RESUMEN
VEXAS syndrome (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is a monogenic disease of adulthood caused by somatic mutations in UBA1 in hematopoietic progenitor cells. Patients develop inflammatory and hematologic symptoms. Myeloid-driven autoinflammation and progressive bone marrow failure lead to substantial morbidity and mortality. Effective medical treatments need to be identified. Reports in the current issue of Blood describe novel UBA1 genetic variants, treatment options, and insight into disease pathophysiology. VEXAS syndrome represents a prototype for a new class of diseases.
Asunto(s)
Genes Ligados a X , Enfermedades Genéticas Congénitas , Mutación , Trastornos Mieloproliferativos , Enzimas Activadoras de Ubiquitina/genética , Células Eritroides/enzimología , Enfermedades Genéticas Congénitas/diagnóstico por imagen , Enfermedades Genéticas Congénitas/enzimología , Enfermedades Genéticas Congénitas/genética , Humanos , Masculino , Células Mieloides/enzimología , Trastornos Mieloproliferativos/diagnóstico por imagen , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/genética , SíndromeAsunto(s)
Células de la Médula Ósea , Células Eritroides , Genes Ligados a X , Mutación , Células Mieloides , Células Progenitoras Mieloides , Trastornos Mieloproliferativos , Enzimas Activadoras de Ubiquitina/genética , Vacuolas , Adulto , Anciano , Anciano de 80 o más Años , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/patología , Niño , Células Eritroides/enzimología , Células Eritroides/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Mieloides/enzimología , Células Mieloides/patología , Células Progenitoras Mieloides/enzimología , Células Progenitoras Mieloides/patología , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Síndrome , Vacuolas/enzimología , Vacuolas/genética , Vacuolas/patologíaRESUMEN
Diamond-Blackfan anemia (DBA) is a rare, inherited bone marrow failure syndrome, characterized by red blood cell aplasia, developmental abnormalities, and enhanced risk of malignancy. However, the underlying pathogenesis of DBA is yet to be understood. Recently, mutations in the gene encoding ribosomal protein (RP) L18 were identified in DBA patients. RPL18 is a crucial component of the ribosomal large subunit but its role in hematopoiesis remains unknown. To genetically model the ribosomal defect identified in DBA, we generated a rpl18 mutant line in zebrafish, using CRISPR/Cas9 system. Molecular characterization of this mutant line demonstrated that Rpl18 deficiency mirrored the erythroid defects of DBA, namely a lack of mature red blood cells. Rpl18 deficiency caused an increase in p53 activation and JAK2-STAT3 activity. Furthermore, we found inhibitors of JAK2 or STAT3 phosphorylation could rescue anemia in rpl18 mutants. Our research provides a new in vivo model of Rpl18 deficiency and suggests involvement of signal pathway of JAK2-STAT3 in the DBA pathogenesis.
Asunto(s)
Anemia de Diamond-Blackfan/enzimología , Células Eritroides/enzimología , Eritropoyesis , Janus Quinasa 2/metabolismo , Proteínas Ribosómicas/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteínas de Pez Cebra/metabolismo , Anemia de Diamond-Blackfan/sangre , Anemia de Diamond-Blackfan/tratamiento farmacológico , Anemia de Diamond-Blackfan/genética , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Células Eritroides/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Janus Quinasa 2/antagonistas & inhibidores , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Ribosómicas/genética , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/antagonistas & inhibidoresRESUMEN
Aged hematopoietic stem cells (HSCs) undergo biased lineage priming and differentiation toward production of myeloid cells. A comprehensive understanding of gene regulatory mechanisms causing HSC aging is needed to devise new strategies to sustainably improve immune function in aged individuals. Here, a focused short hairpin RNA screen of epigenetic factors reveals that the histone acetyltransferase Kat6b regulates myeloid cell production from hematopoietic progenitor cells. Within the stem and progenitor cell compartment, Kat6b is highly expressed in long-term (LT)-HSCs and is significantly decreased with aging at the transcript and protein levels. Knockdown of Kat6b in young LT-HSCs causes skewed production of myeloid cells at the expense of erythroid cells both in vitro and in vivo. Transcriptome analysis identifies enrichment of aging and macrophage-associated gene signatures alongside reduced expression of self-renewal and multilineage priming signatures. Together, our work identifies KAT6B as a novel epigenetic regulator of hematopoietic differentiation and a target to improve aged immune function.
Asunto(s)
Envejecimiento/metabolismo , Diferenciación Celular , Células Eritroides/enzimología , Regulación Enzimológica de la Expresión Génica , Histona Acetiltransferasas/biosíntesis , Células Progenitoras Mieloides/enzimología , Envejecimiento/genética , Envejecimiento/patología , Animales , Epigénesis Genética , Células Eritroides/patología , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Histona Acetiltransferasas/genética , Masculino , Ratones , Ratones Transgénicos , Células Progenitoras Mieloides/patología , TranscriptomaRESUMEN
Cancer-related anemia is present in more than 60% of newly diagnosed cancer patients and is associated with substantial morbidity and high medical costs. Drugs that enhance erythropoiesis are urgently required to decrease transfusion rates and improve quality of life. Clinical studies have observed an unexpected improvement in hemoglobin and RBC transfusion-independence in patients with acute myeloid leukemia (AML) treated with the isocitrate dehydrogenase 2 (IDH2) mutant-specific inhibitor enasidenib, leading to improved quality of life without a reduction in AML disease burden. Here, we demonstrate that enasidenib enhanced human erythroid differentiation of hematopoietic progenitors. The phenomenon was not observed with other IDH1/2 inhibitors and occurred in IDH2-deficient CRISPR-engineered progenitors independently of D-2-hydroxyglutarate. The effect of enasidenib on hematopoietic progenitors was mediated by protoporphyrin accumulation, driving heme production and erythroid differentiation in committed CD71+ progenitors rather than hematopoietic stem cells. Our results position enasidenib as a promising therapeutic agent for improvement of anemia and provide the basis for a clinical trial using enasidenib to decrease transfusion dependence in a wide array of clinical contexts.
Asunto(s)
Aminopiridinas/farmacología , Diferenciación Celular/efectos de los fármacos , Células Eritroides/enzimología , Células Madre Hematopoyéticas/enzimología , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Triazinas/farmacología , Células Eritroides/citología , Células Madre Hematopoyéticas/citología , Humanos , Isocitrato Deshidrogenasa/metabolismo , Protoporfirinas/metabolismoRESUMEN
PI-PLCß1 is involved in cell proliferation, differentiation, and myelodysplastic syndrome (MDS) pathogenesis. Moreover, the increased activity of PI-PLCß1 reduces the expression of PKC-α, which, in turn, delays the cell proliferation and is linked to erythropoiesis. Lenalidomide is currently used in low-risk patients with MDS and del(5q), where it can suppress the del(5q) clone and restore normal erythropoiesis. In this study, we analyzed the effect of lenalidomide on 16 patients with low-risk del(5q) MDS, as well as del(5q) and non-del(5q) hematopoietic cell lines, mainly focusing on erythropoiesis, cell cycle, and PI-PLCß1/PKC-α signaling. Overall, 11 patients were evaluated clinically, and 10 (90%) had favorable responses; the remaining case had a stable disease. At a molecular level, both responder patients and del(5q) cells showed a specific induction of erythropoiesis, with a reduced γ/ß-globin ratio, an increase in glycophorin A, and a nuclear translocation of PKC-α. Moreover, lenalidomide could induce a selective G0/G1 arrest of the cell cycle in del(5q) cells, slowing down the rate proliferation in those cells. Altogether, our results could not only better explain the role of PI-PLCß1/PKC-α signaling in erythropoiesis but also lead to a better comprehension of the lenalidomide effect on del(5q) MDS and pave the way to innovative, targeted therapies.-Poli, A., Ratti, S., Finelli, C., Mongiorgi, S., Clissa, C., Lonetti, A., Cappellini, A., Catozzi, A., Barraco, M., Suh, P.-G., Manzoli, L., McCubrey, J. A., Cocco, L., Follo, M. Y. Nuclear translocation of PKC-α is associated with cell cycle arrest and erythroid differentiation in myelodysplastic syndromes (MDSs).
Asunto(s)
Diferenciación Celular , Núcleo Celular/enzimología , Células Eritroides/enzimología , Eritropoyesis , Puntos de Control de la Fase G1 del Ciclo Celular , Síndromes Mielodisplásicos/enzimología , Proteína Quinasa C-alfa/metabolismo , Transducción de Señal , Transporte Activo de Núcleo Celular , Anciano , Anciano de 80 o más Años , Línea Celular , Núcleo Celular/genética , Núcleo Celular/patología , Células Eritroides/patología , Femenino , Humanos , Masculino , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Proteína Quinasa C-alfa/genética , Fase de Descanso del Ciclo CelularRESUMEN
During terminal differentiation, the global protein complement is remodeled, as epitomized by erythrocytes, whose cytosol is ~98% globin. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving programmed elimination of preexisting cytosolic proteins are unclear. We found that a mutation in the murine Ube2o gene, which encodes a ubiquitin-conjugating enzyme induced during erythropoiesis, results in anemia. Proteomic analysis suggested that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, ribosome elimination, a hallmark of reticulocyte differentiation, was defective in Ube2o-/- mutants. UBE2O recognized ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.
Asunto(s)
Células Eritroides/citología , Eritropoyesis/fisiología , Proteínas Ribosómicas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Anemia/genética , Anemia Hipocrómica/genética , Animales , Eritrocitos/citología , Eritrocitos/enzimología , Células Eritroides/enzimología , Eritropoyesis/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteoma/metabolismo , Proteómica , Reticulocitos/citología , Reticulocitos/enzimología , Ribosomas/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Globinas beta/genética , Globinas beta/metabolismoRESUMEN
Anemia due to attenuated erythroid terminal differentiation is one of the most common hematological disorders occurring at all stages of life. We previously demonstrated that catalytic subunit α of protein phosphatase 2A (PP2Acα) modulates fetal liver erythropoiesis. However the corresponding PP2A regulatory subunit in this process remains unknown. In this study, we report that chemical inhibition of PP2A activity with okadaic acid impairs hemin-induced erythroid differentiation. Interestingly, B56 family member B56ß is the only regulatory subunit whose expression is induced by both erythropoietin in fetal liver cells and hemin in erythroleukemia K562 cells. Finally, knockdown of B56ß attenuates hemin-induced K562 erythroid differentiation. Collectively, our data identify B56ß as the potential functional regulatory subunit of PP2A in erythroid differentiation, shedding light on new target for precise modulation of PP2A activity for treatment of anemia and related diseases.
Asunto(s)
Diferenciación Celular , Células Eritroides/citología , Células Eritroides/enzimología , Proteínas de la Membrana/metabolismo , Proteína Fosfatasa 2/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Hemina/farmacología , Humanos , Células K562 , Ratones Endogámicos C57BL , Modelos Biológicos , Ácido Ocadaico/farmacologíaRESUMEN
Human blood cell counts are tightly maintained within narrow physiologic ranges, largely controlled by cytokine-integrated signaling and transcriptional circuits that regulate multilineage hematopoietic specification. Known genetic loci influencing blood cell production account for <10% of platelet and red blood cell variability, and thrombopoietin/cellular myeloproliferative leukemia virus liganding is dispensable for definitive thrombopoiesis, establishing that fundamentally important modifier loci remain unelucidated. In this study, platelet transcriptome sequencing and extended thrombocytosis cohort analyses identified a single loss-of-function mutation (BLVRB(S111L)) causally associated with clonal and nonclonal disorders of enhanced platelet production. BLVRB(S111L) encompassed within the substrate/cofactor [α/ß dinucleotide NAD(P)H] binding fold is a functionally defective redox coupler using flavin and biliverdin (BV) IXß tetrapyrrole(s) and results in exaggerated reactive oxygen species accumulation as a putative metabolic signal leading to differential hematopoietic lineage commitment and enhanced thrombopoiesis. These data define the first physiologically relevant function of BLVRB and implicate its activity and/or heme-regulated BV tetrapyrrole(s) in a unique redox-regulated bioenergetic pathway governing terminal megakaryocytopoiesis; these observations also define a mechanistically restricted drug target retaining potential for enhancing human platelet counts.
Asunto(s)
Hemo/metabolismo , Redes y Vías Metabólicas , Mutación/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Trombopoyesis/genética , Alelos , Antígenos CD34/metabolismo , Plaquetas/metabolismo , Linaje de la Célula , Estudios de Cohortes , Células Eritroides/citología , Células Eritroides/enzimología , Estudios de Asociación Genética , Hematopoyesis , Humanos , Megacariocitos/citología , Megacariocitos/enzimología , Oxidación-Reducción , Polimorfismo de Nucleótido Simple/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Riesgo , Análisis de Secuencia de ARN , Trombocitosis/genéticaRESUMEN
Oxidative stress due to a superoxide dismutase 1 (SOD1) deficiency causes anemia and autoimmune responses, which are phenotypically similar to autoimmune hemolytic anemia (AIHA) and systemic lupus erythematosus (SLE) in C57BL/6 mice and aggravates AIHA pathogenesis in New Zealand black (NZB) mice. We report herein on an evaluation of the role of reactive oxygen species (ROS) in a model mouse with inherited SLE, that is, F1 mice of the NZB × New Zealand white (NZW) strain. The ROS levels within red blood cells (RBCs) of the F1 mice were similar to the NZW mice but lower compared to the NZB mice throughout adult period. Regarding SLE pathogenesis, we examined the effects of an SOD1 deficiency or the overexpression of human SOD1 in erythroid cells by establishing corresponding congenic F1 mice. A SOD1 deficiency caused an elevation in ROS production, methemoglobin content, and hyperoxidation of peroxiredoxin in RBC of the F1 mice, which were all consistent with elevated oxidative stress. However, while the overexpression of human SOD1 in erythroid cells extended the life span of the congenic F1 mice, the SOD1 deficiency had no effect on life span compared to wild-type F1 mice. It is generally recognized that NZW mice possess a larval defect in the immune system and that NZB mice trigger an autoimmune reaction in the F1 mice. Our results suggest that the oxidative insult originated from the NZB mouse background has a functional role in triggering an aberrant immune reaction, leading to fatal responses in F1 mice.
Asunto(s)
Lupus Eritematoso Sistémico/genética , Superóxido Dismutasa-1/genética , Animales , Modelos Animales de Enfermedad , Células Eritroides/citología , Células Eritroides/enzimología , Femenino , Lupus Eritematoso Sistémico/enzimología , Lupus Eritematoso Sistémico/inmunología , Masculino , Ratones , Ratones Endogámicos NZB , Fenotipo , Superóxido Dismutasa-1/biosíntesis , Superóxido Dismutasa-1/metabolismo , Tasa de Supervivencia , TransgenesRESUMEN
5-Aminolevulinate synthase (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme, catalyzes the initial step of heme biosynthesis in non-plant eukaryotes. The precursor form of the enzyme is translated in the cytosol, and upon mitochondrial import, the N-terminal targeting presequence is proteolytically cleaved to generate mature ALAS. In bone marrow-derived erythroid cells, a mitochondrial- and site-specific endoprotease of yet unknown primary structure, produces a protein shorter than mature erythroid ALAS (ALAS2) found in peripheral blood erythroid cells. This truncated ALAS2 lacks the presequence and the N-terminal sequence (corresponding to ~7 KDa molecular mass) present in ALAS2 from peripheral blood erythroid cells. How the truncation affects the structural topology and catalytic properties of ALAS2 is presently not known. To address this question, we created a recombinant, truncated, murine ALAS2 (ΔmALAS2) devoid of the cleavable N-terminal region and examined its catalytic and biophysical properties. The N-terminal truncation of mALAS2 did not significantly affect the organization of the secondary structure, but a subtle reduction in the rigidity of the tertiary structure was noted. Furthermore, thermal denaturation studies revealed a decrease of 4.3°C in the Tm value of ΔmALAS2, implicating lower thermal stability. While the kcat of ΔmALAS2 is slightly increased over that of the wild-type enzyme, the slowest step in the ΔmALAS2-catalyzed reaction remains dominated by ALA release. Importantly, intrinsic disorder algorithms imply that the N-terminal region of mALAS2 is highly disordered, and thus susceptible to proteolysis. We propose that the N-terminal truncation offers a cell-specific ALAS2 regulatory mechanism without hindering heme synthesis.
Asunto(s)
5-Aminolevulinato Sintetasa/química , 5-Aminolevulinato Sintetasa/genética , Hemo/biosíntesis , Relación Estructura-Actividad , 5-Aminolevulinato Sintetasa/metabolismo , Animales , Células de la Médula Ósea/enzimología , Catálisis , Células Eritroides/enzimología , Hemo/genética , RatonesRESUMEN
The C-terminal domain (CTD) of the RNA polymerase II (Pol II) large subunit contains tandem repeats of the heptapeptide, Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. The CTD is subject to dynamic phosphorylation during transcription, mainly at serine residues (Ser2, Ser5 and Ser7). Regulation of CTD phosphorylation by specific kinases and phosphatases is crucial for coordinating transcription with RNA processing and histone modification. Human small CTD phosphatase 4 (SCP4), also called CTDSPL2 or HSPC129, is a putative CTD phosphatase belonging to the FCP/SCP family and implicated in control of ε- and γ-globin gene expression. Here, we report the biochemical and functional characterization of SCP4. SCP4 exhibited Ser5-preferential CTD phosphatase activity in vitro, while small interfering RNA-mediated SCP4 knockdown in HeLa cells increased phosphorylation levels of Pol II at Ser5 and Ser7, but not at Ser2. Furthermore, cell fractionation, chromatin immunoprecipitation and immunofluorescence assays revealed an exclusive localization for SCP4 in the chromatin, particularly at transcriptionally silenced chromosomal regions. Interestingly, SCP4 was gradually released from the chromatin fraction during hemin-induced erythroid differentiation of K562 cells, with concomitant cytoplasmic accumulation. Therefore, SCP4 is a unique chromatin-associated, Ser5-preferential CTD phosphatase that preferentially distributes to transcriptionally silenced gene regions and may participate in gene regulation during erythroid differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Cromatina/enzimología , Células Eritroides/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Cromatina/genética , Células Eritroides/citología , Células HeLa , Humanos , Células K562 , Fosfoproteínas Fosfatasas/genética , Transporte de Proteínas/fisiologíaRESUMEN
5-Aminolevulinate synthase (ALAS) catalyzes the first step in mammalian heme biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent and reversible reaction between glycine and succinyl-CoA to generate CoA, CO2, and 5-aminolevulinate (ALA). Apart from coordinating the positioning of succinyl-CoA, Rhodobacter capsulatus ALAS Asn-85 has a proposed role in regulating the opening of an active site channel. Here, we constructed a library of murine erythroid ALAS variants with substitutions at the position occupied by the analogous bacterial asparagine, screened for ALAS function, and characterized the catalytic properties of the N150H and N150F variants. Quinonoid intermediate formation occurred with a significantly reduced rate for either the N150H- or N150F-catalyzed condensation of glycine with succinyl-CoA during a single turnover. The introduced mutations caused modifications in the ALAS active site such that the resulting variants tipped the balance between the forward- and reverse-catalyzed reactions. Although wild-type ALAS catalyzes the conversion of ALA into the quinonoid intermediate at a rate 6.3-fold slower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA, the N150F variant catalyzes the forward reaction at a mere 1.2-fold faster rate than that of the reverse reaction, and the N150H variant reverses the rate values with a 1.7-fold faster rate for the reverse reaction than that for the forward reaction. We conclude that the evolutionary selection of Asn-150 was significant for optimizing the forward enzymatic reaction at the expense of the reverse, thus ensuring that ALA is predominantly available for heme biosynthesis.
Asunto(s)
5-Aminolevulinato Sintetasa/química , 5-Aminolevulinato Sintetasa/genética , Sustitución de Aminoácidos , Células Eritroides/enzimología , 5-Aminolevulinato Sintetasa/metabolismo , Acilcoenzima A , Ácido Aminolevulínico/química , Ácido Aminolevulínico/metabolismo , Animales , Asparagina/genética , Asparagina/metabolismo , Biocatálisis , Dominio Catalítico , Células Eritroides/química , Glicina/metabolismo , Cinética , Ratones , Especificidad por SustratoRESUMEN
BACKGROUND: The transcription factor GATA-2 is predominantly expressed in hematopoietic stem and progenitor cells and counteracts the erythroid-specific transcription factor GATA-1, to modulate the proliferation and differentiation of hematopoietic cells. During hematopoietic cell differentiation, GATA-2 exhibits dynamic expression patterns, which are regulated by multiple transcription factors. METHODS: Stable LSD1-knockdown cell lines were established by growing murine erythroleukemia (MEL) or mouse embryonic stem cells together with virus particles, in the presence of Polybrene(®) at 4 µg/mL, for 24-48 hours followed by puromycin selection (1 µg/mL) for 2 weeks. Real-time polymerase chain reaction (PCR)-based quantitative chromatin immunoprecipitation (ChIP) analysis was used to test whether the TAL1 transcription factor is bound to 1S promoter in the GATA-2 locus or whether LSD1 colocalizes with TAL1 at the 1S promoter. The sequential ChIP assay was utilized to confirm the role of LSD1 in the regulation of H3K4me2 at the GATA-2 locus during erythroid differentiation. Western blot analysis was employed to detect the protein expression. The alamarBlue(®) assay was used to examine the proliferation of the cells, and the absorbance was monitored at optical density (OD) 570 nm and OD 600 nm. RESULTS: In this study, we showed that LSD1 regulates the expression of GATA-2 during erythroid differentiation. Knockdown of LSD1 results in increased GATA-2 expression and inhibits the differentiation of MEL and embryonic stem cells. Furthermore, we demonstrated that LSD1 binds to the 1S promoter of the GATA-2 locus and suppresses GATA-2 expression, via histone demethylation. CONCLUSION: Our data revealed that LSD1 mediates erythroid differentiation, via epigenetic modification of the GATA-2 locus.
Asunto(s)
Células Madre Embrionarias/enzimología , Células Eritroides/enzimología , Eritropoyesis , Factor de Transcripción GATA2/metabolismo , Histona Demetilasas/metabolismo , Leucemia Eritroblástica Aguda/enzimología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular , Metilación de ADN , Regulación hacia Abajo , Epigénesis Genética , Factor de Transcripción GATA2/genética , Técnicas de Silenciamiento del Gen , Histona Demetilasas/genética , Histonas/metabolismo , Leucemia Eritroblástica Aguda/genética , Ratones , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda , Factores de TiempoRESUMEN
The methyltransferase activity of the trithorax group (TrxG) protein MLL1 found within its COMPASS (complex associated with SET1)-like complex is allosterically regulated by a four-subunit complex composed of WDR5, RbBP5, Ash2L, and DPY30 (also referred to as WRAD). We report structural evidence showing that in WRAD, a concave surface of the Ash2L SPIa and ryanodine receptor (SPRY) domain binds to a cluster of acidic residues, referred to as the D/E box, in RbBP5. Mutational analysis shows that residues forming the Ash2L/RbBP5 interface are important for heterodimer formation, stimulation of MLL1 catalytic activity, and erythroid cell terminal differentiation. We also demonstrate that a phosphorylation switch on RbBP5 stimulates WRAD complex formation and significantly increases KMT2 (lysine [K] methyltransferase 2) enzyme methylation rates. Overall, our findings provide structural insights into the assembly of the WRAD complex and point to a novel regulatory mechanism controlling the activity of the KMT2/COMPASS family of lysine methyltransferases.
Asunto(s)
Histonas/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Animales , Diferenciación Celular , Línea Celular Tumoral , Cristalización , Análisis Mutacional de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/genética , Células Eritroides/citología , Células Eritroides/enzimología , N-Metiltransferasa de Histona-Lisina/metabolismo , Metilación/efectos de los fármacos , Metiltransferasas/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Protein tyrosine phosphatases (PTPs) are involved in hematopoiesis, but the function of many PTPs is not well characterized in vivo. Here, we have identified Ptpn9a, an ortholog of human PTPN9, as a crucial regulator of erythroid cell development in zebrafish embryos. ptpn9a, but not ptpn9b, was expressed in the posterior lateral plate mesoderm and intermediate cell mass - two primitive hematopoietic sites during zebrafish embryogenesis. Morpholino-mediated knockdown of ptpn9a caused erythrocytes to be depleted by inhibiting erythroid cell maturation without affecting erythroid proliferation and apoptosis. Consistently, both dominant-negative PTPN9 (with mutation C515S) and siRNA against PTPN9 inhibited erythroid differentiation in human K562 cells. Mechanistically, depletion of ptpn9 in zebrafish embryos in vivo or in K562 cells in vitro increased phosphorylated STAT3, and the hyper-phosphorylated STAT3 entrapped and prevented the transcription factors GATA1 and ZBP-89 (also known as ZNF148) from regulating erythroid gene expression. These findings imply that PTPN9 plays an important role in erythropoiesis by disrupting an inhibitory complex of phosphorylated STAT3, GATA1 and ZBP-89, providing new cellular and molecular insights into the role of ptpn9a in developmental hematopoiesis.
Asunto(s)
Células Eritroides/enzimología , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Fosfatasas no Receptoras/fisiología , Factor de Transcripción STAT3/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/fisiología , Pez Cebra/fisiología , Animales , Embrión no Mamífero/citología , Embrión no Mamífero/enzimología , Eritropoyesis , Factor de Transcripción GATA1/metabolismo , Gastrulación , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Células K562 , Fosforilación , Factores de Transcripción/metabolismo , Pez Cebra/embriologíaRESUMEN
To clarify the role of HDACs in erythropoiesis, expression, activity and function of class I (HDAC1, HDAC2, HDAC3) and class IIa (HDAC4, HDAC5) HDACs during in vitro maturation of human erythroblasts were compared. During erythroid maturation, expression of HDAC1, HDAC2 and HDAC3 remained constant and activity and GATA1 association (its partner of the NuRD complex), of HDAC1 increased. By contrast, HDAC4 content drastically decreased and HDAC5 remained constant in content but decreased in activity. In erythroid cells, pull down experiments identified the presence of a novel complex formed by HDAC5, GATA1, EKLF and pERK which was instead undetectable in cells of the megakaryocytic lineage. With erythroid maturation, association among HDAC5, GATA1 and EKLF persisted but levels of pERK sharply decreased. Treatment of erythroleukemic cells with inhibitors of ERK phosphorylation reduced by >90% the total and nuclear content of HDAC5, GATA1 and EKLF, suggesting that ERK phosphorylation is required for the formation of this complex. Based on the function of class IIa HDACs as chaperones of other proteins to the nucleus and the erythroid-specificity of HDAC5 localization, this novel HDAC complex was named nuclear remodeling shuttle erythroid (NuRSERY). Exposure of erythroid cells to the class II-selective HDAC inhibitor (HDACi) APHA9 increased γ/(γ+ß) globin expression ratios (Mai et al., 2007), suggesting that NuRSERY may regulate globin gene expression. In agreement with this hypothesis, exposure of erythroid cells to APHA9 greatly reduced the association among HDAC5, GATA1 and EKLF. Since exposure to APHA9 did not affect survival rates or p21 activation, NuRSERY may represent a novel, possibly less toxic, target for epigenetic therapies of hemoglobinopaties and other disorders.
Asunto(s)
Células Eritroides/metabolismo , Factor de Transcripción GATA1/metabolismo , Histona Desacetilasas/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , eIF-2 Quinasa/metabolismo , Diferenciación Celular/fisiología , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Eritroblastos/citología , Eritroblastos/enzimología , Eritroblastos/patología , Células Eritroides/citología , Células Eritroides/enzimología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células K562 , Megacariocitos/citología , Megacariocitos/enzimología , Megacariocitos/metabolismo , FosforilaciónRESUMEN
Newborn infants are highly susceptible to infection. This defect in host defence has generally been ascribed to the immaturity of neonatal immune cells; however, the degree of hyporesponsiveness is highly variable and depends on the stimulation conditions. These discordant responses illustrate the need for a more unified explanation for why immunity is compromised in neonates. Here we show that physiologically enriched CD71(+) erythroid cells in neonatal mice and human cord blood have distinctive immunosuppressive properties. The production of innate immune protective cytokines by adult cells is diminished after transfer to neonatal mice or after co-culture with neonatal splenocytes. Neonatal CD71(+) cells express the enzyme arginase-2, and arginase activity is essential for the immunosuppressive properties of these cells because molecular inhibition of this enzyme or supplementation with L-arginine overrides immunosuppression. In addition, the ablation of CD71(+) cells in neonatal mice, or the decline in number of these cells as postnatal development progresses parallels the loss of suppression, and restored resistance to the perinatal pathogens Listeria monocytogenes and Escherichia coli. However, CD71(+) cell-mediated susceptibility to infection is counterbalanced by CD71(+) cell-mediated protection against aberrant immune cell activation in the intestine, where colonization with commensal microorganisms occurs swiftly after parturition. Conversely, circumventing such colonization by using antimicrobials or gnotobiotic germ-free mice overrides these protective benefits. Thus, CD71(+) cells quench the excessive inflammation induced by abrupt colonization with commensal microorganisms after parturition. This finding challenges the idea that the susceptibility of neonates to infection reflects immune-cell-intrinsic defects and instead highlights processes that are developmentally more essential and inadvertently mitigate innate immune protection. We anticipate that these results will spark renewed investigation into the need for immunosuppression in neonates, as well as improved strategies for augmenting host defence in this vulnerable population.
Asunto(s)
Antígenos CD/metabolismo , Células Eritroides/inmunología , Infecciones por Escherichia coli/inmunología , Tolerancia Inmunológica/inmunología , Listeriosis/inmunología , Receptores de Transferrina/metabolismo , Animales , Animales Recién Nacidos , Arginasa/genética , Arginasa/metabolismo , Susceptibilidad a Enfermedades/inmunología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células Eritroides/enzimología , Escherichia coli/inmunología , Femenino , Sangre Fetal/citología , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Tolerancia Inmunológica/genética , Listeria monocytogenes/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The unique sensitivity of early red cell progenitors to iron deprivation, known as the erythroid iron restriction response, serves as a basis for human anemias globally. This response impairs erythropoietin-driven erythropoiesis and underlies erythropoietic repression in iron deficiency anemia. Mechanistically, the erythroid iron restriction response results from inactivation of aconitase enzymes and can be suppressed by providing the aconitase product isocitrate. Recent studies have implicated the erythroid iron restriction response in anemia of chronic disease and inflammation (ACDI), offering new therapeutic avenues for a major clinical problem; however, inflammatory signals may also directly repress erythropoiesis in ACDI. Here, we show that suppression of the erythroid iron restriction response by isocitrate administration corrected anemia and erythropoietic defects in rats with ACDI. In vitro studies demonstrated that erythroid repression by inflammatory signaling is potently modulated by the erythroid iron restriction response in a kinase-dependent pathway involving induction of the erythroid-inhibitory transcription factor PU.1. These results reveal the integration of iron and inflammatory inputs in a therapeutically tractable erythropoietic regulatory circuit.
Asunto(s)
Anemia/tratamiento farmacológico , Células Eritroides/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Deficiencias de Hierro , Isocitratos/farmacología , Aconitato Hidratasa/metabolismo , Anemia/metabolismo , Anemia/patología , Animales , Células Cultivadas , Células Eritroides/enzimología , Femenino , Humanos , Interferón gamma/fisiología , Isocitratos/uso terapéutico , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Ratas Endogámicas Lew , Transducción de Señal , Transactivadores/metabolismo , Activación TranscripcionalRESUMEN
Expression of oncogenic Bcr-Abl inhibits cell differentiation of hematopoietic stem/progenitor cells in chronic myeloid leukemia (CML). Differentiation therapy is considered to be a new strategy for treating this type of leukemia. Aclacinomycin A (ACM) is an antitumor antibiotic. Previous studies have shown that ACM induced erythroid differentiation of CML cells. In this study, we investigate the effect of ACM on the sensitivity of human CML cell line K562 to Bcr-Abl specific inhibitor imatinib (STI571, Gleevec). We first determined the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis in K562 cells. Then, pretreatment with this optimal concentration of ACM followed by a minimally toxic concentration of imatinib strongly induced growth inhibition and apoptosis compared to that with simultaneous co-treatment, indicating that ACM-induced erythroid differentiation sensitizes K562 cells to imatinib. Sequential treatment with ACM and imatinib induced Bcr-Abl down-regulation, cytochrome c release into the cytosol, and caspase-3 activation, as well as decreased Mcl-1 and Bcl-xL expressions, but did not affect Fas ligand/Fas death receptor and procaspase-8 expressions. ACM/imatinib sequential treatment-induced apoptosis was suppressed by a caspase-9 inhibitor and a caspase-3 inhibitor, indicating that the caspase cascade is involved in this apoptosis. Furthermore, we demonstrated that ACM induced erythroid differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. The inhibition of erythroid differentiation by p38MAPK inhibitor SB202190, p38MAPK dominant negative mutant or p38MAPK shRNA knockdown, reduced the ACM/imatinib sequential treatment-mediated growth inhibition and apoptosis. These results suggest that differentiated K562 cells induced by ACM-mediated p38MAPK pathway become more sensitive to imatinib and result in down-regulations of Bcr-Abl and anti-apoptotic proteins, growth inhibition and apoptosis. These results provided a potential management by which ACM might have a crucial impact on increasing sensitivity of CML cells to imatinib in the differentiation therapeutic approaches.