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1.
Antioxidants (Basel) ; 10(2)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669171

RESUMO

Sickle cell disease (SCD) is the most common hereditary disorder of hemoglobin (Hb), which affects approximately a million people worldwide. It is characterized by a single nucleotide substitution in the ß-globin gene, leading to the production of abnormal sickle hemoglobin (HbS) with multi-system consequences. HbS polymerization is the primary event in SCD. Repeated polymerization and depolymerization of Hb causes oxidative stress that plays a key role in the pathophysiology of hemolysis, vessel occlusion and the following organ damage in sickle cell patients. For this reason, reactive oxidizing species and the (end)-products of their oxidative reactions have been proposed as markers of both tissue pro-oxidant status and disease severity. Although more studies are needed to clarify their role, antioxidant agents have been shown to be effective in reducing pathological consequences of the disease by preventing oxidative damage in SCD, i.e., by decreasing the oxidant formation or repairing the induced damage. An improved understanding of oxidative stress will lead to targeted antioxidant therapies that should prevent or delay the development of organ complications in this patient population.

2.
Adv Hematol ; 20102010.
Artigo em Inglês | MEDLINE | ID: mdl-20827391

RESUMO

In recent years there have been major advances in our knowledge of the regulation of iron metabolism that have had implications for understanding the pathophysiology of some human disorders like beta-thalassemia and other iron overload diseases. However, little is known about the relationship among ineffective erythropoiesis, the role of iron-regulatory genes, and tissue iron distribution in beta-thalassemia. The principal aim of this paper is an update about the role of Ferroportin during human normal and pathological erythroid differentiation. Particular attention will be given to beta-thalassemia and other diseases with iron overload. Recent discoveries indicate that there is a potential for therapeutic intervention in beta-thalassemia by means of manipulating iron metabolism.

3.
J Mol Cell Cardiol ; 45(6): 761-9, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18851973

RESUMO

The treatment with beta-blockers causes an enhancement of the norepinephrine-induced fetal gene response in cultured cardiomyocytes. Here, we tested whether the activation of cAMP-mediated beta-adrenergic signaling antagonizes alpha(1)-adrenergic receptor (AR)-mediated fetal gene response. To address this question, the fetal gene program, of which atrial natriuretic peptide (ANP) and the beta-isoform of myosin heavy chain are classical members, was induced by phenylephrine (PE), an alpha(1)-AR agonist. In cultured neonatal rat cardiomyocytes, we found that stimulation of beta-ARs with isoproterenol, a beta-AR agonist, inhibited the fetal gene expression induced by PE. Similar results were also observed when cardiomyocytes were treated with forskolin (FSK), a direct activator of adenylyl cyclase, or 8-CPT-6-Phe-cAMP, a selective activator of protein kinase A (PKA). Conversely, the PE-induced fetal gene expression was further upregulated by H89, a selective PKA inhibitor. To evaluate whether these results could be generalized to Gq-mediated signaling and not specifically to alpha(1)-ARs, cardiomyocytes were treated with prostaglandin F(2)alpha, another Gq-coupled receptor agonist, which is able to promote fetal gene expression. This treatment caused an increase of both ANP mRNA and protein levels, which was almost completely abolished by FSK treatment. The capability of beta-adrenergic signaling to regulate the fetal gene expression was also evaluated in vivo conditions by using beta1- and beta2-AR double knockout mice, in which the predominant cardiac beta-AR subtypes are lacking, or by administering isoproterenol (ISO), a beta-AR agonist, at a subpressor dose. A significant increase of the fetal gene expression was found in beta(1)- and beta(2)-AR gene deficient mice. Conversely, we found that ANP, beta-MHC and skACT mRNA levels were significantly decreased in ISO-treated hearts. Collectively, these data indicate that cAMP-mediated beta-adrenergic signaling negatively regulates Gq cascade activation-induced fetal gene expression in cultured cardiomyocytes and that this inhibitory regulation is already operative in the mouse heart under physiological conditions.


Assuntos
Fator Natriurético Atrial/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , AMP Cíclico/metabolismo , Feto/metabolismo , Regulação da Expressão Gênica/fisiologia , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais/fisiologia , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Fator Natriurético Atrial/genética , Colforsina/farmacologia , AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprosta/farmacologia , Ativadores de Enzimas/farmacologia , Feto/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Isoproterenol/farmacologia , Isoquinolinas/farmacologia , Camundongos , Miócitos Cardíacos/citologia , Cadeias Pesadas de Miosina/biossíntese , Cadeias Pesadas de Miosina/genética , Miosina não Muscular Tipo IIB/biossíntese , Miosina não Muscular Tipo IIB/genética , Fenilefrina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos beta/genética , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia
4.
Blood Cells Mol Dis ; 39(1): 82-91, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17428703

RESUMO

Different proteins ensure the fine control of iron metabolism at the level of various tissues. Among these proteins, it was discovered a second transferrin receptor (TfR2), that seems to play a key role in the regulation of iron homeostasis. Its mutations are responsible for type 3 hemochromatosis (Type 3 HH). Although TfR2 expression in normal tissues was restricted at the level of liver and intestine, we observed that TfR2 was frequently expressed in tumor cell lines. Particularly frequent was its expression in ovarian cancer, colon cancer and glioblastoma cell lines; less frequent was its expression in leukemic and melanoma cell lines. Interestingly, in these tumor cell lines, TfR2 expression was inversely related to that of receptor 1 for transferrin (TfR1). Experiments of in vitro iron loading or iron deprivation provided evidence that TfR2 is modulated in cancer cell lines according to cellular iron levels following two different mechanisms: (i) in some cells, iron loading caused a downmodulation of total TfR2 levels; (ii) in other cell types, iron loading caused a downmodulation of membrane-bound TfR2, without affecting the levels of total cellular TfR2 content. Iron deprivation caused in both conditions an opposite effect compared to iron loading. These observations suggest that TfR2 expression may be altered in human cancers and warrant further studies in primary tumors. Furthermore, our studies indicate that, at least in tumor cells, TfR2 expression is modulated by iron through different biochemical mechanisms, whose molecular basis remains to be determined.


Assuntos
Regulação Neoplásica da Expressão Gênica , Ferro/metabolismo , Leucemia/metabolismo , Melanoma/metabolismo , Receptores da Transferrina/biossíntese , Antígenos CD/biossíntese , Antígenos CD/genética , Regulação Neoplásica da Expressão Gênica/genética , Células HL-60 , Hemocromatose/genética , Hemocromatose/metabolismo , Homeostase/genética , Humanos , Mucosa Intestinal/metabolismo , Células K562 , Leucemia/genética , Fígado/metabolismo , Melanoma/genética , Mutação , Receptores da Transferrina/genética , Células U937
5.
J Cell Sci ; 119(Pt 21): 4486-98, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17046995

RESUMO

Transferrin receptor 2 (TfR2) possesses a YQRV motif similar to the YTRF motif of transferrin receptor 1 (TfR1) responsible for the internalization and secretion through the endosomal pathway. Raft biochemical dissection showed that TfR2 is a component of the low-density Triton-insoluble (LDTI) plasma membrane domain, able to co-immunoprecipitate with caveolin-1 and CD81, two structural raft proteins. In addition, subcellular fractionation experiments showed that TfR1, which spontaneously undergoes endocytosis and recycling, largely distributed to intracellular organelles, whereas TfR2 was mainly associated with the plasma membrane. Given the TfR2 localization in lipid rafts, we tested its capability to activate cell signalling. Interaction with an anti-TfR2 antibody or with human or bovine holotransferrin showed that it activated ERK1/ERK2 and p38 MAP kinases. Integrity of lipid rafts was required for MAPK activation. Co-localization of TfR2 with CD81, a raft tetraspanin exported through exosomes, prompted us to investigate exosomes released by HepG2 and K562 cells into culture medium. TfR2, CD81 and to a lesser extent caveolin-1, were found to be part of the exosomal budding vesicles. In conclusion, the present study indicates that TfR2 localizes in LDTI microdomains, where it promotes cell signalling, and is exported out of the cells through the exosome pathway, where it acts as an intercellular messenger.


Assuntos
Microdomínios da Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores da Transferrina/metabolismo , Transdução de Sinais , Vesículas Transportadoras/metabolismo , Antígenos CD/metabolismo , Western Blotting , Carcinoma Hepatocelular/metabolismo , Caveolina 1/metabolismo , Membrana Celular/metabolismo , Células Cultivadas/metabolismo , Endocitose , Citometria de Fluxo , Humanos , Imunoprecipitação , Células K562/metabolismo , Neoplasias Hepáticas/metabolismo , Fosforilação , Receptores da Transferrina/genética , Frações Subcelulares , Tetraspanina 28
6.
Haematologica ; 90(12): 1595-606, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16330432

RESUMO

BACKGROUND AND OBJECTIVES: Ferroportin-1 (FPN1) is expressed in various types of cells that play critical roles in mammalian iron metabolism and appears to act as an iron exporter in these tissues. The aim of this study was to investigate whether erythroid cells possess specific mechanisms for iron export. DESIGN AND METHODS: The expression of FPN1 during human erythroid differentiation, the characterization of alternative transcripts, the modulation by iron and the subcellular localization of this protein were studied. RESULTS: FPN1 mRNA and protein are highly expressed during human erythroid differentiation. The iron-responsive element (IRE) in the 5'- untranslated region (UTR) of FPN1 mRNA is functional but, in spite of that, FPN1 protein expression, as well as mRNA level and half-life, seem not to be affected by iron. To explain these apparenthy discordant results we searched for alternative transcripts of FPN1 and found at least three different types of transcripts, displaying alternative 5' ends. Most of the FPN1 transcripts code for the canonical protein, but only half of them contain an IRE in the 5'-UTR and have the potential to be translationally regulated by iron. Expression analysis shows that alternative FPN1 transcripts are differentially expressed during erythroid differentiation. Finally, sustained expression of alternative FPN1 transcripts is apparently observed only in erythroid cells. INTERPRETATION AND CONCLUSIONS: This is the first report describing the presence of FPN1 in erythroid cells at all stages of differentiation, providing evidence that erythroid cells possess specific mechanisms of iron export. The existence of multiple FPN1 transcripts indicates a complex regulation of the FPN1 gene in erythroid cells.


Assuntos
Processamento Alternativo , Proteínas de Transporte de Cátions/biossíntese , Células Eritroides/metabolismo , Regulação da Expressão Gênica , Ferro/sangue , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/fisiologia , Diferenciação Celular/genética , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Desferroxamina/farmacologia , Células Eritroides/citologia , Eritropoese , Éxons/genética , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Meia-Vida , Humanos , Quelantes de Ferro/farmacologia , Células K562/citologia , Células K562/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Especificidade de Órgãos , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Frações Subcelulares/química , Frações Subcelulares/ultraestrutura , Transcrição Gênica , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/metabolismo
7.
Biochem J ; 381(Pt 3): 629-34, 2004 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-15084147

RESUMO

Human TFR2 (transferrin receptor 2) is a membrane-bound protein homologous with TFR1. High levels of TFR2 mRNA were found mainly in the liver and, to a lesser extent, in erythroid precursors. However, although the presence of the TFR2 protein in hepatic cells has been confirmed in several studies, evidence is lacking about the presence of the TFR2 protein in normal erythroid cells. Using two anti-TFR2 monoclonal antibodies, G/14C2 and G/14E8, we have provided evidence that TFR2 protein is not expressed in normal erythroid cells at any stage of differentiation, from undifferentiated CD34+ cells to mature orthochromatic erythroblasts. In contrast, erythroleukaemic cells (K562 cells) exhibited a high level of expression of TFR2 at both the mRNA and the protein level. We can therefore conclude that an elevated expression of TFR2 protein is observed in leukaemic cells, but not in normal erythroblasts. The implications of this observation for the understanding of the phenotypic features of haemochromatosis due to mutation of the TFR2 gene are discussed.


Assuntos
Células Eritroides/química , Células Eritroides/metabolismo , Receptores da Transferrina/biossíntese , Anticorpos/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Células Eritroides/citologia , Células Eritroides/patologia , Células Precursoras Eritroides/química , Células Precursoras Eritroides/metabolismo , Humanos , Células K562 , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Peso Molecular , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/química , Isoformas de Proteínas/imunologia , RNA Mensageiro/biossíntese , Receptores da Transferrina/química , Receptores da Transferrina/imunologia , Frações Subcelulares/química
8.
Blood ; 101(7): 2826-32, 2003 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-12424200

RESUMO

Mechanisms underlying fetal hemoglobin (HbF) reactivation in stress erythropoiesis have not been fully elucidated. We suggested that a key role is played by kit ligand (KL). Because glucocorticoids (GCs) mediate stress erythropoiesis, we explored their capacity to potentiate the stimulatory effect of KL on HbF reactivation, as evaluated in unilineage erythropoietic culture of purified adult progenitors (erythroid burst-forming units [BFU-Es]). The GC derivative dexamethasone (Dex) was tested in minibulk cultures at graded dosages within the therapeutical range (10(-6) to 10(-9) M). Dex did not exert significant effects alone, but synergistically it potentiated the action of KL in a dose-dependent fashion. Specifically, Dex induced delayed erythroid maturation coupled with a 2-log increased number of generated erythroblasts and enhanced HbF synthesis up to 85% F cells and 55% gamma-globin content at terminal maturation (ie, in more than 80%-90% mature erythroblasts). Equivalent results were obtained in unicellular erythroid cultures of sibling BFU-Es treated with KL alone or combined with graded amounts of Dex. These results indicate that the stimulatory effect of KL + Dex is related to the modulation of gamma-globin expression rather than to recruitment of BFU-Es with elevated HbF synthetic potential. At the molecular level, Id2 expression is totally suppressed in control erythroid culture but is sustained in KL + Dex culture. Hypothetically, Id2 may mediate the expansion of early erythroid cells, which correlates with HbF reactivation. These studies indicate that GCs play an important role in HbF reactivation. Because Dex acts at dosages used in immunologic disease therapy, KL + Dex administration may be considered to develop preclinical models for beta-hemoglobinopathy treatment.


Assuntos
Dexametasona/farmacologia , Células Precursoras Eritroides/metabolismo , Hemoglobina Fetal/biossíntese , Fator de Células-Tronco/farmacologia , Técnicas de Cultura de Células , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Hemoglobina Fetal/efeitos dos fármacos , Globinas/análise , Humanos , Hipóxia , Cinética , Masculino , Fatores de Transcrição/análise
9.
Leuk Lymphoma ; 43(8): 1645-50, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12400608

RESUMO

Interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 5 (IL-5) exert their biological activities through interaction with cell-surface receptors that consist of two subunits, a specific a subunit and a common beta transducing subunit (beta c). We have evaluated the effect of growth factors on the expression of beta c in normal monocytes. Addition of either GM-CSF or M-CSF to monocytes elicited a marked increase of beta c chain expression, a phenomenon seemingly related to a stimulation of the transcriptional activity of this gene mediated through an enhancement of the PU.1 DNA binding activity. Interestingly, during the activation of beta c chain expression by growth factors a switch from the synthesis of the truncated betaIT to the full-length beta c was observed. Similar observations have been made also in the growth factor-dependent erythroleukemic cell line TF-1, showing that GM-CSF deprivation elicited a marked decrease of beta c chain expression.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Interleucina-3/genética , Células Cultivadas , Humanos , Regiões Promotoras Genéticas , Subunidades Proteicas , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo
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