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1.  Hepcidin response to acute iron intake and chronic iron loading in dysmetabolic iron overload syndrome 
Background
The pathogenesis of dysmetabolic iron overload syndrome (DIOS) is still unclear. Hepcidin is the key regulator of iron homeostasis controlling iron absorption and macrophage release.
Aim
To investigate hepcidin regulation by iron in DIOS.
Methods
We analysed urinary hepcidin at baseline and 24 h after a 65 mg oral iron dose in 24 patients at diagnosis and after iron depletion (n=13) and compared data with those previously observed in 23 healthy controls. Serum iron indices, liver histology and metabolic data were available for all patients.
Results
At diagnosis, hepcidin values were significantly higher than in controls (P<0.001). After iron depletion, hepcidin levels decreased to normal values in all patients. At baseline, a significant response of hepcidin to iron challenge was observed only in the subgroup with lower basal hepcidin concentration (P=0.007). In iron-depleted patients, urinary hepcidin significantly increased after oral iron test (P=0.006).
Conclusions
Ours findings suggest that in DIOS, the progression of iron accumulation is counteracted by the increase in hepcidin production and progressive reduction of iron absorption, explaining why these patients develop a mild–moderate iron overload that tends to a plateau.
doi:10.1111/j.1478-3231.2011.02520.x
PMCID: PMC4048852  PMID: 21733088
2.  HEPCIDIN AND IRON HOMEOSTASIS 
Biochimica et biophysica acta  2012;1823(9):1434-1443.
Despite fluctuations in dietary iron intake and intermittent losses through bleeding, the plasma iron concentrations in humans remain stable at 10–30 μM. While most of the iron entering blood plasma comes from recycling, appropriate amount of iron is absorbed from the diet to compensate for losses and maintain nontoxic amounts in stores. Plasma iron concentration and iron distribution are similarly regulated in laboratory rodents. The hepatic peptide hepcidin was identified as the systemic iron-regulatory hormone. In the efferent arc, hepcidin regulates intestinal iron absorption, plasma iron concentrations, and tissue iron distribution by inducing degradation of its receptor, the cellular iron exporter ferroportin. Ferroportin exports iron into plasma from absorptive enterocytes, from macrophages that recycle the iron of senescent erythrocytes, and from hepatocytes that store iron. In the more complex and less well understood afferent arc, hepatic hepcidin synthesis is transcriptionally regulated by extracellular and intracellular iron concentrations through a molecular complex of bone morphogenetic protein receptors and their iron-specific ligands, modulators and iron sensors. Through as yet undefined pathways, hepcidin is also homeostatically regulated by the iron requirements of erythroid precursors for hemoglobin synthesis. In accordance with the role of hepcidin-mediated iron redistribution in host defense, hepcidin production is regulated by inflammation as well. Increased hepcidin concentrations in plasma are pathogenic in iron-restrictive anemias including anemias associated with inflammation, chronic kidney disease and some cancers. Hepcidin deficiency causes iron overload in hereditary hemochromatosis and ineffective erythropoiesis. Hepcidin, ferroportin and their regulators represent potential targets for the diagnosis and treatment of iron disorders and anemias.
doi:10.1016/j.bbamcr.2012.01.014
PMCID: PMC4048856  PMID: 22306005
Iron overload; iron deficiency; anemia
3.  The Hepcidin-Ferroportin System as a Therapeutic Target in Anemias and Iron Overload Disorders 
The review summarizes the current understanding of the role of hepcidin and ferroportin in normal iron homeostasis and its disorders. It further discusses the various approaches to therapeutic targeting of hepcidin and ferroportin in iron overload diseases (mainly hereditary hemochromatosis and β-thalassemia) and iron-restrictive anemias (anemias associated with infections, inflammatory disorders and certain malignancies, anemia of chronic kidney diseases, iron-refractory iron deficiency anemia).
doi:10.1182/asheducation-2011.1.538
PMCID: PMC4034574  PMID: 22160086
4.  Mouse Models of Anemia of Cancer 
PLoS ONE  2014;9(3):e93283.
Anemia of cancer (AC) may contribute to cancer-related fatigue and impair quality of life. Improved understanding of the pathogenesis of AC could facilitate better treatment, but animal models to study AC are lacking. We characterized four syngeneic C57BL/6 mouse cancers that cause AC. Mice with two different rapidly-growing metastatic lung cancers developed the characteristic findings of anemia of inflammation (AI), with dramatically different degrees of anemia. Mice with rapidly-growing metastatic melanoma also developed a severe anemia by 14 days, with hematologic and inflammatory parameters similar to AI. Mice with a slow-growing peritoneal ovarian cancer developed an iron-deficiency anemia, likely secondary to chronically impaired nutrition and bleeding into the peritoneal cavity. Of the four models, hepcidin mRNA levels were increased only in the milder lung cancer model. Unlike in our model of systemic inflammation induced by heat-killed Brucella abortus, ablation of hepcidin in the ovarian cancer and the milder lung cancer mouse models did not affect the severity of anemia. Hepcidin-independent mechanisms play an important role in these murine models of AC.
doi:10.1371/journal.pone.0093283
PMCID: PMC3969362  PMID: 24681760
5.  Molecular and clinical correlates in iron overload associated with mutations in ferroportin 
Haematologica  2006;91(8):1092-1095.
Mutations in ferroportin (Fpn) result in iron overload. We correlate the behavior of three Fpn mutants in vitro with patients’ phenotypes. Patients with Fpn mutations A77D or N174I showed macrophage iron loading. In cultured cells, FpnA77D did not reach the cell surface and cells did not export iron. Fpn mutant N174I showed plasma membrane and intracellular localization, and did not transport iron. Fpn mutation G80S was targeted to the cell surface and was transport competent, however patients showed macrophage iron. We suggest that FpnG80S represents a class of Fpn mutants whose behavior in vitro does not explain the patients’ phenotype.
PMCID: PMC3718284  PMID: 16885049
ferroportin; hemochromatosis; hepcidin; iron transport
6.  Inhibition of hepcidin transcription by growth factors 
Hepatology (Baltimore, Md.)  2012;56(1):291-299.
The hepatic peptide hormone hepcidin controls the duodenal absorption of iron, its storage and its systemic distribution. Hepcidin production is often insufficient in chronic hepatitis C and alcoholic liver disease, leading to hyperabsorption of iron and its accumulation in the liver. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) mediate the hepatic regeneration after liver injury. We examined the effect of the growth factors on hepcidin synthesis by hepatocytes. Results: HGF and EGF treatment of primary mouse hepatocytes, as well as EGF administration in mice, suppressed hepcidin mRNA synthesis. The suppression of hepcidin by these growth factors was transcriptional, and was mediated by a direct effect of HGF and EGF on the BMP pathway regulating hepcidin synthesis. We further showed that growth factors interfered with nuclear localization of activated Smads and increased the nuclear pool of the BMP transcriptional co-repressor TG-interacting factor (TGIF). In a kinase screen with small-molecule kinase inhibitors, inhibitors in the PI3 kinase pathway and in the MEK/ERK pathway prevented HGF suppression of hepcidin in primary mouse hepatocytes. Conclusion: HGF and EGF suppress hepatic hepcidin synthesis, in part through PI3 kinase MEK/ERK kinase pathways which may be modulating the nuclear localization of BMP pathway transcriptional regulators including activated Smads1/5/8 and the co-repressor TGIF. EGF, HGF and possibly other growth factors that activate similar pathways may contribute to hepcidin suppression in chronic liver diseases, promote iron accumulation in the liver and exacerbate the destructive disease processes.
doi:10.1002/hep.25615
PMCID: PMC3362690  PMID: 22278715
Chronic liver disease; iron overload; hepatocyte growth factor; epidermal growth factor; bone morphogenetic protein pathway
7.  Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination 
Cell Metabolism  2012;15(6):918-924.
SUMMARY
Ferroportin exports iron into plasma from absorptive enterocytes, erythrophagocytosing macrophages, and hepatic stores. The hormone hepcidin controls cellular iron export and plasma iron concentrations by binding to ferroportin and causing its internalization and degradation. We explored the mechanism of hepcidin-induced endocytosis of ferroportin, the key molecular event in systemic iron homeostasis. Hepcidin binding caused rapid ubiquitination of ferroportin in cell lines overexpressing ferroportin and in murine bone marrow-derived macrophages. No hepcidin-dependent ubiquitination was observed in C326S ferroportin mutant which does not bind hepcidin. Substitutions of lysines between residues 229 and 269 in the third cytoplasmic loop of ferroportin prevented hepcidin-dependent ubiquitination and endocytosis of ferroportin, and promoted cellular iron export even in the presence of hepcidin. The human ferroportin mutation K240E, previously associated with clinical iron overload, caused hepcidin resistance in vitro by interfering with ferroportin ubiquitination. Our study demonstrates that ubiquitination is the functionally-relevant signal for hepcidin-induced ferroportin endocytosis.
doi:10.1016/j.cmet.2012.03.018
PMCID: PMC3372862  PMID: 22682227
8.  Cellular Catabolism of the Iron-Regulatory Peptide Hormone Hepcidin 
PLoS ONE  2013;8(3):e58934.
Hepcidin, a 25-amino acid peptide hormone, is the principal regulator of plasma iron concentrations. Hepcidin binding to its receptor, the iron exporter ferroportin, induces ferroportin internalization and degradation, thus blocking iron efflux from cells into plasma. The aim of this study was to characterize the fate of hepcidin after binding to ferroportin. We show that hepcidin is taken up by ferroportin-expressing cells in a temperature- and pH-dependent manner, and degraded together with its receptor. When Texas red-labeled hepcidin (TR-Hep) was added to ferroportin-GFP (Fpn-GFP) expressing cells, confocal microscopy showed co-localization of TR-Hep with Fpn-GFP. Using flow cytometry, we showed that the peptide was almost completely degraded by 24 h after its addition, but that lysosomal inhibitors completely prevented degradation of both ferroportin and hepcidin. In addition, using radio-labeled hepcidin and HPLC analysis we show that hepcidin is not recycled, and that only degradation products are released from the cells. Together these results show that the hormone hepcidin and its receptor ferroportin are internalized together and trafficked to lysosomes where both are degraded.
doi:10.1371/journal.pone.0058934
PMCID: PMC3594189  PMID: 23536837
9.  Hepcidin Expression in Iron Overload Diseases Is Variably Modulated by Circulating Factors 
PLoS ONE  2012;7(5):e36425.
Hepcidin is a regulatory hormone that plays a major role in controlling body iron homeostasis. Circulating factors (holotransferrin, cytokines, erythroid regulators) might variably contribute to hepcidin modulation in different pathological conditions. There are few studies analysing the relationship between hepcidin transcript and related protein expression profiles in humans. Our aims were: a. to measure hepcidin expression at either hepatic, serum and urinary level in three paradigmatic iron overload conditions (hemochromatosis, thalassemia and dysmetabolic iron overload syndrome) and in controls; b. to measure mRNA hepcidin expression in two different hepatic cell lines (HepG2 and Huh-7) exposed to patients and controls sera to assess whether circulating factors could influence hepcidin transcription in different pathological conditions. Our findings suggest that hepcidin assays reflect hepatic hepcidin production, but also indicate that correlation is not ideal, likely due to methodological limits and to several post-trascriptional events. In vitro study showed that THAL sera down-regulated, HFE-HH and C-NAFLD sera up-regulated hepcidin synthesis. HAMP mRNA expression in Huh-7 cells exposed to sera form C-Donors, HFE-HH and THAL reproduced, at lower level, the results observed in HepG2, suggesting the important but not critical role of HFE in hepcidin regulation.
doi:10.1371/journal.pone.0036425
PMCID: PMC3346721  PMID: 22586470
10.  Iron Metabolism: Interactions with Normal and Disordered Erythropoiesis 
Hemoglobinopathies and other disorders of erythroid cells are often associated with abnormal iron homeostasis. We review the molecular physiology of intracellular and systemic iron regulation, and the interactions between erythropoiesis and iron homeostasis. Finally, we discuss iron disorders that affect erythropoiesis as well as erythroid disorders that cause iron dysregulation.
Homeostatic mechanisms maintain plasma iron concentrations at 10–30 μm. Chronically low or high concentrations lead to ineffective erythropoiesis, anemia, generation of nontransferrin-bound iron, and tissue damage.
doi:10.1101/cshperspect.a011668
PMCID: PMC3331689  PMID: 22553501
11.  Evidence for distinct pathways of hepcidin regulation by acute and chronic iron loading 
Hepatology (Baltimore, Md.)  2011;53(4):1333-1341.
In response to iron loading, hepcidin synthesis is homeostatically increased to limit further absorption of dietary iron and its release from stores. Mutations in HFE, transferrin receptor 2 (Tfr2), hemojuvelin (HJV) or bone morphogenetic protein 6 (BMP6) prevent appropriate hepcidin response to iron, allowing increased absorption of dietary iron, and eventually iron overload. To understand the role each of these proteins plays in hepcidin regulation by iron, we analyzed hepcidin mRNA responsiveness to short and long-term iron challenge in iron-depleted Hfe, Tfr2, Hjv and Bmp6 mutant mice. After 1-day (acute) iron challenge, Hfe−/− showed a smaller hepcidin increase than their wild-type strain-matched controls, Bmp6−/− nearly no increase, and Tfr2 and Hjv mutants no increase in hepcidin expression, indicating that all four proteins participate in hepcidin regulation by acute iron changes. After a 21-day (chronic) iron challenge, Hfe and Tfr2 mutants increased hepcidin expression to nearly wild-type levels but a blunted increase of hepcidin was seen in Bmp6−/− and Hjv−/− mice. BMP6, whose expression is also regulated by iron, may mediate hepcidin regulation by iron stores. None of the mutant strains (excepting Bmp6−/− mice) had impaired BMP6 mRNA response to chronic iron loading. Conclusion: TfR2, HJV and BMP6 and, to a lesser extent, HFE, are required for the hepcidin response to acute iron loading, but are partially redundant for hepcidin regulation during chronic iron loading, and are not involved in the regulation of BMP6 expression. Our findings support a model in which acute increases in holotransferrin concentrations transmitted through HFE, TfR2 and HJV augment BMP receptor sensitivity to BMPs. A distinct regulatory mechanism that senses hepatic iron may modulate hepcidin response to chronic iron loading.
doi:10.1002/hep.24178
PMCID: PMC3074982  PMID: 21480335
Hereditary hemochromatosis; bone morphogenetic protein 6; hemojuvelin; HFE; transferrin receptor 2
12.  Understanding the Structure/Activity Relationships of the Iron Regulatory Peptide Hepcidin 
Chemistry & biology  2011;18(3):336-343.
Summary
The peptide hormone hepcidin is a key homeostatic regulator of iron metabolism and involved in pathological regulation of iron in response to infection, inflammation, hypoxia and anaemia. It acts by binding to the iron exporter ferroportin, causing it to be internalised and degraded; however, little is known about the structure/activity relationships of the interaction of hepcidin with ferroportin. Here we show that there are key residues within the N-terminal region of hepcidin that influence its interaction with ferroportin, and we explore the structure/function relationships at these positions. We found that the interaction is primarily hydrophobic with critical stereochemical requirements at positions 4 and 6. In addition, a series of hepcidin mutants in which disulfide bonds had been replaced with diselenide bonds showed no change in biological activity compared to native hepcidin. The results provide mechanistic insight into the interaction between hepcidin and ferroportin and identify important constraints for the development of hepcidin congeners for the treatment of hereditary iron overload.
doi:10.1016/j.chembiol.2010.12.009
PMCID: PMC3073735  PMID: 21439478
13.  Minihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload 
The Journal of Clinical Investigation  2011;121(12):4880-4888.
Iron overload is the hallmark of hereditary hemochromatosis and a complication of iron-loading anemias such as β-thalassemia. Treatment can be burdensome and have significant side effects, and new therapeutic options are needed. Iron overload in hereditary hemochromatosis and β-thalassemia intermedia is caused by hepcidin deficiency. Although transgenic hepcidin replacement in mouse models of these diseases prevents iron overload or decreases its potential toxicity, natural hepcidin is prohibitively expensive for human application and has unfavorable pharmacologic properties. Here, we report the rational design of hepcidin agonists based on the mutagenesis of hepcidin and the hepcidin-binding region of ferroportin and computer modeling of their docking. We identified specific hydrophobic/aromatic residues required for hepcidin-ferroportin binding and obtained evidence in vitro that a thiol-disulfide interaction between ferroportin C326 and the hepcidin disulfide cage may stabilize binding. Guided by this model, we showed that 7–9 N-terminal amino acids of hepcidin, including a single thiol cysteine, comprised the minimal structure that retained hepcidin activity, as shown by the induction of ferroportin degradation in reporter cells. Further modifications to increase resistance to proteolysis and oral bioavailability yielded minihepcidins that, after parenteral or oral administration to mice, lowered serum iron levels comparably to those after parenteral native hepcidin. Moreover, liver iron concentrations were lower in mice chronically treated with minihepcidins than those in mice treated with solvent alone. Minihepcidins may be useful for the treatment of iron overload disorders.
doi:10.1172/JCI57693
PMCID: PMC3225996  PMID: 22045566
14.  Neutrophil α-Defensins Cause Lung Injury by Disrupting the Capillary–Epithelial Barrier 
Rationale: The involvement of neutrophil activation in the sentinel, potentially reversible, events in the pathogenesis of acute lung injury (ALI) is only partially understood. α-Defensins are the most abundant proteins secreted by activated human neutrophils, but their contribution to ALI in mouse models is hindered by their absence from murine neutrophils and the inability to study their effects in isolation in other species.
Objectives: To study the role of α-defensins in the pathogenesis of ALI in a clinically relevant setting using mice transgenic for polymorphonuclear leukocyte expression of α-defensins.
Methods: Transgenic mice expressing polymorphonuclear leukocyte α-defensins were generated. ALI was induced by acid aspiration. Pulmonary vascular permeability was studied in vivo using labeled dextran and fibrin deposition. The role of the low-density lipoprotein–related receptor (LRP) in permeability was examined.
Measurements and Main Results: Acid aspiration induced neutrophil migration and release of α-defensins into lung parenchyma and airways. ALI was more severe in α-defensin–expressing mice than in wild-type mice, as determined by inspection, influx of neutrophils into the interstitial space and airways, histological evidence of epithelial injury, interstitial edema, extravascular fibrin deposition, impaired oxygenation, and reduced survival. Within 4 hours of insult, α-defensin–expressing mice showed greater disruption of capillary–epithelial barrier function and ALI that was attenuated by systemic or intratracheal administration of specific inhibitors of the LRP.
Conclusions: α-Defensins mediate ALI through LRP-mediated loss of capillary–epithelial barrier function, suggesting a potential new approach to intervention.
doi:10.1164/rccm.200907-1128OC
PMCID: PMC2862305  PMID: 20093642
acute lung injury; capillary–epithelial barrier; α-defensins; low-density lipoprotein–related receptor; receptor-associated protein
15.  Reduced serum hepcidin levels in patients with chronic hepatitis C 
Journal of hepatology  2009;51(5):845-852.
Background/Aims
Patients with chronic hepatitis C (CHC) often have increased liver iron, a condition associated with reduced sustained response to antiviral therapy, more rapid progression to cirrhosis, and development of hepatocellular carcinoma. The hepatic hormone hepcidin is the major regulator of iron metabolism and inhibits iron absorption and recycling from erythrophagocytosis. Hepcidin decrease is a possible pathophysiological mechanism of iron overload in CHC, but studies in humans have been hampered so far by the lack of reliable quantitative assays for the 25-amino acid bioactive peptide in serum (s-hepcidin).
Methods
Using a recently validated immunoassay, we measured s-hepcidin levels in 81 untreated CHC patients and 57 controls with rigorous definition of normal iron status. All CHC patients underwent liver biopsy with histological iron score.
Results
S-hepcidin was significantly lower in CHC patients than in controls (geometric means with 95% confidence intervals: 33.7, 21.5–52.9 vs. 90.9, 76.1–108.4 ng/mL, respectively; p < 0.001). In CHC patients, s-hepcidin significantly correlated with serum ferritin and histological total iron score, but not with s-interleukin-6. After stratification for ferritin quartiles, s-hepcidin increased significantly across quartiles in both controls and CHC patients (chi for trend, p < 0.001). However, in CHC patients, s-hepcidin was significantly lower than in controls for each corresponding quartile (analysis of variance, p < 0.001).
Conclusions
These results, together with very recent studies in animal and cellular models, indicate that although hepcidin regulation by iron stores is maintained in CHC, the suppression of this hormone by hepatitis C virus is likely an important factor in liver iron accumulation in this condition.
doi:10.1016/j.jhep.2009.06.027
PMCID: PMC2761995  PMID: 19729219
Chronic hepatitis C; Hemochromatosis; Hepcidin; Iron overload; Ferritin
16.  The Role of Hepcidin in Iron Metabolism 
Acta Haematologica  2009;122(2-3):78-86.
Hepcidin is the central regulator of systemic iron homeostasis. Dysregulation of hepcidin production results in a variety of iron disorders. Hepcidin deficiency is the cause of iron overload in hereditary hemochromatosis, iron-loading anemias, and hepatitis C. Hepcidin excess is associated with anemia of inflammation, chronic kidney disease and iron-refractory iron deficiency anemia. Diagnostic and therapeutic applications of this new knowledge are beginning to emerge. Dr. Ernest Beutler played a significant role in advancing our understanding of the function of hepcidin. This review is dedicated to his memory.
doi:10.1159/000243791
PMCID: PMC2855274  PMID: 19907144
Anemia of inflammation; Bone morphogenetic protein; Hemochromatosis; Hepcidin; Iron-loading anemia
17.  Iron sequestration and anemia of inflammation 
Seminars in hematology  2009;46(4):387-393.
Anemia of chronic disease, also called anemia of inflammation, is characterized by hypoferremia due to iron sequestration that eventually results in iron-restricted erythropoiesis. During the last decade, the molecular mechanisms of iron sequestration have been found to center on cytokine-stimulated overproduction of the iron-regulatory hormone hepcidin. The inflammatory cytokine IL-6 is a particularly prominent inducer of hepcidin but other cytokines are likely to contribute as well. Hepcidin excess causes the endocytosis and proteolysis of the sole known cellular iron exporter, ferroportin, trapping iron in macrophages and iron-absorbing enterocytes. The supply of iron to hemoglobin synthesis becomes limiting, eventually resulting in anemia. Depending on the details of the underlying disease, other inflammation-related mechanisms may also contribute to anemia.
doi:10.1053/j.seminhematol.2009.06.001
PMCID: PMC2755591  PMID: 19786207
18.  Animal Models of Anemia of Inflammation 
Seminars in hematology  2009;46(4):351-357.
Anemia of inflammation (AI) is a complex multi-organ response to inflammatory disorders. Because AI can result from many infectious and non-infectious inflammatory diseases, multiple mechanisms may contribute to its pathogenesis including iron restriction, direct erythropoietic suppression, shortened red cell survival or frank hemolysis. Animal models have been helpful in the study of the mechanisms of AI and its potential treatments but each model reflects distinct aspects of this heterogeneous syndrome. It is therefore important to study a variety of models of AI. This review focuses on the use of infectious and noninfectious mouse models of inflammation that have been shown to manifest anemia. We review many of the models reported in the literature or developed in our laboratory, and discuss their respective merits and drawbacks.
doi:10.1053/j.seminhematol.2009.06.003
PMCID: PMC2755601  PMID: 19786203
19.  Iron in Innate Immunity: Starve the Invaders 
Current opinion in immunology  2009;21(1):63-67.
Summary
Iron is essential for nearly all living organisms. Innate immunity effectively restricts iron availability to microbial invaders. Some microbes have evolved effective countermeasures that blunt the effect of iron restriction. Recent epidemiologic studies have highlighted the potentiating effect of iron on microbial infections. Laboratory studies have focused on specific immune mechanisms that mediate iron withholding from microbes constitutively and in response to infections. Specialized inflammation-regulated proteins chelate iron, trap siderophores, and transport iron or modulate its transport to alter its tissue distribution during infections.
doi:10.1016/j.coi.2009.01.011
PMCID: PMC2668730  PMID: 19231148
20.  The hepcidin-binding site on ferroportin is evolutionarily conserved 
Cell metabolism  2008;8(2):146-156.
Summary
Mammalian iron homeostasis is regulated by the interaction of the liver-produced peptide hepcidin and its receptor, the iron transporter ferroportin. Hepcidin binds to ferroportin resulting in degradation of ferroportin and decreased cellular iron export. We identify the hepcidin-binding domain (HBD) on ferroportin and show that a synthetic 19 amino acid peptide corresponding to the HBD recapitulates the characteristics and specificity of hepcidin binding to cell surface ferroportin. The binding of mammalian hepcidin to ferroportin or the HBD shows an unusual temperature dependency with an increased rate of dissociation at temperatures below 15°C. The increased rate of dissociation is due to temperature dependent changes in hepcidin structure. In contrast, hepcidin from poikilothermic vertebrates, such as fish or frogs, binds the HBD in a temperature independent fashion. The affinity of hepcidin for the HBD permits a rapid, sensitive assay of hepcidin from all species and yields insights into the evolution of hepcidin.
doi:10.1016/j.cmet.2008.07.002
PMCID: PMC2660598  PMID: 18680715
21.  Co-Regulation and Interdependence of the Mammalian Epidermal Permeability and Antimicrobial Barriers 
Human epidermis elaborates two small cationic, highly hydrophobic antimicrobial peptides (AMP), β-defensin 2 (hBD2), and the carboxypeptide cleavage product of human cathelicidin (hCAP18), LL-37, which are co-packaged along with lipids within epidermal lamellar bodies (LBs) before their secretion. Because of their colocalization, we hypothesized that AMP and barrier lipid production could be coregulated by altered permeability barrier requirements. mRNA and immunostainable protein levels for mBD3 and cathelin-related antimicrobial peptide (CRAMP) (murine homologues of hBD2 and LL-37, respectively) increase 1–8 hours after acute permeability barrier disruption and normalize by 24 hours, kinetics that mirror the lipid metabolic response to permeability barrier disruption. Artificial permeability barrier restoration, which inhibits the lipid-synthetic response leading to barrier recovery, blocks the increase in AMP mRNA/protein expression, further evidence that AMP expression is linked to permeability barrier function. Conversely, LB-derived AMPs are also important for permeability barrier homeostasis. Despite an apparent increase in mBD3 protein, CRAMP−/− mice delayed permeability barrier recovery, attributable to defective LB contents and abnormalities in the structure of the lamellar membranes that regulate permeability barrier function. These studies demonstrate that (1) the permeability and antimicrobial barriers are coordinately regulated by permeability barrier requirements and (2) CRAMP is required for permeability barrier homeostasis.
doi:10.1038/sj.jid.5701099
PMCID: PMC2671223  PMID: 17943185
22.  Functional Analysis of the Host Defense Peptide Human Beta Defensin-1: New Insight into Its Potential Role in Cancer 
Molecular immunology  2007;45(3):839-848.
Although it is known that innate immunity is key for protecting the body against foreign agents such as bacteria, little is known about elements of the innate immune system that have anti-tumor activity. Human Beta Defensin-1 (hBD-1), an important component of the innate immune response, is lost at high frequencies in malignant prostatic tissue, while high levels of expression are maintained in adjacent benign regions. In prostate carcinoma, frequent genetic alterations occur in the 8p22-23 region and several studies indicate there may be multiple tumor suppressor genes present within this region. The high incidence of loss of hBD-1 expression in prostate cancer, along with its chromosomal location of 8p23.2, raised the possibility that it may play a role in tumor suppression. To gain insight as to its function in prostate cancer, hBD-1 was cloned and ectopically expressed in four prostate cancer cell lines. Induction of hBD-1 expression resulted in a decrease in cellular growth in DU145 and PC3 cells. However, hBD-1 has no effect on the growth of androgen receptor (AR) positive LNCaP prostate cancer cells, but was again growth suppressive to PC3 cells with ectopic AR expression (PC3/AR+). hBD-1 also caused rapid induction of cytolysis and caspase-mediated apoptosis in DU145 and PC3 prostate cancer cells. Although the regulation of hBD-1 was not addressed in this study, our preliminary data demonstrated that the pathways involoved may include cMYC and/or PAX2. Data presented here are the first to provide evidence of its potential role in prostate cancer cell death.
doi:10.1016/j.molimm.2006.11.026
PMCID: PMC2131727  PMID: 17868871
Apoptosis; Defensin; Function; hBD-1; prostate cancer; tumor immunity
23.  POSTTRANSLATIONAL PROCESSING OF HEPCIDIN IN HUMAN HEPATOCYTES IS MEDIATED BY THE PROHORMONE CONVERTASE FURIN 
Blood cells, molecules & diseases  2007;40(1):132-138.
Hepcidin is encoded as an 84 amino acid prepropeptide containing a typical N-terminal 24 amino acid endoplasmic reticulum targeting signal sequence, and a 35 amino acid proregion (pro) with a consensus furin cleavage site immediately followed by the C-terminal 25 amino acid bioactive iron-regulatory hormone (mature peptide). We performed pulse-chase studies of posttranslational processing of hepcidin in human hepatoma HepG2 cells and in primary human hepatocytes induced with bone morphogenic protein (BMP-9). In some experiments, the cells were treated with the furin protease inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK) or furin siRNA. In the absence of furin inhibitor, hepcidin was found to be processed in less than 1 hour and secreted as a 3 kD form reactive with anti-mature but not anti-pro antibody. In the presence of furin inhibitors or furin siRNA, a 6 kD form reactive with both anti-pro and anti-mature antibody was rapidly secreted into the medium. Processing was not affected by inhibitors of the hypoxia inducible factor (HIF) pathway, or by treatment with 30 μM holo- or apo-transferrin.
Conclusion
The hepatic prohormone convertase furin mediates the posttranslational processing of hepcidin. The proteolytic cleavage of prohepcidin to hepcidin is not regulated by iron-transferrin or the HIF pathway.
doi:10.1016/j.bcmd.2007.07.009
PMCID: PMC2211381  PMID: 17905609
Liver; iron regulation; pro; hepcidin; pulse; chase study; metabolic protein labeling
24.  Soluble hemojuvelin is released by proprotein convertase-mediated cleavage at a conserved polybasic RNRR site 
Blood cells, molecules & diseases  2007;40(1):122-131.
As the principal iron-regulatory hormone, hepcidin plays an important role in systemic iron homeostasis. The regulation of hepcidin expression by iron loading appears to be unexpectedly complex and has attracted much interest. The GPI-linked membrane protein hemojuvelin (GPI-hemojuvelin) is an essential upstream regulator of hepcidin expression. A soluble form of hemojuvelin (s-hemojuvelin) exists in blood and acts as antagonist of GPI-hemojuvelin to downregulate hepcidin expression. The release of s-hemojuvelin is negatively regulated by both transferrin-bound iron (holo-Tf) and non-transferrin bound iron (FAC), indicating s-hemojuvelin could be one of the mediators of hepcidin regulation by iron. In this report, we investigate the proteinase involved in the release of s-hemojuvelin and show that s-hemojuvelin is released by a proprotein convertase through the cleavage at a conserved polybasic RNRR site.
doi:10.1016/j.bcmd.2007.06.023
PMCID: PMC2211380  PMID: 17869549
hepcidin; iron; furin convertase inhibitor; GPI anchor; repulsive guidance molecule C
25.  A Model for Antimicrobial Gene Therapy: Demonstration of Human β-Defensin 2 Antimicrobial Activities In Vivo 
Human gene therapy  2002;13(17):2017-2025.
We transfected host cells with an antimicrobial peptide/protein-encoding gene as a way to enhance host defense mechanisms against infection. The human b-defensin 2 (HBD-2) gene was chosen as a model because its protein does not require cell type-specific processing. Using a retroviral vector carrying HBD-2 cDNA, we treated several mouse or human cell lines and primary cell cultures including fibroblasts, salivary gland cells, endothelial cells, and T cells. All transduced cells produced detectable HBD-2. In Escherichia coli gel overlay experiments, secreted HBD-2 from selected cell lines showed potent antimicrobial activity electrophoretically identical to that of purified HBD-2. We then used a mouse model (nonobese diabetic/severely compromised immunodeficient [NOD/SCID]) to test HBD-2 antimicrobial activities in vivo. HT-1080 cells carrying HBD-2 or control vector were implanted subcutaneously into NOD/SCID mice to allow tumor formation. Escherichia coli was then injected into each tumor mass. Tumors were resected after 16 hr and homogenized for bacterial colony-forming unit analysis. Compared with control tumors, HBD-2-bearing tumors contained only 7.8 6 3.3% viable bacteria. On the basis of this demonstration of HBD-2 in vivo antimicrobial activity, enhancement of antibacterial host defense by HBD-2 gene therapy may be feasible.
doi:10.1089/10430340260395875
PMCID: PMC1790959  PMID: 12489997

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