RESUMO
Acute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications-driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.
Assuntos
Leucemia Mieloide Aguda , Ubiquitina-Proteína Ligases , Humanos , Animais , Camundongos , Ubiquitina-Proteína Ligases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Transdução de Sinais/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , MutaçãoRESUMO
The hematopoietic system is highly sensitive to stress from both aging and radiation exposure, and the hematopoietic acute radiation syndrome (H-ARS) should be modeled in the geriatric context separately from young for development of age-appropriate medical countermeasures (MCMs). Here we developed aging murine H-ARS models, defining radiation dose response relationships (DRRs) in 12-month-old middle-aged and 24-month-old geriatric male and female C57BL/6J mice, and characterized diverse factors affecting geriatric MCM testing. Groups of approximately 20 mice were exposed to â¼10 different doses of radiation to establish radiation DRRs for estimation of the LD50/30. Radioresistance increased with age and diverged dramatically between sexes. The LD50/30 in young adult mice averaged 853 cGy and was similar between sexes, but increased in middle age to 1,005 cGy in males and 920 cGy in females, with further sex divergence in geriatric mice to 1,008 cGy in males but 842 cGy in females. Correspondingly, neutrophils, platelets, and functional hematopoietic progenitor cells were all increased with age and rebounded faster after irradiation. These effects were higher in aged males, and neutrophil dysfunction was observed in aged females. Upstream of blood production, hematopoietic stem cell (HSC) markers associated with age and myeloid bias (CD61 and CD150) were higher in geriatric males vs. females, and sex-divergent gene signatures were found in HSCs relating to cholesterol metabolism, interferon signaling, and GIMAP family members. Fluid intake per gram body weight decreased with age in males, and decreased after irradiation in all mice. Geriatric mice of substrain C57BL/6JN sourced from the National Institute on Aging were significantly more radiosensitive than C57BL/6J mice from Jackson Labs aged at our institution, indicating mouse source and substrain should be considered in geriatric radiation studies. This work highlights the importance of sex, vendor, and other considerations in studies relating to hematopoiesis and aging, identifies novel sex-specific functional and molecular changes in aging hematopoietic cells at steady state and after irradiation, and presents well-characterized aging mouse models poised for MCM efficacy testing for treatment of acute radiation effects in the elderly.
Assuntos
Síndrome Aguda da Radiação , Animais , Modelos Animais de Doenças , Feminino , Hematopoese/efeitos da radiação , Células-Tronco Hematopoéticas/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tolerância a RadiaçãoRESUMO
Polycomb group protein Bmi1 is essential for hematopoietic stem cell (HSC) self-renewal and terminal differentiation. However, its target genes in hematopoietic stem and progenitor cells are largely unknown. We performed gene expression profiling assays and found that genes of the Wnt signaling pathway are significantly elevated in Bmi1 null hematopoietic stem and progenitor cells (HSPCs). Bmi1 is associated with several genes of the Wnt signaling pathway in hematopoietic cells. Further, we found that Bmi1 represses Wnt gene expression in HSPCs. Importantly, loss of ß-catenin, which reduces Wnt activation, partially rescues the HSC self-renewal and differentiation defects seen in the Bmi1 null mice. Thus, we have identified Bmi1 as a novel regulator of Wnt signaling pathway in HSPCs. Given that Wnt signaling pathway plays an important role in hematopoiesis, our studies suggest that modulating Wnt signaling may hold potential for enhancing HSC self-renewal, thereby improving the outcomes of HSC transplantation.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Via de Sinalização Wnt , Animais , Hematopoese/genética , Células-Tronco Hematopoéticas , Camundongos , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
Medical countermeasures (MCMs) for hematopoietic acute radiation syndrome (H-ARS) should be evaluated in well-characterized animal models, with consideration of at-risk populations such as pediatrics. We have developed pediatric mouse models of H-ARS and delayed effects of acute radiation exposure (DEARE) for efficacy testing of MCMs against radiation. Male and female C57BL/6J mice aged 3, 4, 5, 6, 7 and 8 weeks old (±1 day) were characterized for baseline hematopoietic and gastrointestinal parameters, radiation response, efficacy of a known MCM, and DEARE at six and 12 months after total-body irradiation (TBI). Weanlings (age 3 weeks) were the most radiosensitive age group with an estimated LD50/30 of 712 cGy, while mice aged 4 to 8 weeks were more radioresistant with an estimated LD50/30 of 767-787 cGy. Female weanlings were more radiosensitive than males at 3 and 4 weeks old but became significantly more radioresistant after the pubertal age of 5 weeks. The most dramatic increase in body weight, RBC counts and intestinal circumference length occurred from 3 to 5 weeks of age. The established radiomitigator Neulasta® (pegfilgrastim) significantly increased 30-day survival in all age groups, validating these models for MCM efficacy testing. Analyses of DEARE among pediatric survivors revealed depressed weight gain in males six months post-TBI, and increased blood urea nitrogen at 12 months post-TBI which was more severe in females. Hematopoietic DEARE at six months post-TBI appeared to be less severe in survivors from the 3- and 4-week-old groups but was equally severe in all age groups by 12 months of age. Similar to our other acute radiation mouse models, there was no appreciable effect of Neulasta used as an H-ARS MCM on the severity of DEARE. In summary, these data characterize a pediatric mouse model useful for assessing the efficacy of MCMs against ARS and DEARE in children.
Assuntos
Síndrome Aguda da Radiação/tratamento farmacológico , Filgrastim/farmacologia , Sistema Hematopoético/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Síndrome Aguda da Radiação/etiologia , Síndrome Aguda da Radiação/fisiopatologia , Animais , Modelos Animais de Doenças , Sistema Hematopoético/fisiopatologia , Sistema Hematopoético/efeitos da radiação , Humanos , Camundongos , Pediatria , Tolerância a Radiação/efeitos da radiação , Irradiação Corporal Total/efeitos adversosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Clonal hematopoiesis of indeterminate potential (CHIP) increases with age and is associated with increased risks of hematological malignancies. While TP53 mutations have been identified in CHIP, the molecular mechanisms by which mutant p53 promotes hematopoietic stem and progenitor cell (HSPC) expansion are largely unknown. Here we discover that mutant p53 confers a competitive advantage to HSPCs following transplantation and promotes HSPC expansion after radiation-induced stress. Mechanistically, mutant p53 interacts with EZH2 and enhances its association with the chromatin, thereby increasing the levels of H3K27me3 in genes regulating HSPC self-renewal and differentiation. Furthermore, genetic and pharmacological inhibition of EZH2 decreases the repopulating potential of p53 mutant HSPCs. Thus, we uncover an epigenetic mechanism by which mutant p53 drives clonal hematopoiesis. Our work will likely establish epigenetic regulator EZH2 as a novel therapeutic target for preventing CHIP progression and treating hematological malignancies with TP53 mutations.
Assuntos
Epigênese Genética , Doenças Hematológicas/metabolismo , Hematopoese , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Doenças Hematológicas/genética , Doenças Hematológicas/fisiopatologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Metilação , Camundongos Endogâmicos C57BL , Mutação , Ligação ProteicaRESUMO
Accurate analyses of the delayed effects of acute radiation exposure in survivors of the hematopoietic acute radiation syndrome are hampered by low numbers of mice for examination due to high lethality from the acute syndrome, increased morbidity and mortality in survivors, high cost of husbandry for long-term studies, biological variability, and inconsistencies of models from different laboratories complicating meta-analyses. To address this, a compilation of 38 similar hematopoietic acute radiation syndrome studies conducted over a 7-y period in the authors' laboratory, comprising more than 1,500 irradiated young adult C57BL/6 mice and almost 600 day-30 survivors, was assessed for hematopoietic delayed effects of acute radiation exposure at various times up to 30 mo of age. Significant loss of long-term repopulating potential of phenotypically defined primitive hematopoietic stem cells was documented in hematopoietic acute radiation syndrome survivors, as well as significant decreases in all hematopoietic lineages in peripheral blood, prominent myeloid skew, significantly decreased bone marrow cellularity, and numbers of lineage-negative Sca-1+ cKit+ CD150+ cells (KSL CD150+; the phenotype known to be enriched for hematopoietic stem cells), and increased cycling of KSL CD150+ cells. Studies interrogating the phenotype of bone marrow cells capable of initiation of suspension cultures and engraftment in competitive transplantation assays documented the phenotype of hematopoietic stem cells in hematopoietic acute radiation syndrome survivors to be the same as that in nonirradiated age-matched controls. This compilation study adds rigor and validity to our initial findings of persistent hematopoietic dysfunction in hematopoietic acute radiation syndrome survivors that arises at the level of the hematopoietic stem cell and which affects all classes of hematopoietic cells for the life of the survivor.
Assuntos
Síndrome Aguda da Radiação/mortalidade , Medula Óssea/efeitos da radiação , Hematopoese/efeitos da radiação , Lesões Experimentais por Radiação/mortalidade , Síndrome Aguda da Radiação/patologia , Animais , Medula Óssea/patologia , Ciclo Celular/efeitos da radiação , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Lesões Experimentais por Radiação/patologiaAssuntos
Autorrenovação Celular , Leucemia Experimental/etiologia , Células-Tronco Neoplásicas/patologia , Proteína Supressora de Tumor p53/fisiologia , Tirosina Quinase 3 Semelhante a fms/fisiologia , Animais , Proliferação de Células , Leucemia Experimental/patologia , Camundongos , Camundongos Knockout , Células-Tronco Neoplásicas/metabolismo , Sequências de Repetição em TandemRESUMO
Manipulations of lethally-irradiated animals, such as for administration of pharmaceuticals, blood sampling, or other laboratory procedures, have the potential to induce stress effects that may negatively affect morbidity and mortality. To investigate this in a murine model of the hematopoietic acute radiation syndrome, 20 individual survival efficacy studies were grouped based on the severity of the administration (Admn) schedules of their medical countermeasure (MCM) into Admn 1 (no injections), Admn 2 (1-3 injections), or Admn 3 (29 injections or 6-9 oral gavages). Radiation doses ranged from LD30/30 to LD95/30. Thirty-day survival of vehicle controls in each group was used to construct radiation dose lethality response relationship (DRR) probit plots, which were compared statistically to the original DRR from which all LDXX/30 for the studies were obtained. The slope of the Admn 3 probit was found to be significantly steeper (5.190) than that of the original DRR (2.842) or Admn 2 (2.009), which were not significantly different. The LD50/30 for Admn 3 (8.43 Gy) was less than that of the original DRR (8.53 Gy, p < 0.050), whereas the LD50/30 of other groups were similar. Kaplan-Meier survival curves showed significantly worse survival of Admn 3 mice compared to the three other groups (p = 0.007). Taken together, these results show that stressful administration schedules of MCM can negatively impact survival and that dosing regimens should be considered when constructing DRR to use in survival studies.
Assuntos
Síndrome Aguda da Radiação/etiologia , Síndrome Aguda da Radiação/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Protetores contra Radiação/administração & dosagem , Irradiação Corporal Total/efeitos adversos , Animais , Relação Dose-Resposta a Droga , Feminino , Dose Letal Mediana , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Doses de Radiação , Radiometria/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The threat of radiation exposure from warfare or radiation accidents raises the need for appropriate animal models to study the acute and chronic effects of high dose rate radiation exposure. The goal of this study was to assess the late development of fibrosis in multiple organs (kidney, heart, and lung) in survivors of the C57BL/6 mouse model of the hematopoietic-acute radiation syndrome (H-ARS). Separate groups of mice for histological and functional studies were exposed to a single uniform total body dose between 8.53 and 8.72 Gy of gamma radiation from a Cs radiation source and studied 1-21 mo later. Blood urea nitrogen levels were elevated significantly in the irradiated mice at 9 and 21 mo (from â¼22 to 34 ± 3.8 and 69 ± 6.0 mg dL, p < 0.01 vs. non-irradiated controls) and correlated with glomerosclerosis (29 ± 1.8% vs. 64 ± 9.7% of total glomeruli, p < 0.01 vs. non-irradiated controls). Glomerular tubularization and hypertrophy and tubular atrophy were also observed at 21 mo post-total body irradiation (TBI). An increase in interstitial, perivascular, pericardial and peribronchial fibrosis/collagen deposition was observed from â¼9-21 mo post-TBI in kidney, heart, and lung of irradiated mice relative to age-matched controls. Echocardiography suggested decreased ventricular volumes with a compensatory increase in the left ventricular ejection fraction. The results indicate that significant delayed effects of acute radiation exposure occur in kidney, heart, and lung in survivors of the murine H-ARS TBI model, which mirrors pathology detected in larger species and humans at higher radiation doses focused on specific organs.
Assuntos
Síndrome Aguda da Radiação/etiologia , Síndrome Aguda da Radiação/fisiopatologia , Modelos Animais de Doenças , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/fisiopatologia , Irradiação Corporal Total/efeitos adversos , Síndrome Aguda da Radiação/diagnóstico , Animais , Relação Dose-Resposta à Radiação , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos/efeitos da radiação , Doses de Radiação , Fatores de Tempo , Irradiação Corporal Total/métodosRESUMO
Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Since diminished ECFC survival is known to dampen the vasculogenic response in vivo, we tested how long implanted ECFC survive and generate vessels in three-dimensional (3D) type I collagen matrices in vitro and in vivo. We hypothesized that human platelet lysate (HPL) would promote cell survival and enhance vasculogenesis in the 3D collagen matrices. We report that the percentage of ECFC co-cultured with HPL that were alive was significantly enhanced on days 1 and 3 post-matrix formation, compared to ECFC alone containing matrices. Also, co-culture of ECFC with HPL displayed significantly more vasculogenic activity compared to ECFC alone and expressed significantly more pro-survival molecules (pAkt, p-Bad and Bcl-xL) in the 3D collagen matrices in vitro. Treatment with Akt1 inhibitor (A-674563), Akt2 inhibitor (CCT128930) and Bcl-xL inhibitor (ABT-263/Navitoclax) significantly decreased the cell survival and vasculogenesis of ECFC co-cultured with or without HPL and implicated activation of the Akt1 pathway as the critical mediator of the HPL effect on ECFC in vitro. A significantly greater average vessel number and total vascular area of human CD31(+) vessels were present in implants containing ECFC and HPL, compared to the ECFC alone implants in vivo. We conclude that implantation of ECFC with HPL in vivo promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC.
Assuntos
Plaquetas/citologia , Plaquetas/metabolismo , Colágeno/química , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Sangue Fetal/citologia , Neovascularização Fisiológica/fisiologia , Animais , Apoptose , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células-Tronco/citologiaRESUMO
While Polycomb group protein Bmi1 is important for stem cell maintenance, its role in lineage commitment is largely unknown. We have identified Bmi1 as a novel regulator of erythroid development. Bmi1 is highly expressed in mouse erythroid progenitor cells and its deficiency impairs erythroid differentiation. BMI1 is also important for human erythroid development. Furthermore, we discovered that loss of Bmi1 in erythroid progenitor cells results in decreased transcription of multiple ribosomal protein genes and impaired ribosome biogenesis. Bmi1 deficiency stabilizes p53 protein, leading to upregulation of p21 expression and subsequent G0/G1 cell cycle arrest. Genetic inhibition of p53 activity rescues the erythroid defects seen in the Bmi1 null mice, demonstrating that a p53-dependent mechanism underlies the pathophysiology of the anemia. Mechanistically, Bmi1 is associated with multiple ribosomal protein genes and may positively regulate their expression in erythroid progenitor cells. Thus, Bmi1 promotes erythroid development, at least in part through regulating ribosome biogenesis. Ribosomopathies are human disorders of ribosome dysfunction, including Diamond-Blackfan anemia (DBA) and 5q- syndrome, in which genetic abnormalities cause impaired ribosome biogenesis, resulting in specific clinical phenotypes. We observed that BMI1 expression in human hematopoietic stem and progenitor cells from patients with DBA is correlated with the expression of some ribosomal protein genes, suggesting that BMI1 deficiency may play a pathological role in DBA and other ribosomopathies.
Assuntos
Células Eritroides/citologia , Células Eritroides/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ribossomos/metabolismo , Animais , Diferenciação Celular/fisiologia , Eritropoese/fisiologia , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genéticaRESUMO
Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs), and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription, is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK) whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis.
Assuntos
Carcinogênese/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Transcrição STAT5/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Benzotiazóis/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , Mastocitose Sistêmica/patologia , Camundongos Endogâmicos C57BL , Proteínas Mutantes/metabolismo , Mutação/genética , Compostos de Fenilureia/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Quinases Ativadas por p21/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony-forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel-forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >10(8) ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Células-Tronco Pluripotentes/citologia , Animais , Proliferação de Células/genética , Células Endoteliais/metabolismo , Sangue Fetal/citologia , Humanos , Camundongos , Neuropilina-1/metabolismo , Células-Tronco/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The fetal liver is a major hematopoietic site containing progenitor cells that give rise to nearly all blood cells, including B-1 cells. Because the fetal liver is not a de novo site of hematopoietic stem cell (HSC) or progenitor-cell emergence, it must be seeded by yolk sac (YS)-derived erythromyeloid progenitors at embryonic day (E) 8.5-E10 and aorta-gonado-mesonephros (AGM)-derived HSCs at E10.5-E11.5. Although the B-1 progenitor cell pool in the fetal liver is considered to be of HSC origin, we have previously proposed that YS-derived B-1 progenitors may also contribute to this pool. Until now, it has been impossible to determine whether HSC-independent B-1 progenitor cells exist in the fetal liver. Here, we demonstrate the presence of transplantable fetal-liver B-1 and marginal zone B progenitor cells in genetically engineered HSC-deficient embryos. HSC-deficient YS and AGM tissues produce B-1 progenitors in vitro and thus may serve as sites of origin for the B-1 progenitors that seed the fetal liver. Furthermore, we have found that core-binding factor beta (Cbfß) expression is required for fetal-liver B-1 progenitor cell maturation and expansion. Our data provide, to our knowledge, the first evidence for the presence of B-1 progenitor cells in the fetal liver that arise independently of HSCs and implicate Cbfß as a critical molecule in the development of this lineage.
Assuntos
Subunidade beta de Fator de Ligação ao Core/genética , Células-Tronco Hematopoéticas/citologia , Fígado/embriologia , Animais , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Reação em Cadeia da PolimeraseRESUMO
Angiopoietin-like 2 (ANGPTL2) has been reported to induce sprouting angiogenesis; however, its role in vasculogenesis, the de novo lumenization of endothelial cells (EC), remains unexplored. We sought to investigate the potential role of ANGPTL2 in regulating human cord blood derived endothelial colony forming cell (ECFC) vasculogenesis through siRNA mediated inhibition of ANGPTL2 gene expression. We found that ECFCs in which ANGPTL2 was diminished displayed a threefold decrease in in vitro lumenal area whereas addition of exogenous ANGPTL2 protein domains to ECFCs lead to increased lumen formation within a 3 dimensional (3D) collagen assay of vasculogenesis. ECFC migration was attenuated by 36 % via ANGPTL2 knockdown (KD) although proliferation and apoptosis were not affected. We subsequently found that c-Jun NH2-terminal kinase (JNK), but not ERK1/2, phosphorylation was decreased upon ANGPTL2 KD, and expression of membrane type 1 matrix metalloproteinase (MT1-MMP), known to be regulated by JNK and a critical regulator of EC migration and 3D lumen formation, was decreased in lumenized structures in vitro derived from ANGPTL2 silenced ECFCs. Treatment of ECFCs in 3D collagen matrices with either a JNK inhibitor or exogenous rhTIMP-3 (an inhibitor of MT1-MMP activity) resulted in a similar phenotype of decreased vascular lumen formation as observed with ANGPTL2 KD, whereas stimulation of JNK activity increased vasculogenesis. Based on gene silencing, pharmacologic, cellular, and biochemical approaches, we conclude that ANGPTL2 positively regulates ECFC vascular lumen formation likely through its effects on migration and in part by activating JNK and increasing MT1-MMP expression.
Assuntos
Angiopoietinas/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/genética , Angiopoietinas/farmacologia , Ensaios de Migração Celular , Movimento Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1(-/-) and ROCK2(-/-) mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1(-/-) MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2(-/-) MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.
Assuntos
Quinases Associadas a rho/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/metabolismo , Amidas/farmacologia , Animais , Antibióticos Antineoplásicos/farmacologia , Miosinas Cardíacas/metabolismo , Caspases/metabolismo , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cofilina 1/metabolismo , Citocalasina D/farmacologia , Doxorrubicina/farmacologia , Camundongos , Camundongos Knockout , Cadeias Leves de Miosina/metabolismo , Fosforilação , Piridinas/farmacologia , Quinases Associadas a rho/deficiência , Quinases Associadas a rho/genéticaRESUMO
Erythropoiesis is a dynamic, multistep process whereby hematopoietic stem cells differentiate toward a progressively committed erythroid lineage through intermediate progenitors. Although several downstream signaling molecules have been identified that regulate steady-state erythropoiesis, the major regulators under conditions of stress remain poorly defined. Rho kinases (ROCKs) belong to a family of serine/threonine kinases. Using gene-targeted ROCK1-deficient mice, we show that lack of ROCK1 in phenylhydrazine-induced oxidative stress model results in enhanced recovery from hemolytic anemia as well as enhanced splenic stress erythropoiesis compared with control mice. Deficiency of ROCK1 also results in enhanced survival, whereas wild-type mice die rapidly in response to stress. Enhanced survivability of ROCK1-deficient mice is associated with reduced level of reactive oxygen species. BM transplantation studies revealed that enhanced stress erythropoiesis in ROCK1-deficient mice is stem cell autonomous. We show that ROCK1 binds to p53 and regulates its stability and expression. In the absence of ROCK1, p53 phosphorylation and expression is significantly reduced. Our findings reveal that ROCK1 functions as a physiologic regulator of p53 under conditions of erythroid stress. These findings are expected to offer new perspectives on stress erythropoiesis and may provide a potential therapeutic target in human disease characterized by anemia.
Assuntos
Anemia Hemolítica/mortalidade , Anemia Hemolítica/prevenção & controle , Apoptose , Eritropoese/fisiologia , Estresse Oxidativo/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Quinases Associadas a rho/fisiologia , Anemia Hemolítica/induzido quimicamente , Animais , Antimetabólitos Antineoplásicos/toxicidade , Western Blotting , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patologia , Eritropoese/efeitos dos fármacos , Eritropoetina/sangue , Feminino , Citometria de Fluxo , Fluoruracila/toxicidade , Imunoprecipitação , Masculino , Camundongos , Camundongos Knockout , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Fenil-Hidrazinas/toxicidade , Fosforilação , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia , Taxa de Sobrevida , Proteína Supressora de Tumor p53/genéticaRESUMO
Sox17 is essential for both endoderm development and fetal hematopoietic stem cell (HSC) maintenance. While endoderm-derived organs are well known to originate from Sox17-expressing cells, it is less certain whether fetal HSCs also originate from Sox17-expressing cells. By generating a Sox17(GFPCre) allele and using it to assess the fate of Sox17-expressing cells during embryogenesis, we confirmed that both endodermal and a part of definitive hematopoietic cells are derived from Sox17-positive cells. Prior to E9.5, the expression of Sox17 is restricted to the endoderm lineage. However, at E9.5 Sox17 is expressed in the endothelial cells (ECs) at the para-aortic splanchnopleural region that contribute to the formation of HSCs at a later stage. The identification of two distinct progenitor cell populations that express Sox17 at E9.5 was confirmed using fluorescence-activated cell sorting together with RNA-Seq to determine the gene expression profiles of the two cell populations. Interestingly, this analysis revealed differences in the RNA processing of the Sox17 mRNA during embryogenesis. Taken together, these results indicate that Sox17 is expressed in progenitor cells derived from two different germ layers, further demonstrating the complex expression pattern of this gene and suggesting caution when using Sox17 as a lineage-specific marker.
Assuntos
Células-Tronco Fetais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/genética , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição SOXF/genética , Animais , Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos , Desenvolvimento Embrionário , Endoderma/citologia , Endoderma/metabolismo , Células-Tronco Fetais/citologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas HMGB/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Transgênicos , RNA Mensageiro/biossíntese , Fatores de Transcrição SOXF/metabolismoRESUMO
The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.