RESUMO
BACKGROUND: Hemochromatosis gene (HFE)-associated hereditary hemochromatosis (HH) is characterized by downregulation of hepcidin synthesis, leading to increased intestinal iron absorption. OBJECTIVES: The objectives were to characterize and elucidate a possible association between gene expression profile, hepcidin levels, disease severity, and markers of inflammation in HFE-associated HH patients. METHODS: Thirty-nine HFE-associated HH patients were recruited and assigned to 2 groups according to genetic profile: C282Y homozygotes in 1 group and patients with H63D, as homozygote or in combination with C282Y, in the other group. Eleven healthy first-time blood donors were recruited as controls. Gene expression was characterized from peripheral blood cells, and inflammatory cytokines and hepcidin-25 isoform were quantified in serum. Biochemical disease characteristics were recorded. RESULTS: Elevated levels of interleukin 8 were observed in a significant higher proportion of patients than controls. In addition, compared to controls, gene expression of ζ-globin was significantly increased among C282Y homozygote patients, while gene expression of matrix metalloproteinase 8, and other neutrophil-secreted proteins, was significantly upregulated in patients with H63D. CONCLUSION: Different disease signatures may characterize HH patients according to their HFE genetic profile. Studies on larger populations, including analyses at protein level, are necessary to confirm these findings.
Assuntos
Citocinas/sangue , Proteína da Hemocromatose/genética , Hemocromatose/patologia , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Estudos de Casos e Controles , Genótipo , Hemocromatose/genética , Proteína da Hemocromatose/metabolismo , Hepcidinas/sangue , Hepcidinas/metabolismo , Homozigoto , Humanos , Interleucina-8/sangue , Metaloproteinase 8 da Matriz/genética , Metaloproteinase 8 da Matriz/metabolismo , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/sangue , Isoformas de Proteínas/metabolismo , Índice de Gravidade de Doença , Transcriptoma , Regulação para Cima , Globinas zeta/genética , Globinas zeta/metabolismoRESUMO
HepG2-conditioned medium (CM) facilitates early differentiation of murine embryonic stem cells (mESCs) into hematopoietic cells in two-dimensional cultures through formation of embryoid-like colonies (ELCs), bypassing embryoid body (EB) formation. We now demonstrate that three-dimensional (3D) cultures of alginate-encapsulated mESCs cultured in a rotating wall vessel bioreactor can be differentially driven toward definitive erythropoiesis and cardiomyogenesis in the absence of ELC formation. Three groups were evaluated: mESCs in maintenance medium with leukemia inhibitory factor (LIF, control) and mESCs cultured with HepG2 CM (CM1 and CM2). Control and CM1 groups were cultivated for 8 days in early differentiation medium with murine stem cell factor (mSCF) followed by 10 days in hematopoietic differentiation medium (HDM) containing human erythropoietin, m-interleukin (mIL)-3, and mSCF. CM2 cells were cultured for 18 days in HDM, bypassing early differentiation. In CM1, a fivefold expansion of hematopoietic colonies was observed at day 14, with enhancement of erythroid progenitors, hematopoietic genes (Gata-2 and SCL), erythroid genes (EKLF and ß-major globin), and proteins (Gata-1 and ß-globin), although ζ-globin was not expressed. In contrast, CM2 primarily produced beating colonies in standard hematopoietic colony assay and expressed early cardiomyogenic markers, anti-sarcomeric α-actinin and Gata-4. In conclusion, a scalable, automatable, integrated, 3D bioprocess for the differentiation of mESC toward definitive erythroblasts has been established. Interestingly, cardiomyogenesis was also directed in a specific protocol with HepG2 CM and hematopoietic cytokines making this platform a useful tool for the study of erythroid and cardiomyogenic development.
Assuntos
Alginatos/metabolismo , Diferenciação Celular/fisiologia , Citocinas/metabolismo , Células-Tronco Embrionárias/fisiologia , Eritropoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Desenvolvimento Muscular/fisiologia , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/metabolismo , Células-Tronco Embrionárias/metabolismo , Fatores de Ligação de DNA Eritroide Específicos/metabolismo , Ácido Glucurônico/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Células Hep G2 , Ácidos Hexurônicos/metabolismo , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Interleucina-3/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Fator de Células-Tronco/metabolismo , Globinas beta/metabolismo , Globinas zeta/metabolismoRESUMO
A variant Hb ζ2ß2(s) that is formed from sickle hemoglobin (Hb S; α2ß2(s)) by exchanging adult α-globin with embryonic ζ-globin subunits shows promise as a therapeutic agent for sickle-cell disease (SCD). Hb ζ2ß2(s) inhibits the polymerization of deoxygenated Hb S in vitro and reverses characteristic features of SCD in vivo in mouse models of the disorder. When compared with either Hb S or with normal human adult Hb A (α2ß2), Hb ζ2ß2(s) exhibits atypical properties that include a high oxygen affinity, reduced cooperativity, a weak Bohr effect and blunted 2,3-diphosphoglycerate allostery. Here, the 1.95â Å resolution crystal structure of human Hb ζ2ß2(s) that was expressed in complex transgenic knockout mice and purified from their erythrocytes is presented. When fully liganded with carbon monoxide, Hb ζ2ß2(s) displays a central water cavity, a ζ1-ß(s)2 (or ζ2-ß(s)1) interface, intersubunit salt-bridge/hydrogen-bond interactions, C-terminal ßHis146 salt-bridge interactions, and a ß-cleft, that are highly unusual for a relaxed hemoglobin structure and are more typical of a tense conformation. These quaternary tense-like features contrast with the tertiary relaxed-like conformations of the ζ1ß(s)1 dimer and the CD and FG corners, as well as the overall structures of the heme cavities. This crystallographic study provides insights into the altered oxygen-transport properties of Hb ζ2ß2(s) and, moreover, decouples tertiary- and quaternary-structural events that are critical to Hb ligand binding and allosteric function.
Assuntos
Anemia Falciforme/sangue , Anemia Falciforme/genética , Hemoglobina Falciforme/genética , Globinas zeta/química , Adulto , Regulação Alostérica/genética , Animais , Cristalografia por Raios X , Variação Genética , Hemoglobina Falciforme/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Oxigênio/metabolismo , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica/genética , alfa-Globinas/genética , alfa-Globinas/metabolismo , Globinas zeta/metabolismoRESUMO
Embryonic stem cell (ESC) differentiation via embryoid body (EB) formation is an established method that generates the 3 germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Previously, we have enhanced mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from mESCs without standard formation of EBs. Two predifferentiation conditions were tested: (1) mESCs cultured 3 days in standard predifferentiation medium (control) and (2) mESCs cultured 3 days in HepG2 CM (CM-mESCs). Both groups were then exposed to primary differentiation for 8 days (ELC-formation period) and 14 days of hematopoietic differentiation. Enhanced mesoderm formation was observed in the CM-mESC group with an almost 5-fold increase in ELC formation (P ≤ 0.05) and higher expression of mesoderm genes-Brachyury-T, Goosecoid, and Flk-1-compared with those of control mESCs. Hematopoietic colony formation by CM-mESCs was also enhanced by 2-fold at days 7 and 14 with earlier colony commitment compared with those of control mESCs (P ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in CM-mESC culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (ß-globin) erythroid genes and proteins was also observed by day 7 in CM-mESC cultures. These data indicate that hematopoietic cells more quickly differentiate from CM-mESCs, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Eritrócitos/citologia , Células Precursoras Eritroides/citologia , Animais , Técnicas de Cultura de Células/métodos , Linhagem da Célula , Ensaio de Unidades Formadoras de Colônias/métodos , Meios de Cultivo Condicionados/metabolismo , Células-Tronco Embrionárias/metabolismo , Eritrócitos/metabolismo , Células Precursoras Eritroides/metabolismo , Eritropoese , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Células Hep G2 , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Tempo , Globinas beta/genética , Globinas beta/metabolismo , Globinas zeta/genética , Globinas zeta/metabolismoRESUMO
The mammalian embryonic zeta-globin genes, including that of humans, are expressed at the early embryonic stage and then switched off during erythroid development. This autonomous silencing of the zeta-globin gene transcription is probably regulated by the cooperative work of various protein-DNA and protein-protein complexes formed at the zeta-globin promoter and its upstream enhancer (HS-40). We present data here indicating that a protein-binding motif, ZF2, contributes to the repression of the HS-40-regulated human zeta-promoter activity in erythroid cell lines and in transgenic mice. Combined site-directed mutagenesis and EMSA suggest that repression of the human zeta-globin promoter is mediated through binding of the zinc finger factor RREB1 to ZF2. This model is further supported by the observation that human zeta-globin gene transcription is elevated in the human erythroid K562 cell line or the primary erythroid culture upon RNA interference (RNAi)(2) knockdown of RREB1 expression. These data together suggest that RREB1 is a putative repressor for the silencing of the mammalian zeta-globin genes during erythroid development. Because zeta-globin is a powerful inhibitor of HbS polymerization, our experiments have provided a foundation for therapeutic up-regulation of zeta-globin gene expression in patients with severe hemoglobinopathies.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Globinas zeta/genética , Animais , Sequência de Bases , Sítios de Ligação , Western Blotting , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Células K562 , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Globinas zeta/antagonistas & inibidores , Globinas zeta/metabolismoRESUMO
OBJECTIVE: To explore a rapid method for diagnosing alpha-thalassemia-1 of Southeast Asia type. METHODS: Seventy-six patients were detected by polymerase chain reaction (PCR). RESULTS: Thirty-one patients were diagnosed as alpha-thalassemia-1 of Southeast Asia type. PCR was compared with polyacrylamide gel electropheresis for detecting zeta-globin chain in 56 patients and the correspondance rate was 83.93%. Ten cases of prenatal diagnosis were performed by PCR, and the results showed that one was Hb bart's hydrop fetalis, four were carriers of alpha-thalassemia-1 of Southeast Asia type, and the others were normal fetalis or carrier of alpha-thalassemia-2. CONCLUSION: The PCR method is simple and accurate for diagnosing alpha-thalassemia of Southeast Asia type, and it provides a new approach to the prenatal diagnosis of alpha-thalassemia fetalis and the detection of alpha-globin gene cluster.