrin ß2 deficiency skews granulocyte/macrophage progenitor (GMP) proliferation. FACS analysis demonstrated that GMP frequency and cell number were higher and megakaryocyte/erythrocyte progenitor frequency and cell number were lower in ß2-/- mice than ß2+/+ mice. However, the common myeloid progenitors (CMP) frequency and cell number were similar between the two groups. The increased GMP number was due to GMP proliferation as evidenced by the percentage of BrdU-incorporating GMP. Whole genome transcriptome analysis identified increased FcεRIα expression in ß2-/- CMP compared to ß2+/+ CMP. FcεRIα expression on ß2-/- GMP was detected increased in ß2-/- mice by qRT-PCR and FACS. Although transplantation of FcεRIαhi GMP or FcεRIαlo GMP into lethally irradiated CD45.1 recipient resulted in comparable myeloid cell production, transplantation of ß2 deficient FcεRIαhi GMP generated more myeloid cells than ß2+/+ FcεRIαhi GMP. GATA2 expression was increased in ß2-/- GMP. Using a luciferase reporter assay, we demonstrated that mutation of the GATA2 binding site in the FcεRIα promoter region diminished FcεRIα transcription. In vitro, the addition of IgE, the ligand of FcεRIα, promoted GMP expansion, which was abrogated by inhibition of JNK phosphorylation. Integrin ß2 deficiency promoted GMP proliferation and myeloid cell production, which was mediated via FcεRIα/IgE-induced JNK phosphorylation in GMP. Stem Cells 2019;37:430-440.


Assuntos
Antígenos CD18/metabolismo , Proliferação de Células , Células Progenitoras de Granulócitos e Macrófagos/metabolismo , Animais , Antígenos CD18/genética , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica , MAP Quinase Quinase 4 , Camundongos , Camundongos Knockout , Receptores de IgE/biossíntese , Receptores de IgE/genética , Transcrição Genética
15.
Cell Rep ; 25(10): 2821-2835.e7, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30517869

RESUMO

During development, hematopoietic stem and progenitor cells (HSPCs) arise from specialized endothelial cells by a process termed endothelial-to-hematopoietic transition (EHT). The genetic program driving human HSPC emergence remains largely unknown. We previously reported that the generation of hemogenic precursor cells from mouse fibroblasts recapitulates developmental hematopoiesis. Here, we demonstrate that human fibroblasts can be reprogrammed into hemogenic cells by the same transcription factors. Induced cells display dynamic EHT transcriptional programs, generate hematopoietic progeny, possess HSPC cell surface phenotype, and repopulate immunodeficient mice for 3 months. Mechanistically, GATA2 and GFI1B interact and co-occupy a cohort of targets. This cooperative binding is reflected by engagement of open enhancers and promoters, initiating silencing of fibroblast genes and activating the hemogenic program. However, GATA2 displays dominant and independent targeting activity during the early phases of reprogramming. These findings shed light on the processes controlling human HSC specification and support generation of reprogrammed HSCs for clinical applications.


Assuntos
Reprogramação Celular , Hemangioblastos/citologia , Hemangioblastos/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Sequência de Bases , Elementos Facilitadores Genéticos/genética , Fibroblastos/metabolismo , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Recém-Nascido , Fenótipo , Regiões Promotoras Genéticas/genética , Ligação Proteica
16.
Cell Rep ; 25(3): 571-584.e5, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30332639

RESUMO

Wnt/ß-catenin signaling is necessary for lymphatic vascular development. Oscillatory shear stress (OSS) enhances Wnt/ß-catenin signaling in cultured lymphatic endothelial cells (LECs) to induce expression of the lymphedema-associated transcription factors GATA2 and FOXC2. However, the mechanisms by which OSS regulates Wnt/ß-catenin signaling and GATA2 and FOXC2 expression are unknown. We show that OSS activates autocrine Wnt/ß-catenin signaling in LECs in vitro. Tissue-specific deletion of Wntless, which is required for the secretion of Wnt ligands, reveals that LECs and vascular smooth muscle cells are complementary sources of Wnt ligands that regulate lymphatic vascular development in vivo. Further, the LEC master transcription factor PROX1 forms a complex with ß-catenin and the TCF/LEF transcription factor TCF7L1 to enhance Wnt/ß-catenin signaling and promote FOXC2 and GATA2 expression in LECs. Thus, our work defines Wnt sources, reveals that PROX1 directs cell fate by acting as a Wnt signaling component, and dissects the mechanisms of PROX1 and Wnt synergy.


Assuntos
Células Endoteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Músculo Liso Vascular/citologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Células Cultivadas , Células Endoteliais/metabolismo , Feminino , Fatores de Transcrição Forkhead/metabolismo , Fator de Transcrição GATA2/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Via de Sinalização Wnt
17.
Nat Commun ; 9(1): 4421, 2018 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-30356064

RESUMO

Mammalian pregnancy depends on the ability of the uterus to support embryo implantation. Previous studies reveal the Sox17 gene as a downstream target of the Pgr-Gata2-dependent transcription network that directs genomic actions in the uterine endometrium receptive for embryo implantation. Here, we report that ablating Sox17 in the uterine epithelium impairs leukemia inhibitory factor (LIF) and Indian hedgehog homolog (IHH) signaling, leading to failure of embryo implantation. In vivo deletion of the SOX17-binding region 19 kb upstream of the Ihh locus by CRISPR-Cas technology reduces Ihh expression specifically in the uterus and alters proper endometrial epithelial-stromal interactions, thereby impairing pregnancy. This SOX17-binding interval is also bound by GATA2, FOXA2, and PGR. This cluster of transcription factor binding is common in 737 uterine genes and may represent a key regulatory element essential for uterine epithelial gene expression.


Assuntos
Proteínas HMGB/metabolismo , Fatores de Transcrição SOXF/metabolismo , Útero/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Endométrio/metabolismo , Feminino , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Proteínas HMGB/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Camundongos , Fatores de Transcrição SOXF/genética , Transcriptoma/genética
18.
Stem Cell Reports ; 11(4): 1009-1020, 2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30197119

RESUMO

Mast cells are tissue-resident immune cells. Their overgrowth/overactivation results in a range of common distressing, sometimes life-threatening disorders, including asthma, psoriasis, anaphylaxis, and mastocytosis. Currently, drug discovery is hampered by use of cancer-derived mast cell lines or primary cells. Cell lines provide low numbers of mature mast cells and are not representative of in vivo mast cells. Mast cell generation from blood/bone marrow gives poor reproducibility, requiring 8-12 weeks of culture. Here we report a method for the rapid/robust production of mast cells from pluripotent stem cells (PSCs). An advantageous Gata2Venus reporter enriches mast cells and progenitors as they differentiate from PSCs. Highly proliferative mouse mast cells and progenitors emerge after 2 weeks. This method is applicable for rapid human mast cell generation, and could enable the production of sufficient numbers of physiologically relevant human mast cells from patient induced PSCs for the study of mast cell-associated disorders and drug discovery.


Assuntos
Técnicas de Cultura de Células/métodos , Fator de Transcrição GATA2/metabolismo , Genes Reporter , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Humanos , Mastócitos/citologia , Mastócitos/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Peptídeo Hidrolases/metabolismo , Fenótipo , Receptores de Superfície Celular/metabolismo
19.
Cell ; 174(5): 1200-1215.e20, 2018 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30100187

RESUMO

Nuclear pore complexes (NPCs) regulate nuclear-cytoplasmic transport, transcription, and genome integrity in eukaryotic cells. However, their functional roles in cancer remain poorly understood. We interrogated the evolutionary transcriptomic landscape of NPC components, nucleoporins (Nups), from primary to advanced metastatic human prostate cancer (PC). Focused loss-of-function genetic screen of top-upregulated Nups in aggressive PC models identified POM121 as a key contributor to PC aggressiveness. Mechanistically, POM121 promoted PC progression by enhancing importin-dependent nuclear transport of key oncogenic (E2F1, MYC) and PC-specific (AR-GATA2) transcription factors, uncovering a pharmacologically targetable axis that, when inhibited, decreased tumor growth, restored standard therapy efficacy, and improved survival in patient-derived pre-clinical models. Our studies molecularly establish a role of NPCs in PC progression and give a rationale for NPC-regulated nuclear import targeting as a therapeutic strategy for lethal PC. These findings may have implications for understanding how NPC deregulation contributes to the pathogenesis of other tumor types.


Assuntos
Fator de Transcrição E2F1/metabolismo , Glicoproteínas de Membrana/metabolismo , Poro Nuclear/fisiologia , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Carcinogênese , Núcleo Celular/metabolismo , Proliferação de Células , Fator de Transcrição GATA2/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Membrana Nuclear , Complexo de Proteínas Formadoras de Poros Nucleares , Transdução de Sinais
20.
J Cell Mol Med ; 22(10): 4709-4720, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30044048

RESUMO

Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor with diverse functions. It has been reported that NR4A1, as a transcriptional activator, is implicated in glucose and lipid metabolism. The aim of this study was to investigate the regulatory role of NR4A1 in adipogenesis and explore the underlying mechanisms. Quantitative real-time PCR and Western blotting were used to analyse the expression of genes involved in synthesis and mobilization of fats in vivo and in vitro. Dual-luciferase reporter assay was conducted to study the regulatory mechanisms of NR4A1. Our data from in vivo study confirmed that NR4A1 knockout (KO) mice fed with high-fat diet were more prone to obesity, and gene expression levels of PPARγ and FAS were increased in KO mice compared to controls; our data from in vitro study showed that NR4A1 overexpression in 3T3-L1 pre-adipocytes inhibited adipogenesis. Moreover, NR4A1 enhanced GATA binding protein 2 (GATA2) expression, which in turn inhibited peroxisome proliferator-activated receptor γ (PPARγ); NR4A1 inhibited sterol regulatory element binding transcription factor 1 (SREBP1) and its downstream gene fatty acid synthase (FAS) by up-regulating p53. NR4A1 inhibits the differentiation and lipid accumulation of adipocytes by enhancing the expression of GATA2 and p53.


Assuntos
Adipócitos/metabolismo , Adipogenia/genética , Fator de Transcrição GATA2/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Obesidade/genética , Proteína Supressora de Tumor p53/genética , Células 3T3-L1 , Adipócitos/citologia , Animais , Sequência de Bases , Diferenciação Celular/genética , Dieta Hiperlipídica/efeitos adversos , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Metabolismo dos Lipídeos/genética , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , PPAR gama/genética , PPAR gama/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína Supressora de Tumor p53/metabolismo
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