Hepcidin Expression In Hepatocytes Research Articles

Serum Iron Overload Triggers the SMAD Pathway and Induces Hepcidin Expression in Hepatocytes through SMURF1.

Serum Iron Overload Triggers the SMAD Pathway and Induces Hepcidin Expression in Hepatocytes through SMURF1.

FKBP12 inhibits hepcidin expression by modulating BMP receptors interaction and ligand responsiveness in hepatocytes.

The expression of the iron regulatory hormone hepcidin in hepatocytes is regulated by the BMP-SMAD pathway through the type I receptors ALK2 and ALK3, the type II receptors ACVR2A and BMPR2, and the ligands BMP2 and BMP6. We previously identified the immunophilin FKBP12 as a new hepcidin inhibitor that acts by blocking ALK2. Both the physiologic ALK2 ligand BMP6 and the immunosuppressive drug Tacrolimus (TAC) displace FKBP12 from ALK2 and activate the signaling. However, the molecular mechanism whereby FKBP12 regulates BMP-SMAD pathway activity and thus hepcidin expression remains unclear. Here, we show that FKBP12 acts by modulating BMP receptor interactions and ligand responsiveness. We first demonstrate that in primary murine hepatocytes TAC regulates hepcidin expression exclusively via FKBP12. Downregulation of the BMP receptors reveals that ALK2, to a lesser extent ALK3, and ACVR2A are required for hepcidin upregulation in response to both BMP6 and TAC. Mechanistically, TAC and BMP6 increase ALK2 homo-oligomerization and ALK2-ALK3 hetero-oligomerization and the interaction between ALK2 and the type II receptors. By acting on the same receptors, TAC and BMP6 cooperate in BMP pathway activation and hepcidin expression both in vitro and in vivo. Interestingly, the activation state of ALK3 modulates its interaction with FKBP12, which may explain the cell-specific activity of FKBP12. Overall, our results identify the mechanism whereby FKBP12 regulates the BMP-SMAD pathway and hepcidin expression in hepatocytes, and suggest that FKBP12-ALK2 interaction is a potential pharmacologic target in disorders caused by defective BMP-SMAD signaling and characterized by low hepcidin and high BMP6 expression.

Melatonin Regulates Iron Homeostasis by Inducing Hepcidin Expression in Hepatocytes.

The pineal hormone, melatonin, plays important roles in circadian rhythms and energy metabolism. The hepatic peptide hormone, hepcidin, regulates iron homeostasis by triggering the degradation of ferroportin (FPN), the protein that transfers cellular iron to the blood. However, the role of melatonin in the transcriptional regulation of hepcidin is largely unknown. Here, we showed that melatonin upregulates hepcidin gene expression by enhancing the melatonin receptor 1 (MT1)-mediated c-Jun N-terminal kinase (JNK) activation in hepatocytes. Interestingly, hepcidin gene expression was increased during the dark cycle in the liver of mice, whereas serum iron levels decreased following hepcidin expression. In addition, melatonin significantly induced hepcidin gene expression and secretion, as well as the subsequent FPN degradation in hepatocytes, which resulted in cellular iron accumulation. Melatonin-induced hepcidin expression was significantly decreased by the melatonin receptor antagonist, luzindole, and by the knockdown of MT1. Moreover, melatonin activated JNK signaling and upregulated hepcidin expression, both of which were significantly decreased by SP600125, a specific JNK inhibitor. Chromatin immunoprecipitation analysis showed that luzindole significantly blocked melatonin-induced c-Jun binding to the hepcidin promoter. Finally, melatonin induced hepcidin expression and secretion by activating the JNK-c-Jun pathway in mice, which were reversed by the luzindole treatment. These findings reveal a previously unrecognized role of melatonin in the circadian regulation of hepcidin expression and iron homeostasis.

Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling.

Elevations in plasma phosphate concentrations (hyperphosphatemia) occur in chronic kidney disease (CKD), in certain genetic disorders, and following the intake of a phosphate-rich diet. Whether hyperphosphatemia and/or associated changes in metabolic regulators, including elevations of fibroblast growth factor 23 (FGF23) directly contribute to specific complications of CKD is uncertain. Here, we report that similar to patients with CKD, mice with adenine-induced CKD develop inflammation, anemia, and skeletal muscle wasting. These complications are also observed in mice fed high phosphate diet even without CKD. Ablation of pathologic FGF23-FGFR4 signaling did not protect mice on an increased phosphate diet or mice with adenine-induced CKD from these sequelae. However, low phosphate diet ameliorated anemia and skeletal muscle wasting in a genetic mouse model of CKD. Our mechanistic in vitro studies indicate that phosphate elevations induce inflammatory signaling and increase hepcidin expression in hepatocytes, a potential causative link between hyperphosphatemia, anemia, and skeletal muscle dysfunction. Our study suggests that high phosphate intake, as caused by the consumption of processed food, may have harmful effects irrespective of pre-existing kidney injury, supporting not only the clinical utility of treating hyperphosphatemia in CKD patients but also arguing for limiting phosphate intake in healthy individuals.

Hepcidin contributes to Swedish mutant APP-induced osteoclastogenesis and trabecular bone loss

Patients with Alzheimer’s disease (AD) often have lower bone mass than healthy individuals. However, the mechanisms underlying this change remain elusive. Previously, we found that Tg2576 mice, an AD animal model that ubiquitously expresses Swedish mutant amyloid precursor protein (APPswe), shows osteoporotic changes, reduced bone formation, and increased bone resorption. To understand how bone deficits develop in Tg2576 mice, we used a multiplex antibody array to screen for serum proteins that are altered in Tg2576 mice and identified hepcidin, a master regulator of iron homeostasis. We further investigated hepcidin’s function in bone homeostasis and found that hepcidin levels were increased not only in the serum but also in the liver, muscle, and osteoblast (OB) lineage cells in Tg2576 mice at both the mRNA and protein levels. We then generated mice selectively expressing hepcidin in hepatocytes or OB lineage cells, which showed trabecular bone loss and increased osteoclast (OC)-mediated bone resorption. Further cell studies suggested that hepcidin increased OC precursor proliferation and differentiation by downregulating ferroportin (FPN) expression and increasing intracellular iron levels. In OB lineage cells, APPswe enhanced hepcidin expression by inducing ER stress and increasing OC formation, in part through hepcidin. Together, these results suggest that increased hepcidin expression in hepatocytes and OB lineage cells in Tg2576 mice contributes to enhanced osteoclastogenesis and trabecular bone loss, identifying the hepcidin-FPN-iron axis as a potential therapeutic target to prevent AD-associated bone loss.

The Immunophilin FKBP12 Inhibits Hepcidin By Modulating BMP Type I-Type II Receptors Interaction and Ligand Responsiveness

Introduction The liver hormone hepcidin is the master regulator of iron metabolism that modulates iron release into the circulation by binding and blocking the iron exporter ferroportin (Nemeth et al., 2004). Hepcidin expression is under the control of the BMP-SMAD pathway (Babitt et al., 2006), whose activation requires the formation of a hexameric complex composed of a dimer of BMP receptors type I (BMPR-Is), a dimer of BMPR type II (BMPR-IIs) and dimeric ligands. ALK2 and ALK3, as BMPR-Is (Steinbiecker et al., 2011), BMPR2 and ACVR2A, as BMPR-IIs (Mayeur et al., 2014), and BMP2 (Koch et al, 2017) and BMP6 (Meynard et al., 2009), as ligands, control hepcidin expression in vivo. We previously demonstrated that the immunophilin FKBP12 limits hepcidin expression in hepatocytes by binding ALK2 (Colucci et al., 2017). However, the molecular mechanism whereby FKBP12 regulates ALK2 and its relationship with BMPR-IIs and ligands in the regulation of the BMP-SMAD pathway and hepcidin expression are still unclear. Methods: BMPR-Is dimerization was evaluated by co-immunoprecipitation (CoIP) experiments performed in the HuH7 human hepatoma cell line. BMP-SMAD pathway and hepcidin promoter activation were analyzed by using a reporter vector with the luciferase under the control of BMP responsive elements or of the human hepcidin promoter, respectively. Endogenous hepcidin expression was measured by qRT-PCR. Results: Since BMPRIs act as dimers, we first tested whether FKBP12 modulates the dimerization process. MYC- and FLAG-tagged ALK2 or ALK3 were transfected in HuH7 cells in the presence of FKBP12. Cells were treated or not with tacrolimus (TAC), an immunosuppressive drug that sequesters FKBP12 from ALK2. FKBP12 promotes ALK2 homodimers, functionally inactive in the absence of ligands, with no effect on ALK3 homodimerization. TAC promotes increased ALK2 homodimerization and SMAD1/5/8 phosphorylation, demonstrating that in the absence of FKBP12, ALK2 homodimers are stabilized and functionally active. We next focused on BMP6, the physiologic ligand that binds preferentially ALK2 and plays a fundamental role in hepcidin regulation in vivo. In HuH7 cells transfected with FKBP12 and ALK2, BMP6 treatment reduced FKBP12-ALK2 binding and increased ALK2 homodimers. In agreement, SMAD1/5/8 phosphorylation was increased, indicating that FKBP12 displacement allows the formation of functional receptor complexes responsive to BMP6. BMPR-Is activate SMAD1/5/8 following BMPR-IIs phosphorylation. Since TAC induces SMAD1/5/8 phosphorylation in the absence of ligands, we hypothesized that FKBP12 displacement also affects the formation of BMPR-I/BMPR-II oligomers. HuH7 cells were transfected with ALK2, BMPR2 or ACVR2A and FKBP12, and treated or not with TAC. FKBP12 sequestration by TAC enhances the ALK2-BMPR2 and ALK2-ACVR2A interaction and accordingly activates SMAD1/5/8 signaling. Given that FKBP12 modulates BMP receptor interaction, we wondered how this functionally impacts on the response to BMP ligands, as BMP2, that guarantees basal hepcidin levels by binding ALK3, and BMP6, upregulated in iron overload that signals preferentially through ALK2. ALK3 upregulates the BMP pathway and hepcidin expression in a similar way in response to BMP2 and BMP6, in agreement with the evidence that both ligands bind ALK3. ALK2, which failed to activate the pathway in the absence ligands, leads to a greater response to BMP6, consistent with the fact that it is the BMP6 receptor. Thus FKBP12 quantitatively, rather than qualitatively, modulates the BMP-SMAD pathway activation in response to BMP ligands. Conclusions: Altogether our results clarify the molecular mechanisms of hepcidin regulation demonstrating that: 1) FKBP12 limits hepcidin expression by inducing the formation of inactive ALK2 homodimers in the absence of ligands. 2) Decreased FKBP12 binding to ALK2, by TAC or BMP6, favors the formation of active ALK2 homodimers. 3) FKBP12 sequestration increases the binding of ALK2 with the BMPR-IIs, thus favoring SMAD1/5/8 phosphorylation and pathway activation. 4) FKBP12 quantitatively modulates the response of BMPRIs to the ligands BMP2 and BMP6. Disclosures Camaschella: Vifor Iron Core: Consultancy; Celgene: Consultancy; Novartis: Consultancy.

Heparin inhibits intracellular Mycobacterium tuberculosis bacterial replication by reducing iron levels in human macrophages

Iron is a crucial micronutrient for both mammals and their associated pathogens, and extensive literature has shown that Mycobacterium tuberculosis (Mtb) bacilli inhibited from acquiring iron from the host are severely attenuated. In contrast, increased dietary iron concentrations or patients with hemochromatosis have long been associated with a more severe tuberculosis (TB) disease outcome. We have observed that upon macrophage infection, Mtb bacilli strongly promote intracellular iron sequestration, both through increased expression of hepcidin, a key mammalian iron regulatory protein, and downregulation of the iron exporter protein, ferroportin. Heparin is a highly sulfated glycosaminoglycan released by mast cells and basophils at sites of tissue injury. During Mtb infection, heparin alters intracellular trafficking in alveolar epithelial cells and decreases extrapulmonary dissemination but recently, heparin also has been reported to inhibit hepcidin expression in hepatocytes, decreasing intracellular iron availability. In this report, we demonstrate that heparin significantly reduces hepcidin expression in macrophages infected with Mtb bacilli. Heparin-treated macrophages have higher ferroportin expression compared to untreated macrophages, promoting iron export and decreasing iron availability to intracellular bacilli. Thus, here we describe a novel immunomodulatory effect and potential therapeutic role for heparin against mycobacterial infection in human macrophages.

Interleukin-1β (IL-1β) transcriptionally activates hepcidin by inducing CCAAT enhancer-binding protein δ (C/EBPδ) expression in hepatocytes

Hepcidin is a liver-derived hormone that negatively regulates serum iron levels and is mainly regulated at the transcriptional level. Previous studies have clarified that in addition to hepatic iron levels, inflammation also efficiently increases hepatic hepcidin expression. The principle regions responsible for efficient hepcidin transcription are bone morphogenetic protein-responsive elements (BMP-REs) 1 and 2 as well as the signal transducer and activator of transcription 3-binding site (STAT-BS). Here, we show that the proinflammatory cytokine interleukin-1β (IL-1β) efficiently increases hepcidin expression in human HepG2 liver-derived cells and primary mouse hepatocytes. The primary region responsible for IL-1β-mediated hepcidin transcription was the putative CCAAT enhancer-binding protein (C/EBP)-binding site (C/EBP-BS) at the hepcidin promoter spanning nucleotides -329 to -320. IL-1β induces the expression of C/EBPδ but neither C/EBPα nor C/EBPβ in hepatocytes, and C/EBPδ bound to the C/EBP-BS in an IL-1β-dependent manner. Lipopolysaccharide (LPS) induced the expression of IL-1β in Kupffer cells and hepatocytes in the mouse liver; furthermore, the culture supernatants from the macrophage-like cell line RAW264.7 treated with LPS potentiated the stimulation of hepcidin expression in hepatocytes. The present study reveals that: 1) inflammation induces IL-1β production in Kupffer cells and hepatocytes; 2) IL-1β increases C/EBPδ expression in hepatocytes; and 3) induction of C/EBPδ activates hepcidin transcription via the C/EBP-BS that has been uncharacterized yet. In cooperation with the other pathways activated by inflammation, IL-1β pathway stimulation leads to excess production of hepcidin, which could be causative to anemia of inflammation.

Hepcidin Upregulation By Inflammation Is Not Causally Related to Liver Activation of Smad1/5/8 Signaling By Activin B

Hepcidin induction during inflammation is partly due to direct transcriptional regulation by the IL6/STAT3 pathway. However, SMAD1/5/8 signaling is also believed to have a role in hepcidin regulation during inflammation, as inhibitors of BMP type I receptors or the BMP ligand antagonist ALK3-Fc block hepcidin induction, increase iron availability, and ameliorate anemia in different animal models of inflammation.We previously observed that LPS stimulates liver Smad1/5/8 signaling even in Bmp6-deficient mice and our data suggested that, rather than Bmp6, activin B could be the activating ligand of this pathway during inflammation. There was indeed a dramatic induction of Inhbb mRNA, encoding activin B, in the liver of mice challenged with LPS, slightly preceding an increase in Smad1/5/8 phosphorylation and hepcidin (Hamp) mRNA. In liver cells in vitro, activin B stimulated not only canonical Smad2/3 but also non-canonical Smad1/5/8 signaling and hepcidin expression. Finally, pretreatment with a BMP type I receptor inhibitor showed that the effect of activin B on hepcidin expression in liver cells was entirely attributable to its effect on non-canonical Smad1/5/8 signaling. However, although these data demonstrate that activin B potently crossactivates non-canonical Smad1/5/8 signaling to induce hepcidin expression in hepatocytes in vitro, they do not definitively prove the role of activin B in hepcidin induction in vivo. Therefore, the goal of the present study was to challenge Inhbb-/- mice (deficient in activin B) with LPS or infect them with E. Coli and examine whether, as expected from the in vitro data, the lack of activin B prevents stimulation of both canonical Smad2/3 and non-canonical Smad1/5/8 signaling and induction of hepcidin in these mice.We first showed that activin B is actually the ligand that in vivo induces hepatic Smad2/3 and Smad1/5/8 phosphorylation in response to inflammatory stimuli such as LPS and bacterial infections. Indeed, these signaling pathways are no longer activated in Inhbb-/- mice (Fig. 1A). Interestingly however, we found that the lack of activin B and, as a consequence, the lack of activation of Smad1/5/8 signaling does not impair the induction of hepatic hepcidin expression by these inflammatory stimuli (Fig. 1B), illustrating the limitations of in vitro studies in simulating what is actually going on inside a liver. In conclusion, although activin B is directly responsible for liver activation of Smad1/5/8 signaling in vivo, this signaling pathway is not governing upregulation of hepcidin production in animals submitted to inflammatory stimuli.We also noticed that the level of Smad1/5/8 phosphorylation in the liver of mice challenged with LPS is not correlated with the expression of hepcidin. Indeed, although LPS-treated Bmp6-/- and wild-type mice have similar activation of Smad1/5/8 (Fig. 2A), the amount of circulating hepcidin in Bmp6-/- mice is about three times lower than in wild-type mice (Fig. 2B). This could indicate that induction of Smad1/5/8 signaling by inflammatory stimuli takes place in non-parenchymal cells rather than in hepatocytes and has no impact on hepcidin expression. Further investigations are necessary to determine in which liver cells activin B activates the canonical Smad2/3 and non-canonical Smad1/5/8 signaling observed in this study, and what are the exact target genes induced by this signaling. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Validation of the Intrinsic Hepcidin IDxTM Test, an LDT for Clinical Hepcidin Assessment

▪Introduction: Hepcidin is the principal regulator of iron absorption and iron recycling, and is required to provide iron for erythropoiesis. Hepcidin is a peptide hormone that binds to the sole iron channel ferroportin (Fpn), and blocks iron efflux by degrading Fpn, trapping iron stored in the cell. Dysregulation of hepcidin by genetic mutations affecting hepcidin expression in hepatocytes is responsible for hereditary hemochromatosis where too little hepcidin leads to iron-overload disease, and iron refractory iron deficiency anemia (IRIDA) where hepcidin expression is inappropriately high leading to anemia. Hepcidin itself is regulated by iron and the inflammatory cytokine IL-6. Thus, hepcidin is dysregulated in most inflammatory diseases and chronic infections. Interest in developing diagnostic applications for hepcidin in iron disorders is high.Objective: We developed a high-affinity monoclonal antibody (mAb) to hepcidin and used it to produce a competitive ELISA (C-ELISA) similar to our first RUO test (Ganz et al. 2008). We determined the performance characteristics using CLIA analytical method validation guidelines. The Intrinsic Hepcidin IDx Test is used for serum and Li-Heparin plasma. We determined the reference range in adult first time blood donors (n=292) and studied an iron deficient (ID) sub-set of the donors.Methods: We developed mouse anti-human hepcidin monoclonal antibodies and coated, blocked, and dried the plates in a temperature and humidity controlled incubator. We used highly purified synthetic hepcidin as reference standard and a biotinylated hepcidin analog as the competitive tracer for the C-ELISA (Peptides International, Louisville, KY). The Intrinsic Hepcidin IDx Test is fully automated on the Biomek FX platform. Serum from first time blood donors (n=292) was obtained, and ferritin, transferrin, plasma iron, CRP, and hepcidin were measured. The reference range for hepcidin was determined and the normal iron status range determined by eliminating iron deficient (ID) donors or those shown to be iron overloaded. Receiver operator characteristic curves (ROC) were plotted to determine the utility of hepcidin and to identify optimal cutoffs of hepcidin as a test of iron deficiency.Results: The mAb used in the Intrinsic Hepcidin IDx Test is specific for human hepcidin. The analytical measurement range (AMR) is 4 - 200 ng/ml. The intra-assay precision CVs range between 7% (at 12 ng/ml) and 4% (at 99 ng/ml). The between-day precision CVs range between 7% (12 ng/ml) and 9% (100 ng/ml). The mean spike recovery across the AMR is 3%. Hepcidin was stable at 4oC for up to 7 days and if specimens are stored at -20oC, for at least 6 months. Hepcidin concentrations determined in blood donors were highly correlated with those measured using our polyclonal RUO C-ELISA (r = 0.95, p<0.001). The reference range is 4.4-54.1 ng/ml for women and 6.1-91.2 ng/ml for men. Clear gender differences were observed: hepcidin was higher in men (median 28.5 ng/ml, n = 143) than in women (median 13.1 ng/ml, n = 149). In addition, the ID group had lower hepcidin (median 6.7 ng/ml) than the normal iron status group (median 21.4 ng/ml, p<0.0001). The area under the ROC curve for hepcidin compared with various ferritin concentrations was greater than 0.90. An optimal hepcidin cutoff less than 10 ng/ml was determined to predict ID with a correct classification rate of greater than 86%.Conclusion: We developed and validated a sensitive, accurate and reproducible C-ELISA, the Intrinsic Hepcidin IDx test, using CLIA validation guidelines. We determined the hepcidin reference ranges by analyzing first-time blood donor serum. We found a significant difference in median hepcidin values between genders. Also, our data show that hepcidin was lower in donors with ID compared to iron replete, healthy donors. DisclosuresGutschow:Intrinsic LifeSciences: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment. Han:Intrinsic Lifesciences.: Employment, Equity Ownership. Ostland:Intrinsic LifeScienc s: Employment, Equity Ownership. Olbina:Intrinsic LifeSciences: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment, Equity Ownership.

Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation.

Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb(th1/th1) (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

Commensal Bacteria-induced Interleukin 1β (IL-1β) Secreted by Macrophages Up-regulates Hepcidin Expression in Hepatocytes by Activating the Bone Morphogenetic Protein Signaling Pathway

The liver hormone hepcidin is the central regulator of systemic iron metabolism. Its increased expression in inflammatory states leads to hypoferremia and anemia. Elucidation of the mechanisms that up-regulate hepcidin during inflammation is essential for developing rational therapies for this anemia. Using mouse models of inflammatory bowel disease, we have shown previously that colitis-associated hepcidin induction is influenced by intestinal microbiota composition. Here we investigate how two commensal bacteria, Bifidobacterium longum and Bacteroides fragilis, representative members of the gut microbiota, affect hepcidin expression. We found that supernatants of a human macrophage cell line infected with either of the bacteria up-regulated hepcidin when added to a human hepatocyte cell line. This activity was abrogated by neutralization of IL-1β. Moreover, purified IL-1β increased hepcidin expression when added to the hepatocyte line or primary human hepatocytes and when injected into mice. IL-1β activated the bone morphogenetic protein (BMP) signaling pathway in hepatocytes and in mouse liver, as indicated by increased phosphorylation of small mothers against decapentaplegic proteins. Activation of BMP signaling correlated with IL-1β-induced expression of BMP2 in human hepatocytes and activin B in mouse liver. Treatment of hepatocytes with two different chemical inhibitors of BMP signaling or with a neutralizing antibody to BMP2 prevented IL-1β-induced up-regulation of hepcidin. Our results clarify how commensal bacteria affect hepcidin expression and reveal a novel connection between IL-1β and activation of BMP signaling. They also suggest that there may be differences between mice and humans with respect to the mechanism by which IL-1β up-regulates hepcidin.

IL-1β secreted by macrophages in response to commensal bacteria acts together with bone morphogenetic protein signals to increase hepcidin expression in hepatocytes (MUC5P.758)

Abstract Elevated expression of the iron regulating hormone hepcidin leads to abnormalities of iron metabolism in inflammatory diseases. The mechanisms involved in inflammation-associated hepcidin upregulation are not well understood. We have shown in mouse IBD models that the commensal microbiota significantly influences hepcidin expression. Here we describe experiments to elucidate how commensal bacteria upregulate hepcidin. Differentiated THP-1 macrophages were infected with the commensal Bifidobacterium infantis. Sterile supernatants of the infected, but not uninfected, THP-1 cells increased hepcidin expression in HuH7 human hepatocytes, whereas direct infection of HuH7 cells failed to induce hepcidin. The infected THP-1 cells secreted IL-1β, neutralization of which significantly inhibited induction of hepcidin expression in the HuH7 cells. Pretreatment of the HuH7 cells with inhibitors of the bone morphogenetic protein (BMP) signaling pathway also prevented the upregulation of hepcidin by the supernatant of B. infantis-infected THP-1 cells. Our findings demonstrate that IL-1β secreted by macrophages in response to commensal bacteria can induce increased expression of hepcidin in hepatocytes. An activator of the BMP signaling pathway is also involved but the identity and source of this molecule are yet to be determined. Furthermore, our results suggest that neutralization of IL-1β may be useful in preventing or treating the dysregulation of iron homeostasis associated with IBD.

Transferrin Receptor 2 and HFE Regulate Hepcidin Levels Independently of Bmp6 and Hemojuvelin

Hereditary hemochromatosis (HH) is a genetically heterogeneous disorder characterized by elevated iron absorption from the diet, with consequent iron overload and tissue injury. Adult-onset forms of HH are caused by mutations in the HFE gene and in the gene for transferrin receptor 2 (TFR2). Human patients and mouse models of TFR2 and HFE-related HH show inappropriately low expression of hepcidin, the central regulator of iron metabolism. However, although these genes have been discovered far more than a decade ago, the mechanisms by which HFE and TFR2 influence hepcidin expression remain unclear.The bone morphogenetic protein BMP6 plays a key role in the regulation of hepcidin expression. BMP6 binds to type I (ALK3) and type II serine threonine kinase receptors, and to the coreceptor hemojuvelin (HJV), which phosphorylates intracellular SMAD proteins. Phosphorylated SMADs then bind to SMAD4 and translocate to the nucleus to induce the transcription of hepcidin. Inactivation of Bmp6 or Hjv in mice leads to considerably reduced hepcidin production and severe hepatic iron overload. However, there are major differences in hepcidin expression and extrahepatic tissue iron loading between Bmp6 or Hjv KO males and females, due to the suppressive effect of testosterone on hepcidin in males. In contrast to males, Bmp6-/- and Hjv-/-females still produce some hepcidin and do not massively accumulate iron in the pancreas, heart, or kidneys.The goal of this study was to investigate the role of Hfe and Tfr2 in the residual hepcidin production observed in the absence of Bmp6 in females. We used Bmp6-/-, Tfr2-/-, and B2m-/- mice to generate wild-type, single KO (Bmp6-/-, Tfr2-/-, or B2m-/-) and double KO (Tfr2-/- and Bmp6-/-, or B2m-/- and Bmp6-/-) mice, and we assessed Smad5 phosphorylation, hepcidin expression, and the sites of iron accumulation in the different groups of mice. Notably, B2m-/- mice develop spontaneously hepatic iron overload with a distribution similar to that seen in the liver of Hfe-/-mice and the lack of CD8+ lymphocytes and the absence of classical class I molecules in these mice are not responsible for their iron phenotype.Interestingly, the lack of functional Hfe or the lack of Tfr2 in Bmp6-/- females leads to a very similar phenotype that is much more severe than the single impairment of Bmp6, with massive iron loading in extrahepatic tissues, most notably the exocrine pancreas, the heart, and the kidney. Hepcidin mRNA and pSmad levels in the two categories of double KO females were much more strongly downregulated than in single Bmp6-/- females and, in contrast to Bmp6-/-females, no protein was detectable by ELISA in the double KO mice.Our findings clearly demonstrate that Hfe and Tfr2 regulate hepcidin production independently of Bmp6. The symmetrical phenotype of double Bmp6 and Tfr2, or double Bmp6 and Hfe KO mice suggests that Hfe and Tfr2 could participate in the same signaling complex affecting pSmad levels, even if they do not physically interact. Two complexes, one made of ALK3 and HJV, and the other made of ALK2, ALK3, HFE, and TFR2, very likely affect pSMAD levels independently of each other. Signaling through the second complex occurs in the absence of BMP6 and is most probably initiated by another BMP, for instance BMP2 or BMP4 that are also expressed in the liver and, although not regulated by iron, are capable of stimulating hepcidin expression in hepatocytes. Regulation of hepcidin expression by these two complexes would explain why, whereas Alk3, double Bmp6/Hfe, and double Bmp6/Tfr2 deficient females present with iron overload in extrahepatic tissues as do hepcidin KO mice, Bmp6 and Hjv single KO females present with early-onset iron overload only in the liver, similarly to patients with juvenile HH, and mice KO for Alk2, Hfe, or Tfr2 have the least severe phenotype, comparable with that of patients with adult-onset HH. Notably, TFR2 was also shown to localize in lipid rafts where it promotes MAPK activation. Thus, in addition to its role in the complex affecting pSMAD levels, TFR2 could also regulate hepcidin through a parallel pathway involving ERK1/2 signaling. This would explain the more severe phenotype of mice with combined deletion of Hfe and Tfr2 and the fact that, whereas Tfr2-/- mice do not respond to acute iron loading, Hfe-/-mice still have a significant, although blunted hepcidin response.This work was funded in part by FRM (DEQ2000326528) and ANR (ANR-13-BSV3-0015-01). DisclosuresNo relevant conflicts of interest to declare.

HFE interacts with the BMP type I receptor ALK3 to regulate hepcidin expression

AbstractMutations in HFE are the most common cause of hereditary hemochromatosis (HH). HFE mutations result in reduced expression of hepcidin, a hepatic hormone, which negatively regulates iron absorption from the duodenum and iron release from macrophages. However, the mechanism by which HFE regulates hepcidin expression in hepatocytes is not well understood. It is known that the bone morphogenetic protein (BMP) pathway plays a central role in controlling hepcidin expression in the liver. Here we show that HFE overexpression increased Smad1/5/8 phosphorylation and hepcidin expression, whereas inhibition of BMP signaling abolished HFE-induced hepcidin expression in Hep3B cells. HFE was found to associate with ALK3, inhibiting ALK3 ubiquitination and proteasomal degradation and increasing ALK3 protein expression and accumulation on the cell surface. The 2 HFE mutants associated with HH, HFE C282Y and HFE H63D, regulated ALK3 protein ubiquitination and trafficking differently, but both failed to increase ALK3 cell-surface expression. Deletion of Hfe in mice resulted in a decrease in hepatic ALK3 protein expression. Our results provide evidence that HFE induces hepcidin expression via the BMP pathway: HFE interacts with ALK3 to stabilize ALK3 protein and increase ALK3 expression at the cell surface.

The Role Of Liver Endothelial Sinusoidal Cells In Iron-Mediated Bmp6 Regulation

IntroductionIron-dependent regulation of hepcidin responds to circulating (holo-Tf) and hepatic iron. Although the signals that modulate hepcidin according to holo-Tf are still unknown, hepatic iron regulates hepcidin expression through transcriptional modulation of Bmp6. Bmp6 is controlled by iron only in the liver. Heterogeneous cell types with different functions in the liver include hepatocytes (HC) and non-parenchymal cells as Kupffer cells (KC), sinusoidal endothelial cells (LSEC), stellate cells. It has been reported that LSEC and KC, but not HC, upregulate Bmp6 independently of intracellular iron (Enns et al., Plos One 2013). Here we extend this observation analyzing Bmp6 regulation in isolated liver cells after acute and chronic changes of the iron status in wild type mice. In addition we studied pathogenic models of iron deregulation due to low (iron loaded Hjv KO mice) and high (iron deficient Tmprss6 KO mice) hepcidin. MethodsMice were maintained in accordance with the European Union guidelines. The study was approved by the IACUC of San Raffaele Scientific Institute, Milan, Italy. For chronic changes of the iron status, four weeks-old male mice were maintained an iron-replete (IB, 200 mg/kg iron), iron-deficient diet (ID, <3 mg /kg iron) or iron-loaded (IL, 8.3 g/kg iron) diet for 3 weeks. To induce acute iron changes, we used two approaches: iron dextran injection (1 g/kg body weight i.p.) or 2 weeks ID diet followed by 1, 3 and 6 days of IB, ID or IL diet. Hjv and Tmprss6 KO mice were studied in basal conditions at 7-8 weeks. Liver cells were isolated according to Liu et al. (Proteomics 2011) and cell purity validated by mRNA expression of specific genes. RNA was extracted using RNeasy Mini kit. Total RNA was retro-transcribed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem). Gene expression levels were measured by quantitative real-time PCR using TaqMan Gene Expression Master Mix (Applied Biosystem). Transferrin saturation (TS) and liver iron content (LIC) was measured as previously described (Pagani et al. Blood 2011). ResultsIn wild type animals hepcidin in basal condition is expressed exclusively in HC, whereas Bmp6 is expressed in non-parenchymal cells, mainly LSEC. In all cell types, Bmp6 levels inversely correlate with Tfr1 expression, suggesting that cellular iron influences Bmp6. In mice chronically maintained an IB, ID and IL diet HC, LSEC and KC modulate Bmp6 according to both cellular iron and TS. To investigate which cell type first regulates Bmp6, we injected wild type mice with a single dose of iron dextran and separated cells 3 and 6 hrs post injection. At 6 hrs, when TS and LIC are increased and Hamp is activated in HC, only LSEC upregulate Bmp6. As for the pathogenic models, Bmp6 is upregulated in HC, LSEC and KC cells in Hjv KO mice and downregulated in Tmprss6 KO animals. However, at difference with HC, regulation of Bmp6 in LSEC and KC is independent on cellular iron (as shown by Tfr1 mRNA), and related to TS in both animal models. To distinguish between the contribution of cellular and circulating iron in the regulation of Bmp6, we fed mice previously treated with an ID diet, with an ID, IB and IL diet for 1-3-6 days. At day 1, TS increases but LIC does not change in IB and ID mice while total liver Bmp6 is modulated according to TS. These results will be analyzed in separated cells. In vitro, exogenous BMP6 transcriptionally activates BMP6 expression in hepatoma-derived cells, even at short time point, suggesting that a paracrine mechanism contributes to BMP6 regulation. ConclusionsIn our hands Bmp6 expression is modulated not only in LSEC and KC but also in HC in animals fed a variable iron content diet. LSEC are the first iron “sensor” that, through Bmp6, activates hepcidin expression in HC. Bmp6 in LSEC and KC responds mainly to TS changes. We also suggest that an autocrine/paracrine mechanism activates liver Bmp6 to amplify signaling for hepcidin increase. Disclosures:No relevant conflicts of interest to declare.

Increased Iron Loading Induces Bmp6 Expression in the Non-Parenchymal Cells of the Liver Independent of the BMP-Signaling Pathway

Bone morphogenetic protein 6 (BMP6) is an essential cytokine for the expression of hepcidin, an iron regulatory hormone secreted predominantly by hepatocytes. Bmp6 expression is upregulated by increased iron-levels in the liver. Both hepatocytes and non-parenchymal liver cells have detectable Bmp6 mRNA. Here we showed that induction of hepcidin expression in hepatocytes by dietary iron is associated with an elevation of Bmp6 mRNA in the non-parenchymal cells of the liver. Consistently, incubation with iron-saturated transferrin induces Bmp6 mRNA expression in isolated hepatic stellate cells, but not in hepatocytes. These observations suggest an important role of the non-parenchymal liver cells in regulating iron-homeostasis by acting as a source of Bmp6.

Treatment with the Small Molecule Genistein Increases Hepcidin Expression In Zebrafish Embryos and Human Hepatocytes

AbstractAbstract 4245Hepcidin, a transcriptionally regulated peptide hormone, produced by hepatocytes in response to iron overload, 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 patients. Among patients with hereditary hemochromatosis, premenopausal women exhibit less severe iron overload than their male counterparts. While this has been attributed to menstrual blood loss, we hypothesized that estrogens may play a role in regulating Hepcidin expression. We tested estradiol and the phytoestrogens, genistein, apigenin, daizdein, and genistin, for their effects on hepcidin expression in zebrafish embryos. In addition to its estrogenic effects, genistein is known to inhibit multiple protein tyrosine kinases. We found that genistein treatment from 28–52 hours post-fertilization in zebrafish embryos enhanced hepcidin transcript levels relative to liver size, as assessed by whole mount in situ hybridization, while estradiol and the other phytoestrogens did not produce a similar effect. To evaluate whether these effects were conserved in human hepatocytes, we treated HepG2 cells with genistein for 24 hours. We found that genistein treatment was associated with a significant increase in Hepcidin transcript levels, which was abrogated by co-treatment with the bone morphogenic protein (BMP) signaling antagonist dorsomorphin, but not by co-treatment with the estrogen receptor antagonist ICI 182,780. In conclusion, genistein promotes Hepcidin expression in hepatocytes in a BMP-dependent, but estrogen receptor independent manner. Future experiments will address the signaling pathway by which genistein exerts its effect on Hepcidin expression. Disclosures:No relevant conflicts of interest to declare.

EPO‐mediated reduction in Hamp expression in vivo corrects iron deficiency anaemia in TMPRSS6 deficiency

Hepcidin, a 25 amino acid liver-derived peptide, has been recognized as a regulator of iron homeostasis. Hepcidin levels are negatively correlated with erythropoietic expansion, consistent with erythrocytes representing the major compartment that utilizes iron and hepcidin the major regulator for limiting iron availability (Nemeth, 2008). However, the molecular pathway transmitting the signal from proliferating erythrocytes to the regulation of hepcidin expression in hepatocytes has not been clearly defined.

Down-regulation of Bmp/Smad signaling by Tmprss6 is required for maintenance of systemic iron homeostasis

AbstractIron-refractory, iron-deficiency anemia (IRIDA) is a familial disorder characterized by iron deficiency anemia unresponsive to oral iron treatment but partially responsive to intravenous iron therapy. Previously, we showed that IRIDA patients harbor loss-of-function mutations in TMPRSS6, a type II transmembrane serine protease primarily expressed by the liver. Both humans and mice with TMPRSS6 mutations show inappropriately elevated levels of the iron-regulatory hormone hepcidin, suggesting that TMPRSS6 acts to negatively regulate hepcidin expression. Here we investigate the relationship between Tmprss6 and the bone morphogenetic protein (BMP)–Smad signaling pathway, a key pathway promoting hepcidin transcription in hepatocytes. We show that livers from mice deficient for Tmprss6 have decreased iron stores and decreased Bmp6 mRNA, but markedly increased mRNA for Id1, a target gene of Bmp6 signaling. In contrast, mice deficient for both Tmprss6 and hemojuvelin (Hjv), a BMP coreceptor that augments hepcidin expression in hepatocytes, showed markedly decreased hepatic levels of hepcidin and Id1 mRNA, markedly increased hepatic Bmp6 mRNA levels, and systemic iron overload similar to mice deficient for Hjv alone. These findings suggest that down-regulation of Bmp/Smad signaling by Tmprss6 is required for regulation of hepcidin expression and maintenance of systemic iron homeostasis.