RESUMEN
Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin-ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery.
RESUMEN
Iron-refractory iron deficiency anemia (IRIDA) is an autosomal recessive disorder caused by genetic mutations on TMPRSS6 gene which encodes Matriptase2 (MT2). An altered MT2 cannot appropriately suppress hepatic BMP6/SMAD signaling in case of low iron, hence hepcidin excess blocks dietary iron absorption, leading to a form of anemia resistant to oral iron supplementation. In this study, using the IRIDA mouse model Mask, we characterized homozygous (msk/msk) compared to asymptomatic heterozygous (msk/wt) mice, assessing the major parameters of iron status in different organs, at different ages in both sexes. The effect of carbonyl iron diet was analyzed as control iron supplementation being used for many studies in mice. It resulted effective in both anemic control and msk/msk mice, as expected, even if there is no information about its mechanism of absorption. Then, we mainly compared two forms of oral iron supplement, largely used for humans: ferrous sulfate and Sucrosomial iron. In anemic control mice, the two oral formulations corrected hemoglobin levels from 11.40 ± 0.60 to 15.38 ± 1.71 g/dl in 2-4 weeks. Interestingly, in msk/msk mice, ferrous sulfate did not increase hemoglobin likely due to ferroportin/hepcidin-dependent absorption, whereas Sucrosomial iron increased it from 11.50 ± 0.60 to 13.53 ± 0.64 g/dl mainly in the first week followed by a minor increase at 4 weeks with a stable level of 13.30 ± 0.80 g/dl, probably because of alternative absorption. Thus, Sucrosomial iron, already used in other conditions of iron deficiency, may represent a promising option for oral iron supplementation in IRIDA patients.
Asunto(s)
Anemia Ferropénica/terapia , Compuestos Férricos/uso terapéutico , Compuestos Ferrosos/uso terapéutico , Compuestos de Hierro/uso terapéutico , Hierro de la Dieta/uso terapéutico , Administración Oral , Anemia Ferropénica/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Compuestos Férricos/administración & dosificación , Compuestos Ferrosos/administración & dosificación , Humanos , Hierro/metabolismo , Compuestos de Hierro/administración & dosificación , Hierro de la Dieta/administración & dosificación , Masculino , RatonesRESUMEN
Ferritinophagy is a ferritin autophagic degradation process mediated by the selective nuclear receptor coactivator-4 (NCOA4). NCOA4 binds to ferritin and delivers it to nascent autophagosomes, which then merge with the lysosomes for ferritin degradation and iron release. Earlier studies have demonstrated a specific association of NCOA4 with ferritin H-subunits, but not L-subunits. However, neither the thermodynamics of this interaction nor the effect of NCOA4 on iron oxidation, iron mineral core formation, or iron mobilization in ferritin has been explored. Using isothermal titration calorimetry, light absorption spectroscopy, and a soluble fragment (residues 383-522) of human NCOA4 expressed in Escherichia coli, we show that the NCOA4 fragment specifically binds H-rich ferritins with a binding stoichiometry of â¼8 NCOA4 molecules per ferritin shell, and Kd values of â¼0.4 and â¼2 µM for homopolymer H-chain ferritin and heteropolymer H-rich ferritin, respectively. The binding reaction was both enthalpically and entropically favored. Whereas the iron oxidation kinetics were not affected by the presence of NCOA4, iron mobilization from ferritin by two different reducing agents (FMN/NADH and sodium dithionite) showed a strong inhibitory effect that was dependent on the concentration of NCOA4 present in solution. Our results suggest that the binding of NCOA4 to ferritin may interfere in the electron transfer pathway through the ferritin shell and may have important biological implications on cellular iron homeostasis.
Asunto(s)
Apoferritinas/metabolismo , Ferritinas/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , Oxidorreductasas/metabolismo , Apoferritinas/química , Sitios de Unión , Ferritinas/química , Humanos , Cinética , Coactivadores de Receptor Nuclear/química , Oxidorreductasas/química , Unión Proteica , Mapas de Interacción de Proteínas , TermodinámicaRESUMEN
Hepcidin is a liver-derived peptide hormone that controls systemic iron homeostasis. Its expression is regulated by the bone morphogenetic protein 6 (BMP6)/SMAD1/5/8 pathway and by the proinflammatory cytokine interleukin 6 (IL6). Proteoglycans that function as receptors of these signaling proteins in the liver are commonly decorated by heparan sulfate, but the potential role of hepatic heparan sulfate in hepcidin expression and iron homeostasis is unclear. Here, we show that modulation of hepatic heparan sulfate significantly alters hepcidin expression and iron metabolism both in vitro and in vivo Specifically, enzymatic removal of heparan sulfate from primary human hepatocytes, CRISPR/Cas9 manipulation of heparan sulfate biosynthesis in human hepatoma cells, or pharmacological manipulation of heparan sulfate-protein interactions using sodium chlorate or surfen dramatically reduced baseline and BMP6/SMAD1/5/8-dependent hepcidin expression. Moreover inactivation of the heparan sulfate biosynthetic gene N-deacetylase and N-sulfotransferase 1 (Ndst1) in murine hepatocytes (Ndst1f/fAlbCre+) reduced hepatic hepcidin expression and caused a redistribution of systemic iron, leading to iron accumulation in the liver and serum of mice. Manipulation of heparan sulfate had a similar effect on IL6-dependent hepcidin expression in vitro and suppressed IL6-mediated iron redistribution induced by lipopolysaccharide in vivo These results provide compelling evidence that hepatocyte heparan sulfate plays a key role in regulating hepcidin expression and iron homeostasis in mice and in human hepatocytes.
Asunto(s)
Heparitina Sulfato/metabolismo , Hepatocitos/metabolismo , Hepcidinas/genética , Homeostasis , Hierro/metabolismo , Animales , Línea Celular , Perfilación de la Expresión Génica , Hepcidinas/metabolismo , Humanos , Ratones , Ratones Endogámicos , Regiones Promotoras Genéticas/genéticaRESUMEN
Ferritin turnover plays a major role in tissue iron homeostasis, and ferritin malfunction is associated with impaired iron homeostasis and neurodegenerative diseases. In most eukaryotes, ferritin is considered an intracellular protein that stores iron in a nontoxic and bioavailable form. In insects, ferritin is a classically secreted protein and plays a major role in systemic iron distribution. Mammalian ferritin lacks the signal peptide for classical endoplasmic reticulum-Golgi secretion but is found in serum and is secreted via a nonclassical lysosomal secretion pathway. This study applied bioinformatics and biochemical tools, alongside a protein trafficking mouse models, to characterize the mechanisms of ferritin secretion. Ferritin trafficking via the classical secretion pathway was ruled out, and a 2:1 distribution of intracellular ferritin between membrane-bound compartments and the cytosol was observed, suggesting a role for ferritin in the vesicular compartments of the cell. Focusing on nonclassical secretion, we analyzed mouse models of impaired endolysosomal trafficking and found that ferritin secretion was decreased by a BLOC-1 mutation but increased by BLOC-2, BLOC-3, and Rab27A mutations of the cellular trafficking machinery, suggesting multiple export routes. A 13-amino-acid motif unique to ferritins that lack the secretion signal peptide was identified on the BC-loop of both subunits and plays a role in the regulation of ferritin secretion. Finally, we provide evidence that secretion of iron-rich ferritin was mediated via the multivesicular body-exosome pathway. These results enhance our understanding of the mechanism of ferritin secretion, which is an important piece in the puzzle of tissue iron homeostasis.
Asunto(s)
Ferritinas/metabolismo , Vesículas Secretoras/metabolismo , Secuencias de Aminoácidos , Animales , Biomarcadores/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Exosomas/metabolismo , Exosomas/ultraestructura , Ferritinas/sangre , Ferritinas/química , Aparato de Golgi/metabolismo , Lisosomas/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Células RAW 264.7RESUMEN
Objective- Calcific aortic valve disease is a prominent finding in elderly and in patients with chronic kidney disease. We investigated the potential role of iron metabolism in the pathogenesis of calcific aortic valve disease. Approach and Results- Cultured valvular interstitial cells of stenotic aortic valve with calcification from patients undergoing valve replacement exhibited significant susceptibility to mineralization/osteoblastic transdifferentiation in response to phosphate. This process was abrogated by iron via induction of H-ferritin as reflected by lowering ALP and osteocalcin secretion and preventing extracellular calcium deposition. Cellular phosphate uptake and accumulation of lysosomal phosphate were decreased. Accordingly, expression of phosphate transporters Pit1 and Pit2 were repressed. Translocation of ferritin into lysosomes occurred with high phosphate-binding capacity. Importantly, ferritin reduced nuclear accumulation of RUNX2 (Runt-related transcription factor 2), and as a reciprocal effect, it enhanced nuclear localization of transcription factor Sox9 (SRY [sex-determining region Y]-box 9). Pyrophosphate generation was also increased via upregulation of ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase-2). 3H-1, 2-dithiole-3-thione mimicked these beneficial effects in valvular interstitial cell via induction of H-ferritin. Ferroxidase activity of H-ferritin was essential for this function, as ceruloplasmin exhibited similar inhibitory functions. Histological analysis of stenotic aortic valve revealed high expression of H-ferritin without iron accumulation and its relative dominance over ALP in noncalcified regions. Increased expression of H-ferritin accompanied by elevation of TNF-α (tumor necrosis factor-α) and IL-1ß (interleukin-1ß) levels, inducers of H-ferritin, corroborates the essential role of ferritin/ferroxidase via attenuating inflammation in calcific aortic valve disease. Conclusions- Our results indicate that H-ferritin is a stratagem in mitigating valvular mineralization/osteoblastic differentiation. Utilization of 3H-1, 2-dithiole-3-thione to induce ferritin expression may prove a novel therapeutic potential in valvular mineralization.
Asunto(s)
Estenosis de la Válvula Aórtica/metabolismo , Apoferritinas/fisiología , Calcificación Vascular/metabolismo , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Estenosis de la Válvula Aórtica/patología , Apoferritinas/antagonistas & inhibidores , Apoferritinas/farmacología , Transporte Biológico , Núcleo Celular/metabolismo , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Canales Iónicos/biosíntesis , Hierro/farmacología , Lisosomas/metabolismo , Fosfatos/metabolismo , Hidrolasas Diéster Fosfóricas/biosíntesis , Hidrolasas Diéster Fosfóricas/genética , Factor de Transcripción SOX9/metabolismo , Tionas/farmacología , Tiofenos/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Calcificación Vascular/patologíaRESUMEN
Ferritin is a molecule with enormous potentiality in biotechnology that have been already used to encapsulate molecules, as contrast in magnetic resonance imaging and to carry epitopes. We proposed to use it to carry another key protein of iron metabolism, hepcidin that is a small hormone peptide that control systemic iron homeostasis. In this work, we purified the previously produced camel hepcidin and human H-ferritin heteropolymer (HepcH-FTH) and to monitor its binding capability toward J744 cell line in presence or absence of ferric ammonium citrate. Fused camel hepcidin and human H-ferritin monomer (HepcH) as well as the assembled HepcH-FTH heteropolymer (ratio 1:5) was easily purified by a one-step purification using size exclusion chromatography. SDS-PAGE electrophoresis of HepcH, purified from soluble and insoluble fractions, showed a single band of 24 kDa with an estimated purity of at least 90%. The purification yields of HepcH from the soluble and insoluble fractions was, respectively, of about 6.80 and 2 mg/L of bacterial culture. Time curse cellular binding assays of HepcH-FTH revealed its great potential to bind the J774 cells after 15 min of incubation. Furthermore, HepcH-FTH was able to degrade ferroportin, the unique hepcidin receptor, even after 30 min of incubation with J774 cells treated with 100 µM ferric ammonium citrate. In conclusion, we proposed ferritin as a peptide carrier to promote the association of the hybrid HepcH-FTH nanoparticle with a particular type of cell for therapeutic or diagnostic.
Asunto(s)
Ferritinas/metabolismo , Hepcidinas/metabolismo , Macrófagos/metabolismo , Multimerización de Proteína , Proteínas Recombinantes/metabolismo , Animales , Camelus , Línea Celular , Ferritinas/química , Hepcidinas/química , Humanos , Macrófagos/inmunología , Ratones , Unión Proteica , Proteínas Recombinantes/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
In animals, the iron storage and detoxification protein, ferritin, is composed of two functionally and genetically distinct subunit types, H (heavy) and L (light), which co-assemble in various ratios with tissue specific distributions to form shell-like protein structures of 24 subunits within which a mineralized iron core is stored. The H-subunit possesses a ferroxidase center (FC) that catalyzes Fe(II) oxidation, whereas the L-subunit does not. To assess the role of the L-subunit in iron oxidation and core formation, two human recombinant heteropolymeric ferritins, designated H-rich and L-rich with ratios of â¼20H:4L and â¼22L:2H, respectively, were employed and compared to the human homopolymeric H-subunit ferritin (HuHF). These heteropolymeric ferritins have a composition similar to the composition of those found in hearts and brains (i.e., H-rich) and in livers and spleens (i.e., L-rich). As for HuHF, iron oxidation in H-rich ferritin was found to proceed with a 2:1 Fe(II):O2 stoichiometry at an iron level of 2 Fe(II) atoms/H-subunit with the generation of H2O2. The H2O2 reacted with additional Fe(II) in a 2:1 Fe(II):H2O2 ratio, thus avoiding the production of hydroxyl radical. A µ-1,2-peroxo-diFe(III) intermediate was observed at the FC of H-rich ferritin as for HuHF. Importantly, the H-rich protein regenerated full ferroxidase activity more rapidly than HuHF did and additionally formed larger iron cores, indicating dual roles for the L-subunit in facilitating iron turnover at the FC and in mineralization of the core. The L-rich ferritin, while also facilitating iron oxidation at the FC, additionally promoted oxidation at the mineral surface once the iron binding capacity of the FC was exceeded.
Asunto(s)
Apoferritinas/metabolismo , Coenzimas/metabolismo , Ferritinas/metabolismo , Hemo/metabolismo , Hierro/metabolismo , Apoferritinas/química , Apoferritinas/genética , Dominio Catalítico , Coenzimas/química , Electroforesis Capilar , Ferritinas/química , Ferritinas/genética , Hemo/química , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Hierro/química , Cinética , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Ferritin is considered the major iron storage protein which maintains a large iron core in its cavity and has ferroxidase activity. There are many types of ferritin particularly in prokaryotes that include the canonical 24-mer FTN molecules, the heme-containing BFR, the smaller 12-mer DPS and the newly recognized EncFtn of encapsulin that forms a very large iron storage compartment. Recent studies show that ferritin function is more dynamic than previous depicted and new mechanisms of ferritin iron recycling are emerging. They participate to the regulation of cellular iron homeostasis as those of ferritin biosynthesis, cooperating also with the iron-dependent mechanism of cellular iron secretion. Some of these basic processes are in common between unicellular and animal cells, and this review aims at discussing the findings on the connections between iron storage, cellular iron regulation and ferritin iron recycling that have been explored in unicellular organisms and in animals. © 2017 IUBMB Life, 69(6):414-422, 2017.
Asunto(s)
Ferritinas/genética , Homeostasis/genética , Hierro/metabolismo , Células Procariotas/metabolismo , Receptores de Transferrina/genética , Animales , Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Ferritinas/química , Ferritinas/metabolismo , Regulación de la Expresión Génica , Hemo/metabolismo , Proteína 1 Reguladora de Hierro/genética , Proteína 1 Reguladora de Hierro/metabolismo , Oxidación-Reducción , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Transferrina/metabolismo , Transducción de Señal , Especificidad de la EspecieRESUMEN
The high stability and strong self-assembly properties made ferritins the most used proteins for nanotechnological applications. Human ferritins are made of 24 subunits of the H- and L-type that coassemble in an almost spherical nanocage 12nm across, delimiting a large cavity. The mechanism and kinetics of ferritin self-assembly and why H/L heteropolymers formation is favored over the homopolymers remain unclarified. In order to study this, we used the Fluorescence Resonance Energy Transfer (FRET) tool by binding multiple donor or acceptor Alexa Fluor fluorophores on the outer surface of human H and L ferritins and then denaturing and reassembling them in different proportions and conditions. The FRET efficiency increase from <0.3 of the disassembled to >0.7 in the assembled allowed to study the assembly kinetics. We found that their assembly was complete in about one hour, and that the initial rate of self-assembly of H/L heteropolymers was slightly faster than that of the H/H homopolymers. Then, by adding various proportions of unlabeled H or L-chains to the FRET system we found that the presence of the L-chains displaced the formation of H-H dimers more efficiently than that of the H-chains. This favored formation of H/L heterodimers, which is the initial step in ferritin self-assembly, contributes to explain the preferred formation of H/L heteropolymers over the H or L homopolymers. Moreover, we found that the H-chains arrange at distant positions on the heteropolymeric shell until they reach a number above eight, when they start to co-localize.
Asunto(s)
Apoferritinas/química , Polímeros/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Humanos , Hierro/química , CinéticaRESUMEN
Ferritinophagy is the process of autophagic degradation of ferritin that participates in the regulation of cellular iron homeostasis. This process was shown to be mediated by the selective cargo-receptor Nuclear Receptor Coactivator-4 (NCOA4) that binds ferritin and targets it to emerging autophagosome. To characterize some of the biochemical properties of the interaction between the two proteins we cloned and expressed in E. coli the ferritin-binding domain of human NCOA4, fragment 383-522. It was purified and subjected to biochemical analysis. The NCOA4(383-522) fragment was expressed in soluble and dimeric form, and CD spectra indicated low level of secondary structure. The Ferritin binding activity of the fragment was investigated by developing an electrophoretic mobility shift and an ELISA assays. They showed that the NCOA4 fragment binds the H-ferritin with an affinity in the nM range, but not the R23A H-ferritin mutant and the L-ferritin chain, confirming the high specificity for the H-chain. The H-ferritin could bind up to 24 NCOA4(383-522) fragments forming highly stable and insoluble complexes. The binding was partially inhibited only by Fe(II) among the various divalent metal ions analyzed. The iron-dependent, highly-specific formation of the remarkably stable H-ferritin-NCOA4 complex shown in this work may be important for the characterization of the mechanism of ferritinophagy.
Asunto(s)
Autofagia/genética , Ferritinas/química , Coactivadores de Receptor Nuclear/química , Fagosomas/metabolismo , Escherichia coli/genética , Ferritinas/deficiencia , Ferritinas/genética , Ferritinas/metabolismo , Regulación de la Expresión Génica/genética , Homeostasis , Humanos , Hierro/química , Coactivadores de Receptor Nuclear/genética , Coactivadores de Receptor Nuclear/metabolismo , Fagosomas/genética , Unión Proteica , Dominios ProteicosRESUMEN
Mitochondrial ferritin (FtMt) is a functional ferritin targeted to mitochondria that is highly expressed in the testis. To investigate the role of FtMt in the testis we set up a series of controlled matings between FtMt gene-deletion mice (FtMt-/-) with FtMt+/+ mice. We found that the number of newborns per litter and the fertility rate were strongly reduced for the FtMt-/- males, but not for the females, indicating that FtMt has an important role for male fertility. The morphology of the testis and of the spermatozoa of FtMt-/- mice was normal and we did not detect alterations in sperm parameters or in oxidative stress indices. In contrast, we observed that the cauda epididymides of FtMt-/- mice were significantly lighter and contained a lower number of spermatozoa compared with the controls. Also, the ATP content of FtMt-/- spermatozoa was found to be lower than that of FtMt+/+ spermatozoa. These data show that FtMt contributes to sperm epididymis maturation and to male fertility.
Asunto(s)
Ferritinas/genética , Fertilidad/genética , Infertilidad Masculina/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética , Animales , Ferritinas/metabolismo , Infertilidad Masculina/metabolismo , Masculino , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Estrés Oxidativo/genética , Motilidad Espermática/genética , Espermatozoides/metabolismo , Testículo/metabolismoRESUMEN
Recent data have indicated a fundamental role of iron in mediating a non-apoptotic and non-necrotic oxidative form of programmed cell death termed ferroptosis that requires abundant cytosolic free labile iron to promote membrane lipid peroxidation. Different scavenger molecules and detoxifying enzymes, such as glutathione (GSH) and glutathione peroxidase 4 (GPX4), have been shown to overwhelm or exacerbate ferroptosis depending on their expression magnitude. Ferroptosis is emerging as a potential weapon against tumor growth since it has been shown to potentiate cell death in some malignancies. However, this mechanism has been poorly studied in Rhabdomyosarcoma (RMS), a myogenic tumor affecting childhood and adolescence. One of the main drivers of RMS genesis is the Retrovirus Associated DNA Sequences/Extracellular signal Regulated Kinases (RAS/ERK)signaling pathway, the deliberate activation of which correlates with tumor aggressiveness and oxidative stress levels. Since recent studies have indicated that treatment with oxidative inducers can significantly halt RMS tumor progression, in this review we covered different aspects, ranging from iron metabolism in carcinogenesis and tumor growth, to mechanisms of iron-mediated cell death, to highlight the potential role of ferroptosis in counteracting RMS growth.
Asunto(s)
Hierro/metabolismo , Estrés Oxidativo , Rabdomiosarcoma/metabolismo , Animales , Biomarcadores de Tumor , Transformación Celular Neoplásica/metabolismo , Humanos , Peroxidación de Lípido , Neoplasias/etiología , Neoplasias/metabolismo , Neoplasias/patología , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Rabdomiosarcoma/diagnóstico , Rabdomiosarcoma/genética , Transducción de SeñalRESUMEN
The peptide hormone hepcidin is a key controller of systemic iron homeostasis, and its expression in the liver is mainly regulated by bone morphogenetic proteins (BMPs), which are heparin binding proteins. In fact, heparins are strong suppressors of hepcidin expression in hepatic cell lines that act by inhibiting the phosphorylation of SMAD1/5/8 proteins elicited by the BMPs. The inhibitory effect of heparins has been demonstrated in cells and in mice, where subcutaneous injections of non-anticoagulant heparins inhibited liver hepcidin expression and increased iron bioavailability. The chemical characteristics for high anti-hepcidin activity in vitro and in vivo include the 2O-and 6O-sulfation and a molecular weight above 7 kDa. The most potent heparins have been found to be the super-sulfated ones, active in hepcidin suppression with a molecular weight as low as 4 kDa. Moreover, the alteration of endogenous heparan sulfates has been found to cause a reduction in hepcidin expression in vitro and in vivo, indicating that heparins act by interfering with the interaction between BMPs and components of the complex involved in the activation of the BMP/SMAD1/5/8 pathway. This review summarizes recent findings on the anti-hepcidin activity of heparins and their possible use for the treatment of anemia caused by hepcidin excess, including the anemia of chronic diseases.
Asunto(s)
Anemia/tratamiento farmacológico , Anemia/metabolismo , Heparina/farmacología , Heparina/uso terapéutico , Hepcidinas/antagonistas & inhibidores , Anemia/etiología , Animales , Proteína Morfogenética Ósea 6/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Expresión Génica , Heparitina Sulfato/metabolismo , Hepcidinas/genética , Hepcidinas/metabolismo , Homeostasis , Humanos , Hierro/metabolismo , Hígado/metabolismo , Unión ProteicaRESUMEN
Vascular calcification is a frequent complication of atherosclerosis, diabetes and chronic kidney disease. In the latter group of patients, calcification is commonly seen in tunica media where smooth muscle cells (SMC) undergo osteoblastic transformation. Risk factors such as elevated phosphorus levels and vitamin D3 analogues have been identified. In the light of earlier observations by our group and others, we sought to inhibit SMC calcification via induction of ferritin. Human aortic SMC were cultured using ß-glycerophosphate with activated vitamin D3 , or inorganic phosphate with calcium, and induction of alkaline phosphatase (ALP) and osteocalcin as well as accumulation of calcium were used to monitor osteoblastic transformation. In addition, to examine the role of vitamin D3 analogues, plasma samples from patients on haemodialysis who had received calcitriol or paricalcitol were tested for their tendency to induce calcification of SMC. Addition of exogenous ferritin mitigates the transformation of SMC into osteoblast-like cells. Importantly, pharmacological induction of heavy chain ferritin by 3H-1,2-Dithiole-3-thione was able to inhibit the SMC transition into osteoblast-like cells and calcification of extracellular matrix. Plasma samples collected from patients after the administration of activated vitamin D3 caused significantly increased ALP activity in SMC compared to the samples drawn prior to activated vitamin D3 and here, again induction of ferritin diminished the osteoblastic transformation. Our data suggests that pharmacological induction of ferritin prevents osteoblastic transformation of SMC. Hence, utilization of such agents that will cause enhanced ferritin synthesis may have important clinical applications in prevention of vascular calcification.
Asunto(s)
Ferritinas/metabolismo , Miocitos del Músculo Liso/fisiología , Osteoblastos/fisiología , Fosfatasa Alcalina/metabolismo , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/fisiología , Calcitriol/metabolismo , Calcio/metabolismo , Células Cultivadas , Colecalciferol/metabolismo , Ergocalciferoles/metabolismo , Glicerofosfatos/farmacología , Humanos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Fosfatos/metabolismo , Tionas/farmacología , Tiofenos/farmacología , Calcificación Vascular/metabolismo , Calcificación Vascular/fisiopatologíaRESUMEN
BACKGROUND: Three functional ferritin genes have been identified so far in mammals, and they encode the cytosolic Heavy (FTH) and Light chain (FTL) and the mitochondrial ferritin. The expression of a transcript by a fourth ferritin-like gene (Ferritin-Heavy-Polypeptide-Like-17, FTHL17) on the X chromosome was reported in mouse spermatogonia and in early embryonic cells. METHODS: The intronless human FTHL17 gene encodes a protein with 64% identity to human FTH with substitution of key residues of the ferroxidase center. The gene was cloned into vectors for expression in Escherichia coli and mammalian cells, linked to a flag-tag. RESULTS: The recombinant FTHL17 from E. coli purified as an assembled 24-mer ferritin devoid of ferroxidase activity and with a reduced physical stability. When transiently expressed in mammalian cells the flag-FTHL17 assembled in ferritin shells that showed reduced stability to denaturants compared with flag H and L ferritins. Immunocytochemistry with anti-flag antibody decorated the nuclei of flag-FTHL17 transfected COS cells, but not those of the cells transfected with flag-FTH or flag-FTL. CONCLUSIONS: We concluded that FTHL17 encodes a ferritin-like protein without ferroxidase activity. Its restricted embryonic expression and partial nuclear localization suggest that this novel ferritin type may have functions other than iron storage. GENERAL SIGNIFICANCE: The work confirms the presence of a fourth functional human ferritin gene with properties distinct from the canonical cytosolic ones.
Asunto(s)
Apoferritinas/metabolismo , Núcleo Celular/metabolismo , Secuencia de Aminoácidos , Animales , Apoferritinas/química , Apoferritinas/genética , Células COS , Diferenciación Celular , Chlorocebus aethiops , Células Madre Embrionarias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Hep G2 , Humanos , Ratones , Datos de Secuencia Molecular , Estructura Molecular , Desnaturalización Proteica , Estabilidad Proteica , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Hepcidin controls systemic iron availability, and its excess contributes to the anemia of chronic diseases, the most prevalent anemia in hospitalized patients. We previously reported that heparins are efficient hepcidin inhibitors both in vitro and in vivo, but their anticoagulant activity limits therapeutic use. We studied nonanticoagulant heparins produced by N-acetylation and oxidation/reduction (glycol-split) that lost antithrombin-binding affinity. Four nonanticoagulant heparins inhibited hepcidin expression in hepatic HepG2 cells and primary hepatocytes. The 2 most potent ones used in mice suppressed liver hepcidin expression and serum hepcidin in 6 hours, with a significant decrease of spleen iron. This occurred also in lipopolysaccharide (LPS)-treated animals that mimic inflammation, as well as after chronic 1-week treatments, without evident adverse effects on coagulation. Heparin injections increased iron mobilization and facilitated the recovery from the anemia induced by heat-killed Brucella abortus, a model of inflammatory anemia. The heparins were used also in Bmp6(-/-) mice. A single dose of heparin reduced the already low level of hepcidin of these mice and prevented its induction by LPS. These nonanticoagulant compounds impair bone morphogenetic protein /sons of mothers against decapentaplegic signaling with no evident adverse effect in vivo, even when administered chronically. They may offer a strategy for the treatment of diseases with high hepcidin levels.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Heparina/análogos & derivados , Hepcidinas/genética , Anemia/inducido químicamente , Anemia/tratamiento farmacológico , Anemia/genética , Animales , Proteína Morfogenética Ósea 6/genética , Proteína Morfogenética Ósea 6/metabolismo , Línea Celular , Dermatán Sulfato/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Células Hep G2 , Heparina/administración & dosificación , Heparina/farmacología , Hepcidinas/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Proteína 1 Inhibidora de la Diferenciación/genética , Hierro/metabolismo , Lipopolisacáridos/inmunología , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Bazo/efectos de los fármacos , Bazo/metabolismo , Factores de Tiempo , Activación Transcripcional/efectos de los fármacosRESUMEN
Ferritins, the main intracellular iron storage proteins, have been studied for over 60 years, mainly focusing on the mammalian ones. This allowed the elucidation of the structure of these proteins and the mechanisms regulating their iron incorporation and mineralization. However, ferritin is present in most, although not all, eukaryotic cells, comprising monocellular and multicellular invertebrates and vertebrates. The aim of this review is to provide an update on the general properties of ferritins that are common to various eukaryotic phyla (except plants), and to give an overview on the structure, function and regulation of ferritins. An update on the animal models that were used to characterize H, L and mitochondrial ferritins is also provided. The data show that ferritin structure is highly conserved among different phyla. It exerts an important cytoprotective function against oxidative damage and plays a role in innate immunity, where it also contributes to prevent parenchymal tissue from the cytotoxicity of pro-inflammatory agonists released by the activation of the immune response activation. Less clear are the properties of the secretory ferritins expressed by insects and molluscs, which may be important for understanding the role played by serum ferritin in mammals.