Exosomes released by imatinib­resistant K562 cells contain specific membrane markers, IFITM3, CD146 and CD36 and increase the survival of imatinib­sensitive cells in the presence of imatinib.

Chronic myeloid leukemia (CML) is a malignant hematopoietic disorder distinguished by the presence of a BCR­ABL1 fused oncogene with constitutive kinase activity. Targeted CML therapy by specific tyrosine kinase inhibitors (TKIs) leads to a marked improvement in the survival of the patients and their quality of life. However, the development of resistance to TKIs remains a critical issue for a subset of patients. The most common cause of resistance are numerous point mutations in the BCR­ABL1 gene, followed by less common mutations and multiple mutation-independent mechanisms. Recently, exosomes, which are extracellular vesicles excreted from normal and tumor cells, have been associated with drug resistance and cancer progression. The aim of the present study was to characterize the exosomes released by imatinib­resistant K562 (K562IR) cells. The K562IR­derived exosomes were internalized by imatinib­sensitive K562 cells, which thereby increased their survival in the presence of 2 µM imatinib. The exosomal cargo was subsequently analyzed to identify resistance­associated markers using a deep label­free quantification proteomic analysis. There were >3,000 exosomal proteins identified of which, 35 were found to be differentially expressed. From this, a total of 3, namely the membrane proteins, interferon­induced transmembrane protein 3, CD146 and CD36, were markedly upregulated in the exosomes derived from the K562IR cells, and exhibited surface localization. The upregulation of these proteins was verified in the K562IR exosomes, and also in the K562IR cells. Using flow cytometric analysis, it was possible to further demonstrate the potential of CD146 as a cell surface marker associated with imatinib resistance in K562 cells. Taken together, these results suggested that exosomes and their respective candidate surface proteins could be potential diagnostic markers of TKI drug resistance in CML therapy.

Copper and iron metabolism in Ostreococcus tauri - the role of phytotransferrin, plastocyanin and a chloroplast copper-transporting ATPase.

Iron and copper are essential elements for practically all living organisms. Their metabolism is frequently interconnected, and while copper is relatively abundant in the ocean, iron is often a limiting factor for the growth of many marine microorganisms. In the present study, we aimed to elucidate the metabolisms of copper and iron and the connection of both in the marine picoalga Ostreococcus tauri. We show that O. tauri adjusts its copper economy in response to copper deficiency by downregulation of the expression of plastocyanin in favor of cytochrome c oxidase without significant changes in growth and physiology. Copper deprivation leads to increased expression of copper transporting ATPase and proteins involved in tetrapyrrole synthesis, most likely to ensure higher turnover of chlorophyll and/or heme. Elucidation of the effect of copper on the incorporation of iron into O. tauri proteins led us to identify the major iron uptake mediating protein, Ot-Fea1, whose expression and binding of iron is copper dependent. Based on our investigation of the incorporation of iron into Ot-Fea1 and ferritin, we hypothesize that O. tauri possesses another Fea1-independent iron uptake system.

Myocardial iron homeostasis and hepcidin expression in a rat model of heart failure at different levels of dietary iron intake.

BACKGROUND: Up to 50% of patients with chronic heart failure (HF) have systemic iron deficiency, which contributes to symptoms and poor prognosis. Myocardial iron deficiency (MID) in HF patients has been recently documented, but its causes and consequences are unknown. The goal of our study was to address these questions in a well-defined rat HF model induced by volume overload due to aorto-caval fistula. METHODS: Modulation of dietary iron content in a rat model of HF has been used to address how iron status affects cardiac iron levels, heart structure and function, and how the presence of HF affects cardiac expression of hepcidin and other iron-related genes. RESULTS: MID developed in the rat model of heart failure. Iron supplementation did not normalize the myocardial iron content; however, it improved survival of HF animals compared to animals fed diet with normal iron content. We observed marked upregulation of hepcidin mRNA expression in HF animals, which was not associated with systemic or cardiac iron levels but strongly correlated with markers and parameters of heart injury. Identical iron-independent pattern was observed for expression of several iron-related genes. CONCLUSIONS: MID is not caused by defective iron absorption or decreased systemic iron levels, but rather by intrinsic myocardial iron deregulation. Altered cardiac expression of hepcidin and other iron-related genes is driven by iron-independent stimuli in the failing heart. GENERAL SIGNIFICANCE: Understanding of the causes and consequences of MID is critical for finding strategies how to improve cardiac iron stores and in HF patients.

Therapeutic potential of hepcidin - the master regulator of iron metabolism.

Iron is an essential biogenic element for both prokaryotic and eukaryotic cells. In humans iron is present in hundreds of different metalloproteins. The peptide hormone hepcidin serves as a master regulator of iron homeostasis on the level of single cells and whole organism - by altering cell surface expression of cellular iron exporter - protein ferroportin. Altered levels of extracellular hepcidin lead to pathological conditions such as hemochromatosis and iron loading or, on the other side, iron restrictive anemias. Therapeutic modulation of hepcidin is a new and promising approach to treatment of these conditions. In this review, a summary of the current knowledge of hepcidin function, regulation and pathological involvements are provided, followed by a section covering the therapeutic potential of hepcidin and the current strategies how to modulate its levels and biological functions for therapeutic purposes.

Hepcidin bound to α2-macroglobulin reduces ferroportin-1 expression and enhances its activity at reducing serum iron levels.

Hepcidin regulates iron metabolism by down-regulating ferroportin-1 (Fpn1). We demonstrated that hepcidin is complexed to the blood transport protein, α2-macroglobulin (α2M) (Peslova, G., Petrak, J., Kuzelova, K., Hrdy, I., Halada, P., Kuchel, P. W., Soe-Lin, S., Ponka, P., Sutak, R., Becker, E., Huang, M. L., Suryo Rahmanto, Y., Richardson, D. R., and Vyoral, D. (2009) Blood 113, 6225-6236). However, nothing is known about the mechanism of hepcidin binding to α2M or the effects of the α2M·hepcidin complex in vivo. We show that decreased Fpn1 expression can be mediated by hepcidin bound to native α2M and also, for the first time, hepcidin bound to methylamine-activated α2M (α2M-MA). Passage of high molecular weight α2M·hepcidin or α2M-MA·hepcidin complexes (≈725 kDa) through a Sephadex G-25 size exclusion column retained their ability to decrease Fpn1 expression. Further studies using ultrafiltration indicated that hepcidin binding to α2M and α2M-MA was labile, resulting in some release from the protein, and this may explain its urinary excretion. To determine whether α2M-MA·hepcidin is delivered to cells via the α2M receptor (Lrp1), we assessed α2M uptake and Fpn1 expression in Lrp1(-/-) and Lrp1(+/+) cells. Interestingly, α2M·hepcidin or α2M-MA·hepcidin demonstrated similar activities at decreasing Fpn1 expression in Lrp1(-/-) and Lrp1(+/+) cells, indicating that Lrp1 is not essential for Fpn1 regulation. In vivo, hepcidin bound to α2M or α2M-MA did not affect plasma clearance of α2M/α2M-MA. However, serum iron levels were reduced to a significantly greater extent in mice treated with α2M·hepcidin or α2M-MA·hepcidin relative to unbound hepcidin. This effect could be mediated by the ability of α2M or α2M-MA to retard kidney filtration of bound hepcidin, increasing its half-life. A model is proposed that suggests that unlike proteases, which are irreversibly bound to activated α2M, hepcidin remains labile and available to down-regulate Fpn1.

Nutritional hepatic iron overload is not prevented by parenteral hepcidin substitution therapy in mice.

The peptide hormone hepcidin functions as a negative regulator of intestinal Fe absorption and Fe recycling. Since its discovery as a systemic negative regulator of Fe metabolism, hepcidin has attracted enormous interest as a potential drug for the treatment and/or prevention of several forms of Fe overload. We therefore tested whether multiple doses of intraperitoneally administered synthetic renatured hepcidin can prevent hepatic Fe loading in mice concurrently fed an Fe-rich diet, and whether the same treatment affects hepatic Fe stores in mice fed a normal diet. Cohorts of male mice were fed either a normal defined diet (180 parts per million Fe) or an Fe-rich diet (the same diet supplemented with 2 % carbonyl iron for 2 weeks). Concurrently, half of the animals in each diet group received 100 µg of renatured hepcidin intraperitoneally every 12 h, for the same 2-week period. The second half of the animals received PBS only. The renatured synthetic hepcidin demonstrated biological activity by significantly decreasing transferrin saturation, which lasted for up to 24 h after a single hepcidin dose. However, the 14 d intraperitoneal hepcidin therapy did not prevent hepatic Fe overload in mice fed the Fe-rich diet, nor did it affect hepatic Fe stores in mice fed the normal diet. Both hepcidin agonists and antagonists are expected to have broad therapeutic potential. The absence of an effect of biologically active hepcidin on hepatic Fe loading shows the need for thorough future studies on the hepcidin regulation of Fe absorption and tissue distribution.

Human MRCKalpha is regulated by cellular iron levels and interferes with transferrin iron uptake.

Myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha, formally known as CDC42BPA) is a serine/threonine kinase that can regulate actin/myosin assembly and activity. Recently, it has been shown that it possesses a functional iron responsive element (IRE) in the 3'-untranslated region (UTR) of its mRNA, suggesting that it may be involved in iron metabolism. Here we report that MRCKalpha protein expression is also regulated by iron levels; MRCKalpha colocalizes with transferrin (Tf)-loaded transferrin receptors (TfR), and attenuation of MRCKalpha expression by a short hairpin RNA silencing construct leads to a significant decrease in Tf-mediated iron uptake. Our results thus indicate that MRCKalpha takes part in Tf-iron uptake, probably via regulation of Tf-TfR endocytosis/endosome trafficking that is dependent on the cellular cytoskeleton. Regulation of the MRCKalpha activity by intracellular iron levels could thus represent another molecular feedback mechanism cells could use to finely tune iron uptake to actual needs.

Hepcidin, the hormone of iron metabolism, is bound specifically to alpha-2-macroglobulin in blood.

Hepcidin is a major regulator of iron metabolism. Hepcidin-based therapeutics/diagnostics could play roles in hematology in the future, and thus, hepcidin transport is crucial to understand. In this study, we identify alpha2-macroglobulin (alpha2-M) as the specific hepcidin-binding molecule in blood. Interaction of 125I-hepcidin with alpha2-M was identified using fractionation of plasma proteins followed by native gradient polyacrylamide gel electrophoresis and mass spectrometry. Hepcidin binding to nonactivated alpha2-M displays high affinity (Kd 177 +/- 27 nM), whereas hepcidin binding to albumin was nonspecific and displayed nonsaturable kinetics. Surprisingly, the interaction of hepcidin with activated alpha2-M exhibited a classical sigmoidal binding curve demonstrating cooperative binding of 4 high-affinity (Kd 0.3 microM) hepcidin-binding sites. This property probably enables efficient sequestration of hepcidin and its subsequent release or inactivation that may be important for its effector functions. Because alpha2-M rapidly targets ligands to cells via receptor-mediated endocytosis, the binding of hepcidin to alpha2-M may influence its functions. In fact, the alpha2-M-hepcidin complex decreased ferroportin expression in J774 cells more effectively than hepcidin alone. The demonstration that alpha2-M is the hepcidin transporter could lead to better understanding of hepcidin physiology, methods for its sensitive measurement and the development of novel drugs for the treatment of iron-related diseases.

Prion protein modulates cellular iron uptake: a novel function with implications for prion disease pathogenesis.

Converging evidence leaves little doubt that a change in the conformation of prion protein (PrP(C)) from a mainly alpha-helical to a beta-sheet rich PrP-scrapie (PrP(Sc)) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrP(Sc), nor the normal function of PrP(C) is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrP(C) mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrP(C) increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrP(C) on ferritin iron content is enhanced by stimulating PrP(C) endocytosis, and reversed by cross-linking PrP(C) on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP(102L) decreases ferritin iron content significantly relative to PrP(C) expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrP(C) nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrP(C) in cellular iron uptake and transport to ferritin, and dysfunction of PrP(C) as a significant contributing factor of brain iron imbalance in prion disorders.

Déjà vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins.

After reading many 2-DE-based articles featuring lists of the differentially expressed proteins, one starts experiencing a disturbing déjà vu. The same proteins seem to predominate regardless of the experiment, tissue or species. To quantify the occurrence of individual differentially expressed proteins in 2-DE experiment reports, we compiled the identities of differentially expressed proteins identified in human, mouse, and rat tissues published in three recent volumes of Proteomics and calculated the appearance of the most predominant proteins in the dataset. The most frequently identified protein is a highly abundant glycolytic enzyme enolase 1, differentially expressed in nearly every third experiment on both human and rodent tissues. Heat-shock protein 27 (HSP27) and heat-shock protein 60 (HSP60) were differentially expressed in about 30 percent of human and rodent samples, respectively. Considering protein families as units, keratins and peroxiredoxins are the most frequently identified molecules, with at least one member of the group being differentially expressed in about 40 percent of all experiments. We suggest that the frequent identification of these proteins must be considered in the interpretation of any 2-DE studies. We consider if these commonly observed changes represent common cellular stress responses or are a reflection of the technical limitations of 2-DE.

Proteomic analysis of hearts from frataxin knockout mice: marked rearrangement of energy metabolism, a response to cellular stress and altered expression of proteins involved in cell structure, motility and metabolism.

A frequent cause of death in Friedreich's ataxia patients is cardiomyopathy, but the molecular alterations underlying this condition are unknown. We performed 2-DE to characterize the changes in protein expression of hearts using the muscle creatine kinase frataxin conditional knockout (KO) mouse. Pronounced changes in protein expression profile were observed in 9 week-old KO mice with severe cardiomyopathy. In contrast, only several proteins showed altered expression in asymptomatic 4 week-old KO mice. In hearts from frataxin KO mice, components of the iron-dependent complex-I and -II of the mitochondrial electron transport chain and enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) displayed decreased expression. Interestingly, the KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilization and pyruvate decarboxylation. This constitutes evidence of metabolic compensation due to decreased expression of electron transport proteins. There was also pronounced up-regulation of proteins involved in stress protection, such as a variety of chaperones, as well as altered expression of proteins involved in cellular structure, motility and general metabolism. This is the first report of the molecular changes at the protein level which could be involved in the cardiomyopathy of the frataxin KO mouse.

Iron-independent specific protein expression pattern in the liver of HFE-deficient mice.

Hereditary hemochromatosis type I is an autosomal-recessive iron overload disease associated with a mutation in HFE gene. The most common mutation, C282Y, disrupts the disulfide bond necessary for the association of HFE with beta-2-microglobulin and abrogates cell surface HFE expression. HFE-deficient mice develop iron overload indicating a central role of the protein in the pathogenesis of hereditary hemochromatosis type I. However, despite significant effort, the role of the HFE protein in iron metabolism is still unknown. To shed a light on the molecular mechanism of HFE-related hemochromatosis we studied protein expression changes elicited by HFE-deficiency in the liver which is the organ critical for the regulation of iron metabolism. We undertook a proteomic study comparing protein expression in the liver of HFE deficient mice with control animals. We compared HFE-deficient animals with control animals with identical iron levels obtained by dietary treatment to identify changes specific to HFE deficiency rather than iron loading. We found 11 proteins that were differentially expressed in the HFE-deficient liver using two-dimensional electrophoresis and mass spectrometry identification. Of particular interest were urinary proteins 1, 2 and 6, glutathione-S-transferase P1, selenium binding protein 2, sarcosine dehydrogenase and thioredoxin-like protein 2. Our data suggest possible involvement of lipocalins, TNF-alpha signaling and PPAR alpha regulatory pathway in the pathogenesis of hereditary hemochromatosis and suggest future targeted research addressing the roles of the identified candidate genes in the molecular mechanism of hereditary hemochromatosis.

Proteomic analysis of hepatic iron overload in mice suggests dysregulation of urea cycle, impairment of fatty acid oxidation, and changes in the methylation cycle.

Liver iron overload can be found in hereditary hemochromatosis, chronic liver diseases such as alcoholic liver disease, and chronic viral hepatitis or secondary to repeated blood transfusions. The excess iron promotes liver damage, including fibrosis, cirrhosis, and hepatocellular carcinoma. Despite significant research effort, we remain largely ignorant of the cellular consequences of liver iron overload and the cellular processes that result in the observed pathological changes. In addition, the variability in outcome and the compensatory response that likely modulates the effect of increased iron levels are not understood. To provide insight into these critical questions, we undertook a study to determine the consequences of iron overload on protein levels in liver using a proteomic approach. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we studied hepatic iron overload induced by carbonyl iron-rich diet in mice and identified 30 liver proteins whose quantity changes in condition of excess liver iron. Among the identified proteins were enzymes involved in several important metabolic pathways, namely the urea cycle, fatty acid oxidation, and the methylation cycle. This pattern of changes likely reflects compensatory and pathological changes associated with liver iron overload and provides a window into these processes.

Native proteomic analysis of protein complexes in murine intestinal brush border membranes.

Intestinal epithelial cell protrusions referred as microvilli or brush border membranes (BBMs) are specialized in the digestion, uptake, and transport of nutrients, trace elements and vitamins from intestinal lumen into the circulation. Disorders of intestinal absorption are common in human pathology and include serious defects such as malabsorption. A detailed description of native digestive protein complexes in BBMs is therefore essential for understanding the physiology and pathology of digestion and absorption. In this study, we employed blue native PAGE (BN-PAGE) technique to separate protein complexes from purified mouse intestinal BBMs. We found 23 distinct protein complexes, which were cut off from the gel, and their protein composition was determined by LC-MS/MS. A total of 55 individual proteins were identified including peptidases, enzymes of carbohydrate metabolism, membrane transporters, cytoskeletal proteins, chaperones, and regulatory enzymes. From the identified proteins, 50% represent molecules with at least one predicted transmembrane domain as predicted by SOSUI software. To the best of our knowledge, this work is the first attempt aimed to characterize the native membrane proteome of intestinal BBM. As demonstrated here, BN-PAGE is a powerful tool for the separation of not only mitochondrial, but also membrane hydrophobic proteins in general. In addition, BN-PAGE technique preserves metal-protein interactions, as shown by the presence of 65Zn in metalloprotein complexes, isolated from zinc-radiolabeled BBMs.

Proteomic analysis of erythroid differentiation induced by hexamethylene bisacetamide in murine erythroleukemia cells.

OBJECTIVE: Murine erythroleukemia (MEL) cells are transformed erythroid precursors that are arrested in an immature and proliferating state. These leukemic cells can be grown in cell cultures and induced to terminal erythroid differentiation by a treatment with a specific chemical inducer such as N,N'-hexamethylene bisacetamide. MEL cells then re-enter their original erythroid program and differentiate along the erythroid pathway into non-dividing hemoglobin-rich cells resembling orthochromatophilic normoblasts. To deepen our understanding of the molecular mechanisms underlying and erythroid differentiation and leukemia we monitored changes in protein expression in differentiating MEL cells. METHODS: In our effort to find new candidate proteins involved in the differentiation of MEL cells, we embraced a proteomic approach. Employing two-dimensional (2D) electrophoresis combined with mass spectrometry, we compared protein expression in non-induced MEL cells with MEL cells exposed to N,N'-hexamethylene bisacetamide for 48 h. RESULTS: From 700 proteins spots observed, 31 proteins were differentially expressed. We successfully identified 27 of the differentially expressed molecules by mass spectrometry (MALDI-MS). CONCLUSION: In addition to proteins involved in heme biosynthesis, protein metabolism, stress defense and cytoskeletal organization, we identified 3 proteins engaged in regulation of cellular trafficking and 7 proteins important for regulation of gene expression and cell cycle progression including 3 components of chromatin remodeling complexes. Many of the identified molecules are associated with erythroid differentiation or leukemia for the first time. To our knowledge, this is the first study applying a modern proteomic approach to the direct analysis of erythroid differentiation of leukemic cells.

Proteomic analysis of iron overload in human hepatoma cells.

Iron-mediated organ damage is common in patients with iron overload diseases, namely, hereditary hemochromatosis. Massive iron deposition in parenchymal organs, particularly in the liver, causes organ dysfunction, fibrosis, cirrhosis, and also hepatocellular carcinoma. To obtain deeper insight into the poorly understood and complex cellular response to iron overload and consequent oxidative stress, we studied iron overload in liver-derived HepG2 cells. Human hepatoma HepG2 cells were exposed to a high concentration of iron for 3 days, and protein expression changes initiated by the iron overload were studied by two-dimensional electrophoresis and mass spectrometry. From a total of 1,060 spots observed, 21 spots were differentially expressed by iron overload. We identified 19 of them; 11 identified proteins were upregulated, whereas 8 identified proteins showed a decline in response to iron overload. The differentially expressed proteins are involved in iron storage, stress response and protection against oxidative stress, protein folding, energy metabolism, gene expression, cell cycle regulation, and other processes. Many of these molecules have not been previously suggested to be involved in the response to iron overload and the consequent oxidative stress.

Hepcidin: a direct link between iron metabolism and immunity.

Hepcidin, originally discovered in urine as a bactericidal peptide synthesized by hepatocytes was later proved to be a key regulator of iron metabolism at the whole body level, namely, in conditions of altered iron demand such as the increased or decreased total amount of body iron, inflammation, hypoxia and anemia. The major mechanism of hepcidin function seems to be the regulation of transmembrane iron transport. Hepcidin binds to its receptor, protein ferroportin, which serves as a transmembrane iron channel enabling iron efflux from cells. The hepcidin-ferroportin complex is then degraded in lysosomes and iron is locked inside the cells (mainly enterocytes, hepatocytes and macrophages). This leads to lowering of iron absorption in the intestine and to a decrease in serum iron concentration. Utilizing this mechanism, hepcidin regulates serum iron levels during inflammation, infection and possibly also in cancer. Under these conditions iron is shifted from circulation into cellular stores in hepatocytes and macrophages, making it less available for invading microorganisms and tumor cells. In anemia and hypoxia, hepcidin regulates the availability of iron for erythropoiesis. Hepcidin or hepcidin-related therapeutics could find a place in the treatment of various diseases such as hemochromatosis and anemia of chronic disease.

Hephaestin--a ferroxidase of cellular iron export.

Hephaestin is a transmembrane copper-dependent ferroxidase necessary for effective iron transport from intestinal enterocytes into the circulation. Hephaestin is mutated in sex-linked anemia (sla) mice. The initial uptake of iron from the diet in these animals is normal, but the basolateral export of iron from enterocytes is defective, resulting in iron deficiency and microcytic hypochromic anemia. In addition to the small intestine, hephaestin is expressed to a lesser extent in colon, spleen, placenta and kidney but its role in these tissues remains unknown. So far, hephaestin has not been linked to a human disease.

Identification of heme binding protein complexes in murine erythroleukemic cells: study by a novel two-dimensional native separation -- liquid chromatography and electrophoresis.

In the current postgenomic era there is a growing interest in analysis of protein complexes in their native state. Here we present a novel two-dimensional separation technique for assessment of native protein complexes. The method combines native chromatography with native electrophoresis. The approach was used to study heme-binding protein complexes in murine erythroleukemia cells. The cells were metabolically labeled with [(59)Fe]-heme and cellular lysates were separated by anion-exchange chromatography. Fractions containing the (59)Fe isotope were collected, concentrated and further separated by native gel electrophoresis. A total of 13 radioactive protein bands were detected and analyzed by liquid chromatography-tandem mass spectrometry. Thirty-three individual proteins were identified and attributed to four novel multiprotein complexes representing four different 'snapshots' of cellular events involved in hemoglobin biosynthesis.

Different expression pattern of hepcidin genes in the liver and pancreas of C57BL/6N and DBA/2N mice.

BACKGROUND/AIMS: Male C57BL/6 and DBA/2 mice differ in their liver iron content. The aim of this study was to examine possible differences in the expression of hepcidin genes (Hamp and Hamp2) between the two strains. METHODS: Hepatic mRNAs were quantified by real-time PCR. RESULTS: Ferroportin1, transferrin receptor 2 and HAMP mRNA levels displayed no significant strain differences. However, HAMP2 mRNA levels were higher in DBA/2N mice. In both strains, HAMP2 mRNA content was sex-dependent, with higher values in female animals. Both hepatic HAMP and HAMP2 mRNA levels were elevated by iron overload, but treatment with lipopolysaccharide increased only HAMP mRNA. Lipopolysaccharide also elevated the amount of HAMP mRNA in the pancreas, while pancreatic HAMP2 mRNA levels were decreased. Sequence analysis of hepcidin amplicons from DBA/2N mice predicted an Asn-->Lys substitution at position 73 of the HAMP peptide and a Ser-->Phe substitution at position 76 of the HAMP2 peptide. CONCLUSIONS: Hepatic Hamp2 expression displays considerable strain- and sex-dependent variation. Lipopolysaccharide increases expression of Hamp both in the liver and pancreas, but Hamp2 does not respond to lipopolysaccharide treatment. The significance of the amino acid substitutions in hepcidin peptides in DBA/2N mice is at present unknown.