Anthrancenyl and dansyl fluorophore ligands [AnCH2pipCS(2)K (1), Ds-Hen (2), Ds-HAMP (3), Ds-HAQ (4), and Ds-HAPP (5)] were prepared for coordination to Cu(II). Five Cu complexes, [Cu(AnCH2pipCS2)2] (6), [Cu(Ds-en)2] (7), [Cu(Ds-AMP)2] (8), [Cu(Ds-AQ)2] (9), and [Cu(Ds-APP)(OTf)] (10), were synthesized as candidates for detecting nitric oxide (NO) by fluorescence and characterized by X-ray crystallography. A decrease in fluorescence compared to that of the free ligands (1-5) was measured following the formation of the corresponding five Cu(II) complexes 6-10. Fluorescence was restored in the presence of NO in CH3OH/CH2Cl2 solutions of the compounds. Complexes 7, 8, and 10 exhibited a fluorescence response to NO in pH 7.0 or 9.0 buffered aqueous solutions. Spectroscopic studies revealed that NO-induced fluorescence enhancement in these Cu(II) complexes occurs by reduction to Cu(I). The present studies demonstrate that Cu(II) complexes are effective as fluorescent probes for detecting NO in both organic and aqueous environments.

Extensive investigation into the molecular basis of iron overload disorders has provided new insights into the complexity of iron metabolism and related cellular pathways. The possible involvement of genes affecting iron homeostasis, including HFE, SLC40A1, HAMP and CYBRD1, was investigated in individuals who were referred for confirmation or exclusion of a diagnosis of haemochromatosis, but who tested negative or were heterozygous for the causative HFE mutation, C282Y. Denaturing high performance liquid chromatography analysis of these genes revealed a unique spectrum of mutations in the South African study population, including 67 unrelated patients and 70 population-matched controls. Two novel CYBRD1 gene mutations, R226H and IVS1-4C->>G, were identified in 11% of South African Caucasian patient referrals. We identified a novel D270V mutation in the SLC40A1 gene in a Black South African female with iron overload. These mutations were absent in the control population. In Africans with iron overload not related to the HFE gene, the possible involvement of the SLC40A1 and CYBRD1 genes was demonstrated for the first time. This study confirms the genetic heterogeneity of haemochromatosis and highlights the significance of CYBRD1 mutations in relation to iron overload.

Mutations of the TMPRSS6 gene, which encodes Matriptase-2, are responsible for iron-refractory iron-deficiency anemia. Matriptase-2 is a transmembrane protease that downregulates hepcidin expression. We report one frameshift (p.Ala605ProfsX8) and four novel missense mutations (p.Glu114Lys, p.Leu235Pro, p.Tyr418Cys, p.Pro765Ala) found in IRIDA patients. These mutations lead to changes in both the catalytic and noncatalytic domains of Matriptase-2. Analyses of the mutant proteins revealed a reduction of autoactivating cleavage and the loss of N-Boc-Gln-Ala-Arg-p-nitroanilide hydrolysis. This resulted either from a direct modification of the active site or from the lack of the autocatalytic cleavage that transforms the zymogen into an active protease. In a previously described transfection assay measuring the ability of Matriptase-2 to repress the hepcidin gene (HAMP) promoter, all mutants retained some, if not all, of their transcriptional repression activity. This suggests that caution is called for in interpreting the repression assay in assessing the functional relevance of Matriptase-2 substitutions. We propose that Matriptase-2 activity should be measured directly in the cell medium of transfected cells using the chromogenic substrate. This simple test can be used to determine whether a sequence variation leading to an amino acid substitution is functionally relevant or not.

Hepcidin, a hormone mainly synthesized by hepatocytes and secreted in plasma, controls iron bioavailability. Thus, by inducing the internalization of the iron exporter ferroportin, it regulates iron release from macrophages, enterocytes and hepatocytes towards plasma. Abnormal levels of hepcidin expression alter plasma iron parameters and lead to iron metabolism disorders. Understanding the mechanisms controlling hepcidin (HAMP encodes hepcidin) gene expression is therefore an important goal. We identified a potential GATA-binding site within the human hepcidin promoter. Indeed, in hepatic HepG2 cells, luciferase experiments demonstrated that mutation of this GATA-binding site impaired the hepcidin promoter transcriptional activity in basal conditions. Gel-retardation experiments showed that GATA-4 could bind to this site. Co-transfection of a GATA-4 expression vector with a hepcidin promoter reporter construct enhanced hepcidin promoter transcriptional activity. Furthermore, modulation of GATA4 mRNA expression using specific siRNAs (small interfering RNAs) down-regulated endogenous hepcidin gene expression. Finally, we found that mutation of the GATA-binding site impaired the interleukin-6 induction of hepcidin gene expression, but did not prevent the bone morphogenetic protein-6 response. In conclusion, the findings of the present study (i) indicate that GATA-4 may participate in the control of hepcidin expression, and (ii) suggest that alteration of its expression could contribute to the development of iron-related disorders.

Ammonia is a toxic by-product of amino acid catabolism and a common environmental pollutant that has been associated with increased disease susceptibility in fish although the mechanism is not well understood. We addressed the hypothesis that elevated environmental ammonia acts by impairing the acute phase response (APR). Specifically, we determined the impact of sub-lethal acute (24 h) and chronic (14 d) ammonia exposure on acute phase protein gene expression in zebrafish (Danio rerio) in response to a challenge with bacterial lipopolysaccharide (LPS: i.p. 10 μg/g after 24h). A panel of LPS-responsive genes (SAA, HAMP, LECT2, Hp and IL1β) were identified and evaluated by real-time quantitative PCR. Ammonia was found to impair induction of SAA, HAMP and LECT2 by 50-90%. Both short (15 min, 1h and 24h) and long-term (14 days) exposure to high environmental ammonia concentrations significantly elevated whole-body cortisol levels compared with control fish. Our results reveal for the first time that exposure to high environmental levels of ammonia suppresses the innate immune response in fish. We hypothesize that high environmental ammonia-mediated elevation of cortisol levels in zebrafish may be playing a key role in this immunosuppression, while the mechanisms involved remains to be elucidated.

To develop new treatments for beta-thalassemia, it is essential to identify the genes involved in the relevant pathophysiological processes. Iron metabolism in thalassemia mice being investigated, focusing on the expression of a gene called hepcidin (Hamp), which is expressed in the liver and whose product (Hamp) is secreted into the bloodstream. In mice, iron overload leads to overexpression of Hamp, while Hamp-knockout mice suffer from hemochromatosis. The aim of this study is to investigate Hamp in the mouse model of beta-thalassemia and to address the potential gene transfer of Hamp to prevent abnormal iron absorption.

Immune responses in the testis are regulated in a way that provides protection for the developing male germ cells, while permitting qualitatively normal inflammatory responses and protection against infection. In addition, germ cells are potent targets for the growth factors and cytokines which regulate the reproductive process. Our study analyzes for the first time the pattern of expression of several immune-relevant genes in the gonad of a seasonal breeding teleost fish. The immune molecules analyzed include (i) inflammatory molecules, such as interleukin-1b (il1b), il6, tumor necrosis factor-a (tnfa), cyclooxygenase-2 (cox2) and the NADPH oxidase subunit p40(phox) (ncf4 gene); (ii) the anti-inflammatory cytokine transforming growth factor-b1 (tgfb1) and its type 2 receptor tgfbr2; (iii) innate immune receptors, including toll-like receptor 9 (tlr9), tlr5, tlr22 and macrophage-colony stimulating factor receptor (mcsfr); (iv) lymphocyte receptors, such as the beta subunit of T-cell receptor (Tcrb) and the heavy chain of immunoglobulin M (ighm); (v) the anti-bacterial molecules lysozyme (lyz), hepcidin (hamp) and complement component 3 (c3); (vi) the anti-viral molecule myxovirus (influenza) resistance protein (mx); and (vii) molecules related to leukocyte infiltration, including the CC chemokine ccl4, the CXC chemokine il8 and the leukocyte adhesion molecule E-selectin (Sele). Notably, all of them show a pattern of expression that depends on the reproductive stage of the first two reproductive cycles when the fish develop and function as males. Furthermore, we demonstrate that some of these immune-relevant molecules, such as Il1b and Mcsfr, are produced by germ cells (Il1b) and ovarian and testicular somatic cells (Mcsfr). These data suggest that, as occurs in mammals, there is a critical balance between immune molecules and that these may play an essential role in the orchestration of gametogenesis and the maintenance of gonad tissue homeostasis in fish.

The use of adult stem cells for tissue and organ regeneration constitutes a promising alternative therapy in many human diseases that are currently not treatable. We have isolated a new cell type from mouse adult uterine biopsies (murine adult myometrial precursors or mAMPs) by means of using a simple and non-invasive approach. These cells have been characterized by surface markers, being positive for CD31, CD34, CD44, CD117, Stro-1 and Sca-1. A similar cell population (hAMPs) was isolated from human biopsies. AMPs can differentiate in vitro into a number of mesodermal (smooth and skeletal muscle, osteoblasts and adipocytes) as well as epidermal lineages (all neural lineages). AMPs are unusual adult stem cells as they still express some embryonic antigens and remain undifferentiated through a high number of passages before entering senescence. Importantly, when injected into animal models of muscular disease, AMPs can regenerate new muscle fibers, and promote functional muscular recovery. Moreover, these cells can regenerate the uterine lining after wound healing, reconstructing the uterine muscular architecture. In addition, these cells can form new vessels both in vitro and in vivo. We believe that these cells have superior features to other known adult stem cells and, consequently, their use holds great promise for regenerative medicine, drug development and basic research.

The HAMP domain is present in a large number of transmembrane proteins in prokaryotes including histidine kinases, adenylyl cyclases, chemotaxis receptors, and phosphatases. In this issue of Cell, Hulko et al. (2006) report the NMR structure of a HAMP domain and present data suggesting that it transduces signals through a simple rotation of its four-helix parallel coiled coil.

Hepcidin antimicrobial peptide (<em>HAMP</em>) is a small cysteine-rich peptide and a key molecule of the innate immune system against bacterial infections. Molecular cloning and genomic characterization of <em>HAMP</em> gene in the miiuy croaker (Miichthys miiuy) were reported in this study. The miiuy croaker <em>HAMP</em> was predicted to encode a prepropeptide of 99 amino acids, a tentative RX(K/R)R cleavage motif and eight characteristic cysteine residues were also identified. The gene organization is also similar to corresponding genes in mammals and fish consisting of three exons and two introns. Sequence polymorphism analysis showed that only two different sequences were identified and encoded two proteins in six individuals. As reported for most other species, the expression level was highest in liver and an up-regulation of transcription was seen in spleen, intestine and kidney examined at 24 h after injection of pathogenic bacteria, Vibrio anguillarum, the expression pattern implied that miiuy croaker <em>HAMP</em> is an important component of the first line defense against invading pathogens. In addition, we report on the underlying mechanism that maintains sequences diversity among fish and mammalian species, respectively. A series of site-model tests implemented in the CODEML program revealed that moderate positive Darwinian selection is likely to cause the molecular evolution in the fish <em>HAMP</em>2 genes and it also showed that the fish <em>HAMP</em>1 genes and <em>HAMP</em>2 genes under different selection pressures.

BACKGROUND

New genetic forms of hereditary hemochromatosis (HH) or hereditary hyperferritinemia (HF) have been identified over the last few years, and abnormalities of various genes may interact in a single patient. This study aimed to develop a rapid automated method for sequencing the main genes involved.

METHODS

We used a standard 96-well microplate with a single PCR condition in an adaptation of the SCAIP (single-condition amplification with internal primer) method to sequence the HFE (hemochromatosis), HAMP (hepcidin antimicrobial peptide), HFE2/HJV [hemochromatosis type 2 (juvenile)], SLC40A1 (ferroportin), and TFR2 (transferrin receptor 2) genes, and the 5' untranslated region of the FTL (ferritin, light polypeptide) gene. To further simplify the method, we adjusted PCR conditions to avoid the use of an internal primer and applied this single-condition amplification method to 38 selected, unrelated patients. We tailored the genetic investigation according to the clinical picture, with the patients falling into 2 groups. Group 1 consisted of patients with hyperferritinemia and high transferrin saturation (TS) (classic adult and juvenile HH forms, groups 1A and 1B, respectively), and group 2 consisted of patients with hyperferritinemia and low, typical, or slightly increased TS, with or without iron overload (groups 2A and 2B, respectively).

RESULTS

With this strategy we identified single-gene and multigene abnormalities, including 6 previously undescribed abnormalities in HFE (c.794dupA), HFE2 (c.-89-4dupT), and SLC40A1 (c.262A>G, c.533G>A, c.1468G>A, and c.-59_-45del).

CONCLUSIONS

This method is a simple approach for investigating hereditary iron overload or HF and allows rapid evaluation of patients.

Multiple myeloma (MM) is a malignant disease of plasma cells and is often accompanied by anemia which may influence its progression and survival. The mechanism of anemia of chronic disease (ACD) in which iron homeostasis is impaired underlies that of MM-related anemia. In this study, we analyzed the role of hepcidin which is the main mediator of ACD and ACD-related cytokines in peripheral blood of MM patients. We showed that HAMP mRNA and growth differentiation factors 15 (GDF15) mRNA expressions in peripheral blood mononuclear cells (PBMCs) and plasma hepcidin, GDF15, interleukin-6 and erythropoietin in MM patients all increased significantly as compared to those in controls. In MM patients, the expression of HAMP mRNA showed a positive correlation with serum ferritin level, and a negative correlation with hemoglobin level. The levels of plasma hepcidin and GDF15 were significantly decreased in MM patients who achieved complete remission after six cycles VD (bortezomib + dexamethasone) regimen chemotherapy. These data indicated that overexpression of HAMP mRNA in PBMCs significantly correlated with increased plasma hepcidin level and may be involved in the pathogenesis of MM-related anemia. Furthermore, the levels of plasma hepcidin and GDF15 may be valuable in assessing the progress of MM.

The transmembrane Tsr protein of Escherichia coli mediates chemotactic responses to environmental serine gradients. Serine binds to the periplasmic domain of the homodimeric Tsr molecule, promoting a small inward displacement of one transmembrane helix (TM2). TM2 piston displacements, in turn, modulate the structural stability of the Tsr-HAMP domain on the cytoplasmic side of the membrane to control the autophosphorylation activity of the signaling CheA kinase bound to the membrane-distal cytoplasmic tip of Tsr. A five-residue control cable segment connects TM2 to the AS1 helix of HAMP and transmits stimulus and sensory adaptation signals between them. To explore the possible role of control cable helicity in transmembrane signaling by Tsr, we characterized the signaling properties of mutant receptors with various control cable alterations. An all-alanine control cable shifted Tsr output toward the kinase-on state, whereas an all-glycine control cable prevented Tsr from reaching either a fully on or fully off output state. Restoration of the native isoleucine (I214) in these synthetic control cables largely alleviated their signaling defects. Single amino acid replacements at Tsr-I214 shifted output toward the kinase-off (L, N, H, and R) or kinase-on (A and G) states, whereas other control cable residues tolerated most amino acid replacements with little change in signaling behavior. These findings indicate that changes in control cable helicity might mediate transitions between the kinase-on and kinase-off states during transmembrane signaling by chemoreceptors. Moreover, the Tsr-I214 side chain plays a key role, possibly through interaction with the membrane interfacial environment, in triggering signaling changes in response to TM2 piston displacements.

OBJECTIVE

The Tsr protein of E. coli mediates chemotactic responses to environmental serine gradients. Stimulus signals from the Tsr periplasmic sensing domain reach its cytoplasmic kinase control domain through piston displacements of a membrane-spanning helix and an adjoining five-residue control cable segment. We characterized the signaling properties of Tsr variants to elucidate the transmembrane signaling role of the control cable, an element present in many microbial sensory proteins. Both the kinase-on and kinase-off output states of Tsr depended on control cable helicity, but only one residue, I214, was critical for triggering responses to attractant inputs. These findings suggest that signal transmission in Tsr involves modulation of control cable helicity through interaction of the I214 side chain with the cytoplasmic membrane.

Transmembrane receptors are integral components of sensory pathways in prokaryotes. These receptors share a common dimeric architecture, consisting in its basic form of an N-terminal extracellular sensor, transmembrane helices, and an intracellular effector. As an exception, we have identified an archaeal receptor family--exemplified by Af1503 from Archaeoglobus fulgidus--that is C-terminally shortened, lacking a recognizable effector module. Instead, a HAMP domain forms the sole extension for signal transduction in the cytosol. Here, we examine the gene environment of Af1503-like receptors and find a frequent association with transmembrane transport proteins. Furthermore, we identify and define a closely associated new protein domain family, which we characterize structurally using Af1502 from A. fulgidus. Members of this family are found both as stand-alone proteins and as domains within extant receptors. In general, the latter appear as connectors between the solute carrier 5 (SLC5)-like transmembrane domains and two-component signal transduction (TCST) domains. This is seen, for example, in the histidine kinase CbrA, which is a global regulator of metabolism, virulence, and antibiotic resistance in Pseudomonads. We propose that this newly identified domain family mediates signal transduction in systems regulating transport processes and name it STAC, for SLC and TCST-Associated Component.

Our aim was to contribute to a better understanding of the pathophysiology of anemia in elderly, by studying how aging affects renal function, iron metabolism, erythropoiesis and the inflammatory response, using an experimental animal model. The study was performed in male Wistar, a group of young rats with 2 months age and an old one with 18 months age. Old rats presented a significant higher urea, creatinine, interferon (INF)-gamma, ferritin and soluble transferrin receptor serum levels, as well as increased counts of reticulocytes and RDW. In addition, these rats showed significant lower erythropoietin (EPO) and iron serum levels. Concerning gene expression of iron regulatory proteins, old rats presented significantly higher mRNA levels of hepcidin (Hamp), transferrin (TF), transferrin receptor 2 (TfR2) and hemojuvelin (HJV); divalent metal transporter 1 (DMT1) mRNA levels were significantly higher in duodenal tissue; EPO gene expression was significantly higher in liver and lower in kidney, and the expression of the EPOR was significantly higher in both liver and kidney. Our results showed that aging is associated with impaired renal function, which could be in turn related with the inflammatory process and with a decline in EPO renal production. Moreover, we also propose that aging may be associated with INF-gamma-induced inflammation and with alterations upon iron regulatory proteins gene expression.

Objective- Inflammatory stimuli enhance the progression of atherosclerotic disease. Inflammation also increases the expression of hepcidin, a hormonal regulator of iron homeostasis, which decreases intestinal iron absorption, reduces serum iron levels and traps iron within macrophages. The role of macrophage iron in the development of atherosclerosis remains incompletely understood. The objective of this study was to investigate the effects of hepcidin deficiency and decreased macrophage iron on the development of atherosclerosis. Approach and Results- Hepcidin- and LDL (low-density lipoprotein) receptor-deficient ( Hamp^-/-/ Ldlr^-/-) mice and Hamp^+/+/ Ldlr^-/- control mice were fed a high-fat diet for 21 weeks. Compared with control mice, Hamp^-/-/ Ldlr^-/- mice had decreased aortic macrophage activity and atherosclerosis. Because hepcidin deficiency is associated with both increased serum iron and decreased macrophage iron, the possibility that increased serum iron was responsible for decreased atherosclerosis in Hamp^-/-/ Ldlr^-/- mice was considered. Hamp^+/+/ Ldlr^-/- mice were treated with iron dextran so as to produce a 2-fold increase in serum iron. Increased serum iron did not decrease atherosclerosis in Hamp^+/+/ Ldlr^-/- mice. Aortic macrophages from Hamp^-/-/ Ldlr^-/- mice had less labile free iron and exhibited a reduced proinflammatory (M1) phenotype compared with macrophages from Hamp^+/+/ Ldlr^-/- mice. THP1 human macrophages treated with an iron chelator were used to model hepcidin deficiency in vitro. Treatment with an iron chelator reduced LPS (lipopolysaccharide)-induced M1 phenotypic expression and decreased uptake of oxidized LDL. Conclusions- In summary, in a hyperlipidemic mouse model, hepcidin deficiency was associated with decreased macrophage iron, a reduced aortic macrophage inflammatory phenotype and protection from atherosclerosis. The results indicate that decreasing hepcidin activity, with the resulting decrease in macrophage iron, may prove to be a novel strategy for the treatment of atherosclerosis.

Phenylpyrroles are chemical analogs of the natural antifungal compound pyrrolnitrin. Fenpiclonil, but mainly fludioxonil are registered against multiple fungal crop diseases since over 25 years for seed or foliar treatment. They have severe physiological impacts on the pathogen, including membrane hyperpolarization, changes in carbon metabolism and the accumulation of metabolites leading to hyphal swelling and burst. The selection and characterization of mutants resistant to phenylpyrroles have revealed that these fungicides activate the fungal osmotic signal transduction pathway through their perception by a typical fungal hybrid histidine kinase (HHK). The HHK is prone to point mutations that confer fungicide resistance and affect its sensor domain, composed of tandem repeats of HAMP motifs. Fludioxonil resistant mutants have been selected in many fungal species under laboratory conditions. Generally they present severe impacts on fitness parameters. Since only few cases of field resistance specific to phenylpyrroles have been reported one may suspect that the fitness penalty of phenylpyrrole resistance is the reason for the lack of field resistance.

Iron is an essential metal for cell survival that is regulated by the peptide hormone hepcidin. However, its influence on certain diseases is directly related to iron metabolism or secondary to underlying diseases. Genetic alterations influence the serum hepcidin concentration, which can lead to an iron overload in tissues, as observed in haemochromatosis, in which serum hepcidin or defective hepcidin synthesis is observed. Another genetic imbalance of iron is iron-refractory anaemia, in which serum concentrations of hepcidin are increased, precluding the flow and efflux of extra- and intracellular iron. During the pathogenesis of certain diseases, the resulting oxidative stress, as well as the increase in inflammatory cytokines, influences the transcription of the HAMP gene to generate a secondary anaemia due to the increase in the serum concentration of hepcidin. To date, there is no available drug to inhibit or enhance hepcidin transcription, mostly due to the cytotoxicity described in the in vitro models. The proposed therapeutic targets are still in the early stages of clinical trials. Some candidates are promising, such as heparin derivatives and minihepcidins. This review describes the main pathways of systemic and genetic regulation of hepcidin, as well as its influence on the disorders related to iron metabolism.

Emerging evidence has demonstrated that chronic ethanol exposure induces iron overload, enhancing ethanol-mediated liver damage. The purpose of this study was to explore the effects of the naturally occurring compound quercetin on ethanol-induced iron overload and liver damage, focusing on the signaling pathway of the iron regulatory hormone hepcidin. Adult male C57BL/6J mice were pair-fed with isocaloric-Lieber De Carli diets containing ethanol (accounting for 30% of total calories) and/or carbonyl iron (0.2%) and treated with quecertin (100 mg/kg body weight) for 15 weeks. Mouse primary hepatocytes were incubated with ethanol (100 mM) and quercetin (100 μM) for 24 h. Mice exposed to either ethanol or iron presented significant fatty infiltration and iron deposition in the liver; these symptoms were exacerbated in mice cotreated with ethanol and iron. Quercetin attenuated the abnormity induced by ethanol and/or iron. Ethanol suppressed BMP6 and intranuclear SMAD4 as well as decreased hepcidin expression. These effects were partially alleviated by quercetin supplementation in mice and hepatocytes. Importantly, ethanol caused suppression of SMAD4 binding to the HAMP promoter and of hepcidin messenger RNA expression. These effects were exacerbated by anti-BMP6 antibody and partially alleviated by quercetin or human recombinant BMP6 in cultured hepatocytes. In contrast, co-treatment with iron and ethanol, especially exposure of iron alone, activated BMP6/SMAD4 pathway and up-regulated hepcidin expression, which was also normalized by quercetin in vivo. Quercetin prevented ethanol-induced hepatic iron overload different from what carbonyl iron diet elicited in the mechanism, by regulating hepcidin expression via the BMP6/SMAD4 signaling pathway.

Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. The expression of the hepcidin gene, HAMP, is increased in patients with anemia of chronic disease. Previously, the synthetic compound K7174 was identified through chemical screening as a novel inhibitor of the adhesion of monocytes to cytokine-stimulated endothelial cells. K7174 also ameliorated anemia induced by inflammatory cytokines in mice, which suggests a possible involvement of hepcidin regulation. The present study was performed to assess the impact of K7174 on hepcidin expression in a human hematoma cell line and in mice in vivo. We first demonstrated that K7174 treatment in HepG2 cells significantly decreased HAMP expression. Then, we conducted microarray analysis to determine the molecular mechanism by which K7174 inhibits HAMP expression. Transcriptional profiling confirmed the downregulation of HAMP. Surprisingly, we found that K7174 strongly induced GDF15, known as a negative regulator of HAMP expression. Western blotting analysis as well as ELISA confirmed the induction of GDF15 by K7174 treatment. Furthermore, K7174-mediated HAMP suppression was rescued by the silencing of GDF15 expression. Interestingly, we found that K7174 also upregulates CEBPB. Promoter analysis and chromatin immunoprecipitation analysis revealed that CEBPB could contribute to K7174-mediated transcriptional activation of GDF15. Subsequently, we also examined whether K7174 inhibits hepcidin expression in mice. Quantitative RT-PCR analysis with liver samples from K7174-treated mice demonstrated significant upregulation of Gdf15 and downregulation of Hamp expression, as compared to control mice. Furthermore, serum hepcidin concentration was also significantly decreased in K7174-treated mice. In conclusion, K7174 inhibits hepcidin expression partly by inducing GDF15. K-7174 may be a potential therapeutic option to treat anemia of chronic disease.

BACKGROUND

The peptide hepcidin plays a central role in regulating dietary iron absorption and body iron distribution. This 25-amino acid hormone is produced and secreted predominantly by hepatocytes. Hepcidin has been suggested as a promising diagnostic marker for iron-related disorders. However, its accurate quantification for clinical use remains so far challenging. In this report we describe a highly specific and quantitative serum hepcidin method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).

METHODS

The analytical validation included the determination of the limit of detection, of quantification, repeatability, reproducibility and linearity. This assay was developed for human and mouse hepcidin. The human assay was performed on serum patients with unexplained microcytic anemia. We applied our LC-MS/MS method for quantifying hepcidin-1 in mouse in various conditions: inflammation, hemolytic anemia, Hamp-1, Hjv and Hfe KO mice.

RESULTS

We show that the LC-MS/MS is suitable for accurate determination of hepcidin-25 in clinical samples, thereby representing a useful tool for the clinical diagnosis and follow-up of iron-related diseases. In mouse, a strong correlation between hepatic Hamp-1 mRNA expression and serum hepcidin-1 levels was found (r=0.88; p=0.0002) and the expected variations in mouse models of iron disorders were observed.

CONCLUSIONS

Therefore, we propose this adaptive LC-MS/MS method as a suitable method for accurate determination of hepcidin-25 in clinical samples and as a major tool contributing to the clinical diagnosis, follow-up and management of iron-related disorders. It also opens new avenues to measure hepcidin in animal models without interspecies antigenic limitations.

Herbivory is fundamental to the regulation of both global food webs and the extent of agricultural crop losses. Induced plant responses to herbivores promote resistance and often involve the perception of specific herbivore-associated molecular patterns (HAMPs); however, precisely defined receptors and elicitors associated with herbivore recognition remain elusive. Here, we show that a receptor confers signaling and defense outputs in response to a defined HAMP common in caterpillar oral secretions (OS). Staple food crops, including cowpea (Vigna unguiculata) and common bean (Phaseolus vulgaris), specifically respond to OS via recognition of proteolytic fragments of chloroplastic ATP synthase, termed inceptins. Using forward-genetic mapping of inceptin-induced plant responses, we identified a corresponding leucine-rich repeat receptor, termed INR, specific to select legume species and sufficient to confer inceptin-induced responses and enhanced defense against armyworms (Spodoptera exigua) in tobacco. Our results support the role of plant immune receptors in the perception of chewing herbivores and defense.

Keywords: HAMP; LRR-RLP; PRR; herbivory; receptor.

Dehydrated hereditary stomatocytosis (DHS), or xerocytosis, is an autosomal dominant hemolytic anemia. Most patients with DHS carry mutations in the PIEZO1 gene encoding a mechanosensitive cation channel. We here demonstrate that patients with DHS have low levels of hepcidin and only a slight increase of ERFE, the erythroid negative regulator of hepcidin. We demonstrated that at the physiological level, PIEZO1 activation induced Ca²⁺ influx and suppression of HAMP expression in primary hepatocytes. In two hepatic cellular models expressing PIEZO1 WT and two PIEZO1 gain-of-function mutants (R2456H and R2488Q), we highlight altered expression of a few genes/proteins involved in iron metabolism. Mutant cells showed increased intracellular Ca²⁺ compared to WT, which was correlated to increased phosphorylation of ERK1/2, inhibition of the BMP-SMADs pathway, and suppression of HAMP transcription. Moreover, the HuH7 cells, treated with PD0325901, a potent inhibitor of ERK1/2 phosphorylation, reduced the phosphorylation of ERK1/2 with the consequent increased phosphorylation of SMAD1/5/8, confirming the link between the two pathways. Another 'proof of concept' for the mechanism that links PIEZO1 to HAMP regulation was obtained by mimicking PIEZO1 activation by cell Ca²⁺ overload, by the Ca²⁺ ionophore A23187. There was strong down-regulation of HAMP gene expression after this Ca²⁺ overload. Finally, the inhibition of PIEZO1 by GsMTx4 leads to phenotype rescue. This is the first demonstration of a direct link between PIEZO1 and iron metabolism, which defines the channel as a new hepatic iron metabolism regulator and as a possible therapeutic target of iron overload in DHS and other iron-loading anemias. This article is protected by copyright. All rights reserved.

Hereditary iron overload includes several disorders characterized by iron accumulation in tissues, organs, or even single cells or subcellular compartments. They are determined by mutations in genes directly involved in hepcidin regulation, cellular iron uptake, management and export, iron transport and storage. Systemic forms are characterized by increased serum ferritin with or without high transferrin saturation, and with or without functional iron deficient anemia. Hemochromatosis includes five different genetic forms all characterized by high transferrin saturation and serum ferritin, but with different penetrance and expression. Mutations in HFE, HFE2, HAMP and TFR2 lead to inadequate or severely reduced hepcidin synthesis that, in turn, induces increased intestinal iron absorption and macrophage iron release leading to tissue iron overload. The severity of hepcidin down-regulation defines the severity of iron overload and clinical complications. Hemochromatosis type 4 is caused by dominant gain-of-function mutations of ferroportin preventing hepcidin-ferroportin binding and leading to hepcidin resistance. Ferroportin disease is due to loss-of-function mutation of SLC40A1 that impairs the iron export efficiency of ferroportin, causes iron retention in reticuloendothelial cell and hyperferritinemia with normal transferrin saturation. Aceruloplasminemia is caused by defective iron release from storage and lead to mild microcytic anemia, low serum iron, and iron retention in several organs including the brain, causing severe neurological manifestations. Atransferrinemia and DMT1 deficiency are characterized by iron deficient erythropoiesis, severe microcytic anemia with high transferrin saturation and parenchymal iron overload due to secondary hepcidin suppression. Diagnosis of the different forms of hereditary iron overload disorders involves a sequential strategy that combines clinical, imaging, biochemical, and genetic data. Management of iron overload relies on two main therapies: blood removal and iron chelators. Specific therapeutic options are indicated in patients with atransferrinemia, DMT1 deficiency and aceruloplasminemia.