RESUMO
The current treatments applied in aquaculture to limit disease dissemination are mostly based on the use of antibiotics, either as prophylactic or therapeutic agents, with vaccines being available for a limited number of fish species and pathogens. Antimicrobial peptides are considered as promising novel substances to be used in aquaculture, due to their antimicrobial and immunomodulatory activities. Hepcidin, the major iron metabolism regulator, is found as a single gene in most mammals, but in certain fish species, including the European sea bass (Dicentrarchus labrax), two different hepcidin types are found, with specialized roles: the single type 1 hepcidin is involved in iron homeostasis trough the regulation of ferroportin, the only known iron exporter; and the various type 2 hepcidins present antimicrobial activity against a number of different pathogens. In this study, we tested the administration of sea bass derived hepcidins in models of infection and iron overload. Administration with hamp2 substantially reduced fish mortalities and bacterial loads, presenting itself as a viable alternative to the use of antibiotics. On the other hand, hamp1 seems to attenuate the effects of iron overload. Further studies are necessary to test the potential protective effects of hamp2 against other pathogens, as well as to understand how hamp2 stimulate the inflammatory responses, leading to an increased fish survival upon infection.
Assuntos
Peptídeos Antimicrobianos/uso terapêutico , Bass/imunologia , Doenças dos Peixes/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/veterinária , Hepcidinas/uso terapêutico , Sobrecarga de Ferro/veterinária , Photobacterium , Sequência de Aminoácidos , Animais , Apoferritinas/biossíntese , Apoferritinas/genética , Carga Bacteriana , Bass/microbiologia , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Transporte de Cátions/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Perfilação da Expressão Gênica , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/imunologia , Hepcidinas/biossíntese , Hepcidinas/genética , Ferro/análise , Sobrecarga de Ferro/tratamento farmacológico , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/imunologia , Fígado/química , Photobacterium/isolamento & purificaçãoRESUMO
Overexpression of ferritin heavy chain (FTH1) often associates with good prognosis in breast cancer (BCa), particularly in the triple-negative subtype (triple-negative breast cancer). However, the mechanism by which FTH1 exerts its possible tumor suppressor effects in BCa is not known. Here, we examined the bearing of FTH1 silencing or overexpression on several aspects of BCa cell growth in vitro. FTH1 silencing promoted cell growth and mammosphere formation, increased c-MYC expression, and reduced cell sensitivity to chemotherapy. In contrast, FTH1 overexpression inhibited cell growth, decreased c-MYC expression, and sensitized cancer cells to chemotherapy; silencing of c-MYC recapitulated the effects of FTH1 overexpression. These findings show for the first time that FTH1 suppresses tumor growth by inhibiting the expression of key oncogenes, such as c-MYC.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ferritinas/metabolismo , Oxirredutases/metabolismo , Apoferritinas/biossíntese , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Ferritinas/biossíntese , Ferritinas/genética , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Oxirredutases/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
PM2.5 is becoming a worldwide environmental problem, which profoundly endangers public health, thus progressively capturing public attention this decade. As a fragile target of PM2.5, the underlying mechanisms of endothelial cell damage are still obscure. According to the previous microarray data and signaling pathway analysis, a new form of cell death termed ferroptosis in the current study is proposed following PM2.5 exposure. In order to verify the vital role of ferroptosis in PM2.5-induced endothelial lesion and further understand the potential mechanism involved, intracellular iron content, ROS release and lipid peroxidation, as well as biomarkers of ferroptosis were detected, respectively. As a result, uptake of particles increases cellular iron content and ROS production. Meanwhile, GSH depletion, and the decrease of GSH-Px and NADPH play significant roles in PM2.5-induced endothelial cell ferroptosis. Moreover, significantly changed expression of TFRC, FTL and FTH1 hinted that dysfunction of iron uptake and storage is a major inducer of ferroptosis. Importantly, index monitored above can be partially rescued by lipid peroxidation inhibitor ferrostatin-1 and iron chelator deferoxamine mesylate, which mediated antiferroptosis activity mainly depends on the restoration of antioxidant activity and iron metabolism. In conclusion, our data basically show that PM2.5 enhances ferroptosis sensitivity with increased ferroptotic events in endothelial cells, in which iron overload, lipid peroxidation and redox imbalance act pivotal roles.
Assuntos
Células Endoteliais/metabolismo , Ferroptose/fisiologia , Sobrecarga de Ferro/patologia , Ferro/toxicidade , Material Particulado/toxicidade , Antígenos CD/biossíntese , Apoferritinas/biossíntese , Apoptose/efeitos dos fármacos , Cicloexilaminas/farmacologia , Desferroxamina/farmacologia , Ferritinas/biossíntese , Glutationa/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Oxirredutases , Fenilenodiaminas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores da Transferrina/biossíntese , Transdução de Sinais/efeitos dos fármacosRESUMO
AIMS: Combined magnetic resonance imaging (MRI) of molecular and morpho-functional changes might prove highly valuable for the elucidation of pathological processes involved in the development of cardiac diseases. Our aim was to test a novel MRI reporter gene for in vivo assessment of the canonical Wnt/ß-catenin/TCF pathway activation, an important regulator of post-ischaemic cardiac remodelling. METHODS AND RESULTS: We designed and developed a chimeric construct encoding for both of iron-binding human ferritin heavy chain (hFTH) controlled by the ß-catenin-responsive TCF/lymphoid-enhancer binding factor (Lef) promoter and constitutively expressed green fluorescent protein (GFP). It was carried by adeno-associated virus serotype 9 (rAAV9) vectors and delivered to the peri-infarct myocardium of rats subjected to coronary ligation (n = 11). By 1.5 T MRI and a multiecho T2* gradient echo sequence, we detected iron accumulation only in the border zone of the transduced infarcted hearts. In the same cardiac area, post-mortem histological analysis confirmed the co-existence of iron accumulation and GFP. The iron signal was absent when rats (n = 6) were chronically treated with SEN195 (10 mg/kg/day), a small-molecular inhibitor of ß-catenin/TCF-dependent gene transcription. Canonical Wnt pathway inhibition attenuated the post-ischaemic remodelling process, as demonstrated by the significant preservation of cardiac function, the 42 ± 1% increase of peri-infarct arteriolar density and 43 ± 3% reduction in infarct scar size compared with untreated animals. CONCLUSIONS: The TCF/Lef promoter-hFTH construct is a novel and reliable MRI reporter gene for in vivo detection of the canonical Wnt/ß-catenin/TCF activation state in response to cardiac injury and therapeutic interventions.
Assuntos
Genes Reporter , Imagem Cinética por Ressonância Magnética/métodos , Imagem Molecular/métodos , Infarto do Miocárdio/diagnóstico por imagem , Miocárdio/metabolismo , Fatores de Transcrição TCF/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Via de Sinalização Wnt , Animais , Apoferritinas/biossíntese , Apoferritinas/genética , Dependovirus/genética , Modelos Animais de Doenças , Vetores Genéticos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Ferro/metabolismo , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Valor Preditivo dos Testes , Regiões Promotoras Genéticas , Ratos Wistar , Reprodutibilidade dos Testes , Fatores de Transcrição TCF/genética , TransfecçãoRESUMO
Iron accumulation in the retina is associated with the development of age-related macular degeneration (AMD). IV iron is a common method to treat iron deficiency anemia in adults, and its retinal manifestations have not hitherto been identified. To assess whether IV iron formulations can be retina-toxic, we generated a mouse model for iron-induced retinal damage. Male C57BL/6J mice were randomized into groups receiving IV iron-sucrose (+Fe) or 30% sucrose (-Fe). Iron levels in neurosensory retina (NSR), retinal pigment epithelium (RPE), and choroid were assessed using immunofluorescence, quantitative PCR, and the Perls' iron stain. Iron levels were most increased in the RPE and choroid while levels in the NSR did not differ significantly in +Fe mice compared to controls. Eyes from +Fe mice shared histological features with AMD, including Bruch's membrane (BrM) thickening with complement C3 deposition, as well as RPE hypertrophy and vacuolization. This focal degeneration correlated with areas of high choroidal iron levels. Ultrastructural analysis provided further detail of the RPE/photoreceptor outer segment vacuolization and Bruch's membrane thickening. Findings were correlated with a clinical case of a 43-year-old patient who developed numerous retinal drusen, the hallmark of AMD, within 11 months of IV iron therapy. Our results suggest that IV iron therapy may have the potential to induce or exacerbate a form of retinal degeneration. This retinal degeneration shares features with AMD, indicating the need for further study of AMD risk in patients receiving IV iron treatment.
Assuntos
Compostos Férricos/efeitos adversos , Ácido Glucárico/efeitos adversos , Ferro/metabolismo , Degeneração Macular/induzido quimicamente , Estresse Oxidativo/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Animais , Apoferritinas/biossíntese , Apoferritinas/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Compostos Férricos/administração & dosagem , Óxido de Ferro Sacarado , Regulação da Expressão Gênica , Ácido Glucárico/administração & dosagem , Injeções Intravenosas , Degeneração Macular/genética , Degeneração Macular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores da Transferrina/biossíntese , Receptores da Transferrina/genética , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismoRESUMO
Iron cardiomyopathy is the leading cause of death in iron overload. Men have twice the mortality rate of women, though the cause is unknown. In hemojuvelin-knockout mice, a model of the disease, males load more cardiac iron than females. We postulated that sex differences in cardiac iron import cause differences in cardiac iron concentration. Reverse transcription polymerase chain reaction was used to measure mRNA of cardiac iron transporters in hemojuvelin-knockout mice. No sex differences were discovered among putative importers of nontransferrin-bound iron (L-type and T-type calcium channels, ZRT/IRT-like protein 14 zinc channels). Transferrin-bound iron transporters were also analyzed; these are controlled by the iron regulatory element/iron regulatory protein (IRE/IRP) system. There was a positive relationship between cardiac iron and ferroportin mRNA in both sexes, but it was significantly steeper in females (p < 0.05). Transferrin receptor 1 and divalent metal transporter 1 were more highly expressed in females than males (p < 0.01 and p < 0.0001, respectively), consistent with their lower cardiac iron levels, as predicted by IRE/IRP regulatory pathways. Light-chain ferritin showed a positive correlation with cardiac iron that was nearly identical in males and females (R(2) = 0.41, p < 0.01; R(2) = 0.56, p < 0.05, respectively), whereas heavy-chain ferritin was constitutively expressed in both sexes. This represents the first report of IRE/IRP regulatory pathways in the heart. Transcriptional regulation of ferroportin was suggested in both sexes, creating a potential mechanism for differential set points for iron export. Constitutive heavy-chain-ferritin expression suggests a logical limit to cardiac iron buffering capacity at levels known to produce heart failure in humans.
Assuntos
Apoferritinas/genética , Regulação da Expressão Gênica , Sobrecarga de Ferro/genética , Proteínas Reguladoras de Ferro/biossíntese , Proteínas Reguladoras de Ferro/genética , Ferro/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/biossíntese , Transcrição Gênica , Animais , Apoferritinas/biossíntese , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Transporte de Cátions/genética , Modelos Animais de Doenças , Feminino , Proteínas Ligadas por GPI , Hormônios Esteroides Gonadais/administração & dosagem , Hemocromatose/genética , Hemocromatose/metabolismo , Proteína da Hemocromatose , Hepcidinas/biossíntese , Hepcidinas/genética , Terapia de Reposição Hormonal , Sobrecarga de Ferro/etiologia , Sobrecarga de Ferro/metabolismo , Ferro da Dieta/toxicidade , Fígado/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Orquiectomia , Tamanho do Órgão , Ovariectomia , Subunidades Proteicas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Caracteres SexuaisRESUMO
Ferritin heavy chain (FTH1) is a 21-kDa subunit of the ferritin complex, known for its role in iron metabolism, and which has recently been identified as a favorable prognostic protein for triple negative breast cancer (TNBC) patients. Currently, it is not well understood how FTH1 contributes to an anti-tumor response. Here, we explored whether expression and cellular compartmentalization of FTH1 correlates to an effective immune response in TNBC patients. Analysis of the tumor tissue transcriptome, complemented with in silico pathway analysis, revealed that FTH1 was an integral part of an immunomodulatory network of cytokine signaling, adaptive immunity, and cell death. These findings were confirmed using mass spectrometry (MS)-derived proteomic data, and immunohistochemical staining of tissue microarrays. We observed that FTH1 is localized in both the cytoplasm and/or nucleus of cancer cells. However, high cytoplasmic (c) FTH1 was associated with favorable prognosis (Log-rank p = 0.001), whereas nuclear (n) FTH1 staining was associated with adverse prognosis (Log-rank p = 0.019). cFTH1 staining significantly correlated with total FTH1 expression in TNBC tissue samples, as measured by MS analysis (Rs = 0.473, p = 0.0007), but nFTH1 staining did not (Rs = 0.197, p = 0.1801). Notably, IFN γ-producing CD8+ effector T cells, but not CD4+ T cells, were preferentially enriched in tumors with high expression of cFTH1 (p = 0.02). Collectively, our data provide evidence toward new immune regulatory properties of FTH1 in TNBC, which may facilitate development of novel therapeutic targets.
Assuntos
Apoferritinas/metabolismo , Biomarcadores Tumorais/metabolismo , Linfócitos T CD8-Positivos/imunologia , Ferritinas/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Adulto , Idoso , Apoferritinas/biossíntese , Apoferritinas/imunologia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Feminino , Ferritinas/biossíntese , Ferritinas/imunologia , Humanos , Interferon gama/biossíntese , Interferon gama/imunologia , Pessoa de Meia-Idade , Oxirredutases , Prognóstico , Mapas de Interação de Proteínas , Proteômica , Análise Serial de Tecidos , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/mortalidadeRESUMO
Abnormal scarring results from the expression and composition of extracellular matrix molecules. The transcription and translation of collagens I and III, fibronectin, laminin, periostin, and tenascin are all increased in raised dermal scar tissue. However, human keloid development is not fully defined. In this study, we identified proteins expressed differentially between normal skin and keloid scar tissues and examined their function in keloid formation using fibroblasts. Skin specimens from normal volunteers and patients with keloids were obtained by skin biopsy. Whole proteins were isolated by two-dimensional electrophoresis, and differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. Protein function was determined by proliferation assay using annexin A2-overexpressing keloid fibroblasts. The expression of 11 protein spots was altered by at least 1.5-fold in patients with keloids than in normal volunteers. Of these proteins, annexin A2, a pre-serum amyloid P component, serum albumin precursor, and tryptase-I, were down-regulated in keloid tissue compared to normal skin. Collagen alpha 1(V) chain precursor, collagen alpha 1(I) chain precursor, ferritin light subunit, alpha 1(III) collagen, 6-phosphogluconolactonase, and calponin 2 were up-regulated. Diminished expression of annexin A2 was confirmed by immunoblotting and immunohistochemistry. Treatment with the recombinant human epidermal growth factor increased proliferation of keloid fibroblasts, which was more inhibited in annexin A2-overexpressing fibroblasts than in non-transfected control cells. These results imply that annexin A2 may participate in keloid formation by inhibiting keloid fibroblast proliferation. Therefore, it is concluded that annexin A2 may be a valuable therapeutic target for keloid lesions.
Assuntos
Anexina A2/biossíntese , Proliferação de Células , Fibroblastos , Queloide/metabolismo , Pele/metabolismo , Adolescente , Adulto , Idoso , Apoferritinas/biossíntese , Hidrolases de Éster Carboxílico/biossíntese , Células Cultivadas , Colágeno Tipo I/biossíntese , Colágeno Tipo III/biossíntese , Colágeno Tipo V/biossíntese , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Proteínas dos Microfilamentos/biossíntese , Pessoa de Meia-Idade , Interferência de RNA , RNA Interferente Pequeno , Albumina Sérica/biossíntese , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triptases/biossíntese , Regulação para Cima , Adulto JovemRESUMO
Unconjugated bilirubin (UCB) is responsible for neonatal jaundice and high level of free bilirubin (Bf) can lead to kernicterus. Previous studies suggest that oxidative stress is a critical component of UCB-induced neurotoxicity. The Nrf2 pathway is a powerful sensor for cellular redox state and is activated directly by oxidative stress and/or indirectly by stress response protein kinases. Activated Nrf2 translocates to nucleus, binds to Antioxidant Response Element (ARE), and enhances the up-regulation of cytoprotective genes that mediate cell survival. The aim of the present study was to investigate the role of Nrf2 pathway in cell response to bilirubin mediated oxidative stress in the neuroblastoma SH-SY5Y cell line. Cells exposed to a toxic concentration of UCB (140 nM Bf) showed an increased intracellular ROS levels and enhanced nuclear accumulation of Nrf2 protein. UCB stimulated transcriptional induction of ARE-GFP reporter gene and induced mRNA expression of multiple antioxidant response genes as: xCT, Gly1, γGCL-m, γGCL-c, HO-1, NQO1, FTH, ME1, and ATF3. Nrf2 siRNA decreased UCB induced mRNA expression of HO1 (75%), NQO1 (54%), and FTH (40%). The Nrf2-related HO-1 induction was reduced to 60% in cells pre-treated with antioxidant (NAC) or specific signaling pathway inhibitors for PKC, P38α and MEK1/2 (80, 40 and 25%, respectively). In conclusion, we demonstrated that SH-SY5Y cells undergo an adaptive response against UCB-mediated oxidative stress by activation of multiple antioxidant response, in part through Nrf2 pathway.
Assuntos
Elementos de Resposta Antioxidante/genética , Antioxidantes/farmacologia , Bilirrubina/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Acetilcisteína/farmacologia , Fator 3 Ativador da Transcrição/biossíntese , Apoferritinas/biossíntese , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sequestradores de Radicais Livres/farmacologia , Heme Oxigenase-1/biossíntese , Células Hep G2 , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/biossíntese , Fator 2 Relacionado a NF-E2/genética , Proteína Quinase C/antagonistas & inibidores , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
Decreased serum and increased hepatic iron uptake is the hallmark of acute-phase (AP) response. Iron uptake is controlled by iron transport proteins such as transferrin receptors (TfRs) and lipocalin 2 (LCN-2). The current study aimed to understand the regulation of iron uptake in primary culture hepatocytes in the presence/absence of AP mediators. Rat hepatocytes were stimulated with different concentrations of iron alone (0.01, 0.1, 0.5 mM) and AP cytokines (interleukin 6 [IL-6], IL-1ß, tumor necrosis factor α) in the presence/absence of iron (FeCl3: 0.1 mM). Hepatocytes were harvested at different time points (0, 6, 12, 24 h). Total mRNA and proteins were extracted for reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. A significant iron uptake was detected with 0.1 mM iron administration with a maximum (133.37 ± 4.82 µg/g of protein) at 24 h compared with control and other iron concentrations. This uptake was further enhanced in the presence of AP cytokines with a maximum iron uptake (481 ± 25.81 µg/g of protein) after concomitant administration of IL-6 + iron to cultured hepatocytes. Concomitantly, gene expression of LCN-2 and ferritin subunits (light- and heavy-chain ferritin subunits) was upregulated by iron or/and AP cytokines with a maximum at 24 h both at mRNA and protein levels. In contrast, a decreased TfR1 level was detected by IL-6 and iron alone, whereas combination of iron and AP cytokines (mainly IL-6) abrogated the downregulation of TfR1. An increase in LCN-2 release into the supernatant of cultured hepatocytes was observed after addition of iron/AP cytokines into the medium. This increase in secretion was further enhanced by combination of IL-6 + iron. In conclusion, iron uptake is tightly controlled by already present iron concentration in the culture. This uptake can be further enhanced by AP cytokines, mainly by IL-6.
Assuntos
Reação de Fase Aguda/metabolismo , Citocinas/farmacologia , Hepatócitos/metabolismo , Ferro/farmacocinética , Animais , Apoferritinas/biossíntese , Células Cultivadas , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Hepatócitos/efeitos dos fármacos , Interleucina-6/farmacologia , Ferro/administração & dosagem , Ferro/farmacologia , L-Lactato Desidrogenase/metabolismo , Lipocalina-2 , Lipocalinas/metabolismo , Masculino , RNA Mensageiro/genética , Ratos , Ratos Wistar , Receptores da Transferrina/biossíntese , Regulação para Cima/efeitos dos fármacosRESUMO
DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5 mC to 5 hmC in vivo, and increase 5 hmC in 5751 genes in cells. 5 hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein ferritin light chain showed a significant increase at both mRNA and protein levels indicating a role of iron regulation in stimulating Tet-mediated 5 mC oxidation. Consistently, the deprivation of cellular labile iron using specific chelator blocked the 5 hmC increase, and a delivery of labile iron increased the 5 hmC level. Moreover, both Tet1/Tet2 knockout and dimethyloxalylglycine-induced Tet inhibition diminished the 5 hmC increase. These results suggest an iron-regulated Tet-dependent DNA demethylation mechanism mediated by redox-active biomolecules.
Assuntos
Metilação de DNA , Dioxigenases/metabolismo , Ferro/metabolismo , Quinonas/farmacologia , 5-Metilcitosina/metabolismo , Animais , Apoferritinas/biossíntese , Apoferritinas/genética , Linhagem Celular , Linhagem Celular Tumoral , Cloranila/farmacologia , Citosina/análogos & derivados , Citosina/metabolismo , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Genoma , Humanos , Camundongos , Oxirredução , Proteínas Proto-Oncogênicas/genética , Quinonas/químicaRESUMO
Despite numerous researches and improvements in the past few years, the precise mechanisms underlying secondary brain injury after trauma remain obscure. Iron is essential for almost all types of cells, including nerve cells. However, excess of iron has been proved to contribute to the brain injury following trauma in animal models. As a key iron-handling protein in the brain, ferritin might be involved in iron-induced pathophysiological process of various brain disorders. Therefore, the current study was aimed to investigate the expression of ferritin in the human contused brain. Nineteen contused brain samples were obtained from 19 patients undergoing surgery for brain contusions 3 h-17 d after trauma, and three normal temporal pole samples from 3 patients with petroclival meningioma were collected as controls. Expression of ferritin-H-chain was measured by quantitative real-time polymerase chain reaction (PCR), western blot and immunohistochemistry, respectively. Perl's reaction was taken for iron staining. The results showed that human traumatic brain injury (TBI) could up-regulate ferritin-H-chain in pericontusional cortex. A marked increase of ferritin was detected in the early group (≤12 h), and remained elevated for a long time till after 48 h post-injury. The location of ferritin-H-chain was found mainly at the neuron-like cells and seldom at glia-like cells. Perl's reaction showed that most of the iron-positive cells were glia-like cells. These findings suggested that iron and ferritin might be involved in the secondary brain injury and could be therapeutic targets for patients with TBI.
Assuntos
Apoferritinas/biossíntese , Lesões Encefálicas/diagnóstico , Lesões Encefálicas/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Regulação da Expressão Gênica , Adulto , Apoferritinas/genética , Lesões Encefálicas/cirurgia , Córtex Cerebral/cirurgia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regulação para Cima/genética , Adulto JovemRESUMO
This study investigated the expression and functions of ferritin, which is involved in osteoblastogenesis, in the periodontal ligament (PDL). The PDL is one of the most important tissues for maintaining the homeostasis of teeth and tooth-supporting tissues. Real-time PCR analyses of the human PDL revealed abundant expression of ferritin light polypeptide (FTL) and ferritin heavy polypeptide (FTH), which encode the highly-conserved iron storage protein, ferritin. Immunohistochemical staining demonstrated predominant expression of FTL and FTH in mouse PDL tissues in vivo. In in vitro-maintained mouse PDL cells, FTL and FTH expressions were upregulated at both the mRNA and protein levels during the course of cytodifferentiation and mineralization. Interestingly, stimulation of PDL cells with exogenous apoferritin (iron-free ferritin) increased calcified nodule formation and alkaline phosphatase activity as well as the mRNA expressions of mineralization-related genes during the course of cytodifferentiation. On the other hand, RNA interference of FTH inhibited the mineralized nodule formation of PDL cells. This is the first report to demonstrate that ferritin is predominantly expressed in PDL tissues and positively regulates the cytodifferentiation and mineralization of PDL cells.
Assuntos
Apoferritinas/fisiologia , Calcificação Fisiológica , Diferenciação Celular , Ligamento Periodontal/citologia , Animais , Apoferritinas/biossíntese , Apoferritinas/genética , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ligamento Periodontal/metabolismo , Interferência de RNARESUMO
BACKGROUND: An emerging MRI reporter, ferritin heavy chain (FTH1), is recently applied to enhance the contrast and increase the sensitivity of MRI in the monitoring of solid tumors. However, FTH1-overexpression-related cytotoxicity is required to be explored. METHODS: By using the Tet-Off system, FTH1 overexpression was semi-quantitativiely and dynamicly regulated by doxycycline in a NPC cell line. Effects of FTH1 overexpression on the proliferation, cytotoxicity, apoptosis and migration of NPC cells were investigated in vitro, and MR relaxation rate was measured in vitro and in vivo. RESULTS: In vitro and in vivo overexpression of FTH1 significantly increased the transverse relaxivity (R(2)), which could be enhanced by iron supplementation. In vitro, overexpression of FTH1 reduced cell growth and migration, which were not reduced by iron supplementation. Furthermore, cells were subcutaneously inoculated into the nude mice. Results showed FTH1 overexpression decreased tumor growth in the absence of iron supplementation but not in the presence of iron supplementation. CONCLUSION: To maximize R(2) and minimize the potential adverse effects, supplementation of iron at appropriate dose is recommended during the application of FTH1 as a reporter gene in the monitoring of NPC by MRI.
Assuntos
Apoferritinas/genética , Compostos Férricos/administração & dosagem , Imageamento por Ressonância Magnética/métodos , Neoplasias Nasofaríngeas/genética , Compostos de Amônio Quaternário/administração & dosagem , Animais , Apoferritinas/biossíntese , Apoferritinas/metabolismo , Apoptose/genética , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Doxiciclina/farmacologia , Feminino , Ferritinas/biossíntese , Ferritinas/genética , Genes Reporter , Humanos , Luciferases/genética , Camundongos , Camundongos Nus , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Oxirredutases , Receptores da Transferrina/metabolismo , TransfecçãoRESUMO
Cellular MRI with a reporter gene offers the opportunity to track small numbers of tumor cells and to study metastatic processes in their earliest developmental stages in the target organs of interest. This study demonstrates the feasibility of using the MR reporter ferritin for the noninvasive imaging and quantification of metastatic melanoma cells in the lymph nodes (LNs) of living mice. A B16F10 murine melanoma cell line expressing human ferritin heavy chain (hFTH) and green fluorescent protein (GFP) was constructed to allow the detection of cells by MRI and fluorescence imaging. Stable overexpression of hFTH and GFP in B16F10 murine melanoma cells was feasible and showed no cellular toxicity. In addition, hFTH cells were detectable by 9.4-T MRI in vitro and in vivo, yielding significant changes in T(2)* relative to control cells. In BALB/c nude mice, the presence of hFTH- and GFP-expressing metastatic melanoma cells in deep-seated axillary LNs was demonstrated as areas of low T(2)* on MRI, but the same LNs were not visible by fluorescence imaging because the light was unable to penetrate the tissue. Furthermore, the metastatic volume of each LN, which was assessed by cumulative histogram analysis of the T(2)* MRI data, correlated well with tumor burden, which was determined by histology (r = -0.8773, p = 0.0001). This study is the first to use MRI and an MR reporter gene for both the visualization and quantification of metastatic cancer cells in LNs.
Assuntos
Apoferritinas/análise , Linfonodos/patologia , Imageamento por Ressonância Magnética/métodos , Melanoma Experimental/patologia , Animais , Apoferritinas/biossíntese , Apoferritinas/genética , Linhagem Celular Tumoral , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Linfonodos/metabolismo , Metástase Linfática , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência/métodos , TransfecçãoRESUMO
Genetic ablation of Iron Regulatory Protein 2 (Irp2, Ireb2), which post-transcriptionally regulates iron metabolism genes, causes a gait disorder in mice that progresses to hind-limb paralysis. Here we have demonstrated that misregulation of iron metabolism from loss of Irp2 causes lower motor neuronal degeneration with significant spinal cord axonopathy. Mitochondria in the lumbar spinal cord showed significantly decreased Complex I and II activities, and abnormal morphology. Lower motor neurons appeared to be the most adversely affected neurons, and we show that functional iron starvation due to misregulation of iron import and storage proteins, including transferrin receptor 1 and ferritin, may have a causal role in disease. We demonstrated that two therapeutic approaches were beneficial for motor neuron survival. First, we activated a homologous protein, IRP1, by oral Tempol treatment and found that axons were partially spared from degeneration. Secondly, we genetically decreased expression of the iron storage protein, ferritin, to diminish functional iron starvation. These data suggest that functional iron deficiency may constitute a previously unrecognized molecular basis for degeneration of motor neurons in mice.
Assuntos
Deleção de Genes , Deficiências de Ferro , Proteína 2 Reguladora do Ferro/deficiência , Proteína 2 Reguladora do Ferro/genética , Mitocôndrias/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Animais , Apoferritinas/biossíntese , Atrofia/metabolismo , Axônios/efeitos dos fármacos , Axônios/metabolismo , Axônios/patologia , Biomarcadores/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Óxidos N-Cíclicos/farmacologia , Homeostase/efeitos dos fármacos , Homeostase/genética , Ferro/metabolismo , Proteína 1 Reguladora do Ferro/deficiência , Proteína 1 Reguladora do Ferro/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/patologia , Neurônios Motores/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Marcadores de Spin , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologiaAssuntos
Regulação da Expressão Gênica/fisiologia , Ferro/metabolismo , Ativação de Macrófagos/fisiologia , Macrófagos/metabolismo , Animais , Apoferritinas/biossíntese , Apoferritinas/imunologia , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Transporte de Cátions/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/imunologia , Inflamação/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Interferon gama/farmacologia , Interleucina-4/imunologia , Interleucina-4/metabolismo , Interleucina-4/farmacologia , Ferro/imunologia , Proteína 1 Reguladora do Ferro/biossíntese , Proteína 1 Reguladora do Ferro/imunologia , Proteína 2 Reguladora do Ferro/biossíntese , Proteína 2 Reguladora do Ferro/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/fisiologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Receptores da Transferrina/biossíntese , Receptores da Transferrina/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
A combination of reporter genes for magnetic resonance imaging (MRI) and optical imaging can provide an additional level of noninvasive and quantitative information about biological processes occurring in deep tissues. We developed a bimodal lentiviral vector to monitor deep tissue events using MRI to detect myc-tagged human ferritin heavy chain (myc-hFTH) expression and fluorescence imaging to detect green fluorescent protein (GFP) expression. The transgene construct was stably transfected into MCF-7 and F-98 cells. After transplantation of the cells expressing myc-hFTH and GFP into mice or rats, serial MRI and fluorescence imaging were performed with a human wrist coil on a 1.5T MR scanner and optical imaging analyzer for 4 weeks. No cellular toxicity due to overexpression of myc-hFTH and GFP was observed in MTT and trypan blue exclusion assays. Iron accumulation was observed in myc-hFTH cells and tumors by Prussian blue staining and iron binding assays. The myc-hFTH cells and tumors had significantly lower signal intensities in T(2)-weighted MRI than mock-transfected controls (P ≤ 0.05). This is direct evidence that myc-hFTH expression can be visualized noninvasively with a 1.5T clinical MR scanner. This study shows that MRI and fluorescence imaging of transplanted cells at molecular and cellular levels can be performed simultaneously using our bimodal lentiviral vector system. Our techniques can be used to monitor tumor growth, metastasis, and regression during cell and gene-based therapy in deep tissues.
Assuntos
Apoferritinas/genética , Neoplasias da Mama/genética , Glioma/genética , Proteínas de Fluorescência Verde/genética , Animais , Apoferritinas/biossíntese , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Vetores Genéticos/genética , Glioma/metabolismo , Glioma/patologia , Proteínas de Fluorescência Verde/biossíntese , Humanos , Lentivirus/genética , Imageamento por Ressonância Magnética/instrumentação , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transdução GenéticaRESUMO
An unmet need in cardiac cell therapy is a noninvasive imaging technique capable of tracking changes in graft size over time and monitoring cell dynamics such as replication and death, factors to which commonly used superparamagnetic nanoparticles are insensitive. Our goal was to explore if overexpression of ferritin, a nontoxic iron-binding protein, can be used for noninvasive magnetic resonance imaging (MRI) of cells transplanted into the infarcted heart. Mouse skeletal myoblasts (C2C12 cells) were engineered to overexpress ferritin. Ferritin overexpression did not interfere with cell viability, proliferation, or differentiation into multinucleated myotubes. Ferritin overexpression caused a 25% decrease in T2 relaxation time in vitro compared to wild-type cells. Transgenic grafts were detected in vivo 3 weeks after transplantation into infarcted hearts of syngeneic mice as areas of hypointensity caused by iron accumulation in overexpressed ferritin complexes. Graft size evaluation by MRI correlated tighly with histologic measurements (R2 = .8). Our studies demonstrated the feasibility of ferritin overexpression in mouse skeletal myoblasts and the successful detection of transgenic cells by MRI in vitro and in vivo after transplantation into the infarcted mouse heart. These experiments lay the groundwork for using the MRI gene reporter ferritin to track stem cells transplanted to the heart.
Assuntos
Apoferritinas/biossíntese , Imageamento por Ressonância Magnética/métodos , Mioblastos Esqueléticos/fisiologia , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismo , Animais , Apoferritinas/genética , Apoferritinas/farmacologia , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/fisiologia , Modelos Lineares , Camundongos , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Infarto do Miocárdio/terapia , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Células-Tronco/citologia , TransfecçãoRESUMO
BACKGROUND: Macrophages play a key role in iron homeostasis. In peripheral tissues, they are known to polarize into classically activated (or M1) macrophages and alternatively activated (or M2) macrophages. Little is known on whether the polarization program influences the ability of macrophages to store or recycle iron and the molecular machinery involved in the processes. DESIGN AND METHODS: Inflammatory/M1 and alternatively activated/M2 macrophages were propagated in vitro from mouse bone-marrow precursors and polarized in the presence of recombinant interferon-γ or interleukin-4. We characterized and compared their ability to handle radioactive iron, the characteristics of the intracellular iron pools and the expression of molecules involved in internalization, storage and export of the metal. Moreover we verified the influence of iron on the relative ability of polarized macrophages to activate antigen-specific T cells. RESULTS: M1 macrophages have low iron regulatory protein 1 and 2 binding activity, express high levels of ferritin H, low levels of transferrin receptor 1 and internalize--albeit with low efficiency -iron only when its extracellular concentration is high. In contrast, M2 macrophages have high iron regulatory protein binding activity, express low levels of ferritin H and high levels of transferrin receptor 1. M2 macrophages have a larger intracellular labile iron pool, effectively take up and spontaneously release iron at low concentrations and have limited storage ability. Iron export correlates with the expression of ferroportin, which is higher in M2 macrophages. M1 and M2 cells activate antigen-specific, MHC class II-restricted T cells. In the absence of the metal, only M1 macrophages are effective. CONCLUSIONS: Cytokines that drive macrophage polarization ultimately control iron handling, leading to the differentiation of macrophages into a subset which has a relatively sealed intracellular iron content (M1) or into a subset endowed with the ability to recycle the metal (M2).