RESUMO
Hemoglobin H disease (Hb H) arises through the loss or dysfunction of three of the four alpha globin genes through the co-inheritance of either gross gene deletions or an abnormal hemoglobin which causes a non-deletional loss of α-globin expression. This study sought to investigate erythropoiesis in Hb H-Constant Spring (Hb H-CS) disease, a common form of Hb H disease in Southeast Asia, caused by the inheritance of the Constant Spring variant hemoglobin together with deletion of two of the alpha globin genes. In comparison to normal erythroblasts, Hb H-CS erythroblasts showed reduced cell expansion although no difference in differentiation was observed. Proteomic analysis revealed the increased expression of both chaperone and chaperonin proteins as well as down regulation of proteins regulating apoptosis. Both chaperone and chaperonin mediated folding require ATP, and evidence of increase energy demand was seen in the form of increased expression of enzymes involved in purine biosynthesis and increased levels of reactive oxygen species. A significant increase in apoptosis was seen in Hb H-CS erythroblasts, and the results from the proteomic analysis suggest that this arises at least in part from the consequences of increased folding requirements in the Hb H-CS erythroblast.
Assuntos
Eritroblastos/metabolismo , Hemoglobinas Anormais/metabolismo , Proteoma/metabolismo , Antígenos CD34/metabolismo , Apoptose , Diferenciação Celular , Eritroblastos/citologia , Feminino , Humanos , Masculino , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Erythropoiesis in ß0-thalassaemia/Hb E patients, the most common variant form of ß-thalassaemia in Southeast Asia, is characterized by accelerated differentiation and over-expansion of erythroid precursor cells. The mechanism driving this accelerated expansion and differentiation remain unknown. To address this issue a proteomic analysis was undertaken to firstly identify proteins differentially expressed during erythroblast differentiation and a second analysis was undertaken to identify proteins differentially expressed between ß0-thalassaemia/Hb E erythroblasts and control erythroblasts. The majority of proteins identified as being differentially expressed between ß0-thalassaemia/Hb E and control erythroblasts were constituents of the glycolysis/TCA pathway and levels of oxidative stress correlated with the degree of erythroid expansion. A model was constructed linking these observations with previous studies showing increased phosphorylation of ERK1/2 in thalassemic erythroblasts which predicted the increased activation of PKA, PKB and PKC which Western analysis confirmed. Inhibition of PKA or PKC reduced ß0-thalassaemia/Hb E erythroblast differentiation and/or expansion. We propose that increased expansion and differentiation of ß0-thalassaemia/Hb E erythroblasts occur as a result of feedback loops acting through increased oxidative metabolism.
Assuntos
Eritroblastos , MAP Quinases Reguladas por Sinal Extracelular , Hemoglobina E/metabolismo , Talassemia beta/fisiopatologia , Adolescente , Adulto , Apoptose/fisiologia , Western Blotting/métodos , Estudos de Casos e Controles , Diferenciação Celular , Ativação Enzimática , Eritroblastos/citologia , Eritroblastos/metabolismo , Eritropoese/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Glicólise , Humanos , Células K562 , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Fosforilação , Proteômica , Espécies Reativas de Oxigênio/análise , Células U937 , Talassemia beta/sangueRESUMO
Erythropoiesis in ß-thalassemia patients is ineffective, primarily because of death of the erythroid progenitor cells at the polychromatic normoblast stage. While it is known that autophagy plays a critical role during erythropoiesis by removing organelles from erythroid cells during terminal differentiation, its role in erythroid cells whose function is impaired remains to be explored. To investigate this, CD34+ erythroid progenitor cells from normal controls and ß-thalassemia/Hb E patients were isolated from peripheral blood and cultured under conditions driving differentiation into an erythroid lineage, and levels of autophagy and apoptosis were analyzed both directly and after biochemical manipulation with L: -asparagine. A significantly higher level of autophagy was seen in ß-thalassemia/Hb E erythroblasts as compared to normal control erythroblasts during erythropoiesis. Interestingly, this activation was mediated in part by the presence of high levels of Ca(2+) as modulation of Ca(2+) levels significantly reduced the level of autophagy in these cells. Inhibition of autophagic flux in normal erythroblasts significantly increased apoptosis in normal erythroblasts, but not in thalassemic erythroblasts, although sensitivity to autophagic flux inhibition was restored by reduction of Ca(2+) levels. These results suggest that high levels of autophagy in ß-thalassemia/HbE erythroblasts may contribute to the increased levels of apoptosis that lead to ineffective erythropoiesis in ß-thalassemia/Hb E erythroblasts.
Assuntos
Autofagia/fisiologia , Eritroblastos/fisiologia , Eritropoese/fisiologia , Hemoglobina E/metabolismo , Talassemia beta/sangue , Apoptose/fisiologia , Cálcio/metabolismo , Diferenciação Celular , Eritroblastos/citologia , Citometria de Fluxo , HumanosRESUMO
BACKGROUND: Cells respond to stress stimuli through a number of response pathways, of which one of the most important and well characterized is the unfolded protein response. Despite a large body of work which suggests that stress in erythroblasts may play a pivotal role in the pathogenesis of beta-thalassemia/Hb E disease, this pathway remains uninvestigated. DESIGN AND METHODS: Day 10 erythroblasts from normal controls and beta-thalassemia/Hb E patients were subjected to internal (treatment with tunicamycin) and external (serum and growth factor withdrawal) stress stimuli and the activation of the unfolded protein response pathway was investigated. RESULTS: Normal erythroblasts responded to both internal and external stress by activating the unfolded protein response (UPR) pathway while in contrast, erythroblasts from beta-thalassemia/Hb E patients only showed activation of the unfolded protein response pathway in response to internal stress. This was reflected by a markedly increased induction of apoptosis in serum and growth factor deprived beta-thalassemia/Hb E erythroblasts as compared to control cells. Modulation of the levels of intracellular Ca(2+) in thalassemic erythroblasts restored UPR activation during serum deprivation and significantly reduced the level of serum deprivation induced apoptosis to control levels. CONCLUSIONS: These results suggest the failure of thalassemic erythroblasts to cope with cellular stress caused by an impaired UPR function as a result of high Ca(2+) levels may exacerbate thalassemic cell death during erythropoiesis.
Assuntos
Eritroblastos/patologia , Eritropoese/fisiologia , Hemoglobina E/metabolismo , Talassemia beta/sangue , Talassemia beta/patologia , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Eritroblastos/química , Feminino , Hemoglobina E/química , Hemoglobina E/genética , Humanos , Masculino , Pessoa de Meia-Idade , Estabilidade Proteica , Desdobramento de Proteína , Resposta a Proteínas não Dobradas/genética , Talassemia beta/genéticaRESUMO
Beta-thalassaemia is one of the most common inherited anaemias, arising from a partial or complete loss of beta-globin chain synthesis. In severe cases, marked bone marrow erythroid hyperplasia, believed to result from erythropoietin (EPO)-mediated feedback from the anaemic condition is common, however, as yet, no study has investigated EPO-mediated signal transduction in thalassaemic erythroid cells. Using proerythroblasts generated from peripheral blood circulating CD34+ haematopoietic progenitor cells, the activation of the mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERKs) pathway was examined under conditions of steady state growth, cytokine deprivation and post-EPO stimulation. Levels of cellular cyclic adenosine monophosphate (cAMP) and Ca2+ were determined as was the degree of erythroid expansion. A significantly higher basal level of phosphorylation of ERK1/2 was observed in beta-thalassaemia/Hb E proerythroblasts as compared to normal controls, which was coupled with significantly higher levels of both cAMP and Ca2+. Modulation of either cAMP or Ca2+ or direct inhibition of MAPK/ERK kinase (MEK) reduced basal levels of ERK1/2 phosphorylation, as well as significantly reducing the level of erythroid expansion. These results suggest that, in contrast to current models, hyper proliferation of beta-thalassaemia/Hb E proerythroblasts is an intrinsic process driven by higher basal levels of ERK1/2 phosphorylation resulting from deregulation of levels of cAMP and Ca2+.