Modulate Hepcidin Expression Research Articles

Lactate as a regulator of iron homeostasis

Abstract The key metabolic intermediate lactate can increase expression of the liver-derived peptide hepcidin, the central regulator of body iron homeostasis. A new paper by Liu et al. shows that lactate achieves this by binding to and activating soluble adenylyl cyclase, thereby increasing cellular cyclic adenosine monophosphate (cAMP) (cAMP) and enhancing signaling through the bone morphogenetic protein (BMP) pathway to modulate hepcidin expression.

Lobe specificity of iron binding to transferrin modulates murine erythropoiesis and iron homeostasis

AbstractTransferrin, the major plasma iron-binding molecule, interacts with cell-surface receptors to deliver iron, modulates hepcidin expression, and regulates erythropoiesis. Transferrin binds and releases iron via either or both of 2 homologous lobes (N and C). To test the hypothesis that the specificity of iron occupancy in the N vs C lobe influences transferrin function, we generated mice with mutations to abrogate iron binding in either lobe (TfN-bl or TfC-bl). Mice homozygous for either mutation had hepatocellular iron loading and decreased liver hepcidin expression (relative to iron concentration), although to different magnitudes. Both mouse models demonstrated some aspects of iron-restricted erythropoiesis, including increased zinc protoporphyrin levels, decreased hemoglobin levels, and microcytosis. Moreover, the TfN-bl/N-bl mice demonstrated the anticipated effect of iron restriction on red cell production (ie, no increase in red blood cell [RBC] count despite elevated erythropoietin levels), along with a poor response to exogenous erythropoietin. In contrast, the TfC-bl/C-bl mice had elevated RBC counts and an exaggerated response to exogenous erythropoietin sufficient to ameliorate the anemia. Observations in heterozygous mice further support a role for relative N vs C lobe iron occupancy in transferrin-mediated regulation of iron homeostasis and erythropoiesis.

The Iron Metabolism Receptors, TfR1 and TfR2, Bind and Stabilize Transferrin Through Non‐Conserved Interactions

Hereditary hemochromatosis, a disease marked by chronic iron overload from insufficient expression of the hormone hepcidin, is one of the most common genetic diseases in people of Northern European ancestry. One form of HH (Type III) results from mutations in the transferrin receptor‐2 (TfR2). How TfR2 functions to promote hepcidin expression remains unclear, though it appears to be an important part of a mechanism to modulate hepcidin expression based on systemic iron levels. This responsiveness may be achieved through TfR2's competition with its homolog, TfR1, for their ligand, the iron transport protein, transferrin (Tf). TfR1 is expected to be the preferred receptor for diferric, or holo‐, Tf and that TfR2 will bind holo‐Tf when Tf‐iron saturation levels are elevated. To better understand the nature of these protein‐protein interactions, a surface plasmon resonance binding study with the full‐length receptors was conducted for the first time. In agreement with previous studies, holo‐Tf binds to TfR1 significantly stronger than TfR2. However, the binding constant for Tf‐TfR2 is still far below that of physiological holo‐Tf levels, inconsistent with the hypothetical model and suggests that other factors mediate the interaction. Apo‐Tf only weakly binds TfR2 at serum pH, thus is not be able to effectively compete with holo‐Tf under these conditions. Like TfR1, TfR2 is able to bind apo‐Tf with higher affinity at the lower pH of endosomes, suggesting that it could function to take up iron, where stabilization of the apo‐Tf‐TfR complex is key. Tf‐binding Tf‐binding to a TfR2 chimera containing the TfR1‐helical domain indicates that the differences in the helical domain account for differences in on‐rate of Tf, but non‐conserved inter‐receptor interactions are necessary stabilize the Tf‐TfR1/TfR2 chimera complex. One possible site of stabilization, the apical arm junction, is not important for TfR1‐Tf complex stabilization. The junction is critical for the TfR2‐Tf interaction, however; highlighting one non‐conserved stabilization mechanism. Our results highlight the differences in Tf interactions with the two TfRs.Support or Funding InformationNIH T‐32 Training Grant GMMBI0231C; NIH R01DK072166This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transferrin Receptors TfR1 and TfR2 Bind Transferrin through Differing Mechanisms.

Hereditary hemochromatosis (HH), a disease marked by chronic iron overload from insufficient expression of the hormone hepcidin, is one of the most common genetic diseases. One form of HH (type III) results from mutations in transferrin receptor-2 (TfR2). TfR2 is postulated to be a part of signaling system that is capable of modulating hepcidin expression. However, the molecular details of TfR2's role in this system remain unclear. TfR2 is predicted to bind the iron carrier transferrin (Tf) when the iron saturation of Tf is high. To better understand the nature of these TfR-Tf interactions, a binding study with the full-length receptors was conducted. In agreement with previous studies with truncated forms of these receptors, holo-Tf binds to the TfR1 homologue significantly stronger than to TfR2. However, the binding constant for Tf-TfR2 is still far above that of physiological holo-Tf levels, inconsistent with the hypothetical model, suggesting that other factors mediate the interaction. One possible factor, apo-Tf, only weakly binds TfR2 at serum pH and thus will not be able to effectively compete with holo-Tf. Tf binding to a TfR2 chimera containing the TfR1 helical domain indicates that the differences in the helical domain account for differences in the on rate of Tf, and nonconserved inter-receptor interactions are necessary for the stabilization of the complex. Conserved residues at one possible site of stabilization, the apical arm junction, are not important for TfR1-Tf binding but are critical for the TfR2-Tf interaction. Our results highlight the differences in Tf interactions with the two TfRs.

Low Serum Retinol Concentration Explain Higher Hepcidin Levels In Mexican Older Adults

BackgroundElevated hepcidin levels have been linked to anemia of inflammation (AI). Retinol deficiency has shown to modulate hepcidin expression in animals; however, no studies have explored low serum retinol (LSR) concentration in older adults (OA) and their possible association with hepcidin concentrations. Anemia affect 27% of Mexican OA and inflammation may play a role in the production of anemia in this population. Understanding the mechanisms that play a role in the control hepcidin secretion is an important goal in the prevention of AI.AimTo explore if low serum retinol is associated with higher serum hepcidin concentrations in Mexican older adults (≥60years).MethodsMexican OA were participants in a crossectional study to identify the main causes of anemia. They were recruited in August 2015. Fasting serum samples were collected in 535 OA. Serum retinol (SR) was measured by HPLC and LSR was defined if SR<20μg/dL. Hepcidin (ng/mL) was measured using a commercial ELISA kit. Due to skewed distribution of hepcidin, a quantil regression model was used to associate the LSR and hepcidin, adjusting by sex, age, BMI, ferritin, transferrin receptor, vitamin B12 status, IL‐6, C reactive protein, Alpha‐1‐acid glycoprotein, diabetes, hypertension, tertil of socioeconomic status, sarcopenia and functional disability.ResultsMean age was 71.5y for male and 70.7y for female. LSR was present in 3.2% of OA. Mexican OA with low serum retinol had 12.2 ng/mL higher hepcidin levels than OA with normal serum retinol levels (p=0.03). Hepcidin adjusted median (by confounders) were (median±EE): 19.4± 1.0 ng/mL in OA with normal serum retinol and 31.9±5.7 ng/mL in OA with LSR. Sarcopenic OA had higher hepcidin levels (coef: 7.5 ng/mL, p=0.03) than OA non‐sarcopenic.ConclusionsLSR levels in OA explain their higher hepcidin concentrations. Retinol concentrations affects iron homeostasis but the mechanism is unclear. This data document for the first time that in humans, low serum retinol concentration is inversely associated with hepcidin concentrations. Further longitudinal studies are needed to understand the role of serum hepcidin and Retinol in the development of AI in OA.Support or Funding InformationCONACYT‐FONSEC SSA/IMSS/ISSSTE SALUD 2014‐1, S0008‐2014‐1‐000000000234157

Hepcidin: A Promising Therapeutic Target for Iron Disorders: A Systematic Review.

Iron is required for most forms of organisms, and it is the most essential element for the functions of many iron-containing proteins involved in oxygen transport, cellular respiration, DNA replication, and so on. Disorders of iron metabolism are associated with diverse diseases, including anemias (e.g., iron-deficiency anemia and anemia of chronic diseases) and iron overload diseases, such as hereditary hemochromatosis and β-thalassemia. Hepcidin (encoded by Hamp gene) is a peptide hormone synthesized by hepatocytes, and it plays an important role in regulating the systematic iron homeostasis. As the systemic iron regulator, hepcidin, not only controls dietary iron absorption and iron egress out of iron storage cells, but also induces iron redistribution in various organs. Deregulated hepcidin is often seen in a variety of iron-related diseases including anemias and iron overload disorders. In the case of iron overload disorders (e.g., hereditary hemochromatosis and β-thalassemia), hepatic hepcidin concentration is significantly reduced.Since hepcidin deregulation is responsible for iron disorder-associated diseases, the purpose of this review is to summarize the recent findings on therapeutics targeting hepcidin.Continuous efforts have been made to search for hepcidin mimics and chemical compounds that could be used to increase hepcidin level. Here, a literature search was conducted in PubMed, and research papers relevant to hepcidin regulation or hepcidin-centered therapeutic work were reviewed. On the basis of literature search, we recapitulated recent findings on therapeutic studies targeting hepcidin, including agonists and antagonists to modulate hepcidin expression or its downstream signaling. We also discussed the molecular mechanisms by which hepcidin level and iron metabolism are modulated.Elevating hepcidin concentration is an optimal strategy to ameliorate iron overload diseases, and also to relieve β-thalassemia phenotypes by improving ineffective erythropoiesis. Relative to the current conventional therapies, such as phlebotomy and blood transfusion, therapeutics targeting hepcidin would open a new avenue for treatment of iron-related diseases.

Homocysteine upregulates hepcidin expression through BMP6/SMAD signaling pathway in hepatocytes

Subjects with severe hyperhomocysteinemia have hypoferric anemia and excessive iron deposition in the liver. Hepcidin, the central regulator of iron homeostasis, plays a key role in iron metabolism. However, the regulation of homocysteine (Hcy) on hepcidin is largely unclear. We conducted experiments in HepG2 cells to identify the mechanisms with which Hcy modulates hepcidin expression. We found that treatment with Hcy dose-dependently increased both hepcidin transcript levels and protein levels, as assessed by quantitative real-time reverse-transcriptase polymerase chain reaction and western blotting, respectively. Hcy also activated BMP6 signaling and increased the phosphorylation of SMAD1/5/8 in HepG2 cells. We found that Hcy's effect on hepcidin expression was impaired by the knockdown of BMP6 and its receptors ALK2/3/6 with siRNAs. These results demonstrated that Hcy up-regulated hepcidin expression through the BMP6/SMAD pathway, suggesting a novel mechanism underlying the hyperhomocysteinemia-associated perturbation of iron homeostasis.

Phytoestrogens modulate hepcidin expression by Nrf2: Implications for dietary control of iron absorption

Hepcidin is a liver-derived antimicrobial peptide that regulates iron absorption and is also an integral part of the acute phase response. In a previous report, we found evidence that this peptide could also be induced by toxic heavy metals and xenobiotics, thus broadening its teleological role as a defensin. However it remained unclear how its sensing of disparate biotic and abiotic stressors might be integrated at the transcriptional level. We hypothesized that its function in cytoprotection may be regulated by NFE2-related factor 2 (Nrf2), the master transcriptional controller of cellular stress defenses. In this report, we show that hepcidin regulation is inextricably linked to the acute stress response through Nrf2 signaling. Nrf2 regulates hepcidin expression from a prototypical antioxidant response element in its promoter, and by synergizing with other basic leucine-zipper transcription factors. We also show that polyphenolic small molecules or phytoestrogens commonly found in fruits and vegetables including the red wine constituent resveratrol can induce hepcidin expression in vitro and post-prandially, with concomitant reductions in circulating iron levels and transferrin saturation by one such polyphenol quercetin. Furthermore, these molecules derepress hepcidin promoter activity when its transcription by Nrf2 is repressed by Keap1. Taken together, the data show that hepcidin is a prototypical antioxidant response or cytoprotective gene within the Nrf2 transcriptional circuitry. The ability of phytoestrogens to modulate hepcidin expression in vivo suggests a novel mechanism by which diet may impact iron homeostasis.

Endofin, a novel BMP-SMAD regulator of the iron-regulatory hormone, hepcidin

BMP-SMAD signalling plays a crucial role in numerous biological processes including embryonic development and iron homeostasis. Dysregulation of the iron-regulatory hormone hepcidin is associated with many clinical iron-related disorders. We hypothesised that molecules which mediate BMP-SMAD signalling play important roles in the regulation of iron homeostasis and variants in these proteins may be potential genetic modifiers of iron-related diseases. We examined the role of endofin, a SMAD anchor, and show that knockdown of endofin in liver cells inhibits basal and BMP-induced hepcidin expression along with other BMP-regulated genes, ID1 and SMAD7. We show for the first time, the in situ interaction of endofin with SMAD proteins and significantly reduced SMAD phosphorylation with endofin knockdown, suggesting that endofin modulates hepcidin through BMP-SMAD signalling. Characterisation of naturally occurring SNPs show that mutations in the conserved FYVE domain result in mislocalisation of endofin, potentially affecting downstream signalling and modulating hepcidin expression. In conclusion, we have identified a hitherto unrecognised link, endofin, between the BMP-SMAD signalling pathway, and the regulation of hepcidin expression and iron homeostasis. This study further defines the molecular network involved in iron regulation and provides potential targets for the treatment of iron-related disorders.

A HAMP promoter bioassay system for identifying chemical compounds that modulate hepcidin expression

Hepcidin is the central regulator of systemic iron homeostasis; dysregulation of hepcidin expression causes various iron metabolic disorders, including hereditary hemochromatosis and anemia of inflammation. To identify molecules that modulate hepcidin expression, we developed a bioassay system for hepcidin gene (HAMP) promoter activity by stable transfection of Hep3B hepatoma cells with an expression plasmid in which EGFP was linked to a 2.5-kb human HAMP promoter. Interleukin 6, bone morphogenetic protein 6 (BMP-6), and oncostatin M, well-characterized stimulators of the HAMP promoter, strongly enhanced the green fluorescence intensity of these cells. Dorsomorphin, heparin, and cobalt chloride, known inhibitors of hepcidin expression, significantly suppressed green fluorescence intensity, and these inhibitory effects were more prominent when the cells were stimulated with BMP-6. Employing this system, we screened 1,280 biologically active small molecules and found several candidate inhibitors of hepcidin expression. Apomorphine, benzamil, etoposide, CGS-15943, kenpaullone, and rutaecarpine (all at 10μmol/L) significantly inhibited hepcidin mRNA expression by Hep3B cells without affecting cell viability. CGS-15943 was the strongest suppressor of BMP-6-induced hepcidin-25 secretion in these cells. We conclude that our newly developed hepcidin promoter bioassay system is useful for identifying and evaluating compounds that modulate hepcidin expression.

Cell surface serine protease matriptase-2 suppresses fetuin-A/AHSG-mediated induction of hepcidin.

Matriptase-2 is a type II transmembrane serine protease controlling the expression of hepcidin, the key regulator of iron homeostasis. By cleaving hemojuvelin, matriptase-2 suppresses bone morphogenetic protein/sons of mothers against decapentaplegic signaling. So far, the only known putative substrates of matriptase-2 are hemojuvelin and matriptase-2 itself. In this study, fetuin-A (α2-Heremans-Schmid glycoprotein) was identified in vitro as a substrate of matriptase-2. The protease-substrate interaction was validated by isolating matriptase-2 via the affinity to fetuin-A. Fetuin-A is a liver-derived plasma protein with multiple functions, which is proteolytically processed to yield a disulfide-linked two-chain form. In co-transfected cells, a matriptase-2-dependent conversion of unprocessed fetuin-A into a two-chain form was detected. Conversely, downregulation of endogenously expressed matriptase-2 stabilized fetuin-A. Arg and Lys residues located within the 40 residue spanning connecting peptide of fetuin-A were identified as cleavage sites for matriptase-2. Analysis of hepcidin expression revealed an inductive effect of fetuin-A, which was abolished by matriptase-2. Fetuin-A deficiency in mice resulted in decreased hepcidin mRNA levels. These findings implicate a role of fetuin-A in iron homeostasis and provide new insights into the mechanism of how matriptase-2 might modulate hepcidin expression.

A chemical screen identifies small molecules that regulate hepcidin expression.

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Bone morphogenic protein and Stat3 signaling regulate Hepcidin's transcription. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 2.7kb of the human Hepcidin promoter upstream of a firefly reporter gene. We used high throughput methods to screen 10,169 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1 standard deviation below the mean of the other chemicals (z-score >3 or <1), while not adversely affecting cell viability, quantified by fluorescence assay. Following validation assays, we identified 16 chemicals in a broad range of functional classes that promote Hepcidin expression. All of the chemicals identified increased expression of bone morphogenic protein-dependent and/or Stat3-dependent genes, however none of them strongly increased phosphorylation of Smad1,5,8 or Stat3.

Liver iron modulates hepcidin expression during chronically elevated erythropoiesis in mice

The liver-derived peptide hepcidin controls the balance between iron demand and iron supply. By inhibiting the iron export activity of ferroportin, hepcidin modulates iron absorption and delivery from the body's stores. The regulation of hepcidin, however, is not completely understood and includes a variety of different signals. We studied iron metabolism and hepcidin expression in mice constitutively overexpressing erythropoietin (Epo) (Tg6 mice), which leads to excessive erythropoiesis. We observed a very strong down-regulation of hepcidin in Tg6 mice that was accompanied by a strong increase in duodenal expression of ferroportin and divalent metal tranporter-1, as well as enhanced duodenal iron absorption. Despite these compensatory mechanisms, Tg6 mice displayed marked circulating iron deficiency and low levels of iron in liver, spleen, and muscle. To elucidate the primary signal affecting hepcidin expression during chronically elevated erythropoiesis, we increased iron availability by either providing iron (thus further increasing the hematocrit) or reducing erythropoiesis-dependent iron consumption by means of splenectomy. Both treatments increased liver iron and up-regulated hepcidin expression and the BMP6/SMAD pathway despite continuously high plasma Epo levels and sustained erythropoiesis. This suggests that hepcidin expression is not controlled by erythropoietic signals directly in this setting. Rather, these results indicate that iron consumption for erythropoiesis modulates liver iron content, and ultimately BMP6 and hepcidin. Analysis of the BMP6/SMAD pathway targets showed that inhibitor of DNA binding 1 (ID1) and SMAD7, but not transmembrane serine protease 6 (TMPRSS6), were up-regulated by increased iron availability and thus may be involved in setting the upper limit of hepcidin. We provide evidence that under conditions of excessive and effective erythropoiesis, liver iron regulates hepcidin expression through the BMP6/SMAD pathway.

The small molecule, genistein, increases hepcidin expression in human hepatocytes

Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP6) and interleukin-6 (IL-6) by effects on mothers against decapentaplegic homolog (Smad)4 or signal transducer and activator of transcription (Stat)3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone, genistein, from 28-52 hours postfertilization in zebrafish embryos enhanced Hepcidin transcript levels, as assessed by whole-mount in situ hybridization and quantitative real-time reverse-transcriptase polymerase chain reaction. Genistein's stimulatory effect was conserved in human hepatocytes: Genistein treatment of HepG2 cells increased both Hepcidin transcript levels and promoter activity. We found that genistein's effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either BMP response elements or the Stat3-binding site in the Hepcidin promoter. RNA sequencing of transcripts from genistein-treated hepatocytes indicated that genistein up-regulated 68% of the transcripts that were up-regulated by BMP6; however, genistein raised levels of several transcripts involved in Stat3 signaling that were not up-regulated by BMP6. Chromatin immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells. Conclusion: Genistein is the first small-molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes. (Hepatology 2013;58:1315–1325)

The Small Molecule Genistein Increases Hepcidin Expression by Activating Stat3 and Bone Morphogenic Protein Signaling

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. We conducted a small‐scale chemical screen to identify small molecules that modulate Hepcidin expression. We found that treatment of zebrafish embryos with genistein, a major dietary component of soybeans, enhanced hepcidin transcript levels. By treating human hepatocytes (HepG2), we found that genistein caused a significant increase in Hepcidin transcript levels and promoter activity. We discovered that genistein's effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either the Stat3 binding site or the bone morphogenic protein (BMP) response elements in the Hepcidin promoter. RNA sequencing, chromatinimmunoprecipitation, and ELISA experiments revealed that genistein enhanced BMP and Stat3 signaling in HepG2 cells. In conclusion, genistein is the first nontoxic small molecule identified that increases Hepcidin expression in vivo and in vitro. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes. Funding: March of Dimes, NIDDK, US Army Research Office.

A Chemical Screen Reveals New Modulators of Hepcidin Expression

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing the human Hepcidin promoter upstream of a firefly reporter gene. We then used high throughput methods to screen 10,360 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change &gt;;3 standard deviations above or &gt;;1.5 standard deviations below the mean of the other chemicals, while not adversely affecting cell viability. Using these criteria, we identified 32 small molecules that upregulated and 3 that downregulated Hepcidin expression. On retesting assays, we confirmed 22 of the initial positives (69%) and 1 of the initial negatives as regulators of Hepcidin expression. Functional classification of the positive regulators indicated: 4 anti‐inflammatory agents, 4 antimicrobials, 6 antineoplastic drugs, 4 kinase inhibitors, and 4 with other or unknown functions. We are now evaluating the mechanisms of action in human hepatocytes and in a zebrafish model. The best candidates will subsequently be tested in mouse models of iron overload syndromes. Funding: NIDDK R01 DK085250–01A1.

Hepatocyte growth factor activator inhibitor type 2 (HAI-2) modulates hepcidin expression by inhibiting the cell surface protease matriptase-2

Matriptase-2, a recently identified cell surface protease, is the key enzyme of iron homoeostasis modulating the expression of the liver peptide hormone hepcidin. HAI (hepatocyte growth factor activator inhibitor) types 1 and 2 (HAI-1 and HAI-2 respectively) have been shown to inhibit the close homologue, i.e. matriptase. By co-expressing matriptase-2 and the inhibitor HAI-2 we have identified HAI-2 displaying high inhibitory potential against matriptase-2 at the cell surface as well as in conditioned medium. Accordingly, complex formation between matriptase-2 and HAI-2 was demonstrated by isolation of the complex via immobilizing either HAI-2 or matriptase-2 from lysates and conditioned medium of co-expressing cells. Furthermore, HAI-2 indirectly influences the expression of the hepcidin-encoding gene HAMP. The inhibitor abrogates the matriptase-2-mediated suppression of HAMP expression, presumably by inhibiting the supposed potential of matriptase-2 to cleave membrane-bound HJV (haemojuvelin). Taken together, the results of the present study have characterized HAI-2 as an inhibitor of matriptase-2 that modulates the synthesis of hepcidin and provides new insights into the regulatory mechanism of iron homoeostasis, with clinical importance for a treatment of iron overload diseases.

The Effect of Vitamin D3 on Hepcidin and IL-8 Expression in Monocytes

Background: It has been shown that optimal levels of vitamin D3 offer many health benefits and confer resistance to many diseases, notably those related to metabolic syndrome. Vitamin D3 status is of particular relevance in elderly populations wherein deficiency of this vitamin is increasingly being reported. Anaemia is also a common finding with approximately 10 percent of persons older than 65 years of age suffering from mild to moderate anaemia. In about 1/3 of these patients, the type of anaemia observed is anaemia of inflammation (AI) and is linked to the presence of high levels of the iron regulatory peptide hormone hepcidin. Hepcidin is antimicrobial peptide (AMP) structurally similar to other members of this family, in particular the defensins. Hepcidin negatively regulates iron absorption in the intestine and inhibits reticuloendothelial macrophages from releasing recycled iron. During periods of infection and inflammation, hepcidin levels increase due to the action of many cytokines including IL-8. Hepcidin has therefore a dual role in the immune response. It has been demonstrated that in monocytes, vitamin D3 is also able to induce the expression of the AMP cathelicidin due to the presence of a vitamin D3 response element (VDRE) in promoter regions of the cathelicidin ( Camp ) gene. It is however currently unknown whether vitamin D3 also plays a role in the signalling pathway that controls hepcidin expression. Methods: In the present work, we address the possibility that vitamin D3 is able to modulate hepcidin expression by stimulating monocytic THP-1 cells with varying concentrations of vitamin D3, extracting mRNA and ascertaining hepcidin expression by RT-PCR and gel electrophoresis. We also postulate that vitamin D3 stimulation in these cells is able to regulate the expression of IL-8 and use the supernatant from cell culture to explore this hypothesis. Results: The results presented here confirm previous reports that these cells constitutively express hepcidin mRNA and extend these observations by demonstrating that vitamin D3 can indeed modulate hepcidin expression. Modulation was dependent on the concentration of vitamin D3 used with maximal effect at 10 nM under the conditions used. Results also demonstrate that non-stimulated THP-1 supernatants release low levels of IL-8 and treatment with vitamin D3 at all concentrations tested reduced the levels of IL-8. A further observation was that IL-8 levels were very similar after 4 and 20 hours of vitamin D3 treatment, suggesting that the production and secretion of IL-8 happened at relatively early time points. Conclusion : The present work has demonstrated that THP-1 cells when stimulated with higher concentrations of vitamin D3 (10 and 100 nM) decrease their expression of hepcidin mRNA while no change in the housekeeping gene GAPDH was observed. This downregulation of hepcidin mRNA expression may be translated in vivo with increases in serum iron and therefore may be potentially relevant in the treatment of AI/ACD. The present work also confirms previous reports that stimulation with vitamin D3 causes THP-1 cells to decrease the production of the pro-inflammatory cytokine IL-8, suggesting that vitamin D3 insufficiency may play a role in inflammatory disease and may offer new therapeutic options. doi: http://dx.doi.org/10.4021/jh71e

Hepcidin–Minireview

Iron related disorders are encountered in daily clinical settings. Maintenance of stable extracellular iron concentrations requires the coordinate regulation of iron transport into plasma from dietary sources in the duodenum, from recycled senescent red cells in macrophages and from storage in hepatocytes. Hepcidin acts as a systemic iron-regulatory hormone. Many human diseases are associated with alterations in hepcidin concentrations. This review has focused on hepcidin structure, kinetics and function, its correlation with iron metabolism disorders, the therapeutic potential for modulating hepcidin expression and the diagnostic potential of hepcidin measurements in clinical practice.

A Screen for Small Molecules That Regulate Hepcidin Expression

Abstract 611Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 3 kilobases of the human Hepcidin promoter upstream of a firefly reporter gene. We then used high throughput methods to screen 10,360 chemicals in duplicate from the Harvard Institute of Chemistry and Cell Biology library for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1.5 standard deviations below the mean of the other chemicals (z-score >3 or <-1.5), while not adversely affecting cell viability, quantified by a nonlytic fluorescence assay. Using these criteria, we identified 32 small molecules that upregulated and 3 that downregulated Hepcidin expression. Functional classification of the positive regulators indicated: 4 anti-inflammatory agents, 4 antimicrobials, 6 antineoplastic drugs, 6 kinase inhibitors, and 12 with other or unknown function. Of the positive modulators, two were flavones, consistent with our prior discovery that the isoflavone genistein upregulates Hepcidin expression. Of the negative regulators, one was a kinase inhibitor and two were of unknown function. Experiments are underway to characterize the mechanism of action of these regulators. The best candidates will subsequently be tested in mouse models of iron overload syndromes with the intention of developing new therapies for diseases in which Hepcidin is inappropriately regulated. Disclosures:No relevant conflicts of interest to declare.